Linköping University Medical DissertationsNo. 751

Lysophosphatidic acidPhysiological effects and

structure-activity relationships

Ulrika K. Nilsson

Division of PharmacologyDepartment of Medicine and Care

Faculty of Health Sciences, SE-581 85 Linköping, Sweden

Linköping 2002

© 2002 Ulrika K. Nilsson

ISBN 91-7373-192-7ISSN 0345-0082

Printed in Sweden by UniTryckLinköping 2002

One never notices what has been done;one can only see what remains to be done…

Marie Curie 1894 (1867-1934)

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CONTENTS__________________________________

PAPERS 3

ABSTRACT 5

ABBREVIATIONS 7

INTRODUCTION 9

REVIEW OF THE LITERATURE 11LPA AND OTHER PHOSPHOLIPIDS 11Biochemistry of LPA 11Production, release, and degradation of LPA 12Identification of LPA receptor genes 15Expression patterns of LPA receptors 18Signaling by LPA receptors 18Biological responses of LPA 23LPA in the cardiovascular system 24LPA in the reproductive tract 25Structure-activity relationships of LPA 28

ADRENALINE AND NORADRENALINE 29Identification and expression patterns of α2-ARs 29Signaling by α2-ARs 30α2-ARs in the cardiovascular system 30α2-ARs in the reproductive tract 31

METHODOLOGICAL CONSIDERATIONS 33Cell culturing 33Cell characterization 35Radioligand binding to myometrial SMC membranes 36Reverse transcriptase-polymerase chain reaction 37[3H]thymidine incorporation in SMCs 38Western blot analysis of protein tyrosine kinases 39Measurement of [Ca2+]i in different cell types 40Synthesis of LPA enantiomers and LPA analogues 41Platelet preparation 42Analyzes of platelet aggregation 43Statistical and data analyzes 44Ethical considerations 44

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RESULTS AND DISCUSSION 45HUMAN SMCs 45Cultured myometrial SMCs 45Myometrial SMCs express LPA receptors and α2-ARs 48LPA and noradrenaline stimulate DNA-synthesis 50

in myometrial SMCsGi/o-proteins and cAMP regulate LPA- and noradrenaline- 53

induced DNA synthesisLPA and noradrenaline activate protein tyrosine kinases 55LPA induces cytosolic Ca2+ and CaM kinase responses 56LPA stimulates EGF receptor activation 58PTX inhibits EGF-induced DNA-synthesis 61

HUMAN PLATELETS AND HEL CELLS 64HEL cells express LPA receptors 64LPA induces cytosolic Ca2+ responses 65The structure of LPA is important for cell activation 69LPA and adrenaline can act synergistically 74LPA has potential clinical applications 78

CONCLUSIONS 81

ACKNOWLEDGEMENTS 83

REFERENCES 85

PAPER I-IV

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PAPERS_____________________________________

This thesis is based on the following papers, which will be referred to inthe text by their Roman numerals. Furthermore, some unpublishedobservations are also included in the Results and Discussion section.

I ”Different proliferative responses of Gi/o-protein-coupled receptorsin human myometrial smooth muscle cells: a possible role ofcalcium”Ulrika K. Nilsson, Magnus Grenegård, Göran Berg and Samuel P.S.Svensson.Journal of Molecular Neuroscience 1998, 11: 11-21.

II ”Inhibition of Ca2 +/calmodulin-dependent protein kinase orepidermal growth factor receptor tyrosine kinase abolisheslysophosphatidic acid-mediated DNA-synthesis in humanmyometrial smooth muscle cells”Ulrika K. Nilsson and Samuel P.S. Svensson.Cell Biology International 2002, accepted for publication.

III ”Synergistic activation of human platelets by adrenaline andlysophosphatidic acid”Ulrika K. Nilsson, Magnus Grenegård and Samuel P.S. Svensson.Haematologica 2002, 87: 730-739.

IV ”Lack of stereospecificity in lysophosphatidic acid enantiomer-induced calcium mobilization in human erythroleukemia cells”Ulrika K. Nilsson, Rolf G.G. Andersson, Johan Ekeroth, Elisabeth C.Hallin, Peter Konradsson, Jan Lindberg, and Samuel P.S. SvenssonSubmitted for publication.

The papers are reprinted with the kind permission from Humana Press(I) and Fondazione Ferrata Storti (III).

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ABSTRACT__________________________________

Lipids have previously been considered primarily as building blocks ofthe cell membrane, but are now also recognized as important cellsignaling molecules. Lysophosphatidic acid (LPA) is a glycerophospho-lipid consisting of a phosphate head group, a linker region, and alipophilic tail. LPA has earlier been shown to exert a diversity of cellulareffects such as aggregation, apoptosis, contraction, migration, andproliferation. The effects of LPA are elicited by activation of its cognate Gprotein-coupled receptors LPA1, LPA2, and LPA3. In the present studywe have used cultures of human smooth muscle cells (SMCs) anderythroleukemia cells (HEL), and isolated human platelets tocharacterize physiological effects of LPA compared with adrenaline andnoradrenaline as well as structure-activity relationships of LPA. SMCswere isolated from biopsies of human myometrium obtained at cesareansections. We show that cultured myometrial SMCs express multiple LPAand α2-adrenergic receptor subtypes. Treatment of SMCs with LPA andnoradrenaline resulted in increases in proliferation. However, LPA elicitsa much more pronounced stimulatory effect than noradrenaline. Theability to increase calcium might be one explanation why LPA is moreeffective. Further studies indicated that several pathways mediate thegrowth stimulatory effect of LPA where transactivation of epidermalgrowth factor receptors through matrix metalloproteinases as well ascalcium/calmodulin-dependent protein kinases appears to be important.LPA enantiomers and LPA analogues were synthesized andcharacterized due to their capacity to increase calcium in HEL cells. Ourstudy is the first to show that both natural (R) and unnatural (S) LPAenantiomers are capable of stimulating cells, suggesting LPA receptorsare not stereoselective. Moreover, we have synthesized a LPA analoguewith higher maximal effect than LPA by reducing the hydrocarbon chainlength. In platelets we demonstrated that LPA is a weak calcium-elevating compound which failed to stimulate aggregation. However, incombination with adrenaline, another weak platelet agonist, a completeaggregatory response was obtained in blood from some healthyindividuals. These results are important since platelet activation is a keystep in distinguishing normal from pathological hemostasis. Since LPA ispresent at high concentrations in atherosclerotic lesions, the synergisticeffect of LPA and adrenaline might be a new risk factor for arterialthrombosis.

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ABBREVIATIONS____________________________AC adenylyl cyclaseACD acid citrate dextroseADP adenosine 5’-diphosphateAMP adenosine 5’-monophosphateANOVA analysis of varianceATP adenosine 5’-triphosphateAR adrenergic receptorBmax maximum number of binding sitesCa2+ calcium[Ca2+]i cytosolic Ca2+ concentrationCaM Ca2+/calmodulin-dependent proteincAMP cyclic adenosine 3’, 5’-monophosphatecDNA complementary deoxyribonucleic acidcGMP cyclic guanosine 3’, 5’-monophosphateDAG diacylglycerolDNA deoxyribonucleic acidDMSO dimethyl sulfoxideEC50 the molar concentration of an agonist that produces 50 % of the maximal

possible effect of that agonistEdg endothelial differentiation geneEGF epidermal growth factorFBS fetal bovine serumfura-2 fura-2-acetoxymethylesterGC guanylyl cyclaseG protein heterotrimeric guanine nucleotide-binding regulatory proteinGPCR G protein-coupled receptorGPIIb/IIIa glycoprotein IIb/IIIaGTP guanosine 5’-triphosphateHEL human erythroleukemia cellsHRP horseradish peroxidaseIC50 the molar concentration of an agent that causes a 50 % reduction in the

specific binding of a radioligandIGF insulin-like growth factorKd equilibrium dissociation constantLDL low-density lipoproteinLPA lysophosphatidic acidMAG monoacylglycerolMAP mitogen-activated proteinMMP matrix metalloproteinasemRNA messenger ribonucleic acidOCAF ovarian cancer activating factorPA phosphatidic acidPBS phosphate-buffered salinePCR polymerase chain reactionPKA cAMP-dependent protein kinasePKC protein kinase CPLA1/A2/C/D phospholipase A1/A2/C/DPRP platelet-rich plasmaPTX pertussis toxinRNA ribonucleic acidRT-PCR reverse transcriptase-PCRSDS sodium dodecyl sulfateSEM standard error of the meanSMC smooth muscle cellsPLA2 secretory non-pancreatic type-II PLA2S1P sphingosine 1-phosphateTXA2 thromboxane A2

vzg-1 ventricular zone gene-1

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INTRODUCTION____________________________

The word lipid can bring to mind many distinct associations. Many ofthese are unfortunately often negative, such as the contribution thatexcessive dietary fat makes to body weight and heart disease.Nevertheless lipids are also essential components of the cell membranes.During the past several years, researchers have found that lipidmolecules play many more dynamic roles such as helping to control amajority of cellular activities. This study primarily elucidatesphysiological effects as well as structure-activity relationshipsconcerning one specific phospholipid, namely lysophosphatidic acid(LPA). The physiological effects of LPA were compared with those ofadrenaline and noradrenaline.

Some of the specific aims of this thesis were to

-examine the proliferative effect of LPA and noradrenaline in humanmyometrial smooth muscle cells (SMCs).

-elucidate and characterize the effects of LPA and adrenaline in humanplatelets.

-investigate LPA-induced intracellular signaling.

-evaluate structure-activity relationships of distinct LPA enantiomersand LPA analogues.

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REVIEW OF THE LITERATURE________________

LPA AND OTHER PHOSPHOLIPIDSCell membranes are mainly composed of phospholipids constructedfrom fatty acids and glycerol. The glycerol is linked to a hydrophilicphosphate group and also to a lipophilic fatty acid tail. This makesphospholipids amphipathic, i.e. they consist both of a hydrophilic and ahydrophobic region. Two layers of phospholipids combine tail-to-tail inwater and form a lipid bilayer that is the structural basis of all cellmembranes (Tanford, 1980). Although previously viewed primarily asbuilding blocks of the cell membrane, lipids are now also recognized asimportant cell signaling molecules (Moolenaar, 1999). In 1989 vanCorven et al. established that LPA and other phospholipids are not onlysimply structural components of the cell membrane, but also biologicalmediators. The importance of phospholipid mediators is illustrated bythe steady increase of publications focused on these molecules. Thisthesis will focus on some of the effects of LPA. A few milestones in thisfield of research are: the discovery of biologically active lipid phosphoricacids in 1949, the identification of LPA as a bioactive compound in 1978,the discovery of the growth factor properties of LPA in 1989, and theidentification of LPA receptor genes in 1996 (reviewed in Tigyi, 2001a).

Biochemistry of LPALPA is the simplest of all glycerophospholipids. As can be seen in Figure1, it consists of three substructural domains: the phosphate head group, alinker region (glycerol), and a lipophilic tail (fatty acyl chain) (Hopper etal., 1999). LPA naturally exists in the (R) configuration. Since the fattyacid chain can alter, LPA exists as many different molecular species.According to Xie et al. (2002), the term LPA refers to several classes oflipid metabolites, rather than to a single chemical structure. Either the 1-or 2-position of the glycerol backbone can have conjugated fatty acids. Inaddition, the chain may link to the glycerol backbone through differentchemical linkages. The substituent can be attached to the backbone by anacyl, alkyl or alkenyl linkage at the 1-position, but only by an acyllinkage at the 2-position. The physiological relevance of these differentforms of LPA, if any, remains largely unknown. LPA with an acyllinkage, 18 carbons in the chain, and one unsaturation is the most

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common LPA used experimentally and is also commercially available.This form is commonly referred to as oleoyl-LPA or 18:1 LPA (Figure 1).This LPA is more soluble in water than most other long chainphospholipids, as it has a free hydroxyl and phosphate moiety (reviewedin Jalink et al., 1990; Xu et al., 2001; Xie et al., 2002). If nothing else ismentioned, this is the LPA referred to in the text.

Figure 1. The chemical structure of LPA consisting of three substructuraldomains: the phosphate head group, a linker region, and a lipophilic tail.

Production, release, and degradation of LPACritical concentrations of LPA have to be present extracellulary in orderto induce receptor-dependent biological responses. Enzymes andproteins involved in the synthesis, transport and degradation of LPAcontrol its bioavailability. Some possible pathways for synthesis anddegradation of LPA are illustrated in Figure 2. Historically, LPA hasbeen considered as an intermediate in the cytosolic biosynthesis ofglycerophospholipids. The biological pathways for LPA formationremain to be clarified although several studies indicate that degradationof phosphatidic acid (PA) by phospholipase A (PLA) may be involved(Fourcade et al., 1995; Gaits et al., 1997; le Balle et al., 1999). This pathwaywould be the simplest way to generate LPA since PA accumulatesfrequently in activated cells (Fourcade et al., 1998). LPA synthesis isbelieved to be initiated by the release of membranous microvesiclesenriched in PA (Fourcade et al., 1995). PA is formed by the actions ofphospholipase C (PLC) and diacylglycerol (DAG) kinase (Fourcade et al.,1995). It has been suggested that DAG is deacylated by a lipase tomonoacylglycerol (MAG) that can be further deacylated orphosphorylated to LPA (Gaits et al., 1997). Secretory non-pancreatic type-II PLA2 (sPLA2) can degrade glycerophospholipids when the distributionof phospholipids in the plasma membrane is changed (Fourcade et al.,

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1998). Such change in the distribution of phospholipids can be due toactivation of platelets, cell aging or apoptosis (Fourcade et al., 1998) whenphospholipids are translocated from the internal leaflet to the outerleaflet of the plasma membrane (Budnik and Mukhopadhyay, 2002).Furthermore, sPLA2 is poorly active on intact cells displaying a normaldistribution of phospholipids (le Balle et al., 1999). Small amounts of LPAare, however, associated with membrane biosynthesis in all cells (Contoset al., 2000b). Another possibility of LPA synthesis is a direct productionof PA by phospholipase D (PLD). Bacterial PLD generates LPA in theexternal membrane leaflet of its target cells through hydrolysis oflysophosphatidylcholine (van Dijk et al., 1998). Generation of LPA innormal cells is initiated by physical perturbation or by stimulation ofplasma membrane receptors or other surface proteins, which activatecritical enzymes such as phospholipases (Goetzl, 2001). In contrast,tumor cells may secrete LPA spontaneously (Goetzl, 2001). It has alsobeen shown that LPA is released from certain cell types, e.g. epithelialcells, fibroblasts, macrophages and some tumor cells, followingactivation (Goetzl, 2001). However, only small amounts appear to bereleased into extracellular fluids by fibroblasts (Goetzl, 2001). Plateletsrepresent the best characterized source of LPA (le Balle et al., 1999). Dueto its hydrophilicity, LPA does not necessarily remain membraneassociated after its formation (Moolenaar, 1995a). Activated platelets canproduce significant amounts of extracellular LPA (Eichholtz et al., 1993)and are a major source of LPA in the blood (Morris, 1999). Theconcentration of LPA in serum has been estimated to be in themicromolar range (Eichholtz et al., 1993). The study of Eichholtz et al.(1993) has however been criticized by Sano et al. (2002) who argue thatthe calculations of the amount of LPA were not accurate. Sano et al.(2002) propose that the bulk of LPA, produced through plateletactivation, is generated outside of the platelet. According to theseauthors, only a minor portion of LPA originates within the platelets. Themajority of LPA is the product of released PLA1, PLA2 and lysoPLDthrough sequential cleavage of membrane and serum phospholipids toLPA (Sano et al., 2002). LysoPLD contributes to the conversion of choline-type phospholipid mediators to lipid phosphate-type mediators(Tokumura, 2002). Thus lysoPLD would supply LPA to peripheraltissues (Tokumura, 2002). Until very recently the molecular identity oflysoPLD was not determined (Moolenaar, 2002). However, lysoPLD now

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appears to be identical to autotaxin, a widely expressed transmembraneectoenzyme (Tokumura et al., 2002; Umezu-Goto et al., 2002). LPA canalso be produced intracellulary by acylation of glycerol-3-phosphate, bythe glycerol-3-phosphate-acyltransferase located in mitochondria andendoplasmic reticulum (Pagès et al., 2001). It is however not yet clearwhether this LPA contributes to the extracellulary released LPA.

Figure 2. Some possible pathways for synthesis and degradation of LPA.TAG=triacylglycerol. For other abbreviations and references please seetext.

LPA is bound to and transported by extra- and intracellular lipid-binding proteins. Protein binding regulates the free concentration ofLPA, thus toxic levels can be avoided (Pagès et al., 2001). Plasma LPA isfound largely in albumin- and lipoprotein-bound forms, but the level islower than in serum (Takuwa et al., 2002). Albumin is a high-capacityand low-affinity reservoir of LPA (Goetzl, 2001). Thus, the extracellularpool of free LPA may activate cells locally or via circulating blood.Through its binding to albumin, LPA becomes protected from digestionby serum phospholipases (Tigyi and Miledi, 1992). Consequently,albumin carries LPA in the bloodstream and prolongs its physiologicalhalf-life. Some possible degradation pathways are deacylation by

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lysophospholipase and dephosphorylation by lysophosphatase (Budnikand Mukhopadhyay, 2002). These enzymes can generate new potentialsignaling molecules. LPA may rapidly be converted into PA by an LPA-acyltransferase, into MAG by phosphatidate phosphohydrolase and intoglycerol-3-phosphate by lysophospholipases (Pagès et al., 2001).

Identification of LPA receptor genesFor a long time it was mainly assumed that LPA mediated its effectthrough non-receptor interactions. One possibility was that LPA diffusedin and perturbed the lipid bilayer, and thereby activated heterotrimericguanine nucleotide-binding regulatory proteins (G proteins) (Moolenaarand van Corven, 1990). Another explanation was that LPA exerted itsinfluence through activation of voltage-sensitive calcium (Ca2+) channels(Tokumura et al., 1991). However, it was proposed by Moolenaar et al.(1995a) that LPA might bind to receptors in order to initiate its action.Initially, attempts to identify a specific receptor were complicated byhigh levels of nonspecific binding of LPA (van der Bend et al., 1992;Thomson et al., 1994). Evidence for the existence of a putative receptorcame from photoaffinity labeling studies in which a LPA binding proteinwas identified (van der Bend et al., 1992). The candidate LPA receptorhad an apparent molecular mass of 38-40 kDa and was identified inseveral cell types (reviewed in Moolenaar, 2000). In 1996 Hecht et al.isolated mouse complementary deoxyribonucleic acid (cDNA), termedventricular zone gene-1 (vzg-1), encoding a receptor for LPA. Thereafterthe human homologue of vzg-1, called endothelial differentiation gene(Edg)-2, was identified and later on another subtype called Edg-4 wasdiscovered (An et al., 1997; An et al., 1998a). An additional humansubtype, Edg-7, was characterized by Bandoh et al. in 1999. All these LPAreceptors belong to the family of seven transmembrane spanning Gprotein-coupled receptors (GPCRs). The biological effects of LPA showmany similarities with the effects mediated through other GPCRs, e.g.dose-response relationship, homologous desensitization and kinetic ofCa2+ mobilization. Recently, a new nomenclature for LPA receptorsaccording to Tigyi (2001b) has been proposed. The new names are LPA1

(Edg-2), LPA2 (Edg-4), and LPA3 (Edg-7). These names are consistentwith the guidelines of the International Union of Pharmacology (Chun etal., 2002). The new nomenclature will therefore be used in this text.

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Transmembrane signal transduction in response to hormones andneurotransmitters is mainly mediated through GPCRs. With more than athousand members, this receptor family represents the largest group ofcell surface receptors (Gether, 2000). Seven transmembrane domains,having an extracellular N terminal and a cytoplasmic C terminalcharacterize these receptors. The hydrophobic domains are connected byhydrophilic extracellular and intracellular loops. The way that differentligands bind to GPCRs varies a lot. Small molecules bind to sites in ahydrophobic core between the transmembrane α-helices, whereasbinding sites for larger ligands include the N terminal and theextracellular hydrophilic loops that joins the transmembrane domains.The majority of sequence homologies between GPCR family membersreside in the transmembrane domains. Receptors and G proteins interactthrough the intracellular loops. Therefore, it appears that the character ofthe loops determines to which G protein the receptor preferentiallycouples. The three G protein subunits α, β, and γ can combine in manyways and provide specific signaling linkages between receptors andeffectors. The G protein α subunits are divided into four distinct families,Gs, Gi/o, Gq/11 and G12/13 (reviewed in Hieble et al., 1995; Raymond, 1995;Strader et al., 1995; Gether and Kobilka, 1998; Gutkind, 2000). Like allproteins, receptors can exist in various conformations (Kenakin, 2001).GPCRs often exhibit two states that bind agonists with differentaffinities. The receptors that couple to G proteins are in the high affinitystate. When guanosine 5’-triphosphate (GTP) replaces guanosine 5’-diphosphate on the α-subunit of the G protein, the subunits α and βγ ofthe protein dissociate and mediate the intracellular effects of the receptorligand. Subsequently, the receptor returns to its low affinity state. Inaccordance, the absence of GTP leads to a significant proportion ofreceptors in the high affinity state. Consequently, in the presence of GTPmost receptors will adopt the low affinity state (reviewed in Haylett,1996; Colquhoun, 1998).

The human forms of LPA1, LPA2, and LPA3 have estimated molecularweights of 41.1, 39.1, and 40.1 kDa and consist of 364, 351, and 353 aminoacids, respectively (Contos et al., 2000b). The amino acid homologybetween LPA1, LPA2 and LPA3 is about 55 % (Lynch and Im, 1999; Aokiet al., 2000; Chun et al., 2002). This cluster of receptors is about 35 %identical to a second cluster of receptors, namely sphingosine 1-

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phosphate (S1P)1-5 receptors, formerly Edg-1, Edg-3, Edg-5, Edg-6 andEdg-8 (Lynch and Im, 1999; An, 2000; Chun et al., 2002). The S1Preceptors share about 50 % identical amino acids (Chun et al., 2002). S1Pis the agonist for all these subtypes. A comparison of the amino acidsequences confirm that LPA and S1P receptors are both functionally andstructurally separated into two subfamilies (Takuwa et al., 2002). InFigure 3, a phylogenetic tree based on the amino acids of these distincthuman receptors is shown.

Figure 3. Phylogenetic tree based on human LPA and S1P receptor aminoacid sequences. The tree was constructed with the ClustalW program,available at ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalW. The underlyingamino acid sequences are found associated with the following GenBankfiles: LPA1 u78192, LPA2 ac002306, LPA3 af186380, S1P1 m31210, S1P2

af034780, S1P3 x83864, S1P4 aj000479 and S1P5 NM_030760, available athttp://www.ncbi.nlm.nih.gov /Genbank.

These receptor subfamilies are most closely related to the cannabinoidreceptors among other members of the GPCR family. The LPA, S1P, andcannabinoid receptors share 28 % sequence homology (An, 2000).Furthermore, the LPA1 receptors from amphibians, fish, birds andmammals share remarkably >90 % identical amino acid sequences (Chunet al., 2002). In Xenopus a dissimilar, putative receptor PSP24 has alsobeen reported in addition to the LPA receptors (Guo et al., 1996).However, it remains unclear whether this PSP24 is an LPA receptor since

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there is no independent confirmation. Either PSP24 interacts in afundamentally different way with LPA, as compared to the othersubtypes, or it is simply not a LPA receptor (Chun et al., 1999). Accordingto An (2000) this receptor does not belong to the Edg family due to lowsequence homology. Documented LPA responses in cells lackingmessenger ribonucleic acid (mRNA) expression for the existing subtypes,indicates that there might be LPA receptor subtypes that remainunidentified (Fischer et al., 1998). It has also been proposed that LPA canaffect cells independently of LPA-receptor proteins (Hooks et al., 2001).

Expression patterns of LPA receptorsThe LPA receptor proteins differ with respect to cell distribution andintracellular signal transduction mechanisms (Bandoh et al., 1999). Thehuman LPA1 is widely expressed (brain, colon, heart, small intestine, andprostate) with highest mRNA levels appearing in brain, whereas LPA2 ismost highly expressed in leukocytes and testis (An et al., 1998a). Heart,kidney, lung, pancreas, and prostate have been shown to express LPA3

(Bandoh et al., 1999; Im et al., 2000). All three receptor subtypes havebeen detected in human platelets (Motohashi et al., 2000). Rat hepatoma(RH7777) and neuroblastoma (B103) cell lines do not respond to LPAstimulation and LPA receptors have not been detected. These cells havetherefore been used for heterologous expression of LPA receptors intransfection studies (Fukushima, 1998; Ishii et al., 2000). Malignanttransformation of some cell types results in the appearance and oftenpredominance of one or more LPA receptor subtypes not expressed bythe equivalent non-malignant cell (Goetzl, 2001). For example, manyhuman ovarian cancer cell lines express high levels of LPA2, which is notdetectable in normal ovarian epithelial cells (Goetzl et al., 1999).

Signaling by LPA receptorsEven an amoeba can respond chemotactically to LPA (Jalink et al., 1993).Therefore, it seems that LPA might have been a cellular messenger earlyin evolution. LPA activates cells by stimulating many distinct pathways,which briefly will be described in this section and is illustrated in Figure4. To sort out the signaling pathways mediated by each individual LPAreceptor subtype one would need receptor antagonists specific fordistinct subtypes. Such antagonists are not yet available. Thus some of

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the signaling pathways described here do not distinguish between thethree LPA receptor subtypes. Sections without references are reviewed in(Moolenaar, 1995b; Moolenaar et al., 1997; Moolenaar, 1999; Fukushimaand Chun, 2001; Hla et al., 2001; Siess, 2002; Takuwa et al., 2002). LPAreceptors couple to mitogen-activated protein (MAP) kinase in apertussis toxin (PTX)-sensitive manner, indicating that Gi/o proteinsmediate the response. PTX catalyzes adenosine 5’-diphosphate (ADP)-ribosylation of G proteins negatively coupled to adenylyl cyclase (AC)activation (Katada and Ui, 1982). Generally, heterogeneity exists in themechanisms whereby GPCRs activate MAP kinases. Activation may bemediated by PTX-sensitive or -insensitive G proteins and be either Ras-or protein kinase C (PKC)-dependent, depending upon cell type as wellas receptor subtype (Luttrell et al., 1996). Furthermore, in rat adrenalpheochromocytoma cells, GPCR-mediated PLC activation and Ca2+

influx might mediate MAP kinase activation via proline-rich tyrosinekinase 2-induced tyrosine phosphorylation (Lev et al., 1995). Gi / o-dependent MAP kinase activation is mediated by the βγ-subunits(Crespo et al., 1994). Ras mediates activation of Raf, MEK and MAPkinase (Daaka, 2002). Upon activation, MAP kinase translocates to thenucleus where it phosphorylates and activates nuclear transcriptionfactors involved in DNA synthesis (Daaka, 2002). In SMCs, MAP kinasescan either mediate growth inhibition or proliferation (Bornfeldt et al.,1997). Gi/o proteins can block AC activation in a PTX-sensitive manner.AC catalyzes the formation of cyclic adenosine 3’, 5’-monophosphate(cAMP) from adenosine 5’-triphosphate (ATP). This occurspredominantly via Gs proteins- and/or Ca2+-dependent mechanisms.Most, but not all, effects of cAMP are mediated through activation ofcAMP-dependent protein kinase (PKA) (Bornfeldt and Krebs, 1999).Intracellular concentrations of cAMP are determined both by its rate offormation and the rate of hydrolysis by phosphodiesterases (Yu et al.,1995). Several cyclic nucleotide phosphodiesterases metabolize cAMP to5’-AMP. LPA has been shown to be able to increase the cAMPconcentration and this might be conducted through activation of Gs

proteins and perhaps LPA3 receptors (Bandoh et al., 1999).

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Figure 4. Some signal transduction pathways induced by LPA binding toGPCRs. Lines with arrowheads illustrate activation, whereas the line witha crossbar illustrates inhibition. Gi/o inhibits AC and thus cAMPproduction. Gi/o also activates the MAP kinase cascade, which isresponsible for increased proliferation. Gq activates PLC that results ingeneration of IP3 and DAG. IP3 increases [Ca2+]i, and DAG activates PKC.G12/13 activates Rho that leads to cytoskeletal and morphological changes.For abbreviations and references please see text.

Ca2+ plays an important role as an intracellular messenger in signaltransduction. Upon appropriate cell activation, Ca2+ can enter thecytoplasm from intracellular stores, i.e. endoplasmic reticulum andmitochondria, and from extracellular pools by the action of gatedchannels and transporters. Once in the cytoplasm, Ca2+ binds to targetproteins, especially calmodulin, and regulates their activities.Calmodulin modulates many enzymes as well as protein kinases andone of them is Ca2+/calmodulin (CaM)-dependent protein kinase(reviewed in Clapham, 1995). CaM kinase II regulates the activity of

Gi/o Gq/11 G12/13

Tyrosine kinases

Ras

Raf

MEK

MAP kinase

PLC

ββββγγγγ

PKC [Ca2+]i

IP3Rho

PLD

αααα

AC

[cAMP]i

DNA synthesisCell proliferation

Cytoskeletal changesContractile actions

LLPPAAN

C

DAG

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cellular components involved in many different processes, includingapoptosis, cell cycle control, and gene expression (Muthalif et al., 2001a).The best known Ca2+ signaling pathway triggered by GPCRs is theactivation of PLC which produces inositol 1,4,5-triphosphate (IP3) andDAG (Clapham, 1995). LPA receptors activate PLC both through a PTX-insensitive mechanism that probably involves Gq/11 proteins, andthrough a PTX-sensitive mechanism that probably involves Gi/o proteins.LPA, at nanomolar concentrations, triggers PLC-mediated Ca2+

mobilization in many different cell types (Moolenaar, 1994) via all threereceptor subtypes (An et al., 1998b; Bandoh et al., 1999; Im et al., 2000).Furthermore PLC can activate PKC, and subsequently MAP kinase,through DAG. A third pathway that is activated by LPA receptors is aRho-dependent pathway that mediates remodeling of actin cytoskeletonand is inhibited by Clostridium botulinum C3 toxin. G12/13 proteinsprobably mediate this pathway. LPA3 is not coupled to the Rho pathway.

It has become apparent that GPCRs and receptor tyrosine kinases sharecommon signaling intermediates in the pathway leading to activation ofthe MAP kinase cascade (Luttrell et al., 1999). LPA-induced activation ofthe MAP kinase cascade shows several similarities with activation byepidermal growth factor (EGF) receptor tyrosine kinase. They bothinduce tyrosine phosphorylation of several proteins and activate Ras andRaf (Howe and Marshall, 1993; van Corven et al., 1993; van Biesen et al.,1995). In fact, a transactivation phenomenon has been described whereactivation of GPCRs leads to activation, i.e. tyrosine phosphorylation ofreceptor tyrosine kinases (Buist et al., 1998; Iwasaki et al., 1998; Maudsleyet al., 2000). It has also been proposed that the EGF receptor can betransactivated by stimulation with LPA in rat fibroblasts (Daub et al.,1996). It was concluded that EGF receptor signaling was required for fullproliferative response by LPA (Daub et al., 1996). LPA could notstimulate DNA synthesis in fibroblasts that had no endogenous EGFreceptor (Cunnick et al., 1998). Moreover, in rat fibroblasts expression ofa truncated EGF receptor lacking the cytoplasmic domain abrogatedLPA-stimulated MAP kinase activation (Daub et al., 1996). This resultindicates that the EGF receptor mediates at least a branch of the LPAstimulated MAP kinase activation pathway. Other studies showed thatactivation of LPA receptors resulted in increased tyrosinephosphorylation and activation of EGF receptors (Cunnick et al., 1998;

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Herrlich et al., 1998; Voisin et al., 2002). However, it has not been clearlydefined how GPCRs induce receptor tyrosine kinase transactivation andseveral mechanisms have been proposed. Eguchi et al. (1998) suggest thatCa2+ is necessary for transactivation of the EGF receptor which therebyleads to MAP kinase activation in vascular SMCs. Furthermore,Murasawa et al. (1998) have shown that Ca2+/calmodulin plays a role inthe transactivation of EGF receptor. It has also been suggested that β2-adrenergic receptor (AR)-mediated Src activation precedes EGF receptortyrosine kinase transactivation in monkey kidney fibroblasts (Maudsleyet al., 2000). This transactivation is independent of intracellular Ca2+

release and involves formation of a multi-receptor complex containingboth receptors. In other experiments it was indicated that transactivationcan be mediated by βγ subunits of G proteins (Luttrell et al., 1996). It hasalso been demonstrated that transactivation of the EGF receptor is aresult of release of membrane-bound EGF, i.e. an autocrine activation ofthe receptor and not a ligand-independent transactivation (Dong et al.,1999; Prenzel et al., 1999).

Tyrosine kinases are involved in many signal transduction pathwaysincluding cell proliferation. The state of phosphorylation of intracellulartargets is determined by the interplay between protein kinases andphosphatases. Some enzymes require phosphorylation for activity, whileothers are inactivated by phosphorylation. Protein tyrosine kinases fallinto two general classes (Clark and Brugge, 1996). The receptor tyrosinekinases, e.g. EGF receptors, have intracellular domains with tyrosinekinases that couple external stimuli to intracellular signaling (Wells,1999). EGF binding induces dimerization of monomeric receptorsubunits and subsequent autophosphorylation of tyrosine residues(Zwick et al., 1999a; Daaka, 2002). The activated receptor serves as a corefor assembly of multi-protein complexes that can activate the MAPkinase cascade (Daaka, 2002). The non-receptor tyrosine kinases couplewith membrane receptors or functions downstream in the signalingcascades. Phosphorylation on tyrosine residues of specific target proteinsis a widespread mechanism of signal transduction in cells. It generates anew protein state, resulting in structural alterations that can affect theactivity of the target protein or modify protein-protein interactions.Activation of the G i/o-mediated MAP kinase pathway involvesinteractions of tyrosine kinases with the signaling proteins Shc, Grb2 and

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Sos (van Biesen et al., 1995; Luttrell et al., 1996). LPA can stimulatetyrosine-phosphorylation of EGF receptors which form complexes withShc and Sos in rat adrenal pheochromocytoma cells (Kim et al., 2000). Alltogether, distinct results indicate that the EGF receptor can be activatedby LPA through intracellular crosstalk between signaling pathwaysand/or extracellular released EGF.

Biological responses of LPAAlthough first described as an intermediate in phospholipid synthesis,LPA is now recognized as an extracellular signaling molecule thatevokes many different responses when applied to cells. An interestingaspect of LPA signaling is the wide range of potencies reported. LPA at aconcentration of 0.2 nM produces 50 % of the maximal effect (EC50) ontransient Ca2+ increases in human epidermoid carcinoma cells (Jalink etal., 1995), whereas mitogenesis in rat fibroblasts has an EC50 value of 10-15 µM (van Corven et al., 1989). This wide range is presumably due to acombination of signaling through multiple receptors and degradation ofLPA before cell activation (Lynch and Macdonald, 2002). The LPA3

receptor has a lower affinity for LPA as it shows higher EC50 values invarious assay systems (Bandoh et al., 1999; Heise et al., 2001). Thepotency of LPA depends on its local concentration and the receptordistribution. Furthermore, the levels of precursors, activities of LPA-producing and degrading enzymes and capabilities of LPA-bindingproteins may affect the concentration of LPA. Excessively elevatedproduction of LPA in body fluids and on cell surfaces may lead toundesirable conditions and it might be involved in many disease andinjury states. The level of LPA in fluids surrounding the tissues areelevated in atherosclerosis, corneal injury, lung disease, ovarian cancer,and wound healing (Wang et al., 2001). Until recently there has been alack of direct evidence for the physiological roles of LPA receptors andtheir signaling in living animals. Mice deficient in different receptorsubtypes have provided information on the in vivo roles of thesereceptors (Yang et al., 2002). For example, deleting the gene for the LPA1

receptor resulted in 50 % neonatal lethality and impaired suckling inneonatal pups (Contos et al., 2000a).

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LPA in the cardiovascular systemPlatelets are cell fragments of about 2-4 µm in diameter that circulate inthe blood for about 10 days (George, 2000). Megacaryocytes in the bonemarrow form platelets by pinching of bits of cytoplasm and plateletstherefore contain many granules but no nucleus. There are normally125,000-340,000 platelets per microliter of circulating blood. When ablood vessel wall is injured, platelets adhere to factors in the wall viaintegrins. Binding to collagen initiates platelet activation and results inchanged shape, release of granule contents as well as aggregation toother platelets. The α-granules contain clotting factors and platelet-derived growth factor, whereas dense granules contain ADP, ATP, Ca2+

and serotonin. Activated platelets release compounds such as serotoninand thromboxane A2 (TXA2), which act vasoconstrictive on vascularSMCs and thereby reduce blood loss upon injury. SMCs are 30-200 µmlong with an oval, centrally located nucleus and many distinct filamentsin a non-regular pattern (Chamley-Campbell et al., 1979). Sinceproliferation of vascular SMCs may contribute to atherosclerosis,hypertension, and thickening of the blood vessel walls when theendothelium is damaged, there has been considerable interest in theregulation of smooth muscle growth.

There is experimental evidence indicating that LPA is a potentiallyatherogenic and thrombogenic molecule. Surprisingly, early experimentsshowed that intravenously administrated LPA produced hypotension incats and rabbits, but hypertension in rats and guinea pigs (Tokumura etal., 1978). Furthermore, it was demonstrated that a factor, which wasdeveloped in plasma in vitro after incubation for 18-24 hours, evokedplatelet aggregation after intravenous injection in cats (Schumacher et al.,1979). It was suggested that the mediators were PA and LPA.Aggregation factors, such as thrombin, were shown to lead to an increasein LPA production from isolated platelets (Mauco et al., 1978). In morerecent experiments freshly isolated blood or platelet-poor plasma fromhealthy individuals have been shown to contain a very low or no amountof LPA (Pagès et al., 2001). Conversely, the serum concentration of LPAhas, as already mentioned, been estimated to be in the micromolar range(Eichholtz et al., 1993). The local concentration in the immediatesurrounding of a platelet plug is probably much higher (Gennero et al.,1999). The release of LPA from activated platelets might explain the

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higher levels of LPA in serum as compared to plasma, but other bloodcells, lipoproteins as well as oxidized low-density lipoproteins (LDL) areother possible sources of LPA in plasma and serum (Siess, 2002). Arecent report by Siess et al. (1999) indicates that LPA accumulates in LDLduring mild oxidation. The authors also showed that LPA works as theprimary platelet-activating lipid and accumulates in atheroscleroticplaques. In addition, LPA production can constitute an autocrine loop ofamplification of normal cell proliferation (Pagès et al., 2001). LPAregulates the barrier function of endothelial cell monolayers as well asthe interaction between endothelial cells and leukocytes, which are initialsteps in the development of an atherosclerotic lesion (Tigyi, 2001a). It hasbeen demonstrated that LPA stimulates the closure of a woundedendothelial cell monolayer by increased migration and proliferation ofthese cells (Lee et al., 2000). LPA also stimulates cultured vascular SMCsto proliferate (Gennero et al., 1999). LPA has cell-type specific effects onproliferation as well as on apoptosis and both effects can be induced atdifferent concentrations in some cell types (Ediger and Toews, 2001).LPA may also have beneficial cardioprotective properties by preventingsevere effects of hypoxia on cardiac myocytes (Karliner, 2002). Synthesisand use of inactive LPA analogues, which compete with LPA for bindingon platelets, might be used in the treatment of atherosclerosis. This couldbe appropriate in patients with acute coronary syndromes where onepathological mechanism is rupture of atherosclerotic plaques resulting inthe formation of platelet thrombi (Karliner, 2002).

LPA in the reproductive tractLPA seems to play multiple roles in both female and male reproductivephysiology and pathology. LPA receptor transcripts have for examplebeen detected in human prostate and testis (Bandoh et al., 1999; Im et al.,2000). SMCs from benign prostate hyperplasia have been shown toproliferate when stimulated with LPA (Adolfsson et al., 2002). It has alsobeen suggested that expression of the LPA1 receptor may be involved inthe growth of androgen-independent prostate cancer cells (Daaka, 2002).An increase in serum lysoPLD activity has been indicated during humanpregnancy (Tokumura, 2002). It was suggested that LPA generated bylysoPLD might play an important role in maintenance of pregnancy,perhaps through its involvement in placenta development, fetal growth

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and hyperplasia of myometrial SMCs (Tokumura, 2002). LPA can alsoinduce stress fiber formation in human myometrial SMCs via a pathwayinvolving Rho-kinase, suggesting involvement in the maintenance ofuterine contractions (Gogarten et al., 2001). One report from 1980describes that isolated non-pregnant rat uterine SMCs contractrhythmically on addition of LPA (Tokumura et al., 1980). The uterus iscomposed of three layers, the endometrium is the lining, and themyometrium is the thick, muscular layer, which is covered with a thinserosa. During pregnancy, the human myometrium undergoesconsiderable enlargement both through hypertrophy as well ashyperplasia of SMCs, and actually increases in weight fromapproximately 50 g to about 950 g (Llewellyn-Jones, 1994). Thehyperplasia depends on differentiation of new SMCs fromundifferentiated cells in the connective tissue and also involves mitosisof differentiated SMCs (Bacon and Niles, 1983). Six weeks after deliverythe uterus has almost returned to its original size. The decrease in size isdue to enzymatic removal of collagen, a return of hypertrophic musclefibers to their usual size, and a reduction in number of smooth musclefibers (reviewed in Bacon and Niles, 1983). However, almost nothing isknown about the role of LPA in normal human myometrium. Moreover,little is known about the expression of the LPA receptor subtypes in non-malignant reproductive tissues (Budnik and Mukhopadhyay, 2002).

Ovarian cancer is associated with the production of a large volume ofperitoneal ascites (Xu et al., 2001). The cancer cells usually grow on thesurface of the ovaries, situated in the peritoneal cavity (Xu et al., 2001). Itwas discovered that ascites fluid from ovarian cancer patients contains afactor, termed ovarian cancer activating factor (OCAF), that exhibit apotent growth-promoting activity on cancer cells (Xu et al., 1995a). OCAFappears to be composed of multiple forms of LPA species, whichpresumably are the major proliferative constituents of ascites. LPA wasalso detected at higher levels in plasma from ovarian cancer patientscompared with a control group (Xu et al., 1995a; Xu et al., 1998). Ovariancancer patients had at least 10 times higher plasma levels of LPA whencompared to healthy controls. Some of the LPA had polyunsaturatedfatty acyl chains (Xu et al., 1995b). Mostly because of late detection,ovarian cancer has an extremely poor prognosis. Therefore, it has beenproposed that LPA could be used as a biomarker for different

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gynecologic cancers (Xu et al., 1998). The source of LPA in the ascitesfluid is unclear. A variety of enzymes may either produce high levels oflipids or decrease the degradation rate of lipids, resulting in a LPA poolin the ascites (Xu et al., 2001). Macrophages, mesothelial cells, or ovariancancer cells themselves, could be the potential sources (Westermann etal., 1998). It has been suggested that the receptor subtypes involved inmediating the proliferation signals in the ovarian cancer ascites are LPA2

and possibly LPA3, whereas LPA1 is probably not involved (Contos et al.,2000b). When LPA1 was overexpressed in ovarian cancer cells itappeared to function as a negative growth regulator (Furui et al., 1999).These authors proposed that LPA1 might counterbalance the effects ofother LPA receptors that contribute to cell proliferation. In contrast, ithas been reported that LPA1 receptors are overexpressed in somemalignant ovarian tumor cells, and an autocrine role for the LPA-LPA1

signaling system in ovarian cancer development is suggested (Takuwa etal., 2002). Moreover, LPA activates ovarian cancer cells by increasingcytosolic Ca2+ concentration ([Ca2+]i) and stimulating proliferation (Xu etal., 1995a). Stimulation of ovarian cancer cells with LPA enhances theproduction of the insulin-like growth factor (IGF)-II (Goetzl et al., 1999),which also might be involved in stimulation of cell proliferation. Finallyovarian cancer cells secrete matrix metalloproteinases (MMPs) thatdegrade extracellular matrix proteins and promote invasion into tissues(Stack et al., 1998). LPA upregulates MMP activation in ovarian cancercells (Fishman et al., 2001). As a consequence, this increase in pericellularMMP activity may enable cancer invasion and metastasis (Fishman et al.,2001). All together these data indicate that LPA may be both a markerand a mediator of ovarian cancer progression. On the contrary, Baker etal. (2002) were unable to distinguish patients with ovarian cancer fromhealthy control subjects through determination of plasma LPA levels.This contradiction raises questions about the utility of plasma LPA levelsfor early detection of ovarian cancer. The discrepancies in the studies ofBaker et al. (2002) and Xu et al. (1998) might be caused by differences inpreanalytical conditions, such as use of distinct anticoagulantia.

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Structure-activity relationships of LPAA major limitation in the field of LPA research is the lack of receptorsubtype-specific agonists and antagonists. However, already in 1978Tokumura et al. published the first structure-activity relationship studywith LPA. They noted that the potency of LPA, after intravenousinjection in different species, depended on the hydrocarbon chain lengthand the degree of unsaturation. More recently an interaction between theanionic phosphate group of LPA and its receptor has been suggested(Sardar et al., 2002). Moreover, a dissociable proton near the phosphategroup, provided by the sn-2 hydroxyl in LPA, seems required foroptimal activity (Lynch and Macdonald, 2002). The hydrophilic headgroup of LPA is thought to interact with the amino-terminal of the thirdtransmembrane region of a LPA receptor. However, it is unclear whichareas of the receptor protein that interact with the hydrophobic tail(Chun et al., 2002). The degree of saturation of the hydrocarbon chain ofligands has recently been shown to have little effect on potency, with theexception of LPA3 receptor which has pronounced preference forunsaturated LPAs (Bandoh et al., 1999; Im et al., 2000). However, DNAsynthesis in myeloma cells was inhibited by LPA, and longer acyl chainlengths and higher degrees of unsaturation of LPA tended to increase theantiproliferative effect (Tigyi et al., 1994).

Efforts have been made to identify new LPA receptor agonists as well asantagonists, and the chemical manipulations of the LPA structure havevaried (Hooks et al., 1998; Hopper et al., 1999; Santos et al., 2000). TwoLPA receptor antagonists have recently been described. Fischer et al.(2001) have found a short chain PA analogue to be a competitiveantagonist of the LPA3 receptor. It was suggested that the receptorselectivity is due to antagonist interactions with extracellular loops,which have 40 % homology among the three subtypes (Fischer et al.,2001). This sequence homology can be compared to the approximately 60% homology between the transmembrane domains (Fischer et al., 2001).Furthermore, N-oleoyl ethanolamide phosphate is an antagonist of bothLPA1 and LPA3, but not LPA2 receptors (Heise et al., 2001). However,according to Lynch and Macdonald (2002) these two describedantagonists need improvement before they could be used as morepowerful tools. The LPA receptor has earlier been shown to prefer eithernatural (R) stereochemistry of some ligands (Santos et al., 2000) or

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unnatural (S) stereochemistry of other ligands (Hooks et al., 2001).Moreover, Gueguen et al. (1999) have shown that LPA receptors lackstereospecificity in platelets.

ADRENALINE AND NORADRENALINETogether with dopamine, adrenaline and noradrenaline belong to thefamily of catecholamines, and are formed by hydroxylation anddecarboxylation of the amino acid tyrosine. Dopamine can be convertedto noradrenaline and subsequently to adrenaline (reviewed in Ganong,1995). Both adrenaline and noradrenaline are secreted in the adrenalmedulla but noradrenaline is also released from noradrenergicpostganglionic nerve terminals (Calzada and de Artinano, 2001).Moreover they are metabolized to biological inactive products byoxidation and methylation. Adrenaline and noradrenaline exert theireffects on target tissues through interactions with ARs. As with the LPAreceptors ARs belong to the family of GPCRs. Based on theirpharmacological and biochemical properties the ARs are divided intothree distinct subfamilies, α1, α 2, and β-ARs. These subfamilies arefurther divided into several subgroups of receptors (reviewed in Aantaaet al., 1995; Docherty, 1998). The following sections will focus on α2-ARsand their physiological effects.

Identification and expression patterns of αααα2-ARsThree human α2-AR genes have been cloned (Kobilka et al., 1987; Reganet al., 1988; Lomasney et al., 1990). They were designated α2-C4, α2-C10and α2-C2, according to the locations of the receptor genes onchromosome 4, 10 and 2 respectively. An earlier nomenclature, which isused here, is based on the pharmacological properties of the α2-ARsubtypes where α2A corresponds to α2-C10, α2B corresponds to α2-C2, andα2 C corresponds to α2-C4 (Aantaa et al., 1995). The three receptorsubtypes share a common evolutionary origin and the receptor proteinsare about 50 to 60 % identical on the amino acid level (Link et al., 1996).The receptor proteins consist of 450 to 461 amino acids (Aantaa et al.,1995). The α2-ARs differ from α1 and β-ARs by having shorter amino aswell as carboxyl terminals and a very long third intracellular loop(Hieble et al., 1995). Another subtype, α2D-AR, has been found in rats and

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is a species homologue of the human α2A-AR (Docherty, 1998). Peripheraltissues contain both presynaptic α2-ARs on nerve endings as well aspostsynaptic α2-ARs on target cells (Calzada and de Artinano, 2001).RNA encoding for all three α2-AR subtypes is present in both humanbrain and peripheral tissues (Berkowitz et al., 1994). It was shown byBerkowitz et al. (1994) that the total mRNA levels of α2-ARs are highestin human aorta, heart, kidney and spleen. With the exception of liver,pancreas and small intestine, the α2A-AR subtype represents the majorityof the mRNA present in all human tissue studied. It has beendemonstrated that the level of cAMP in the cells may regulate α2A-ARmRNA and receptor numbers. cAMP promotes a marked increase in α2A-ARs in human colon epithelial cells (Sakaue and Hoffman, 1991).

Signaling by αααα2-ARsAs with LPA receptors, α2-AR coupling to G proteins appears to bedetermined by regions in the second as well as third intracellular loopand juxtamembrane regions of the third cytoplasmic loop. All three α2-ARs appear to couple to the same signaling systems at least in nativetarget cells. Several signaling pathways have been reported includingdecrease in cAMP concentration, stimulation of Na+/H+ exchange,activation of K+ channels, activation of PLA2, PLC, or PLD, mobilizationof Ca2+, regulation of Ca2 + channels, and coupling to MAP kinase(references in Legrand et al., 1993; Saunders and Limbird, 1999).Activation of stable transfected α2A-ARs in rat fibroblasts stimulatesMAP kinase in a PTX-dependent manner (Graham et al., 1996).

αααα2-ARs in the cardiovascular systemα2-ARs are involved in the control of blood pressure homeostasis at anumber of locations. For example, α2A-ARs may play a role in bothperipheral and central cardiovascular regulation (Graham et al., 1996).An antihypertensive therapy target is α2-ARs located in the brain stemsince stimulation of these receptors produce a long lasting drop in bloodpressure (Link et al., 1996). Stimulation of α2-ARs on arterial SMCsincreases paradoxically blood pressure by increasing vascular resistance(Link et al., 1996). Both arterioles and venules respond to α 2A-ARactivation (Hieble and Bond, 1994). However, at the onset of

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hypertension the α2B-ARs play an important role. The α2C-ARs do notseem to play a major role in cardiovascular regulation (Calzada and deArtinano, 2001). Altogether, α2B-ARs are responsible for the initialhypertension, whereas the long-lasting hypotension is mediated by α2A-ARs (Philipp et al., 2002). Moreover, the inhibitory presynaptic feedbackloop that regulates neurotransmitter release from adrenergic nervesrequires α2A- and α2C-ARs (Philipp et al., 2002). Noradrenaline has beenshown to stimulate [3H]thymidine incorporation and growth in rat aorticSMCs (Yu et al., 1996). Yu et al. (1996) did not observe any DNA synthesisfor up to eight hours. Thereafter there was a significant increase in[3H]thymidine incorporation, which peaked after twenty hours. α2A-ARsappear to be the main type of ARs expressed on human platelets. In fact,the first cloned gene for the human α2-ARs was purified from humanplatelets (Kobilka et al., 1987). The main effect of adrenaline on plateletsseems to be potentiating the action of other activators (Zoccarato et al.,1991; Steen et al., 1993).

αααα2-ARs in the reproductive tractThe uterus contains both α- and β-ARs. The ARs mediate excitatory (α)and inhibitory (β) responses to catecholamines. The response of theuterus therefore depends on the proportion of α- and β-ARs. Therelaxing effect of agonists acting via β-ARs can be used clinically toprevent premature labor (Rydén, 1977). Both α1- and α2-ARs have beenidentified in the myometrium of several species (Hoffman et al., 1981;Bottari et al., 1983a; Bottari et al., 1983b; Berg et al., 1986; Phillippe et al.,1990; Legrand et al., 1993; Taneike et al., 1995). According to Fuchs (1995)α1-ARs induce contractions whereas α 2-ARs inhibit relaxation. Inmyometrium from pregnant women α1- and α2-ARs are present in theratio 60:40 (Berg et al., 1986). Although swine myometrium contains bothreceptor types, the α2-ARs are predominant and largely responsible formediating the excitatory responses to noradrenaline (Taneike et al., 1995).Variations in α2-AR number has been reported in ewes myometriumduring pregnancy where progesterone increases the receptor number(Vass-Lopez et al., 1990). In contrast, estrogens increase the number of α2-ARs in rabbit as well as human myometrium (Hoffman et al., 1981;Bottari et al., 1983c), but the physiological function of these receptors andthe consequences of their increase by estrogen remain unclear (Maggi et

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al., 1994). One potential effect of myometrial α2-ARs might be tostimulate cell proliferation, which has been shown in several other celltypes (Tutton and Barkla, 1987; Seuwen et al., 1990; Alblas et al., 1993;Bouloumié et al., 1994). In guinea pigs it has been suggested that uterineα2-ARs are located mostly postjunctionally on myometrial cells, at leastnear term (Arkinstall et al., 1990). Degeneration of adrenergic nervesduring pregnancy has also been demonstrated (Sporrong et al., 1981).Accordingly, the decrease of α2-ARs in rat myometrium duringpregnancy is proposed to be related to a loss of presynaptic receptors(Legrand et al., 1993). In guinea pigs and rats it has been demonstratedthat the expression of α2-ARs increases in the beginning of the pregnancyand after that declines to the basal level (Arkinstall et al., 1990; Legrand etal., 1993). Dahle et al. (1993) showed that the number of α2-ARs in humanmyometrium did not vary during 26 to 42 completed weeks ofpregnancy. However, the mass of human myometrium increases mostduring the first five months of pregnancy. Subsequently, stretching andthinning of the uterine wall occur (Llewellyn-Jones, 1994). Perhaps theα2-ARs are important at this early growth of pregnant myometrium.

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METHODOLOGICAL CONSIDERATIONS______

Methodological aspects that are not included in Paper I-IV areconsidered here. The experimental protocols will not be reiterated in thissection.

Cell culturingThe majority of studies investigating the proliferative effect of GPCRagonists have been performed either in recombinant receptor systems oron cell lines in culture. In vitro overexpression experiments with artificialreceptor systems can complement, but not substitute studies made onprimary cell cultures, since marked alterations of the stoichiometric ratioof receptors, G proteins as well as effector molecules may result indisordered coupling (Kenakin, 1997). Such studies may lead toanomalous cellular responses as compared with naturally occurringsystems. Moreover, since many agonists and antagonists may actdifferently in distinct species (Tokumura et al., 1978) the use of humancell systems is very important.

A first aim of this thesis project was to establish primary cultures ofSMCs. Biopsies of myometrium were obtained from women whounderwent cesarean section at the Division of Obstetrics andGynecology, University Hospital, Linköping, Sweden. Tissue from thelower uterine segment was excised with a scalpel, with the gentle handof a surgeon, from women delivered in 38-39 completed weeks ofpregnancy. The biopsies were immersed in Ringer’s solution (100 mMNaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 30 mM NaAc, pH 7.0, fromB. Braun Medical AB, Bromma, Sweden) and immediately transported tothe laboratory. In the laboratory, the tissue was minced into fragments ofapproximately 1-2 mm3 using a scalpel. As cultures of other types ofSMCs had been established with various methods by other investigators,it seemed valuable to compare the enzymatic method for preparingsingle cells for culture with the attached explant method. All SMCs werecultured in cell culture medium as described in Paper I and II. Casey etal. (1984) first described the collagenase method for isolation andestablishment of human myometrial SMCs. The method was slightlymodified when used in this study. Tissue fragments were transferred to

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Hanks’ balanced salt solution containing collagenase I (2 mg/ml),deoxyribonuclease I (280 U/ml), penicillin (200 U/ml), streptomycin(200 µg/ml), fungizone (0.5 µg/ml), and N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes) buffer (20 mM, pH 7.4). The chemicalswere purchased from Sigma Chemicals (St. Louis, MO, USA) andInvitrogen Ltd. (Paisley, UK). The cell suspension was incubated in awaterbath at 37 °C under gentle agitation for 4 hours. Thereafter, it wasfiltered through a nylon filter (pore size 48 µm, from Bigman AB,Bromma, Sweden) and centrifuged at 600 x g for 10 minutes. The cells inthe pellet were resuspended in Dulbecco’s modified eagle medium andcells were centrifuged a second time as above. The final pellet wasresuspended in medium and cells were placed in plastic cell cultureflasks at a density of approximately 1 x 106 cells/ml. In the secondmethod when using attached explants, cells grew out from small tissuepieces directly placed in a culture dish (Chamley et al., 1977a; Chamley-Campbell et al., 1979). About 20 pieces of the small fragments ofmyometrial tissue were placed separated in a 58 cm2 cell dish. Thesewere then just about covered with medium. The pieces attached to thedish by their own adhesiveness within a few days and a greater volumeof medium was added after aspiration of the old one. When enough cellshad been obtained, these were frozen in medium supplemented with 10% fetal bovine serum (FBS) and 10 % dimethyl sulfoxide (DMSO) andstored in liquid nitrogen. To make sure the cells froze slowly the freezingprocedure was performed in three steps. The ampoules of cells werestored at –20 °C for 30 minutes and subsequently at –70 °C over night.Thereafter, the ampoules were transferred to, and stored in, liquidnitrogen. Freezing and thawing of cells can cause cellular damage thatprobably is caused by intracellular ice crystals and osmotic effects (Hay,1992). Therefore as cryoprotective agent, DMSO is usually added to thecell suspension before freezing. DMSO has earlier been shown tominimize cellular injury (Hay, 1992).

After years of cell culturing, human erythroleukemia (HEL) cells were anew personal experience since these cells do not adhere to cell cultureflasks and have to be cultured in suspension. Frozen HEL cells werepurchased from German Collection of Microorganisms and Cell Cultures(Braunschweig, Germany). The cell culture was established fromperipheral blood of a 30-year-old male with erythroleukemia in relapse

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in 1982 (Martin and Papayannopoulou, 1982). On arrival, the ampoulewas thawed in a 37 °C waterbath and the cells were transferred to aplastic tube. Ten ml of pre-warmed RPMI 1640 medium were addeddropwise and the resulting cell suspension was centrifuged at 200 x g for5 minutes. The supernatant was discarded. Thereafter, the cells wereresuspended and the centrifugation step was repeated. According to thesupplier, cells should thereafter be cultured in medium supplementedwith 10 % FBS. However, after contact with Dr. Jyrki Kukkonen at theDepartment of Physiology, Uppsala University, Sweden, another recipewas used. Dr. Kukkonen kindly contributed with personal tips of howthey had cultured these cells. Instead of 10 % FBS, as low as 3 % could beused without affecting the cells negatively. In our study, the FBS washeat-inactivated at 56 °C for 30 minutes before addition to medium. Tominimize the risk of microbial contamination, penicillin andstreptomycin was also included. As with the SMC cultures the HEL cellculture flasks were kept in a humidified incubator at 37 °C in anatmosphere of 95 % air and 5 % CO2. The cells were grown withoutagitation. Fresh medium was added three times every week. As soon aspossible, after establishing a culture, ampoules of cells, with 10 % FBSand 10 % DMSO included, were frozen in liquid nitrogen to be sure tohave fresh cells whenever needed.

Cell characterizationImmunocytochemical characterization of the SMCs was consideredessential because when cultured, SMCs can have a phenotype that isvery similar to fibroblast morphology. Thus, SMCs and fibroblasts aredifficult to distinguish in a phase-contrast microscope (Chamley et al.,1977b; Chamley-Campbell et al., 1979). However, the humanmyometrium contains very few fibroblasts (Palmberg and Thyberg,1986). The immunocytochemical protocol was kindly provided by Dr.Margaretha Lindroth at the Division of Medical Microbiology at ourfaculty. Cells were seeded onto coverslips in 24 multi-well plates. Whencells were subconfluent, they were fixed with 4 % paraformaldehyde inphosphate-buffered saline (PBS) for 15 minutes at 37 °C. Thereafter, thecells were permeabilized with 0.5 % octyl phenoxy polyethoxy-ethanol(Triton X-100) dissolved in PBS for 5 minutes at room temperature.Subsequently, cells were washed with PBS and incubated with primary

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monoclonal antibodies against α-smooth muscle actin (Sigma Chemicals,St. Louis, MO, USA) for 45 minutes at 37 °C. Antibodies were diluted inPBS containing 10 % goat serum to block non-specific binding. Cellswere washed twice with PBS and incubated with secondary antibodiesfor 45 minutes at 37 °C. As secondary antibodies, goat anti-mouseimmunoglobulins labeled with Texas Red were used (Molecular ProbesInc., Eugene, OR, USA). Finally, the coverslips were washed twice in PBSand once in distilled water before they were inverted and mounted withGelvatol (gift from Dr. Lindroth) on microslides. Negative controls weremade with either primary or secondary antibodies. The antibodies werediluted to different concentrations. The optimal dilutions were 1/400and 1/100 for the primary and secondary antibodies, respectively. Themicroslides were stored overnight at 4 °C, to allow the mountingmedium to harden, and were then examined with a Zeiss Axioskopmicroscope (Carl Zeiss, Oberkochen, Germany). Observations weremade at 400 x and 630 x magnifications.

Radioligand binding to myometrial SMC membranesIn Paper I, pharmacological characterization of α 2-ARs in humanmyometrial SMCs was performed by saturation as well as competitionstudies with [3H]rauwolscine as labeled ligand. [3H]rauwolscine is anantagonist to α2-ARs and binds with nearly equal affinity to all threesubtypes in human tissues (Deupree et al., 1996). The total expression ofα2-ARs was analyzed by saturation experiments. In these experiments,non-specific binding was estimated by measuring the binding of[3H]rauwolscine in the presence of phentolamine in parallel assays. Theantagonist phentolamine was used at a concentration that was calculatedto prevent virtually all specific binding. An excess of unlabeled drugaround 100 times higher than the concentration that causes a 50 %reduction in the specific binding (IC50) has been proposed by Bylund andToews (1993) to be used. Non-specific binding may be attributable toligand bound to other sites in the tissue, e.g. other receptors, enzymes,and cell membranes (Haylett, 1996). Total and non-specific binding wasmeasured over a range of concentrations of [3H]rauwolscine to allowspecific binding to approach saturation. Bmax and Kd values wereobtained when plotting the amount of bound radioligand as a functionof [3H]rauwolscine concentration. Bmax is the maximum number of

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binding sites, here calculated as fmol of receptors/mg protein, and Kd isthe equilibrium dissociation constant of [3H]rauwolscine (Swillens et al.,1995). When drug concentration equals Kd, half the binding sites areoccupied at equilibrium. Similar saturation studies were also performedwith the radioligand [3H]-LPA to characterize the LPA receptors inhuman myometrial SMCs. Competition studies were performed toidentify possible expressions of more than one α2-AR subtype in themyometrial SMCs. In these experiments a fixed concentration of[3H]rauwolscine was incubated with the membrane preparation in thepresence of a range of concentrations of oxymetazoline. The partialagonist, oxymetazoline separates α2A- from α2B- and α2C-AR (Bylund etal., 1992). IC50 values were obtained when plotting the amount ofradioligand bound as a function of log[oxymetazoline]. GTP wasincluded in some competition experiments to be sure that the sites foundwere not due to pre-coupling of receptors to G proteins (Bylund andToews, 1993). Vacuum filtration through glass fiber filters was used toseparate the radioligand-receptor complex, which is retained in the filter,from the free radioligand.

Reverse transcriptase-polymerase chain reactionReverse transcriptase-polymerase chain reaction (RT-PCR) is a methodused to amplify cDNA copies from RNA. As a first step RNA is isolatedfrom cells by using a microprep kit. The next step is a reversetranscription of RNA to single-stranded cDNA. An oligonucleotideprimer is allowed to hybridize to the RNA and is then extended byreverse transcriptase to create a cDNA copy. This copy can thereafter beamplified by PCR (Sambrook and Russell, 2001). In this work, randomhexamer primers were used to synthesize cDNA. These primers arecapable of priming synthesis at many points along messenger RNA(mRNA) templates and will generate fragmentary copies of the entirepopulation of the mRNA molecules present (Sambrook and Russell,2001). The PCRs were run for 40 cycles consisting of 95° C for 30 seconds,55 °C for 30 seconds and 72° C for 30 seconds. Before the first cycle a twominutes denaturation period forces the double-stranded DNA moleculesto separate completely, forming single strands, which become templatesfor primers and DNA polymerase directed DNA synthesis. Lowering ofthe temperature to 55 °C allows the primers to anneal to the

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complementary sequence of the template and DNA synthesis by primerextension can begin. As soon as the primer has been extended by a fewnucleotides the temperature can be raised to 72° C. This temperature isoptimal for the heat-stable Thermus aquaticus DNA polymerase and theDNA synthesis proceeds.

In Paper II and IV, the RT-PCR method was used to elucidate theexpression of LPA receptors in SMCs and HEL cells, respectively. Basedupon known nucleotide sequences of the human LPA receptor subtypesin the GenBank database, oligonucleotide primers were designed andordered according to fit the different subtype mRNAs. However, theseprimers did not work well and identified only the LPA3 subtype. At thesame time an article was published that described other primers thatcould identify all three known LPA receptor subtypes (Motohashi et al.,2000). These new primers were ordered and the experiments could beperformed again. The sense and anti-sense primers used were as follow:

LPA1: 5’-CGGAGACTGACTGTCAGCAC-3’ sense5’-GGTCCAGAACTATGCCGAGA-3’ anti-sense

LPA2: 5’-CCCAACCAACAGGACTGACT-3’ sense5’-GAGCCCTTATCTCTCCCCAC-3’ anti-sense

LPA3: 5’-GGACACCCATGAAGCTAATG-3’ sense5’-TCTGGGTTCTCCTGAGAGAA-3’ anti-sense

[3H]thymidine incorporation in SMCsDuring the process of proliferation, cells replicate their DNA andundergo mitosis and cytokinesis. Incorporation of labeled nucleotidesinto newly synthesized DNA is often used as an indicator of cellproliferation. The most widely used method for assessing cellularproliferation is measurement of [3H]thymidine incorporation into DNA.This method measures the rate of DNA-synthesis but not the number ofcells. In Paper I and II, cellular proliferation was assessed by use of thismethod. It was kindly shared by Rigmor Gidlöf at the Division of CellBiology at our faculty, and ended up to be a slightly modified version ofthe one used by Bornfeldt et al. (1991).

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Mycoplasma is a group of prokaryote microorganisms that cannot beclearly visualized by routine light microscopy and, therefore, theirpresence is not often obvious (Freshney, 1992). However, it is importantto test cell cultures for mycoplasma infection as it can seriously affectcellular biochemistry and growth characteristics (Freshney, 1992).Mycoplasma can also interact with [3H]-thymidine when proliferation isstudied. Evidence has been presented that contaminated cells releaseenzymes, which can degrade the [3H]-thymidine (Sinigaglia andTalmadge, 1985). As no mycoplasma was found in the present workwhen the cultures were tested, cell proliferation of myometrial SMCscould be analyzed by [3H]thymidine incorporation.

To be sure that the inhibiting effects of different drugs did not depend oncell death, the viability of cells was checked by a dye exclusion test. Thisis based on the concept that viable cells do not take up a certain dye,whereas dead cells are permeable to the dye (Griffiths, 1992). Trypanblue was used here to evaluate eventual toxic effects by assessment ofviability. To imitate the protocol used for [3H]thymidine incorporationcells were deprived of FBS for 24 hours and then incubated with drugs inFBS-free medium for another 24 hours. Subsequently, the cells wereincubated with trypan blue. The percentages of dead and viable cellswere counted in a Bürker chamber in a microscope. The dye coloreddead cells blue but left viable cells uncolored. Higher concentrations ofinhibitors, used in the [3H]thymidine incorporation experiments, did notincrease the levels of dead cells.

Western blot analysis of protein tyrosine kinasesIn Paper I and III, western blot was used to analyze the occurrence oftyrosine phosphorylated proteins in SMCs and platelets, respectively.This technique has been widely used to detect specific proteins in cells.Tyrosine phosphorylation was monitored using antibodies binding tophosphorylated tyrosine in any protein. Sodium dodecyl sulfate (SDS)was used in combination with heat to dissociate proteins before loadingonto gels. The denatured polypeptides bound SDS and becamenegatively charged. Subsequently, SDS-polypeptide complexes wereseparated in accordance with the size of the polypeptide by

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polyacrylamide gel electrophoresis. After electrophoresis through gels,proteins were stained with Coomassie Blue or blotted to membranes.

In Paper I, primary mouse anti-phosphotyrosine antibodies (UpstateBiotechnology, Lake Placid, NY, USA) and secondary horseradishperoxidase (HRP)-conjugated goat anti-mouse antibodies (DakopattsA/S, Glostrup, Denmark) were used. However, a search for moresensitive antibodies and western blots with lower background resultedin a change of supplier in Paper III. In that study both the primary(PY99) and secondary antibodies were purchased from Santa CruzBiotechnology (Santa Cruz, CA, USA). These were the same kind ofantibodies but seemed to work better in our system. Both primary aswell as secondary antibody concentrations were optimized to get a highsignal to low background ratio. Enhanced chemiluminescence was usedto visualize the immunostained blots. This is a light emitting non-radioactive method for detection of specific antigens conjugated withHRP-labeled antibodies. The light was detected on X-ray film (Kodak,Rochester, NY, USA) in Paper I, and HyperfilmTM (Amersham PharmaciaBiotech, Uppsala, Sweden) in Paper III.

Measurement of [Ca2+]i in different cell typesMeasurement of [Ca2+] i was performed in Paper I, III and IV with amethod first described in 1985 (Grynkiewicz et al., 1985). The method hasbeen further described (Sage, 1996) and is briefly explained here. This isa convenient method that measures the responses in real-time. Fura-2 isa fluorescent indicator that cannot pass through cell membranes. Insteadan esterified probe, fura-2-acetoxymethylester (fura-2), has to be usedwhen loading cells with this indicator. The ester is lipid soluble andtherefore membrane permeable. Once inside the cell, cytosolic esteraseshydrolyses the ester and the fura-2 is then trapped in the cytosol. Ca2+

binding to fura-2 results in a shift in absorbance and fluorescenceexcitation spectrum. Fluorescence emission at 510 nm and excitation at340 nm increases in proportion to the increase in [Ca2+]i whereas theexcitation at 380 nm decreases. Ca2+-saturated signals are obtained byusing a detergent to permeabilize the plasma membrane in the presenceof millimolar external Ca2 +. Furthermore, the Ca2 +-free signals arethereafter obtained by chelating external Ca2+. Changes in [Ca2+]i were

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calculated using the equation: [Ca2+]i=Kd(R-Rmin)/(Rmax-R)(Fo/Fs)(Grynkiewicz et al., 1985). Kd for fura-2 is 224 nM, and R is the ratio offluorescence determined at 340 nm excitation over that at 380 nm.Maximal (Rmax) as well as minimal (Rmin) ratios were determined byadding Triton X-100 and [ethylenebis(oxy-ethylenenitrilo)] tetraaceticacid (EGTA), respectively. Fs and Fo are the fluorescence at 380 nmexcitation following cell lysis and Ca2+ chelation, respectively. In Paper I,SMCs were adherent to small glass coverslips that were placed crosswisein cuvettes. In Paper III and IV, platelets and HEL cells were used insuspensions during continuously stirring with a magnet.

Changes in [Ca2+]i may result from release of Ca2+ from intracellularstores as well as Ca2+ entry across the plasma membrane. Whenmeasuring [Ca2+]i under physiological conditions, with external Ca2+

present, no information is obtained about the source of Ca2+. Internalrelease can be measured by chelating external Ca2+ with EGTA. In thepresence of EGTA, influx of Ca2+ across the plasma membrane can nolonger take place, and any [Ca2+]i changes observed can be attributed tointracellular release. However, measurement of influx requires the use ofa surrogate for Ca2+. Mn2+ can permeate many Ca2+ entry pathways andmay therefore be used as a surrogate. Mn2+ also binds to fura-2 andquenches the fluorescence. At a wavelength of 360 nm the fluorescenceof fura-2 is Ca2+-insensitive. A fall in fluorescence at this wavelength,when Mn2+ is present extracellulary, can be ascribed Mn2+ entry since thenormal intracellular concentration of Mn2+ is very low.

Synthesis of LPA enantiomers and LPA analoguesPaper IV resulted from collaboration with Prof. Peter Konradsson’sgroup, Division of Chemistry, Institute of Technology at our university.Dr. Johan Ekeroth and Dr. Jan Lindberg have described the chemicalsynthesis of different LPA enantiomers as well as LPA analogues inPaper IV. The chemical structures of the different synthesizedcompounds are shown in Table I on the next page. In the Results anddiscussion section the compounds will be referred to by their capitalletters instead of their chemical names.

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Table I. Structures of synthesized LPA enantiomers and LPA analogues.Compounds contain the denoted functional groups at the R1, R2 and R3

positions. In the Results and discussion section the compounds will bereferred to by their capital letters instead of their chemical names.

R1 R2 R3

(R)-1-O-hexadecyl-3-O-phosphoryl-glycerol D C16H33 OH H

(R)-1-O-oleoyl-3-O-phosphoryl-glycerol G C17H33CO OH H

(S)-1-O-oleoyl-3-O-phosphoryl-glycerol H C17H33CO H OH

(S)-1-O-hexadecyl-3-O-phosphoryl-glycerol I C16H33 H OH

(S)-1-O-phosphoryl-3-O-tetradecyl-glycerol J C14H29 H OH

(S)-1-O-oleyl-3-O-phosphoryl-glycerol K C18H35 H OH

Platelet preparationIn Paper III platelets were prepared from blood donated by healthyblood donors at the Blood Transfusion Centre, University Hospital,Linköping, Sweden. Since the normal survival of platelets is about 10days, all donors were asked if they had taken any medication during theprevious two weeks. For example, non-steroidal anti-inflammatorydrugs are known to irreversibly affect platelet functions (McNicol, 1996).These drugs inhibit cyclooxygenase and thereby reduce TXA2 formationand the second secretion-dependent phase of aggregation. Only bloodfrom donors declaring that they had not taken any medication was used.Blood was collected, from a catheter in an antecubital vein, into two 10ml siliconized tubes. Prior to blood collection, 1.65 ml of acid citratedextrose (ACD) solution had been injected into the tubes with a syringeto maintain the vacuum in the tubes. Platelet activation is inhibited byACD mainly due to its chelation of divalent cations (McNicol, 1996). Thetubes were room temperated during the blood collection. The blood inthe two tubes was gently mixed and transferred to three round-bottomedplastic tubes before centrifugation to obtain platelet-rich plasma (PRP).

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The PRP was carefully transferred with plastic pipettes to one conical-bottomed plastic tube. Apyrase as well as acetylsalicylic acid was addedto minimize platelet activation during the isolation procedure.Acetylsalicylic acid prevents the biosynthesis of TXA2, and apyraseconverts released ADP to inactive adenosine 5’-monophosphate (AMP)(McNicol, 1996). After a second centrifugation a distinct platelet pelletwas obtained. The supernatant, except about 1 ml, was removed and 1ml of a Hepes buffered solution was gently added to the tube. Now twodistinct layers of solutions were obtained, platelet-poor plasma next tothe pellet and buffer above this layer. By using a pasteur pipette theplasma was removed, without touching the pellet, and additional bufferwas added. This procedure, taking away the lower layer of solution, wasrepeated twice. Thereafter the pellet was gently resuspended, in buffercontaining apyrase, with a plastic pipette. The platelet solution wasstored at room temperature until used.

Analyzes of platelet aggregationPlatelet aggregation follows many biochemical and biophysical eventsand is a relatively late index of platelet activation. It is however regardedas an important and sensitive method to measure the activation ofisolated platelets (McNicol, 1996). In Paper III, measuring lighttransmission through a stirred platelet suspension using anaggregometer monitored aggregation. The aggregometer measureschanges in light scatter that occur as platelets change shape, becomeadhesive and aggregate. Glass cuvettes with aliquots of plateletsuspensions were used during the experiments. As washed plateletswere used, the transmission was compared to that through a controlblank, containing only buffer. Platelet activation proceeds in a multi-stepprocess following the addition of an agonist. Many agonists initiallycause shape changes of the platelets which transiently decrease lighttransmission (McNicol, 1996). The shape change is due to that the discoidplatelet becomes sphere-shaped, pseudopodia forms and the surfacemembrane folds (Siess, 1989). This can be recorded as a dip of thetransmission baseline. As aggregation proceeds, the platelets form smallclumps which lead to increased light transmission (McNicol, 1996). Theexperiment can be followed on a recorder in real-time.

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Statistical and data analyzesIn all four papers, GraphPad PrismTM (GraphPad Software, SanDiego,CA, USA) has been used to analyze data and to estimate statisticalsignificance. When data is expressed as means of repeatedmeasurements, the error bars represent standard error of the mean(SEM). In Paper I statistical significance of differences between treatedcells and controls was measured by the use of unpaired Student’s t test.A difference was considered statistically significant when p was 0.05 orless. Furthermore, the saturation and competition studies were analyzedwith non-linear regression models. Comparisons of the fit of twoequations were made. This to investigate if the curves fitted best to oneor two site binding or competition, respectively. Bmax, Kd, and IC50 valueswere also calculated. In Paper II, values were analyzed with one-wayanalysis of variance (ANOVA) followed by Bonferroni’s multiplecomparison test as post hoc test for comparisons between groups. Adifference was considered statistically significant when p was 0.05 orless. In Paper IV the data were analyzed with non-linear regressionmodels. Comparisons were made to investigate if the data fitted to asigmoidal dose-response curve with variable slope or not. Furthermore,the best-fitted EC50 values, representing the concentration causing half-maximal effect, were received. The maximal levels of [Ca2+] i in thesigmoidal dose-response curves were compared between different LPAanalogues using a 95 % confidence interval.

Ethical considerationsThe regional ethical committee for human research at our universityapproved the studies where SMCs were cultured from myometrial tissuebiopsies (Paper I and II). The surgeon informed all delivering womenabout the purpose of the study, and the women gave their consent beforeuterine biopsies were taken. The excision of biopsies was not consideredto cause the women any discomfort or increased risk of complications. InPaper III, blood samples were collected from a catheter in an antecubitalvein from blood donors after regular donation. All blood donors wereinformed about the research and gave their consent before collection ofapproximately 17 ml blood. This was not considered to cause the blooddonors any discomfort or increased risk since a normal blood donation isabout 450 ml.

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RESULTS AND DISCUSSION__________________

HUMAN SMCs

Cultured myometrial SMCsTo start primary cultures we isolated SMCs from human myometriumusing two different methods as described in Methodologicalconsiderations. The enzymatic method was the most convenient as itgave many cells in a short time. After one or two weeks the cell layer wasconfluent and the experiments could start. When the explant methodwas used, cells began to migrate from the explant in about one week.Consequently, it took longer time to get enough cells to startexperiments. However, this method was easier to use at the preparingstep, as one only had to cut the biopsy in small pieces. Casey et al. (1984)incubated minced myometrial tissue in an enzymatic solution for onehour to get a preparation of myometrial fibroblasts, and 12-16 hours forpreparation of SMCs. It has been suggested that fibroblasts are damagedand loose their ability to grow in vitro after collagenase digestion(Palmberg and Thyberg, 1986). In 1987, Richardson et al. incubated tissuefor approximately 4 hours to obtain myometrial SMCs. Since all our cellswere characterized as SMCs (see below) it seemed that 4 hours ofincubation would be appropriate to minimize the risk of havingfibroblasts in the cultures.

In cultures of low cell density, individual cells were spindle-shaped witha distinct nucleus seen in the phase-contrast microscope. Multiplepseudopodia emanated from the cells until they made contact withneighboring cells (Figure 5A). In subconfluent cultures, empty areasseparated a network of elongated cells. This so-called hill-and-valleypattern together with the lack of contact inhibition have been taken asmorphological characteristics of SMCs in cultures (Chamley-Campbell etal., 1979; Fager et al., 1988). According to Chamley-Campbell et al. (1979),cultured SMCs can form as many as 15 cell layers in vitro. However, ithas been shown by Ross and Kariya (1980) that the hill-and-valleypattern is less prominent when cells are cultured in the presence of FBS.In very confluent cultures, the myometrial SMCs form kind of whirls(Figure 5B). This confluent way of growing resembles the cellularorientation in vivo. We did not detect any morphological changes of the

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myometrial SMCs after long storage in culture. The number ofpopulation doublings that can be obtained in vitro has been shown todecline in myometrial SMCs after 30 passages (Rifas et al., 1979). Thecells of Rifas et al. (1979) even failed to reach confluence after such a longperiod of culturing. Such problems were avoided in this study by notletting the myometrial SMCs grow for more than about 10 passages.Fresh cells from the liquid nitrogen tank were thawed regularly. Cellsfrozen in liquid nitrogen showed a high degree of viability whenreturned to culture conditions. There was no apparent change inmorphology after freezing.

Figure 5. Photographs of cultured human myometrial SMCs in a phase-contrast microscope. A ) One irregular-shaped cell. B) Culture atconfluency with long, parallel cells.

Already in 1979 it was described how SMCs change over time in culture(Chamley-Campbell et al., 1979). SMCs from arteries have been reportedto undergo dedifferentiation in culture and loose spontaneouscontractility (Chamley et al., 1977a). Typically, the cells changed fromhaving phenotypic characteristics of contractile cells (largely filled withmyofilaments), to those specialized for synthesis and mitosis (largelyfilled with endoplasmic reticulum, Golgi, mitochondria and ribosomes).Based on the observation that most cells do not proliferate in cell cultureuntil such changes occur, it was proposed that cells must modulate to asynthetic state before proliferating (Chamley-Campbell et al., 1979).Moreover, mitogenic stimulation of human arterial SMCs has beenshown to be associated with proliferation and loss of contractile proteins(Fager et al., 1989). In contrast, results from another study demonstrate

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that loss of cell-specific contractile proteins is not a prerequisite forinitiations of DNA-synthesis in rat vascular SMCs (Owens et al., 1986).

Figure 6. Immunocytochemical characterization of a single humanmyometrial SMC seen in the fluorescence microscope. Cells wereincubated with α-smooth muscle actin as primary antibodies and goatanti-mouse immunoglobulins labeled with Texas Red as secondaryantibodies.

The change from contractile to proliferative cells resembles thephenotypic modulation that SMCs undergo in atherosclerotic lesions(Bornfeldt and Krebs, 1999). Normally, endothelial cells synthesizedifferent ligands that inhibit proliferation and contraction of SMCs in theblood vessels (Bornfeldt and Krebs, 1999). When we performedimmunocytochemical characterization, α-smooth muscle actin stainingoccurred exclusively in long, straight, non-interrupted streaksthroughout the cytoplasm and almost filled the cells (Figure 6). In orderto study if the SMCs were a homogenous population, labeled cells werestudied both with fluorescence and phase-contrast microscopy. In ahomogenous SMC population all cells seen in the phase-contrastmicroscope should also be detected in the fluorescence microscope. Allcells studied were labeled with α-smooth muscle actin and were detectedwith both microscopy techniques, indicating that both the enzymatic aswell as the explant method gave homogenous SMC populations.Consequently, the explant method was used in the following cellpreparation steps since it was easier to use and presumably more gentleto the cells. The monoclonal antibody against α-smooth muscle actin hadpreviously been used by Skalli et al. in 1986 to distinguish SMCs fromfibroblasts in mixed cultures. Even if staining against α-smooth muscle

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actin is a conventional technique used to identify SMCs (Fager et al.,1988; Baskin et al., 1993; Bornfeldt et al., 1997) there has been suggestionsthat fibroblasts also can differentiate and express α-smooth muscle actin(Rønnov-Jessen et al., 1992). However, such smooth muscle-differentiatedfibroblasts were only found in neoplastic tissue. It does not seemplausible that the myometrial tissue cultured in our study consists ofneoplastic cells. Consequently, we consider that the cultures of SMCswere homogenous in the present study.

We conclude from these first experiments that it is possible to obtainhomogenous cultures of SMCs from human myometrium. Now wewanted to elucidate some of the mechanisms involved in the stimulationof proliferation of these cells.

Myometrial SMCs express LPA receptors and αααα2-ARsIn this part of the study, two different groups of GPCRs werecharacterized in the myometrial SMCs. First the myometrial cells werestudied in radioligand binding experiments with the general α2-ARantagonist [3H]rauwolscine to detect if the cells express any α2-ARs whencultured in vitro. α2-ARs have been identified in the human myometriumof pregnant women (Bottari et al., 1983b; Berg et al., 1986; Dahle et al.,1993; Adolfsson et al., 1998), but the physiological importance of thereceptors in regulation of labor is still unclear. When those authorsidentified the receptors, whole biopsies from the myometrial wall wereused. We noticed that binding of [3H]rauwolscine to myometrial SMCmembrane was a saturable process of high affinity. Figure 1A in Paper Ishows one of the saturation curves that lead to the calculated Bmax and Kd

values of 74 fmol/mg protein and 5.3 nM, respectively. Whenmyometrial membranes and not cultured cells were used to performsaturation experiments, Bmax values of 17-262 fmol/mg protein, and Kd

values of 3-5 nM were obtained (Bottari et al., 1983b; Dahle et al., 1993;Adolfsson et al., 1998). Our competition experiments were performedwith increasing concentrations of oxymetazoline and the curves fitted toa two-site model (Figure 1B in Paper I). The high affinity sitecorresponds to the α2A-ARs and had an IC50 value of 2.4 nM. The lowaffinity site corresponds to α2B- and/or α2C-ARs and had an IC50 value of147 nM. Close to 70 % of the total α2-ARs consisted of the α2A-AR

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subtype. GTP was included in some competition experiments to be surethat the two sites found were not due to pre-coupling of receptors to Gproteins (Bylund and Toews, 1993; Bylund et al., 2001). Since no GTPshift occurred and the competition curve still fitted to a two-site model,this showed that the α2-AR population probably consists of α2A-, α 2B-and/or α2C-ARs. Results from our saturation studies fitted best to a one-site model and this could be due to the small difference in Kd for[3H]rauwolscine between the different receptor subtypes (Bylund et al.,1992). Oxymetazoline, prazosin, as well as chlorpromazine are capable ofdiscriminating between the three α2-AR subtypes in ligand bindingassays (Bylund et al., 1992). Prazosin has been shown to have selectiveaffinity for α2B- and α2C-, but low affinity for α2A

1994). Oxymetazoline has high affinity for α2A, and chlorpromazine hashigh affinity for α2B-ARs (Bylund et al., 1992). These three ligands havebeen used to characterize the subtypes of α2-ARs in human myometrialtissue (Bouet-Alard et al., 1997; Adolfsson et al., 1998). It was concludedby Adolfsson et al. (1998) that 50 % of the receptors corresponded to α2A-ARs and that the rest was a mixed population of the other two subtypeswith a dominance of α2B-ARs.

Little is known about the effect of cell culturing on α2-AR expression(Faber et al., 2001). Serendipitously we noticed that culturing myometrialSMCs in FBS-free medium for 48 hours increases the expression of α2-ARs (results not shown). This suggests that something in the FBS mightinhibit α2-AR expression. Similar results have been obtained byDevedjian et al. (1991) who observed that 48 hours serum deprivationcaused an almost two-fold increase in the α2-AR number in human colonepithelial cells, without a change in affinity. The decrease in receptorexpression caused by serum can be mimicked by different growth factorsand is likely due to a decrease in transcription rate of the receptor gene(Devedjian et al., 1991). Moreover, the level of α2D-ARs was reduced incultured rat aorta SMCs compared to in vivo levels (Faber et al., 2001).However, it has been shown that rat hepatocytes acquire α2-ARs duringculturing with high concentrations of insulin (Ogihara, 1995).

It is important to define the receptor(s) involved in LPA responses indifferent cell lines. Molecular identification of LPA receptors has,however, been prevented by the lack of metabolically stable high affinity

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ligands and also by significant levels of non-specific binding of LPA tocultured cells (Morris, 1999). Consequently, our ligand binding studieswith [3H]LPA were hampered by the lipophilic nature of the ligand andresulted in very high levels of non-specific binding (results not shown)which also has been demonstrated by others (van der Bend et al., 1992;Thomson et al., 1994). The lack of high affinity radiolabeled ligands turnthe attention to receptor expression measurement of mRNA (Chun et al.,2002). In our study, RT-PCR was used to characterize which subtypes ofthe LPA receptor that are expressed in human myometrial SMCs. Theresults show, for the first time, that myometrial SMCs express all threeknown LPA receptor subtypes (Figure 1 in Paper II). To our knowledgethis represents the first study aimed at characterizing the expression ofLPA receptors in normal cultured SMCs. mRNA levels do notnecessarily reflect expressed, functional receptor protein. However ingeneral, the presence or absence of mRNA correlates reasonable wellwith the presence or absence of receptor protein in a given tissue(Berkowitz et al., 1994).

We conclude from these experiments that human myometrial SMCs stillexpress α2A-, α2B- and/or α2C-ARs when cultured in vitro. However, the invivo situation with positive and negative regulatory mechanisms,perhaps including distinct growth factors, is difficult to mimic in vitro.More experiments are required to elucidate the expression and functionof α2-ARs in human myometrial SMCs. For many types of experiments itis important to examine receptor expression in defined cultures of cellsand not only in tissues. This is the first study to show that culturedhuman myometrial SMCs also express LPA receptors. Our next step wasto figure out if stimulation of these GPCRs might have a role in theenormous growth of the uterus during pregnancy.

LPA and noradrenaline stimulate DNA-synthesis inmyometrial SMCsThe myometrial SMCs responded to LPA with [3H]thymidineincorporation in a significant dose-dependent fashion, which wasinitiated at 10 nM and reached a maximum at 10 µM (Figure 2A in PaperI, and Figure 7 on page 57). LPA at higher concentration than 10 µMappeared to decrease the DNA synthesis as compared to the maximal

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effect. The mitogenic actions of LPA require micromolar doses, whereasnanomolar concentrations are sufficient for many other responses. Up to100 µM of LPA have been used to induce cell proliferation in fibroblastsas well as vascular SMCs (van Corven et al., 1989; van Corven et al., 1992;Tokumura et al., 1994; Gennero et al., 1999). It has been proposed that therelatively high concentrations needed might be due to a considerableuptake by cells or alternatively metabolism of LPA in the culturemedium and degradation during long-term incubation (Jalink et al.,1990). Cells fail to enter the S phase when LPA is removed several hoursafter stimulation and the peak of [3H]thymidine incorporation occursapproximately 20 hours after stimulation (van Corven et al., 1992).Mitogenic doses of LPA stimulate a biphasic activation of MAP kinase inrat fibroblasts in which the sustained phase persists for several hours(Cook et al., 1999). In contrast, non-mitogenic doses can fully reconstitutethe early peak of MAP kinase but fail to elicit the sustained phase (Cooket al., 1999). Early (5 minutes) as well as late (1-3 hours) phosphorylationof MAP kinase was accompanied by [3H]thymidine incorporationinduced by LPA in vascular SMCs (Gennero et al., 1999). However, a 5minutes exposure of opossum kidney cells to LPA was sufficient to causesignificant subsequent proliferation when measured 24 hours later(Dixon et al., 1999). At higher concentrations than 10 µM, LPA tends toprecipitate in Ca2+ containing solutions (Jalink et al., 1990). Distinctdifferences in [3H]thymidine incorporation were obtained whenstimulating human airway SMCs with LPA in the presence or absence ofextracellular Ca2+ (Ediger and Toews, 2001). When using 100 µM LPA inthe absence of extracellular Ca2+, the SMCs exhibited shape change andloss of viability that was interpreted by the authors as apoptosis. Thiswas suggested to occur because solutions made without Ca2+ containedhigher concentrations of bioactive LPA. When we studied [3H]thymidineincorporation, cells were incubated with LPA in medium without FBS.As the medium had a Ca2+ concentration of 1.8 mM, and as LPA was notused at concentrations over 10 µM, apoptosis due to high LPAconcentration was not likely to occur.

Noradrenaline also induced [3H]thymidine incorporation in quiescentSMCs (Figure 2B in Paper I, and Figure 7 on page 57). However, theincrease in [3H]thymidine incorporation was not as marked as with LPAat the same concentrations. A concentration of 1 µM noradrenaline was

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required to get a significant increase in [3H]thymidine incorporation.Furthermore, a maximum increase in [3H]thymidine incorporationelicited by noradrenaline was obtained at 1 to 10 µM. However, thisresponse was at its maximum only about 25 % of the response generatedby 10 µM LPA. The results also showed that the growth-promoting effectof noradrenaline was blocked by the α2-AR selective antagonistyohimbine, suggesting that the proliferative effect of noradrenaline ismediated through α2-ARs (Figure 3 in Paper I). Interestingly, incubationwith yohimbine even lowered the effect of noradrenaline below the basallevel of non-stimulated cells. A possible explanation for this is thatyohimbine acted as an inverse agonist rather than a pure antagonist inthese cells (Kenakin, 1996). Agonists have higher affinity for the activeform of the receptor (G protein-coupled) than for the inactive form of thereceptor (uncoupled from G protein) (Deupree et al., 1996). A trueantagonist should have equal affinity for both forms of the receptor andan inverse agonist should have higher affinity for the inactive form(Deupree et al., 1996). Inverse agonism activities of several α 2-ARantagonists, including yohimbine, have previously been shown (Tian etal., 1994).

It has been demonstrated that activation of α 2-ARs can induceproliferation in several cell types (Tutton and Barkla, 1987; Seuwen et al.,1990; Alblas et al., 1993; Bouloumié et al., 1994). Furthermore, it has beenproposed that noradrenaline has a proliferative effect on arterial SMCs(Raines and Ross, 1993; Yu et al., 1996). Results from human, rat, andguinea pig suggest that α2-ARs might also have growth promotingeffects in the uterus. This is based on the following findings. Estradiolhas been shown to increase the α2-AR expression in rabbit uteruswithout affecting the contractile response to α2-AR stimulus (Hoffman etal., 1981). α2-AR density in the human uterus seems to be increasedduring the midluteal phase (Bottari et al., 1983c) and the first trimester(Kovács and Falkay, 1993). According to the findings of Kovács andFalkay (1993), the α2-AR density is 10 times higher during earlypregnancy than nearer to term. Similar changes have been reported inthe rat uterus (Legrand et al., 1993). Furthermore, in guinea pigs, theuterine content of noradrenaline is elevated during the first part ofpregnancy and is thereafter declining (Owman, 1981). Since we foundthat the expression of α2-ARs is lower in culture with FBS this could be

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an explanation to why noradrenaline is not as effective as LPA instimulating DNA synthesis. The expression of α2-ARs might be higher invivo and therefore more important in stimulating growth of the uterusthan can be shown in cell culture experiments.

In conclusion, stimulation of either LPA receptors or α2-ARs increasesDNA-synthesis in myometrial SMCs. However, the LPA-induced[3H]thymidine incorporation is much more pronounced than afterstimulation with noradrenaline. Consequently, we wanted to comparethese two agents and study possible intracellular mechanisms that couldaccount for their distinct capacity to stimulate SMCs. Initially, the rolesof G proteins and cAMP on the proliferative effect of LPA andnoradrenaline were evaluated.

Gi/o-proteins and cAMP regulate LPA- and noradrenaline-induced DNA synthesisThe proliferative responses of both LPA and noradrenaline werecompletely blocked by PTX (Figure 2A and 2B in Paper I), whichindicates that Gi/o-proteins are involved in the mitogenic signalinginduced by both agonists (Katada and Ui, 1982). LPA and noradrenalinewere used at concentrations that previously were shown to causemaximal DNA synthesis (10 µM). The selected concentration of PTX (100ng/ml) has commonly been used by others to inhibit responses mediatedby Gi/o-protein (van Corven et al., 1993; Velarde et al., 1999). LPA-stimulated Ca2+ mobilization, MAP kinase phosphorylation, and[3H]thymidine incorporation are all suppressed by PTX treatment indifferent cell types (Moolenaar and van Corven, 1990; Lee et al., 2000). Itis known that LPA receptors can couple to Gi/o-proteins and that ligand-receptor interactions result in, among other things, decreased activity ofAC and thereby a reduction of intracellular cAMP (Moolenaar and vanCorven, 1990). The importance of cAMP in the regulation of cellproliferation has been thoroughly investigated. Numerous reportssupport the dual role of cAMP as a negative and positive regulator forcell growth, depending on the cell type studied. Furthermore, the cellcycle stage seems to be a critical factor for cAMP to exert either astimulatory or an inhibitory effect (Lew et al., 1992). It was shown byLew et al. (1992) that an early transient increase in cAMP accumulation in

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quiescent bovine tracheal SMCs acts as a mitogen, whereas a persistentelevation suppresses mitogenesis. Also, stimulation of AC inhibitsproliferation of rat vascular SMCs by delaying the progression from theG1 into S phase of the cell cycle (Yu et al., 1995). There is little evidenceindicating that a reduction of cAMP is the mitogenic signal for LPAreceptors. However, lowering the activity of AC may still have a positiveeffect on proliferation. Increasing the concentration of cAMP generallyhas a growth-inhibitory effect in airway and vascular SMCs (Assender etal., 1992; Noveral and Grunstein, 1994; Maruno et al., 1995; Yu et al.,1995). The stimulatory effect of LPA on proliferation of rat fibroblasts isassociated with a reduction of cAMP levels (van Corven et al., 1989) andα2-AR agonists inhibit forskolin-stimulated cAMP accumulation in rabbitSMCs (Docherty, 1998). Forskolin is an AC stimulator that rises thecAMP concentration. Our findings indicate that DNA synthesis incultured myometrial SMCs is markedly sensitive to changes in cAMPconcentration. This is based on the observations that forskolin dose-dependently reduced the incorporation of [3H]thymidine (Figure 4A and4C in Paper I). Forskolin, at concentrations of 1 to 10 µM, significantlyreduced [3H]thymidine incorporation in both LPA and noradrenalinestimulated SMCs. Although considerable progress has been made inunderstanding the mechanism of gene regulation by cAMP, little isknown about the molecular mechanisms by which cAMP modulates cellgrowth (Giasson et al., 1997). A number of studies have proposed thatcAMP might inhibit cell proliferation by interfering with the MAP kinasecascade, e.g. in human arterial SMCs and rat fibroblasts (Cook andMcCormick, 1993; Graves et al., 1993; Hordijk et al., 1994a). In arterialSMCs, proliferation is for example inhibited by cAMP at several kinases,both in cytoplasm and nucleus (Koyama et al., 2001). In contrast, cAMPstimulated the MAP kinase pathway in monkey kidney fibroblasts(Faure et al., 1994) and angiotensin II–induced activation of MAP kinaseswas unaffected by cAMP in vascular SMCs (Giasson et al., 1997). Giassonet al. (1997) suggested that stimulation of MEK involve distinct kinds ofRaf that are regulated differently by PKA. It is also possible that, in somecell types, cAMP-mediated inhibition of the MAP kinase cascade is PKA-independent (Bornfeldt and Krebs, 1999). Furthermore, Faure andBourne (1994) presented a model where cAMP effects on Raf inhibitsome cells, whereas other cells are stimulated by cAMP on MAP kinase.

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In summary, inhibition of Gi/o-proteins and stimulation of AC abolishLPA- and noradrenaline-induced DNA synthesis in myometrial SMCs.At this point we could not distinguish between the signalingmechanisms of LPA and noradrenaline. Since phosphorylation of proteinkinases is fundamental for cell proliferation we wanted to furthercharacterize these signals in myometrial SMCs.

LPA and noradrenaline activate protein tyrosinekinasesThe present study shows that LPA and noradrenaline induce tyrosinephosphorylation of proteins in myometrial SMCs (Figure 5 in Paper I).Both agonists appeared to stimulate phosphorylation of proteins of thesame size to approximately the same extent. Although we do not knowthe identity of these tyrosine phosphorylated proteins in myometrialSMCs, their sizes seem to correspond to previously described proteins invascular SMCs (Leduc et al., 1995; Du et al., 1996). LPA also stimulatesrapid tyrosine phosphorylation of several cellular proteins in quiescentfibroblasts (van Corven et al., 1993; Hordijk et al., 1994b; Seufferlein andRozengurt, 1994). Among the major substrates are focal adhesionassociated proteins. Upon binding to its receptor, LPA induces tyrosinephosphorylation of proteins with molecular weights between 42 and 130kDa (Hordijk et al., 1994b; Budnik and Mukhopadhyay, 1997). The LPA-stimulated MAP kinase pathway has been shown to be sensitive to thetyrosine kinase inhibitor genistein (Hordijk et al., 1994b). In the presentstudy, the DNA synthesis in myometrial SMCs was reduced whengenistein was incubated together with LPA and noradrenaline,respectively (Figure 4B and 4D in Paper I). This further supports thecritical role for tyrosine phosphorylations in the mitogenic signaling ofthese agonists. Genistein significantly inhibited the induced DNAsynthesis at 10 µM. It has been proposed that genistein requires a dose of50 µM for full inhibitory effect (van Corven et al., 1993).

To summarize, even if we demonstrated that LPA and noradrenalineinduce tyrosine phosphorylation of proteins in myometrial SMCs, thesignaling mechanisms and outcomes are still unclear. Also, after theseexperiments no distinction could be made between the signaltransduction elicited by LPA and noradrenaline. Since cytosolic Ca2+ is

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important for many cellular mechanisms we wanted to elucidate in thefollowing experiments its eventual role in LPA- and noradrenaline-stimulated SMCs.

LPA induces cytosolic Ca2+ and CaM kinase responsesLPA (10 µM) but not noradrenaline (10 µM), stimulated a rapid andtransient rise in [Ca2+]i in fura-2 loaded SMCs (Figure 6 in Paper I, andFigure 7 on the next page). LPA’s effect on [Ca2+]i was instantaneous as istypically seen with Ca2+-releasing agonists acting through cell surfacereceptors. The Ca2+ signal reached a peak a few seconds after stimulationand returned to the basal level over the next minute. Intracellular Ca2+

and Ca2+-linked protein tyrosine kinases have been proposed to play acritical role in mediating noradrenaline stimulation of MAP kinasepathways in human vascular SMCs (Hu et al., 1999). This effect was,however, mediated through α1-ARs. Already in 1987 it was concludedthat cultured human myometrial SMCs retained properties of SMCs intissue and that they were able to respond to stimuli that increase [Ca2+]i

(Richardson et al., 1987).

The exact role of Ca2+ in the regulation of cell proliferation is unclear andappears to be cell specific. In rat fibroblasts, LPA-stimulated MAP kinaseactivity was not mediated by rises in [Ca2+] i (Cook et al., 1997).Nevertheless, Ca2+ was necessary for expression of MAP kinasephosphatase and to fully reconstitute the response to LPA. Ca2+ alsoappears to be important for GPCR-mediated transactivation of receptortyrosine kinases (Murasawa et al., 1998; Zwick et al., 1999b). Daub et al.(1996) have proposed that the LPA receptor indirectly activates the EGFreceptor. This ligand-independent activation of the EGF receptor can bestimulated through a rapid transient Ca2+ signal (Lev et al., 1995). α2-ARstimulation of MAP kinase activity is mediated through Ca2 + andtyrosine phosphorylation dependent pathways in transfected humanembryonal kidney cells (Della Rocca et al., 1997). Furthermore,Ca2+/calmodulin-stimulated cyclic nucleotide phosphodiesterase ishighly expressed in proliferating but not in quiescent SMCs (Rybalkin etal., 1997). A rise in [Ca2+]i seem also to be an important mechanism bywhich growth factors induce proliferation of vascular SMCs (Magnier-Gaubil et al., 1996). Seewald et al. (1997) reported that LPA can induce an

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increase in [Ca2+]i in vascular SMCs. Increasing evidence suggests thatprogression of the cell cycle is dependent on transient rises of [Ca2+]i.Changes in [Ca2+]i occur at the awakening of cells from quiescence, at theG1/S transition, during the S phase, and at the exit from mitosis(Muthalif et al., 2001b). It has been reported that activation of CaM kinaseII is essential in proliferation of cells by facilitating transition of cellsfrom G1 to S, from G2 to M, and from metaphase to anaphase (Santella,1998). Moreover, a transient rise in [Ca2 +]i might activate genetranscription via kinases directly controlled by CaM kinases (Schulman,1993). Huang et al. (1995) have shown that extracellular Ca2 + canstimulate sustained growth of fibroblasts as well as MAP kinaseactivation in the absence of growth factors.

Figure 7. Differences between LPA- and noradrenaline-stimulated humanmyometrial SMCs. A) Effects of increasing concentration of LPA ornoradrenaline on [3H]thymidine incorporation into cells. Results areexpressed as the percentage increase in [3H]thymidine incorporationcompared to levels in non-stimulated cells. Data points represent means ±SEM of three separate experiments with six samples at each concentration(LPA) or 10 separate experiments in duplicate (noradrenaline). **p<0.01and ***p<0.001 compared with non-stimulated cells. B) Effects of 10 µMLPA or noradrenaline on [Ca2+]i in fura-2-loaded cells. The illustratedtraces are representative of six separate experiments.

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KN-62 is a useful and convenient pharmacological tool for elucidatingphysiological functions of CaM kinase II. This inhibitor prevents theassociation of calmodulin and CaM kinase II (Hidaka and Kobayashi,1994). As we noticed that KN-62 potently inhibited LPA-induced[3H]thymidine incorporation we suggest that CaM kinase II plays animportant role in DNA synthesis in myometrial SMCs (Figure 2 in PaperII). KN-62 significantly reduced the [3H]thymidine incorporation elicitedby LPA at concentrations of 10 and 100 nM. Minami et al. (1994) havestudied the effects of KN-62 in human leukemia cells and showed thatcell growth is inhibited dose-dependently. Their results indicate that theinhibitor blocks cell cycle progression and that cells accumulate in Sphase. Similarly, incubation of lung carcinoma cells with KN-62 potentlyinhibited DNA-synthesis and slowed progression through S phase(Williams et al., 1996). Furthermore, CaM kinase activated cytosolic PLA2

which is involved in proliferation of vascular SMCs (Muthalif et al.,2001a) and human leukemia cells (Muthalif et al., 2001b). However, it hasbeen suggested that LPA-induced increases in [Ca2+]i in ovarian cancercells would not be sufficient for induction of cellular proliferation (Xu etal., 1995b).

To conclude, Ca2+ seems to be an important intracellular messenger inresponse to LPA-stimulated DNA synthesis in myometrial SMCs.Perhaps this capacity of LPA to stimulate many signal transductionpathways can explain why LPA elicits a more powerful stimulation ofDNA synthesis than noradrenaline. However, additional pathwaysmight also be involved in this complex mechanism. Since a potentialtransactivation phenomenon had been described in the literature (Daubet al., 1996; Cunnick et al., 1998; Herrlich et al., 1998; Voisin et al., 2002),we wanted to evaluate possible interactions between LPA stimulationand EGF receptor activation in myometrial SMCs.

LPA stimulates EGF receptor activationStimulated GPCRs can use receptor tyrosine kinases as signaltransduction elements. Since EGF receptors have been reported to bepresent in human myometrial cells (Chegini et al., 1986) we wanted toidentify the possible role of these receptors in LPA-mediated signaling.Based on the fact that treatment with Tyrphostin AG 1478, an inhibitor of

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EGF receptor tyrosine kinase, reduced the LPA-induced DNA-synthesis(Figure 3 in Paper II), we suggested that EGF receptor activity seems tobe important for LPA-mediated DNA-synthesis in myometrial SMCs.The effect of this inhibitor was significant at concentrations of 1 and 10µM. The inactive inhibitor Tyrphostin A1, which was used as a negativecontrol (10 µM), showed no significant effects on the response by LPA.The transactivation phenomenon has mainly been studied inoverexpressed systems. As stated by Della Rocca et al. (1999) it isimportant to study signal transduction pathways in primary cell culturesand not only in transfected and immortalized cell lines. It has beenproposed that the EGF receptor can be transactivated by stimulationwith LPA in rat fibroblasts ( D a u b et al., 1996), and humanneuroepithelioma cells (Buist et al., 1998), and by angiotensin II invascular SMCs (Eguchi et al., 1998). Simultaneous treatment with LPAand EGF exhibited a synergistic activation of mitogenesis in humanairway SMCs (Cerutis et al., 1997; Ediger and Toews, 2000). Furthermore,it was recently shown that LPA up-regulated the expression of EGFreceptors in the same cell types (Ediger et al., 2002). The up-regulationrequired 10 µM of LPA but it was suggested that transactivation of EGFreceptors by LPA was not involved in the up-regulation of receptors.Moreover it was indicated that LPA does not cause ligand-independenttransactivation in human airway SMCs.

Growth factors can be synthesized as transmembrane proteins and mayrequire cleavage as well as release from the membrane to obtain fullactivity (Rifkin et al., 1999). Interestingly, recent results suggest thattransactivation of the EGF receptor is a result of MMP-mediated releaseof membrane-bound EGF, i.e. an autocrine activation of the receptor andnot a ligand-independent transactivation (Dong et al., 1999; Prenzel et al.,1999). Prenzel et al. (1999) have shown that treatment of cells withvarious GPCR agonists rapidly stimulate the conversion of thetransmembrane heparin binding EGF precursor to the soluble ligand forthe EGF receptor. The release is likely to be mediated by MMPs as thetransactivation system is sensitive to Batimastat, an MMP inhibitor.MMPs are Zn2+-dependent endopeptidases that are categorized intothree different functional groups based on their substrate target, and arecollectively capable of degrading essentially all matrix components(Zervos et al., 1997; Rothenberg et al., 1999). Thereby they regulate

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formation, maintenance as well as remodeling of tissues (Shapiro, 1998;Seiki, 1999). These degrading enzymes are fundamental in bothphysiological and pathological states (Woessner, 1991; Botos et al., 1996).For example, major roles for these enzymes in tumor biology arebreakdown of physical barriers, promotion of tumor invasion andmetastasis (Mitsiades et al., 1999). MMPs may also play a role in arterialSMC migration and proliferation (Zempo et al., 1996). In the study ofZempo et al. (1996) inhibition of MMPs suppressed intimal thickeningafter arterial injury, but MMPs did not inhibit DNA-synthesis measuredby [3H]thymidine incorporation. We wanted to examine if MMPs alsocould be involved in the LPA-induced DNA-synthesis in myometrialSMCs. As others had used Batimastat for MMP inhibition we asked theproducer for a kindly gift. However, British Biotech Pharmaceuticals Ltd.(Oxford, UK) proposed us to try BB-3103, another MMP inhibitor,instead. This since Batimastat is very poorly soluble and thereforeunsuited to cell culture assays. BB-3103 is an inhibitor with similaractivity but is much more soluble than Batimastat. As shown in Figure 4in Paper II, the addition of BB-3103 reduced the LPA-induced[3H]thymidine incorporation. BB-3103 was used at concentrationsbetween 1 nM and 10 µM. The inhibition was statistically significant atconcentrations of 0.1 and 10 µM. However, the use of 1 µM BB-3103 didnot significantly reduce the LPA-induced stimulation. The biologicalimportance, if any, of this finding is unknown. Shedding of EGF receptorligands by MMPs may not represent the only mechanism of activation ofMAP kinase, since the MMP cleavage of the ligands require activatedMAP kinase (Gechtman et al., 1999). MMP secretion can be mediated byPLD in human fibrosarcoma cells (Williger et al., 1999). Moreover, PLDactivity has been shown to be stimulated in response to EGF (Slaaby etal., 1998). Furthermore, PLD hydrolyzes phospholipids to generate highconcentrations of PA that subsequently is converted to LPA (Moolenaaret al., 1997; Goetzl and An, 1998; van Dijk et al., 1998). We have in factnoticed that PLD, maybe through conversion of other phospholipids toLPA, can stimulate DNA-synthesis in myometrial SMCs (results notshown). LPA can also stimulate PLD (Moolenaar, 1995a). This indicatesthat EGF, LPA, MMPs, and PLD might influence each other in a loopmechanism (Figure 8).

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Figure 8. Hypothetical illustrationof how these factors mightinfluence each other in a loopmechanism. MMP secretion canbe mediated by PLD, which canbe stimulated by EGF. PLDhydrolyzes phospholipids togenerate PA, which can beconverted to LPA. LPA can alsostimulate PLD. For abbreviationsand references please see text.

From these experiments we conclude that LPA activates the EGF receptorin human myometrial SMCs. At least in part, MMPs may mediate thistransactivation through release of EGF receptor ligands. Now we wantedto examine if also G proteins could act as a link between the two receptorsystems, the LPA receptors and the EGF receptors.

PTX inhibits EGF-induced DNA-synthesisTo study if stimulation by EGF involved a PTX-sensitive G protein inmitogenic signaling, cells were exposed to 100 ng/ml of PTX togetherwith EGF for 24 hours. As shown in Figure 9, PTX inhibited EGF-stimulated DNA-synthesis. For comparisons between cells incubatedwith EGF plus PTX with cells incubated with only EGF statisticalanalysis of the data was performed by one-way ANOVA followed byBonferroni’s multiple comparison test as post hoc test for comparisonsbetween groups. At concentrations of 1 and 10 ng/ml of EGF, PTXsignificantly (p<0.001) inhibited DNA-synthesis. The highestconcentration of EGF (100 ng/ml) was also significantly inhibited byPTX but to a lesser extent (p<0.05). Although EGF binds to a receptorwhich belongs to the family of receptor tyrosine kinases it has beenshown that Gi proteins may have a role in EGF’s activation of MAPkinase (Melien et al., 1998). We have already shown that the proliferativeresponse to LPA can be markedly reduced by PTX (Figure 2A in Paper I).Here we used PTX to evaluate if DNA-synthesis induced by EGF couldbe inhibited as well. We noticed a reduced DNA-synthesis, whichindicates that Gi/o proteins are involved also when EGF is the mitogenicsubstance in myometrial SMCs. In contrast to LPA, EGF in higher

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concentrations was able to overcome some of the inhibitory blockade ofPTX suggesting that EGF receptors in addition to Gi/o proteins use aPTX-insensitive pathway.

Figure 9. Inhibitory effects of 100 ng/ml PTX on the incorporation of[3H]thymidine into human myometrial SMCs stimulated with EGF at theconcentrations 1, 10 and 100 ng/ml. Cells stimulated with 100 ng/ml EGFrepresent 100 %. The bar marked with basal represents cells incubatedwithout FBS and drugs. Data points represent means ± SEM of threeseparate experiments with six samples in each. *p<0.05 and ***p<0.001compared to cells treated only with EGF.

How G-proteins might be involved in EGF-induced signaling is unclear,but Yang et al. (1991) have reported a direct interaction between the EGFreceptor and Gα i2. Furthermore, the juxtamembrane region in thecytosolic domain of the EGF receptor has been shown to increase theGTPase activity of the Gi protein and thereby inactivate the protein (Sunet al., 1995). The cytosolic part of the receptor has also been proposed tobe an important regulatory region of kinase activity andautophosphorylation of the EGF receptor (Poppleton et al., 1999). Forinstance, hormones such as prolactin may regulate EGF receptor activitynegatively by threonine phosphorylation within this region (Fenton andSheffield, 1997). This region also shows sequence homology with thecarboxyl-terminal of the third intracellular loop of the LPA3 receptor, a

basal 1 1 10 10 100 1000

25

50

75

100

125****** *

EGF (ng/ml)

+ PTX (100 ng/ml)

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region that has been shown to interact with G proteins (Schöneberg et al.,1999). We have compared amino acid sequences of the third intracellularloop of the LPA1 receptor and the α2A-AR with the juxtamembrane partof the EGF receptor and found similarities (results not shown). Since thethird intracellular loop is known to interact with G proteins this might bean important link between the GPCRs and EGF receptors. It has beenassumed that direct interactions of EGF receptors with Gi proteins arepossible in rat fibroblasts (Arimura et al., 1998). A possible linkagebetween IGF and Gα i2 signal pathways leading to DNA-synthesis(LaMorte et al., 1992), and insulin-induced phosphorylation of Gαi

proteins has also been found (Kanoh et al., 2000). We have shown thatcalmodulin specifically interacts with the juxtamembrane region of EGFreceptors (Aifa et al., 2002). We propose that this region is essential forEGF-mediated Ca2+-calmodulin signaling as well as signal integrationbetween other signaling pathways (Aifa et al., 2002). Both GPCRs and thejuxtamembrane region of EGF receptors can directly bind calmodulinsuggesting that this may be another link between the two receptorsignaling systems.

To conclude, upon stimulation with EGF Gi/o proteins interact with EGFreceptors and induce DNA synthesis in human myometrial SMCs. Thusthe Gi/o proteins might be an important link between the signalingpathways used by GPCRs and receptor tyrosine kinases during thetremendous growth of pregnant myometrium.

Some results obtained by using different pharmacological tools inhuman myometrial SMCs in this study are summarized in Figure 10 onthe next page.

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Figure 10. Illustration of some of our results obtained by usingpharmacological tools on human myometrial SMCs. Stimulation with LPAor EGF lead to increased DNA synthesis measured as [3H]thymidineincorporation. Several pathways are probably involved in the transfer ofthe effect elicited by LPA. Transactivation of EGF receptors seems to beimportant, presumable through activation of MMPs and release of EGFreceptor ligands. However, it is unclear how CaM kinases could beinvolved in the transactivating phenomenon. DNA synthesis induced byeither LPA or EGF is inhibited by PTX, suggesting that Gi/o proteins mightbe an important link in the crosstalk between GPCRs and receptor tyrosinekinases.

HUMAN PLATELETS AND HEL CELLS

HEL cells express LPA receptorsLPA is considered an agonist of platelet activation, but its mechanism ofaction and its role in physiological and pathological conditions arelargely unknown. Motohashi et al. (2000) have recently used RT-PCR toidentify LPA-receptors in platelet preparations. They found that mRNAfrom all three receptor subtypes was expressed in human platelets. Incontrast, it has been proposed that LPA interacts with plateletsindependently of LPA-receptors (Hooks et al., 2001). HEL cells expressmarkers for different kinds of blood cells, and many distinct receptortypes (Martin and Papayannopoulou, 1982; Schwaner et al., 1992). Withrespect to signal transduction, HEL cells have been used as a model

YYY

YP P

PP

MMP

LLPPAAN

C

DNA synthesis

EEGGFF

EEGGFF

Gi/o

CaM kinase

Ca2+

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system for platelets (references in Schwaner et al., 1992). Wecharacterized the LPA receptor subtypes that are expressed in HEL cellsby using RT-PCR. The results show that HEL cells mainly express LPA1,LPA2 and perhaps also LPA3 (Figure 1 in Paper IV). The expected sizes ofthe PCR products with the primers used were 398, 289 and 256 basepairsfor LPA1, LPA2 and LPA3, respectively. PCR products corresponding tothese sizes were obtained. The LPA1 and LPA2 subtype representativeamplicons were detected in three independent experiments whereasLPA3 amplicons were only detected in one experiment. During all ourRT-PCR experiments with these primers the LPA3 receptorrepresentative has been most difficult to amplify. This might be due tolower mRNA expression and/or non-optimal experimental conditions.

For the first time we show that HEL cells express at least two, perhaps allthree LPA receptors. As we detected LPA receptors, we conclude thatHEL cells provide an important model system for the elucidation of LPAsignaling mechanisms. Receptor-stimulated Ca2+ mobilization in HELcells has properties similar to the corresponding phenomenon inplatelets and can therefore be a valuable system for investigating plateletaspects of LPA signal transduction.

LPA induces cytosolic Ca2+ responsesSince cultured cells can respond to LPA with a transient rise of Ca2+,measurement of [Ca2+]i provides a rapid and appropriate assay for LPA-mediated responses. HEL cells have earlier been used to study Ca2+

mobilization by stimulation of α2-ARs with adrenaline (Schwaner et al.,1992; Jansson et al., 1998; Michel, 1998). We have characterized LPA-induced cytosolic Ca2+ responses both in platelets and HEL cells. Wenoticed that LPA was able to increase [Ca2+]i in fura-2 loaded platelets(Figure 1 in Paper III) and HEL cells (Figure 2 in Paper IV) in a dose-dependent manner. The LPA-induced cytosolic Ca2+ response in plateletswas detectable at LPA concentrations around 0.01 to 0.1 µM and reacheda maximum at 10 µM. At the highest LPA concentration, [Ca2+]i increasedabout 91 ± 7 nM. In HEL cells, LPA was used at concentrations between 1nM and 10 µM. HEL cells increased [Ca2+]i with 150 ± 4 nM at the highestconcentration of LPA. By analyzing the data with a nonlinear regressionmodel an EC50 value of 20 nM was obtained in HEL cells. The complex

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mechanisms underlying receptor-mediated increases in [Ca2+]i are notcompletely understood. However, in almost all cell types it is due to arapid liberation of Ca2+ from intracellular stores with a concomitantinflux of Ca2+ across the plasma membrane, followed by a moresustained influx of Ca2+. Based on the results obtained with the Mn2+

quenching technique and the responses detected in the presence ofEGTA, we conclude that LPA-induced rises in [Ca2+]i involve both arelease of Ca2+ from intracellular stores and an extracellular influx of Ca2+

in platelets and HEL cells (Figure 2A and 2B in Paper III and Figure 3Aand 3B in Paper IV). The response elicited by LPA in platelets wascompared to the more well-known platelet activators thrombin, ADP,and adrenaline. Adrenaline (10 µM) did not affect [Ca2+]i whereasthrombin (0.3 U/ml) and ADP (40 µM) increased [Ca2+]i with about 618 ±48 nM and 245 ± 23 nM, respectively (Figure 4A in Paper III). Takentogether, the results show that LPA stimulated cytosolic Ca2+ signaling ata relatively low concentration in both human platelets and HEL cells.The involvement of cyclic nucleotides, Gi/o proteins and PLC in LPA-stimulated Ca2+ increases was further examined.

Increases in cAMP or cyclic guanosine 3’, 5’-monophosphate (cGMP) areassociated with inhibition of agonist-induced rises in [Ca2+]i in platelets(Geiger et al., 1994). To study if this was the case also in LPA-inducedrises in [Ca2+]i we used forskolin or GEA 3175; a nitric oxide-donor whichrises the cGMP concentration via stimulation of guanylyl cyclase (GC).These drugs, when added separately, markedly inhibited LPA-inducedrises in [Ca2+]i (Figure 3 in Paper III). Forskolin inhibited the LPA-induced [Ca2+]i from 75 ± 15 nM in the absence of inhibitor to 14 ± 9 nMin its presence. In the same way GEA 3175 inhibited the Ca2+ responsefrom 83 ± 12 nM in the absence of inhibitor to 17 ± 9 nM in its presence.Consequently, we propose that LPA-induced increases in [Ca2+]i aresensitive to both cAMP and cGMP. It is tempting to speculate thatinhibition of both AC and GC might be involved in stimulation ofplatelets by LPA. Our result is in agreement with those of Torti et al.(1996). By measuring the intracellular concentration of cAMP they foundthat LPA caused a rapid decrease in the level of cAMP in platelets. It hasbeen proposed that LPA affects platelets independent of LPA-receptorproteins and that LPA disturbs biological membranes (Hooks et al.,2001). Thus, LPA may act as an ionophore and facilitate Ca2+

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translocation independently of GPCRs. Our data do not support a rolefor receptor-independent mechanisms. This conclusion is based on thefollowing findings. First the kinetics of LPA-induced rises in [Ca2+]i

closely resembles that of other receptor-coupled activators, and secondlythe LPA-induced Ca2+ responses were extremely sensitive to elevation ofboth cAMP and cGMP. These patterns are contradictory to thoseestablished for Ca2+ responses triggered by drugs acting independentlyof receptor proteins. For instance, Ca2+ ionophore-induced increase in[Ca2+]i is insensitive both to cAMP and to cGMP elevating compounds(Grenegård et al., 1996).

To further study the characteristics and mechanisms of Ca2+ signalingelicited by LPA in HEL cells, two distinct inhibitors were used. To assessif PTX-sensitive G proteins are involved in LPA-induced Ca2+

mobilization, cells were pretreated with 100 ng/ml PTX for 24 hours. Bycomparing PTX-treated HEL cells with non-treated cells we found thatPTX did not inhibit the ability to increase [Ca2+]i (Figure 4 in Paper IVand Figure 11 on the next page). Instead the increase in [Ca2+]i stimulatedby LPA was even higher when cells had been incubated with the toxin.Cells treated with 10 µM LPA increased [Ca2+]i with approximately 151 ±4 nM (n=3), whereas cells pretreated with PTX elevated [Ca2+]i with 228 ±50 nM (n=3). This indicates the possible involvement of PTX-insensitiveG proteins in the transduction of this response. Maybe the receptors’ability to activate Gq/11 proteins is enhanced when the Gi/o proteins areblocked by PTX. Pretreatment of opossum kidney cells with PTX had noeffect on LPA-induced Ca2+ responses (Dixon et al., 1999). Accordingly, ininsect cells PTX did not inhibit LPA3-transduced Ca2+ responses,indicating that PTX-insensitive G proteins are coupled to LPA3 inmediating the Ca2+ response (Aoki et al., 2000). It has been shown by Anet al. (1998b) that PTX almost completely blocked LPA-induced Ca2+

mobilization by LPA1 in rat hepatoma cells, but only partially blockedthat by LPA2, suggesting that LPA1 use Gi/o proteins, whereas LPA2

requires both Gi/o and Gq/11 proteins. Since we showed that all three LPAreceptor subtypes are expressed in HEL cells further studies withsubtype specific agonists or antagonists are needed to examine the roleof the distinct subtypes in Ca2+ responses.

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As can be seen in Figure 4 on page 20, PLC is important in the Ca2+

signaling pathway triggered by LPA receptors. As this is the best knownpathway inducing Ca2+ mobilization we wanted to examine if it wasincluded in LPA-activated HEL cells. We used U73122, which inhibitsthe coupling of G protein to PLC and thereby inhibits PLC activity. Cellswere pretreated with 4 µM of U73122 for 5 minutes prior to LPAstimulation. As can be seen in Figure 11 below, U73122 inhibited theLPA-induced Ca2+ mobilization. Cells treated with 10 µM LPA increased[Ca2+]i with 169 ± 10 nM (n=3), whereas cells pretreated with U73122elevated [Ca2+]i with 65 ± 16 nM (n=3). These results suggest that theGPCR-mediated PLC-signaling pathway may be involved in LPA-stimulated Ca2+ mobilization in HEL cells. Others have publishedcontradicting results with U73122 on LPA-induced responses. Forexample, in epithelial cells this inhibitor was ineffective in blocking LPA-evoked Ca2+ responses (Thoreson et al., 2002), whereas it markedlyattenuated LPA-induced Ca2+ responses in LPA1 and LPA2 transfected Tlymphocytes (An et al., 1998b). Furthermore it has been shown that LPAmobilize Ca2+ through activation of PLC in various cultured cell types(Moolenaar, 1995a).

Figure 11. Effect of PTX and U73122 on [Ca2+]i in fura-2-loaded HEL cellsstimulated with 10 µM of LPA. Cells were incubated with PTX (100ng/ml) for 24 hours or U73122 (4 µM) for 5 minutes prior to stimulationwith LPA. Fluorescence emission was registered at 510 nm duringsimultaneous excitation at 340 and 380 nm. The data represent means ±SEM from three separate experiments except the bar with only LPA wheren=6.

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200

300 + PTX (100 ng/ml)

+ U73122 (4 µµµµM)

LPA (10 µµµµM)

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To conclude, LPA induces Ca2+ rises in both human platelets and HELcells. In platelets both cAMP and cGMP inhibit LPA-induced rises ofCa2+ and in HEL cells the transduction of Ca2+ rises is possibly mediatedby PTX-insensitive G proteins and PLC. Surprisingly, LPA receptors inplatelets have been shown to lack stereospecificity (Gueguen et al., 1999).However, since chemically modified analogues of LPA have been usedin that and other studies we wanted to further elucidate thestereochemical effects of the (R)- and (S)-enantiomers of LPA. For thispurpose we used HEL cells.

The structure of LPA is important for cell activationThe lack of receptor specific agonists and antagonists obstructstraditional pharmacological analyzes of LPA. The goal with this part ofthe study was to find an antagonist to the LPA receptors.Enantiomerically pure phospholipids were synthesized by starting from(R)- or (S)-glycidol as described by Lindberg et al. (2002). The firstsynthesized LPA analogues were used at 10 µM since this concentrationearlier had been shown to stimulate myometrial SMCs and plateletsmaximally (results not shown). As a screening method we usedmeasurement of changes in [Ca2+]i in fura-2-loaded HEL cells. Theconclusions drawn after some preliminary experiments were as follows.Unnatural (S)-enantiomers worked at least as good as the natural (R)-enantiomers to stimulate cells. Saturated analogues were more efficientthan unsaturated ones. Ester- or ether-linkage of the hydrocarbon chaindid not seem to alter the efficacy. These results made us really interestedand we concluded that further analyzes and new analogues wereneeded. Since (R)-1-O-hexadecyl-3-O-phosphoryl-glycerol (D) seemed tobe the most potent analogue it was chosen as a lead structure. A (S)-enantiomer and analogues with different hydrocarbon chain lengthswere synthesized. Moreover, full dose-response curves were completed.The structures of the analogues can be seen in Table I in Paper IV andTable I in the section Methodological considerations in this text.

To our knowledge this is the first study in which both (R)- and (S)-enantiomers of LPA have been synthesized and analyzed as regardstheir ability to stimulate cells. As can be seen in Figure 5A and 5B inPaper IV, when comparing (R)-1-O-oleoyl-3-O-phosphoryl-glycerol (G)

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with (S)-1-O-oleoyl-3-O-phosphoryl-glycerol (H), and D with (S)-1-O-hexadecyl-3-O-phosphoryl-glycerol (I), agonist potency was almost thesame for both (R)- and (S)-enantiomers. The EC50 values of G and H were73 and 59 nM respectively, while the EC50 values of D and I were 65 and57 nM. Our results are in accordance with those of Simon et al. (1982)who made measurements on analogues D and I and showed that bothanalogues were active. In their study the two enantiomers showedsimilar activity in eliciting platelet aggregation. However, theiranalogues were enzymatically generated and were not analyzedconcerning stereochemistry. Our analogues can be compared to thosechemically synthesized by Yokoyama et al. (2002). They used theseanalogues to induce DNA synthesis as well as intracellular Ca2 +

mobilization in LPA-unresponsive RH7777 cells, i.e. cells not expressingLPA receptors. In contrast to Hooks et al. (2001), Yokoyama et al. (2002)needed LPA1 to be expressed in RH7777 to detect increased[3H]thymidine incorporation.

When comparing the effects of hydrocarbon chain length, we found that(S)-1-O-phosphoryl-3-O-tetradecyl-glycerol (J), the shortest in this study,had the highest ability to activate cells (Figure 6 in Paper IV). I and (S)-1-O-oleyl-3-O-phosphoryl-glycerol (K) stimulated HEL cells in a similarfashion, but to a much lower extent than J. The EC50 values obtained withJ and K were 37 and 42 nM respectively. In contrast to our results, it hasbeen claimed that the biological activities of different LPA speciesdecrease with shorter chain lengths (Jalink et al., 1995; Bandoh et al.,2000). This effect was found when measuring Ca2+ mobilization inhuman epidermoid carcinoma cells and insect cells, respectively.Furthermore, the chain length of different phospholipids determines theability to increase DNA-synthesis in vascular SMCs (Chai et al., 2000).LPAs with hydrocarbon chain lengths shorter than 12 carbons have beenshown not to function as agonists on LPA receptors in insect cells(Bandoh et al., 2000). In addition, it has been proposed that 16:0 and 18:1are the optimal chain lengths of LPA analogues (Santos et al., 2000). Thiswas supported by Wang et al. (2001) who have developed a computermodel of LPA1 where the hydrocarbon chain of LPA occupies ahydrophobic binding pocket. Furthermore, the lipophilic tail of LPAmight facilitate the binding of the phosphate group to the appropriateamino acids at the binding site of the receptor (Jalink et al., 1995).

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According to Xie et al. (2002), 18:1 LPA can activate all three knownreceptor subtypes and is generally most efficient in stimulating cells to avariety of responses. Moreover, 16:0, 18:0 and 18:1 are found in allbiological sources examined e.g. human plasma, platelets and asciticfluids (Xie et al., 2002). According to our results, saturated acyl lengths of12 and 14 carbons were the most effective when stimulating contractionof rat uterine SMCs (Tokumura et al., 1980). The three analogues used forthe purpose of examining chain lengths in our study have an ether groupbetween the chiral center and the lipophilic tail. This can be compared toG , H and the commercial LPA, which have an ester linkage. Possiblythese groups play roles in the LPA analogues’ ability to activate cells.van Corven et al. (1992) suggest that an ester-linked long hydrocarbonchain is essential for mitogenic activity of LPA. Our results show that anether-linked, shorter chain can also stimulate a marked increase in[Ca2+]i. It has been shown that LPA3 prefers a hydrocarbon chainattached by an ester linkage to the sn-2 carbon over one with an esterattached to the sn-1 carbon (Bandoh et al., 2000). However, our LPAenantiomers have their hydrocarbon chain attached to the sn-1 or sn-3carbon, respectively. Whether or not these ether or ester groups areimportant in the analogues ability to increase [Ca2 +]i needs furtherinvestigation.

In our experiments the analogues with 18 carbons in the hydrocarbonchain, i.e. G , H and K, are all unsaturated. Phenotypic modulation ofvascular SMCs can be induced by unsaturated, but not by saturatedLPAs (Hayashi et al., 2001). Hopper et al. (1999) believe that thisdifference can be a consequence of binding to receptors but, moreimportantly, the unsaturated fatty acids solubility in aqueous solutions isincreased compared to the saturated counterpart. Furthermore, eachLPA receptor can be activated differentially by LPA species. The affinityof LPA1 for unsaturated LPA is weak compared to that of LPA2 (Bandohet al., 2000). LPA3 shows even higher affinity for unsaturated LPAs thansaturated ones (Aoki et al., 2000; Bandoh et al., 2000; Heise et al., 2001).

The head group of phospholipids determines the efficacy to stimulateDNA-synthesis in vascular SMCs (Chai et al., 2000). Charge changes aswell as substitutions of head groups, for example to sulfate, give inactivecompounds (Santos et al., 2000). When using 10 µ M of (R)-1-O-

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hexadecyl-3-O-sulfo-glycerol (L), in which the phosphate group ischanged to a sulfate and the hydrocarbon chain consist of 16 carbons, noincrease in [Ca2+]i appeared. L was also tested as an antagonist, i.e. L wasincubated with the HEL cells solution three minutes prior to LPA. Fromthese experiments we concluded that L did not work as an antagonist toLPA and was also ineffective as an agonist. The results show that thephosphate group is not only important for the ligand’s efficacy but isalso necessary for the affinity to the receptor.

As with LPA we wanted to study if stimulation by analogues involved aPTX sensitive G protein in Ca2+ mobilization. When we compared PTX-treated HEL cells with non-treated cells we found that PTX did notinhibit the ability to increase [Ca2+]i. Instead the increases in [Ca2+]i

stimulated by all ligands were even higher when cells had beenincubated with PTX. In accordance with the result of LPA we concludethat the ability of LPA receptors expressed in HEL cells to activate Gq/11

proteins are enhanced when the Gi/o proteins are blocked by PTX.

According to Chai et al. (2000) the effect of LPA may be due to doublemechanisms, in which only one is receptor-dependent. Furthermore, ithas been reported that mitogenic as well as platelet aggregationresponses to LPA are independent of LPA receptors (Hooks et al., 2001).Instead it has been suggested that there is a low affinity LPA-signalingpathway, which mediates the receptor-independent responses andwhich is regulated by lipid phosphate phosphatase enzymes (Hooks etal., 2001). These enzymes normally catalyze the dephosphorylation ofLPA, among other bioactive lipid mediators, and might both terminateLPA signaling and generate further molecules with biological activity(Sciorra and Morris, 2002). On the contrary, our results show that theeffects of LPA as well as its analogues had EC50 values in thephysiological concentration range and were saturable at micromolarconcentrations (Figure 2, 5 and 6 in Paper IV), supporting areceptor–mediated mechanism.

To determine if LPA and the analogues activate the same or distinctreceptors, 10 µM of the ligands were added with three minutes intervals(Figure 7 in Paper IV). LPA and all analogues showed the same response,e.g. at repeated incubation with the same ligand no further increase in

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[Ca2+]i was obtained. However, when combining LPA with J for example,no suppression of the new Ca2+ signal occurred. Furthermore, whenstarting with J followed by LPA a major decrease in [Ca2+]i was obtained.We have not further investigated the underlying mechanism of theseresults. Since HEL cells were shown to express more than one receptorsubtype these might be distinctly activated by the different ligands. Ithas been demonstrated that LPA stimulation of human B lymphoblastsleads to increases in intracellular cAMP and Ca2+ concentrations(Sambrano, 2002). LPA can also elicit a unique signaling cascade inmacrophages and mediate an increased cAMP formation in a dose-dependent manner (Lin et al., 1999). Moreover, we have shown thatincreases in cAMP inhibit LPA-stimulated Ca2+ signals in platelets(Figure 3 in Paper III). Recently it has been shown that selection of Gproteins, activated by the 5-HT4 receptor, is ligand specific (Pindon et al.,2002). LPA and its analogues possibly stimulate Gi/o, Gs, and Gq/11

proteins to a different extent, and stimulated increases in [Ca2+]i mightresult from a balance between G protein activation that differ betweenthe analogues. The outcome of LPA stimulation is probably the net effectof activation of positive and negative receptors, similar to that ofagonists for other GPCRs. Receptor combinatorial functions orsynergism in cells expressing more than one receptor subtype couldmodify cellular responses to LPA. Future work on cells expressingdefined receptor subtypes and/or G proteins is necessary to study thesecomplex Ca2+ mobilizing effects.

Allosteric regulation is another possible mechanism that has been shownrelevant to other GPCRs. Allosteric refers to any mechanism in which aprotein can exist in two distinct conformations, which differ in theiraffinity for a ligand (Colquhoun, 1998). Thomas et al. (1997) havesuggested that the lipid oleamide interacts at an allosteric site on the 5-HT receptor and influences G protein signaling via activation of that site.Oleamide could also enhance signaling at one receptor subtype whileantagonizing the other. According to this, there might be several bindingsites on the LPA receptors that regulate Ca2+ mobilization to differentligands with varying efficacies.

More than a decade ago it was shown that LPA suppresses the transientCa2+ signal on subsequent doses of LPA, i.e. LPA-induced Ca2 +

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mobilization is subject to homologous desensitization (Jalink et al., 1990).More recently it has been shown that repeated application of LPA oncells can completely abolish the response to LPA. This has beensuggested to indicate that homologous desensitization occurs in variouscell types (Xu et al., 1995a; Fischer et al., 1998; Gennero et al., 1999). Thelack of response to a heterologous ligand indicates that two ligands mayshare the same receptor, whereas if the cells were responsive to theheterologous ligand, then the two ligands activate different receptors orbinding sites. GPCRs may undergo ligand-induced desensitization(Freedman and Lefkowitz, 1996). Receptor desensitization can resultfrom either a reduction in agonist-receptor affinity, a reduction in cell-surface receptor number through receptor internalization, or animpairment of coupling the receptor to its effector system (Hieble et al.,1995).

In conclusion, our results show that LPA activity requires a phosphategroup while both ester and ether linkages of the hydrocarbon chain ofLPA are tolerated. LPA with a hydrocarbon chain with 14 carbons is themost potent among the LPA analogues tested in this study. Moreover,we show for the first time that no difference in Ca2+ response is obtainedat stimulation of HEL cells with (R)- or (S)-enantiomers of LPA. In thefuture it could be interesting to further examine the complex Ca2 +

mobilizing effects, induced by LPA and LPA analogues, in cellsexpressing defined receptor subtypes and/or G proteins. Our next stepwas to analyze the effects of LPA in platelets according to aggregationand tyrosine phosphorylation.

LPA and adrenaline can act synergisticallyTo test whether LPA could stimulate platelet aggregation, 10 µM of thedrug was used. When LPA was added to suspensions of isolatedplatelets no increase in light transmission or secretion occurred.However, LPA induced a transient decrease in transmission that wasinterpreted as platelet shape change (Figure 4B in Paper III). In contrastto this, previous studies have shown that LPA stimulates secretion andaggregation (Maschberger et al., 2000; Retzer and Essler, 2000). Thereason for the differences in response to the same agonist is probablygreatly affected by variations in the experimental conditions. More

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specifically, the discrepancy between different studies could be due to;distinct techniques to measure the aggregatory response; use of differentanticoagulants; and use of different kinds of platelet suspensions (i.e.isolated platelets, PRP or whole blood). It has been shown that theconcentration of extracellular Ca2+ might be critical. Addition ofmillimolar Ca2+ concentrations reduced platelet aggregation (Gueguen etal., 1999). Induction of platelet aggregation by lipid phosphoric acids inCa2+-containing medium required the addition of ADP (Sugiura et al.,1994). In the present study we used platelets suspended in aphysiological buffer supplemented with 1 mM Ca2+. The platelets weretreated with acetylsalicylic acid and apyrase to minimize the influence ofthe two important positive feedback loops TXA2 and ADP. Thus plateletaggregation depended solely on release of adhesive proteins, such asfibrinogen, from the α-granules. It should be noted that even in thepresence of extracellular fibrinogen (100 µg/ml), LPA alone did nottrigger platelet aggregation. Therefore, it seems likely that the LPA-induced signal transduction did not affect the current low affinity stateof the fibrinogen binding glycoprotein IIb/IIIa (GPIIb/IIIa). TheGPIIb/IIIa complex is present on the surface of resting platelets but itserves as a receptor for fibrinogen only on activated platelets (Bennettand Vilaire, 1979). It was recently shown that LPA induces platelet shapechange in the absence of an increase in [Ca2+]i and it was suggested thatthis was mediated via Rho/Rho kinase induced myosin light-chain andmoesin phosphorylation (Retzer and Essler, 2000). In the same study,LPA at a concentration between 0.01 and 0.1 µM did not elevate [Ca2+]i.However it has been reported in another study that LPA, atconcentrations from 1 µM, induces rises in [Ca2+] i (Maschberger et al.,2000). We noticed that 0.1 µM of LPA triggered rises in [Ca2+]i and itseems likely that the Ca2+ response contributes to platelet shape change.As with the [Ca2+]i experiments we compared the aggregatory ability ofLPA with that of other ligands. Thrombin (0.3 U/ml) induced acomplete, irreversible aggregatory response, ADP (40 µM) stimulated amarked but reversible increase in light transmission and adrenaline (10µM) had no impact on light transmission (Figure 4B in Paper III). It hasbeen shown that treatment of platelets with thrombin induce theformation of PA, which partly is converted into LPA (le Balle et al., 1999).The released LPA might influence the response of the platelets.

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Moreover, stimulation of α2-ARs in adipocytes provoked release of LPAat concentrations capable of inducing proliferation (Valet et al., 1998).

Several researchers have observed synergistic effects when twoactivators are combined. For example, LPA has been shown not toinduce smooth muscle contraction by itself, but enhances contractioninduced by other ligands in airway SMCs (Toews et al., 1997). Weshowed that a combination of two very weak activators, LPA (10 µM)and adrenaline (10 µM), induced a powerful irreversible aggregation invitro in platelet suspensions from some blood donors (Figure 5B in PaperIII). There was a distinct individual variation in response to thecombination of ligands. The aggregatory response was analyzed inplatelets isolated from 8 blood donors and the results varied from 0 % upto 100 % aggregation (Figure 6 in Paper III). It should also be pointed outthat in the absence of fibrinogen the combination of LPA and adrenalinefailed to induce aggregation. In a previous study, Crouch and Lapetina(1988) showed that adrenaline, added after thrombin, recoupledthrombin-receptor triggered rises in cytosolic Ca2+. Our results show thatadrenaline does not indirectly amplify LPA-induced Ca2+-responses(Figure 5A in Paper III). Based on this, it is unlikely that cytosolic Ca2+ isinvolved in the LPA- and adrenaline-induced synergistic aggregation. Inplatelets, the effects of adrenaline can be mediated by its interaction withα2-ARs as evidenced by their sensitivity to yohimbine (Banga et al., 1986).Both α2- and β-ARs have been shown to be expressed in platelets(Barnett et al., 1985; Kobilka et al., 1987). Variations in expression of thesereceptor subtypes may explain the variability in response of plateletstowards the LPA and adrenaline combination. There are conflictingreports with respect to the ability of adrenaline to induce activation ofplatelets by itself. It has been reported that adrenaline primarily serves toincrease platelet aggregation induced by other autocoids (Steen et al.,1993). As an example, adrenaline can potentiate ADP-inducedaggregation (Nakamura et al., 1997a). According to Freeman et al. (1995),genetic variation and heritability might result in increased adrenaline-mediated platelet aggregation. In another study Nakamura et al. (1997b)compared adrenaline sensitive and insensitive platelets. While studyingthe variability in aggregatory response in Japanese, they found that 16 %of the population had a reduced number of α2-ARs. The number ofreceptors was about 50 % lower than in the rest of the population, but

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synergistic effects of adrenaline were still observed. They also found thatfibrinogen binding to GPIIb/IIIa had a crucial role in activating PLA2,which is required for a full aggregatory response of adrenaline.Furthermore, we found that a combination of LPA and adrenaline insamples from some blood donors could induce an increase in tyrosinephosphorylation (Figure 7 in Paper III). Recently, it was shown thatnonresponders to adrenaline also responded weaker to ADP (Nakahashiet al., 2001). The authors suggest that the reduced platelet responses aredue to impaired signal transduction. Activation of tyrosine kinases iscoupled to fibrinogen binding to GPIIb/IIIa as well as to thrombin andcollagen interactions with their respective receptors (Clark et al., 1994).Agonists such as thrombin, ADP, and collagen induce tyrosinephosphorylations in platelets (Clark and Brugge, 1996). Tyrosine kinasesmay regulate various platelet-signaling pathways, including cytoskeletalreorganization necessary for secretion and aggregation (Wadman et al.,1996). Wang et al. (1997) have been studying adrenaline-potentiatedthrombin-induced aggregation and tyrosine phosphorylation.Adrenaline itself did not cause detectable tyrosine phosphorylations orplatelet aggregation, but could potentiate the effects of thrombin throughinteraction with α2-ARs. In our experimental design, the synergisticactivation of platelet aggregation was dependent on external fibrinogenadded to the samples. Thus, the drug combination did not induce asufficient secretory response of proteins from the α-granules. Oneplausible mechanism could be that the combination of LPA andadrenaline, but not separate treatments, induces a conformationalchange of GPIIb/IIIa to its high affinity state. However, Smyth et al.(1992) have proposed that LPA is a potent activator of fibrinogen bindingto GPIIb/IIIa. Consequently, it could not be excluded that LPA indirectlyfacilitates adrenaline-induced platelet aggregation. Such an action wouldpresumably lead to aggregation-dependent, i.e. integrin-dependent,tyrosine phosphorylation. Tyrosine kinases in platelets are regulatedboth by classical receptor proteins and by integrins (Clark et al., 1994).We have not identified the tyrosine-phosphorylated proteins. However,aggregation-dependent tyrosine phosphorylations of focal adhesionkinase, Src, Syk and p95/97 have previously been reported (Clark et al.,1994). It is likely that some of these proteins are phosphorylated after theaddition of LPA and adrenaline and the subsequent aggregation.

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From these experiments we conclude that isolated platelets from someindividuals respond with a powerful aggregatory effect at stimulationwith a combination of LPA and adrenaline. In the same way acombination of these two weak activators could induce an increase intyrosine phosphorylation in samples from some blood donors. Theintracellular mechanisms of the synergistic effect are not yet clear andclaim further studies. Further analyzes are also needed to examineconceivable clinical applications of these results.

LPA has potential clinical applicationsAs the present study was performed at the Faculty of Health Sciencesone must ask oneself, what health advantages might derive from studiesof LPA? Since LPA binds to and activates multiple GPCRs it emergesthat the beneficial or harmful actions of LPA are critically dependent onthe expression profile of receptor subtypes and coupling to differentsignal transduction pathways in the target cells. Over 60 % of all currentdrugs target the GPCR family of receptors, making the LPA receptorfamily possible targets. There are however difficulties in drugdevelopment because a mixture of different bioactive LPAs, and theirreceptors, characterize many physiological as well as pathologicalsettings. Regulation of synthesis of LPA under physiological andpathological conditions should be a subject of intense investigation.Development of specific antagonists and agonists for LPA receptorsubtypes, and inhibitors of synthesis as well as degradation of LPA,could possibly lead to novel therapeutic strategies for various diseases.The outcome of some severe diseases might also be more beneficial byearly detection of increased levels of LPA.

Women with ovarian cancer have the worst prognosis of anygynecologic malignancy. This is caused by insufficient methods for earlydetection, high metastatic potential and lack of effective treatment for themetastatic disease. The five-year survival prognosis for women with latestages of ovarian cancer is between 15 and 25 % (Rubin and Rockey,1999). However, if cancer is detected at an early stage up to 90 % of thewomen can survive (Rubin and Rockey, 1999). Since the level of LPA inblood has been shown to be increased in women with ovarian cancer (Xuet al., 1995a; Xu et al., 1998), measurement of this lipid might be used for

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the detection of cancer. It is also tempting to speculate that thedevelopment of agonists or antagonists to the LPA receptors may resultin drugs that are capable of preventing the proliferation of ovariancancer cells. Moreover, an easier and faster way for detection ofincreased levels of LPA in the blood must be found if LPA is to be usedas a biomarker for cancer. A first but failed attempt to develop an easyassay for LPA was as follows. Our group has access to a cell line ofmelanophores, which contain dark melanin pigment granules. Inresponse to different agonists, the cells undergo color changes byredistribution of their pigment granules. The melanophores have earlierbeen shown to respond to different stimuli by changing color (Karlssonet al., 2000). The color change can easily be detected in aspectrophotometer by using 96 well plates with cultured cells. Wetransfected these cells with LPA receptors, but we have not yetsucceeded to get a response upon addition of LPA.

Siess et al. (1999) found that the lipid-rich core in carotid atheroscleroticlesions had a high content of LPA. Furthermore, LPA was the mainactivating lipid of platelets in the plaques (Siess et al., 1999). The lipid-rich core in atherosclerotic lesions is very thrombogenic and prone torupture suggesting that LPA could trigger platelet aggregation in thecirculation. Upon plaque rupture LPA might trigger life-threateningthrombosis that might lead to myocardial infarction as well as stroke(Siess, 2002). The prevention or stabilization of plaques offers anadditional or alternative approach to anti-platelet therapy. Theconcentration of LPA in serum has been estimated to be in themicromolar range (Eichholtz et al., 1993). It is also known that LPA canexert such diverse effects as smooth muscle contraction (Vogt, 1963), andcellular growth of both fibroblasts (van Corven et al., 1989) and SMCs(Tokumura et al., 1994); effects all known to be significant for thepathogenesis of atherosclerosis and cardiovascular diseases. It isimportant to try to understand more about what role LPA really plays inthese severe diseases. Since we show that LPA and adrenaline can actsynergistically this might be a new risk factor for thrombosis. In thiscontext, we noticed that the aggregatory response varied considerablybetween individuals. Maybe this could reflect inherited polymorphismwithin platelet membrane receptor genes or other factors important forplatelet aggregation. There are known situations where polymorphism

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may regulate expression levels or alter functional properties. Forexample, the expression and function of collagen receptors on plateletsdiffers markedly among normal subjects and depends uponpolymorphism (Bussel et al., 2000). Increased knowledge aboutconceivable polymorphism might be used in prevention and treatment ofsevere diseases and therefore needs further investigations. It may bepossible to find synergistic effects of LPA and adrenaline also in othercell types or physiological systems. New strategies for prevention andtherapy of cardiovascular diseases could also be targeting specific LPAreceptor subtypes in selective cells in blood and vascular wall. In thefuture LPA might perhaps be used as an early marker for cardiovasculardiseases. Hypertension and psychological stress are major risk factors foratherosclerosis and are also associated with increased levels ofcatecholamines in plasma (Dimsdale and Moss, 1980; Goldstein, 1981).Furthermore, Maes et al. (2002) have demonstrated that psychologicalstress increase platelet α2-AR density, whereas no significant effect on α2-AR affinity could be detected. The authors examined university studentsa few weeks before and a day before a difficult, oral examination. Theirconclusion that academic examination stress induces a significantincrease in α2-AR density might be something to pay attention to duringa dissertation.

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CONCLUSIONS______________________________

Some of the most important findings in this thesis will here beconcluded.

-During pregnancy, the human myometrium increases in weight about20 times. Earlier studies have been concentrated on the growthpromoting effects of sex steroids and peptide growth factors. Our resultssuggest that also stimulation of GPCRs, in particular LPA receptors andα2-ARs, contributes to this proliferative phase. LPA is almost four timesmore effective than noradrenaline to induce DNA synthesis in culturedmyometrial SMCs. The ability to increase [Ca2+]i and to stimulate CaMkinase might be one explanation why LPA produces a more pronouncedproliferative response than noradrenaline.

-We show that human myometrial SMCs express all known LPAreceptor subtypes. The growth stimulatory effect of LPA in these cellsappears to be dependent on several signaling pathways. Transactivationof EGF receptors seems to be important, presumable through activationof MMPs and release of EGF receptor ligands. Whether CaM kinases areinvolved in the transactivating phenomenon is unclear.

-The fact that both responses of LPA and EGF are inhibited by PTX inhuman myometrial SMCs suggests that Gi/o-proteins might be a new andimportant link in the crosstalk between GPCRs and receptor tyrosinekinases.

-Even if it has been suggested that the LPA receptors are unusual in thatthey are not stereoselective, the unnatural (S)-enantiomer of LPA has notpreviously been synthesized and tested. This is the first study to showthat both natural (R) and unnatural (S) LPA enantiomers are capable ofstimulating cells. Structure-activity analyzes are important to understandhow LPA activates cells. Identification of novel lipid mediators might beuseful for the design or discovery of receptor subtype specific agonistsand antagonists. Synthesizing LPA receptor agonists and/or antagonistswill increase our knowledge of physiological and pathologicalsignificance of the lipids.

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-Although LPA belongs to the category of weak platelet activators, itscombination with adrenaline can induce an irreversible plateletaggregation in vitro. However, this synergistic effect is only evident insome healthy subjects. Since atherosclerotic lesions have a high contentof LPA, this synergistic effect might be a new risk factor for arterialthrombosis.

Yesterday is the past, tomorrow is the future, but today is a gift.That is why it is called the present.

Bil Keane

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ACKNOWLEDGEMENTS______________________

Det är väldigt många personer som på ett eller annat sätt har hjälpt mig underavhandlingsarbetet. Till några av dessa vill jag rikta ett speciellt TACK.

Min handledare Samuel Svensson är en av de mest underbara och roliga människorjag har träffat. Han har både som handledare och vän verkligen stöttat ochuppmuntrat mig i med- och motgångar. En fjärdedel av mitt liv har vi delat på 9 m2

så nu förstår jag att han vill se något nytt och ha ett större kontor! Han har inspireratmig och lärt mig varför det heter research och inte bara search!

Chefen och livsnjutaren Rolf Andersson trodde på mig och har låtit mig arbeta påFarmakologen all den tid som behövdes.

Diktaren och uteruskompisen Per Adolfsson har delat cellodlingens vardag med migsamt gärna delat med sig av sina kemikunskaper när mina inte har räckt till.

Svärmorsdrömmen och kakbakaren Fredrik Johansson har alltid funnits där när jaghar behövt ett litet avbrott från forskningen. Han har också tålmodigt hjälpt mig medalla mina möjliga och omöjliga datorproblem.

Exgrododlaren Annika Karlsson var under lång tid min labbänkskompis.Tillsammans hjälptes vi åt att hålla ordning på grabbarna. Hon hjälpte migentusiastiskt under de sista veckornas skrivande.

Med pedagogerna Anna Asplund Persson och Iréne Rydberg har jag haft ett roligt ochlärorikt samarbete när vi har lotsat studenter i fysiologi och farmakologi.

Med händige Dan Linghammar har jag haft små mysiga snack samtidigt som han harautoklaverat åt mig.

In different ways the foreign postdocs Nuraly Avliyakulov and Sami Aifa reallycontributed with their knowledge and culture to the nice atmosphere at our lab.

Förra årets farmakolog Anita Ljungblad och TV-kändisen Lillemor Fransson haralltid viljan att hjälpa till med stort och smått. De är guld värda.

Min nya rumskompis Jan Aydin har under det senaste halvåret fyllt min arbetstidmed exotisk musik. Allt från discopop till syrianska sånger. Han hjälpte mig medmånga saker framför datorn eftersom hans musvana är enorm. Han fick bl.a. ettfylogenetiskt träd att växa.

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Experten Magnus Grenegård har, med ett fåtal ord, lotsat mig i trombocyternas ochkalciumets värld.

Studenterna Daniel Lindstedt, Sitti Hultgren och Elisabeth Hallin har genom sinaarbeten bidragit till forskningen. The foreign students Naomi Tamayama and WendyPowell also helped us with some experiments.

Alla mina tidigare och nuvarande arbetskamrater har hjälpt mig med allt mellanhimmel och jord och har gjort dagarna roliga, trevliga, stökiga och lärorika.

Kemisterna Peter Konradsson, Jan Lindberg och Johan Ekeroth har genom sittsyntetiserande lärt mig lite om stereokemi.

Språkkunnige Jon Jonasson fick det att flyta lite bättre och som tack för det fick hannågot som flyter.

Utan alla frivilliga givare av delar av sin kropp samt de som tog delarna, personalenpå Gyn-Op och Blodcentralen, hade studien inte gått att genomföra.

Med alla mina vänner, ingen nämnd och ingen glömd, har jag alltid kunnat delaglädje och sorg. Nu måste vi ses och höras mycket oftare!

Ostkustens pärlor, mormor Ulla och morfar Nils, har verkligen uppmuntrat mig attläsa. Mormor fick aldrig tillfälle att läsa men hon har poängterat att “kunskap äraldrig tungt att bära”.

De bästa föräldrarna man kan ha, Vivianne och Bo, har lärt mig det mesta jag kanutanför forskningen. De har alltid stöttat mig, trott på mig och hjälpt mig då det harbehövts. Tack vare all läxläsning, alla förhör och “den hårda skolan” har jag nått såhär långt! De är dessutom fina förebilder för Tilda.

Pysslarna lillebror Hans och Elisabet finns alltid till hands vid behov. Tack varederas barnvaktscheckar har jag kunnat jobba i lugn och ro.

Min babe Per och vår pluttinutta Tilda gör livet så mycket roligare. Utan deras hjälpmed både vetenskap och marktjänst hade det aldrig gått. De har ett enormt tålamod.Blöta pussar kan man aldrig få för mycket av!

Genom sina bidrag har finansiärerna Medicinska forskningsrådet, Natur-vetenskapliga forskningsrådet, Östergötlands läns landsting, Stiftelsen Forskningutan djurförsök, Lions forskningsfond mot folksjukdomar, Svenska Föreningen förBiokemi och Molekylärbiologi samt naturligtvis Svenska staten gjort denna studiemöjlig.

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