BRADYKININ AS A MAJOR ENDOGENOUS REGULATOR OFENDOTHELIAL FUNCTION

Ryszard J. Gryglewski, Wojciech Uracz, Stefan Ch l---opicki,and Ewa Marcinkiewicz Jagiellonian University, Cracow, Poland

Healthy vascular endothelium is a powerful generator of nitric oxide (NO), prostacyclin (PGI2), prosta-glandin E2 (PGE2), and plasminogen activator (t-PA). These endothelial products protect vascular wallagainst aggression from activated blood platelets and leukocytes. In particular, they protect against thrombosis,promote thrombolysis, maintain tissue perfusion, and inhibit remodeling of vascular and cardiac walls. En-dothelial dysfunction appears on one hand as suppression in the release of the above mediators, and on the otheras deleterious discharge of prostaglandin endoperoxides (PGH2; PGG2), superoxide anion O–

2 , peroxynitrite(ONOO– ), and plasminogen activator inhibitor (PAI-1).Ourdatapoint to endothelialbradykinin(Bk)asatriggerforprotectiveendothelialmechanisms.Incultured endothelial cells (CEC)Bkthrough kininB2receptorsraised in a concentration-dependentmanner (1pM^10 nM) free cytoplasmic calcium ions [Ca2 ‡]i.This risewas accompanied by the release of NO as quanti¢ed by a porphyrinic sensor. Other endothelial agonists wereweaker stimulators of [Ca2 ‡]i than Bk. In vivo we analyzed the e¡ects of exogenous Bk and of ampli¢ers ofendogenousBk, suchasperindoprilandquinapril (`tissue type’’angiotensinconvertingenzyme inhibitors,ACE-I) on endothelial function using our original thrombolytic bioassay and EIA assays for 6-keto-PGF1aand t-PAantigen.Amajordi¡erencefoundbetweenexogenuousBkand endogenousBk(that rendered by `tissueACE-I’’)wasa prolonged thrombolytic action (>4 h)ofquinapril or perindopril.Moreover, only exogenous Bkevoked animmediateand profound hypotensiveaction. Invivo Bk-induced thrombolysiswasB2kininreceptor-dependent,PGI2-mediated.The unexpected action of Bk came to light in CEC.Then appeared incubated for 4 h increasedexpressionofmRNAsforhaemoxygenase(HO-1), cyclooxygenase 2(COX-2), prostaglandinEsynthase(PGE-S), but hardlyfor nitric oxide synthase 2 (NOS-2). Wehypothesize that a networkof interactions of Bk-inducedenzymes may constitute a delayed phase of Bk e¡ects in the endothelium, whereas the primary phase would beactivation by Bk of [Ca2 ‡]i-dependent constitutive endothelial enzymes. In blood-perfused rat endotoxemiclungs, NO is the most eminent cytoprotective mediator. Summing up, in peripheral circulation endogenous Bk isthemost e¤cientactivatorofprotectiveendothelialfunction.Thrombolyticaction of `tissue-type’’ACE-Isreliesonreceptor B2-mediated, [Ca2 ‡]i-dependent release of PGI2. Bk also may act as a `microcytokine’’by inducingmRNAsfor HO-1,COX-2,orPGE-S.Activation ofHO-1maylead toa de¢ciency in intracellularheme requiredas a cofactor for both COXand NOS.This network of interactions triggered by Bkcall for further studies.

Keywords bradykinin, prostacyclin, nitric oxide, plasminogen activator, hemooxygenase,endothelium

Supported by the State Committee for Scienti¢c Research (Grant No.4 PO5A 05019).We are most gratefulto Professor Tadeusz Malinski, Dr. Danuta Uracz, and Dr. Jozef SwieË s for their scienti¢c input to our paper.Technical help of Barbara Lorkowska, Renata Budzynska, and Lucyna Olejniczak is highly appreciated.We ac-knowledge kind gifts from P¢zer Poland (quinapril, quinaprilat ), Servier Poland (perindopril, perindoprilat),and Hoechst Marion Roussel (icatibant, HOE140).

Address correspondence to Ryszard Gryglewski, Chair of Pharmacology, Jagiellonian University, 31-531Cracow,16 Grzegorzecka, Poland. E-mail: mfgrygle@kinga.cyf-kr.edu.pl

Pediatric Pathology and Molecular Medicine 21: 279^290, 2002Copyright # 2002 Taylor & Francis1522-7952/02 $12.00 + .00DOI:10.1080/0277093 0290056514

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INTRODUCTION

Prostacyclin (PGI2) was discovered as an unstable regionalvasodilator (coeliacand mesenteric arterial strips) and a generalized blood platelet-suppressant madefrom cyclic prostaglandin endoperoxides (PGG2 or PGH2) by aortic microsomes[1, 2]. From the very beginning we realized that its generation by the conductancearteries might constitute a major protective mechanism against thrombosis andatherosclerosis, preventing deleterious invasion of activated platelets onto vascularwall [1]. Moreover, we proposed that a selective inhibition of PGI2 synthase by lipidperoxides would break protective function of endothelial barrier and invite athero-sclerosis [1]. We also described a transcellular mechanism by which blood plateletswould feed arterial wall with their PGH2 to make cytoprotective PGI2 instead ofcytotoxic TXA2 [3]. A similar design of transcellular biosynthesis occurs duringinteraction of activated leukocytes with endothelial cells, but then pro-in£amma-tory cysteinyl-leukotrienes (cyst-LTs) are formed [4, 5].

Originally, the dogma was that among prostanoids only PGI2 would be madeby vascular endothelium. However, generation of prostaglandin E2 (PGE2) overPGI2 in microcirculation was described [6] and an inducible PGE2 synthase(PGE-S) identi¢ed [7]. This distribution of defensive prostanoids (PGI2 in macro-circulation versus PGE2 in microcirculation) makes perfect sense because PGI2

activates adenylate cyclase in platelets while PGE2 does the same in leukocytes.Thisis why vascular endothelium in conductance arteries protects them against athero-thrombosis, whereas vascular endothelium in capillaries appeases acute in£amma-tory response. Garret Fitzgerald et al. [8] not long ago challenged another dogmathat endothelial constitutive cyclooxygenase 1 (COX-1) would be exclusivelyresponsible for making PGI2. Now, also inducible COX-2 has been found inendothelial cells [8] and we con¢rm this ¢nding. In endothelial cells still acts thecytochrome P 450-dependent fourth pathway of arachidonate metabolism. It pro-duces 20-hydroxyeicosatetraenoic acid (20-HETE) [9], and a number of isomericepoxyeicosatrienoic acids (EETs) exert opposite e¡ects on vascular tone. One ofthe EETs is likely to be a vasodilator `endothelium-derived hyperpolarizing factor’’(EDHF) [10].

Endothelium-derived relaxing factor (EDRF) [11], identi¢ed as nitric oxide(NO) [12] shows vasorelaxant and antiplatelet actions mediated by cyclic 30,50-GMP [13]. The abbreviation EDRF(NO) is sometimes used to distinguish NOmade by endothelial constitutive NO synthase (NOS-3) from NO generated byneuronal NOS-1 or by inducible NOS-2. In many biological aspects, EDRF(NO) resembles PGI2, although the PGI2 uses cyclic 30,50-AMP as a second messen-ger. In body £uids EDRF(NO) exists as a lipophylic free radical NO° inactivatedby superoxide anion (O–

2 ) [14]. This reaction may lead to formation of inactivenitrate or to generation of the highly destructive peroxynitrite (ONOO– ) [15].

A de¢ciency of the substrate (L-arginine) or a cofactor (tetrahydrobiopterin)may push NOS-3 to generate O–

2 instead of, or along with NO° [15^17]. In conse-

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quence, the formation of ONOO– is likely to occur, especially because O–2 reacts

more avidly with NO° than with its own scavenging enzymeösuperoxide dysmu-tase [15]. Endothelial cells release PGI2 and NO in a coupled manner [18]; however,interactions between the systems synthesizing PGI2 and NO remain unclear. Bothinhibition [19] and activation [20^22] of COX by NO were reported. Recently,Ulrich et al. (personal communication) proposed that ONOO– (IC50 = 50 nM)would selectively inhibit PGI2 synthase.

In this article we present an overview of our recent data [22a] on a special roleof endothelial bradykinin in setting in motion defensive endothelial mechanismsand discuss di¡erent roles that PGI2 and NO play in pathophysiological events inperipheral and pulmonary circulation.

METHODS

Cytosolic Free Calcium [Ca2+]i in Cultured Endothelial Cellsof Bovine Aorta (BAEC)

BAEC were prepared as described previously [23]. Cells from second passagewere used for experiments.Their homogenity was identi¢ed by their typical cobble-stone morphology under the Axiovert 25 Inverted Microscope (Carl Zeiss JenaGmBH, Germany). [Ca2‡]i assay was accomplished by the method of Grynkie-wicz et al. [24]; namely, BAEC (0.5 £106 cells ml– 1) were loaded with fura-2 byincubation for 60 min at 25¯C with £uorescent dye in its membrane permeant form,i.e., acetoxymethyl ester (fura-2AM) in presence of bovine serum albumin. Thenthe extracellular dye was removed bycentrifugation and the cells were resuspendedin HBS with glucose (5 mM). Fluorescence was measured at 37¯C in a spectro-£uorimeter with a dual wavelength excitation, and magnetic stirring (LS 50B,Perkin-Elmer Corporation, Beacons¢eld, UK) at 500 nm with the excitation wave-length of 340 nm and 380 nm. Calibration was completed using 0.2% Triton X-100for Rmax and 5 mM EGTA for Rmin. Eventually, [Ca2‡]i was calculated accord-ing to the equation of Grynkiewicz et al. [24].

Endothelial Release of NO Measured by Malin ski’sPorphyrinic Electrode

Nitric oxide (NO) was measured using Malinski’s porphyrinic microsensor[25]. The microsensor’s working electrode was placed close (20 § 5 mm) to the sur-face of con£uent monolayer of BAEC along with a platinum wire counterelectrodeand a saturated calomel reference electrode. Voltametric analyzer (PAR model264A) with the current-sensitive preampli¢er (PAR model 181) were used foramperometric measurements at a potential of 0.68 V. NO measurements arestandardized using aqueous NO standard prepared as described byJia and Furch-gott [26]. NO was measured over a single BAEC in Hank’s balance solution at

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37¯C.The response time was 0.1ms and the detection limit 10 – 15 M of NO. Brady-kinin was used at a concentration from 2 £10 – 15 to 1 £10 – 12 M.

Effects of Bradykinin on mRNA Expression for EndothelialEnzymes in HUVEC

Endothelial cells from human umbilical vein (HUVEC) [27] were preparedandcultured as described previously [23]. HUVEC were used for experiments uponreaching 90% con£uency in the third passage, which was carried out in 6-wellplates (1 £106 cells, NUNC, Brand Products, Denmark). Then after 24 h ofHUVEC incubation in OPTI-MEM I without fetal bovine serum, cells werestimulated with Bk (10 nM) for 4 h and total RNA was isolated by the guani-dinum isothocyanate method, as described by Chomczynski and Sacchi [28] usingTRIZOL 1Reagent (Gibco BRL,UK).

Reverse transcription (RT) of total RNA (1 mg) were performed with oligo-(dT)12^18 primer and M-MLV reverse transcriptase (Gibco BRL,UK) for 2 h at42¯C. Then, cDNA (1 ml) was ampli¢ed with HotStar Taq Polimerase (QIAGEN,USA) for both quantitative and qualitative RT-PCR. Speci¢c mRNAs for HO-1,COX-2, COX-1, and NOS-2 were quanti¢ed by Gene Speci¢c Relative RT-PCRKit (Ambion, USA) using multiplex RT-PCR protocol with endogenous18S rRNAstandard and Competimer1 technology. PCR reactions were set up with Gene-Amp 9600 machine (Perkin-Elmer, USA) of the following pro¢le: 95¯C for 15 minand 35 cycles; 94¯Cö30 s, 59¯Cö30 s, 72¯Cö30 s followed by 72¯C for 10 minelongation and then samples were placed on ice. Equal volumes of each sample(5 mL) were run on 3% agarose containing ethidium bromide and images werecaptured electronically with DC40 digital camera (Kodak, USA), and the bandswere quanti¢ed using image analysis software (NIH Image, USA). Results wereexpressed as ratio of pixel density units for speci¢c mRNA to internal standard(18S rRNA). For position and size of observed speci¢c bands, marker M1 (pUC19=Mspl, DNA-Gdansk II, Poland) was run in parallel. For qualitative RT-PCRfor PGE-S following speci¢c primers were used: primer1(sense) 50ATGCCTGCC-CACAGCCTG-30 (40 nM) and primer 2 (antisense): 50-TCACAGGTGGCGG-GCCGC-30. Oligonucleotide primers were constructed by Jakobson et al [7] buttruncated for cloning restriction sites. Primer sets for NOS-2 and internal standard(18S rRNA) were obtained commercially (Ambion, USA).

Thrombolytic Assay for Activation of Entire Endothelial Systemin Rats in Vivo

An in vivo model for studying thrombolysis in cats [29] was adopted to rats [30,31]. Brie£y, male Wistar rats (body weight 300^350 g) were anesthetized (thiopen-tal 30 mg kg– 1 ip) and unfractionated heparin at 800 units kg – 1 iV was adminsi-tered. Extracorporal circulation was established between left carotid artery and

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left jugular vein, and a collagen strip was superfused with arterial blood at a rate of1.5 ml min – 1. Its weight was continuously monitored by an auxotonic Harvardtransducer. Because of deposition of thrombi [29^32] the strip gained in weight by80^120 mg during the ¢rst 20 min of superfusion and stayed at that level duringthe next 3^5 h of the experiment. Mean arterial blood pressure (BP) was monitoredfrom right carotid artery by a Harvard pressure transducer, and the right femoralvein was prepared for drug administration. In the above system, dispersionof thrombi used to occur next to intravenous administration of PGI2 of 0.1^1.0 mgkg – 1 (without concomitant fall in BP) or glyceryl trinitrate at doses of 30^100 mg kg – 1or metacholinehydrochloride (10 mg kg – 1) orkallikrein (100 units kg – 1)(with accompanying fall in BP). Streptokinase (3^30 megaunits kg – 1) producedbiphasic thrombogenic=thrombolytic response [30].

Blood Assays of 6-Keto-PGF1a and t-PA Antigen

Blood samples (500 ml) were collected into Eppendor¡ tubes with indometha-cin to yield its ¢nal concentration of 10 mM, and then stored at – 70¯C not longerthan a week. 6-Keto-PGF1awas assayed using the enzyme immunoassay kit (Cay-man Chemical Co., Ann Arbor, MI, USA) and t-PA antigen was assayed usingthe enzyme immunoassay kit (Biopool TintElize t-PA antigen, Umea, Sweden).All results were expressed in ng ml– 1.

Blood Perfused Rat Endotoxemic Lungs

Lungswere isolated fromWistar ratsweighing 200^250 gand mounted inan iso-lated rat lung set-up (Hugo SachsElektronik) as describedpreviously [33].The lungswere ventilated with negative pressures. The end-expiratory pressure in the cham-ber was set to be – 2 cmH2O and inspiratory pressure was adjusted between – 6 to– 10 cm H2Oto yield the initial tidal volume of about 2.0 ml. Lungs were reperfusedwith rat blood using a peristaltic pump (ISM 834, HSE) at constant £ow of about16 ml=min.The following parameters were measured: arterial and venous pulmon-ary pressures, tidal volume, and lung weight, according to Uhlig’s method [34].

Lipopolysaccharide (LPS) from E. coli was injected 45 min after the beginningof the experiment. L-NAME was administered 15 min prior to LPS. Camona-grelöaTXA2 synthase inhibitor (300 mM),WEB 2170öa PAFreceptor antagonist(100 mM), MK 571öa leucotriene receptor antagonist (100 mM), antiCD62P(1 mg=ml), or sCR1 (100 mg=ml) were added to blood 30 min prior to injection ofLPS.

Statistical Analysis

Arithmetical means are given with s.e. mean (SEM). Di¡erences betweengroups were assessed with unpaired two-tailed t test with Welch’s correctionor paired Student’s t test; p < 0.01 was assumed to denote a signi¢cant di¡erence.

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In isolated lung experiments di¡erence between groups were assessed by one-wayANOVA followed by post hoc Fischer test; p < 0.05 was considered statisticallysigni¢cant.

RESULTS

Effect of Bradykinin on [Ca2+]i and NO Release in BAEC

Basal levels of [Ca2‡]i were between 30 and 150 nM (on average 100 nM). Bkat the concentration of 10 nM increased [Ca2‡]i by 2^10-fold of the basal level. Insome batches of BAEC, Bk produced a rise in [Ca2‡]i at concentrations as low as1fM^10 fM, although on average Bk was active at a range of concentrations 1pM^10 nM. Calcium ionophore (A23187) was e¡ective at concentrations of 1nM^100 nM, adenosine disphosphate (10 nM^1 mM), adrenaline (1 mM^10 mM), acetyl-choline (10 mM^100 mM), as well as tissue-type ACE inhibitors, perindopril, andquinapril (10 mM^30 mM). A potent rise in [Ca2‡]i by Bk (10 nM) was e¡ectivelyblocked by icatibant (30 nM), a B2 kinin receptor antagonist.

Basal NO release over a BAEC single cell was close to 12 nM. Bradykinin(2 fM^10 fM) stimulated NO release from BAEC in a concentration-dependentmanner. Bk at concentrations of 10 fM^1pM evoked NO release at a level of535 § 25 nM.

Effect of Bradykinin on Expression of mRNAs for EndothelialEnzymes in HUVEC

Multiplex RT-PCR was used with two primer sets in one tube for a singlePCR (multiplex system). One set of primers was used to amplify the cDNA ofinterest, i.e., inducible heme oxygenase (HO-1), inducible cyclooxygenase (COX-2), constitutive cyclooxygenase (COX-1), and inducible NO synthase (NOS-2),while a second set of primers was used to amplify invariant endogenous control18S rRNA. In HUVEC incubated for 4 h with Bk (10 nM), a signi¢cant increasein mRNAs for HO-1 (4.5-fold) and COX-2 (2.5-fold) occurred; not so much forCOX-1 (1.35-fold) and NOS-2 (0.3-fold). In separate semiquantitive RT-PCRexperiments, the induction was observed of speci¢c PGE-S mRNA, but not ofNOS-2 mRNA.

In Vivo ACE-I via Endogenous Bk Activate Endothelial Systemin Rats

In rats with extracorporal circulation both ACE-Is (perindopril and quinapril)at doses of 10^30 mg kg – 1produced long lasting (>4 h) thrombolysis with no hypo-tensive e¡ect. Thrombolytic action of perindoprilat and quinaprilat (active meta-bolites of the drugs) resulted in much shorter (<1h) thrombolysis. Pretreatmentwith a B2 kinin receptor antagonistöicatibant (100 mg kg – 1) or with a COXinhibitor (indomethacin 5 mg kg – 1)öabolished thrombolysis by ACE-Is, while

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pretreatment with the NOS inhibitor L-NAME (5 mg kg – 1) only partially andtemporally impeded thrombolytic response to ACE-Is.Within the ¢rst 15 min afterintravenous injection of ACE-Is (30 mg kg – 1) blood levels of 6-keto-PGF1a went upfrom 0.2^0.4 ng ml – 1to 3.8^4.6 ng ml – 1 (n= 6) and were staying at that high levelfor at least the next 4 h while thrombolysis still lasted. At the same time, t-PA anti-gen plasma concentration started to rise signi¢cantly from 1.2^1.4 ng ml – 1 2.8^3.1ng ml – 1 (n= 6) only 1h after administration of ACE-Is, and during the next3 h levels of t-PA antigen continued to rise.

Endogenous NO in Endotoxaemic Blood Perfused Rat Lung

Lipopolysaccharide from E. Coli (LPS, 300 mg=ml) in the isolated blood per-fused rat lungs induced biphasic response. An immediate transient phase of thisresponse consisted of an increase in pulmonary arterial and venous pressures(PAP and PVP) with a simultaneous decrease in tidal volume (TV). After pre-treatment with L-NAME (300 mM), injection of LPS (300 mg=ml) led to theimmediate arrest of lung function. The transient phase became lethal. This irre-versible damage to the lung treated with L-NAME ‡ LPS was partially preventedby pretreatment with a PAF receptor antagonist (WEB 2170 100 mM), a TXA2

synthase inhibitor (Camonagrel, 300 mM), and a leukotriene receptor antagonist(MK 571, 300 mM).

At the morphological level, immediate transient lung response induced by LPSalone did not leave any signs of injury. However, irreversible lethal responseinduced by L-NAME ‡ LPS was associated with alveolar hemorrhagic edemaand alveolar barrier damage as evidenced by light and electron microscopy.

DISCUSSION

Constitutive cyclooxygenase (COX-1) and nitric oxide synthase (NOS-3) areknown to abide endothelial cells of macrocirculation and to generate cytoprotectivePGI2 and NO. In cultured endothelial cells from bovine aorta (BAEC) or fromhuman umbilical vein (HUVEC), these enzymes are activatedbya rise in free cyto-plasmic calcium ions [Ca2‡]i. Here we report that in BAEC calcium ionophore(A23187,1^100 nM), and a number of physiological receptor agonists such as brady-kinin (Bk, 1 pM^10 nM), adenosine diphosphate (ADP, 10 nM^mM), adrenaline(Adr, 1^10 mM), or acetylcholine (Ach, 10^100 mM) may cause 2- 10-fold increasein [Ca2‡]i. Bradykinin is known to release EDRF(NO) from BAEC in a[Ca2‡]i-dependent manner, although calcium channel antagonists do not inhibitthis release [35]. Much less active stimulators of [Ca2‡]i in BAEC are tissue-typeinhibitors of angiotensin converting enzyme (ACE-I), such as quinapril or perindo-pril (30 mM), which, in our opinion, seem to produce thrombolysis as ` bradykininpotentiating factors’’ [36], i.e., as kininase II inhibitors rather than as inhibitors

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of ACE [37]. In high risk patients, ramipril (another ` tissue ACE-I’’) reducesdeath rate from myocardial infraction and ischaemic stroke [38]. We believe thatthe bradykinin-mediated activation of endothelial protective secretory function bytissue ACE-I contributes to this clinical action of ramipril.

As early as 15 years ago we reported that bradykinin (at a concentration of20 nm ) or A23187 (at a concentration of 2 mM) both resulted in a coupled releaseof EDRF(NO) and PGI2 from cultured porcine aortic endothelial cells [18]. Theorder of potencies of bradykinin and A23187 to release PGI2 and EDRF(NO) in acoupled manner [14] is similar to that required for increasing [Ca2‡]i in BAEC,as reported here. ADP also is a potent receptor agonist that raises [Ca2‡]i inendothelial cells. In vivo, the coupled release of PGI2 and EDRF(NO) byADP islikely to occur when endogenous ADP is being released from aggregating platelets,and then ADP-triggered endothelial defense turns up. In contrast with the ADPmode of action, the coupled release of PGI2 and EDRF(NO) by bradykinin isexpected to occur when this peptide is locally released from vascular endothelium,provided that bradykinin would escape destruction by the kininase II function ofendothelial caveolar ACE. Indeed, we show in vivo that inhibition of the endothe-lial ACE by quinapril and perindopril activates the secretory endothelial functionthrough a bradykinin mechanism. In vivo ACE-I-induced thrombolysis is mediatedby bradykinin B2 receptors and then by a powerful outburst of PGI2 generation.EDRF(NO) plays only a permissive role, while t-PA may participate in a late phaseof thrombolysis.

However, the forgoing constitutes only a part of the endothelial action of brady-kinin. We observed that bradykinin (10 nM) when incubated with HUVEC (4 h)would induce transcripts for four enzymes: hemooxygenase1 (HO-1), prostaglandinE synthase (PGE-S), cyclooxygenase 2 (COX-2), and NO synthase 2 (NOS-2).The potency of induction for corresponding mRNAs decreased in order ofHO-1 >PGE-S > COX-2 ¾ NOS-2. Would enzymic activity go along with theappearance of transcripts, then bradykinin should act as a `microcytokin-type’’enzymatic inducer of endothelial enzymes.

Induction of HO-1 is associated not only with increased production of CO, butalso with increased consumption of intracellular heme required as a prosthetic moi-ety both for cyclooxygenase and for NO synthase, as well as for a number of otherhemoprotein enzymes [39]. Anassumption of a regulatory role of HO-1on activitiesof other endothelial enzymes waits for experimental assessment [40]. Recently,the involvement was reported of cytosolic phosholipase A2 in bradykinin-mediatedrelease of PGI2 from HUVEC [41].

The housekeeping function of PGI2 or PGE2 in peripheral circulation and asupportive role of NO emerged from many studies including the present one. How-ever, in pulmonary circulation NO but not prostanoids seems to protect againstlung damage by LPS, both in vivo [42] and in isolated blood perfused rat lung. Inendotoxemia, NO ¢ghts against TXA2 and PAF as well as prevents transcellularformation of cysteinyl leukotrienes [4, 5].This is why the pharmacological blockade

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of NO synthesis during endotoxemia leads to the arrest of all lung functions, potentvascular and bronchial constriction, acute microvascular lung injury, and hemor-rhagic lung edema.

It comes as no surprise that the pneumoprotective role of NO in early endotox-emia cannot be replaced by PGI2. It is NO but not PGI2 that inhibits intercellularadhesion [43, 44]. Although PGI2 is a potent inhibitor of platelet aggregation [45],it hardly in£uences platelet adhesion [46] and has virtually no e¡ect on leukocytefunction [47].

Thus, pulmonary NO appears as the major endogenous mediator that allevi-ates the necessary but dangerous events associated with the removal of bacterialLPS from pulmonary circulation. In peripheral circulation PGI2 is the strongestlink in the chain of events that protect arteries against atherothrombosis. Kallik-rein, kininase II, and B2 receptors of endothelial cells play crucial roles in regulatingtheir own secretory function. Hence, the signi¢cance of the tissue-type ACE (kini-nase II) inhibitors that lately showed their new face as highly e¤cient drugs fortreatment of myocardial infarction and ischemic stroke [38].

REFERENCES

1. Gryglewski RJ, Bunting S, Moncada S, Flower RJ, Vane JR. Arterial walls are pro-tected against deposition of platelet thrombiby a substance (prostaglandin X) whichthey make from prostaglandin endoperoxides. Prostaglandins 1976;12:685^713.

2. Moncada S, Gryglewski R, Bunting S, Vane JR. An enzyme isolated from arteriestransforms prostaglandin endoperoxides to an unstable substance that inhibits pla-telet aggregation. Nature 1976;263:663^665.

3. Bunting S, Gryglewski R, Moncada S, Vane JR. Arterial walls generate from pros-taglandin endoperoxides a substance (prostaglandin X) which relaxes strips ofmesenteric and coeliac ateries and inhibits platelet aggregation. Prostaglandins1976;12:897^913.

4. Sala A, Rossoni G, Berti F, Muller-Peddinghaus R, Folco G. Transcellular synthesisof Cys-LT: from isolated cells to complex organ system. Adv Exp Med Biol1997;433:95^98.

5. Sala A, Buccellati C, Zarini S, Bolla M, Bonazzi A, Folco GC. The polymorpho-nuclear leukocyte: a cell tuned for transcellular biosynthesis of cys-leukotrienes.J Physiol Pharmacol 1997;48:665^673.

6. Gerritsen ME, Printz MP. Sites of prostaglandin synthesis in the bovine heart andisolated bovine coronary microvessels. Cir Res 1981;49:1152^1163.

7. Jakobsson PJ, Thoren S, Morgenstern R, Samuelsson B. Identi¢cation of humanprostaglandin E synthase: a microsomal, glutathione-dependent, inducible enzyme,constituting a potential novel drug target. Proc Nat Acad Sci USA 1999;96:7220^7225.

8. McAdam BF, Catella-Lawson F, Mardini IA, Kapoor S, LawsonJA, F|tzGerald GA.Systemic biosynthesis of prostacyclin by cyclooxygenase (COX)-2: the humanpharmacology of a selective inhibitor of COX-2 [published erratum appears in Proc

Bradykinin and Endothelium 287

Feta

l Ped

iatr

Pat

hol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

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y U

nive

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rary

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echt

on

09/1

1/13

For

pers

onal

use

onl

y.

Natl Acad Sci USA 1999 May 11;96(10):5890]. Proc Nat Acad Sci USA 1999;96:272^277.

9. McGi¡ JC, QuilleyJ. 20-HETE and the kidney: resolution of old problems and newbeginnings. AmJ Physiol 1999;277:R607^R623.

10. Campbell WB, Harder DR. Endothelium-derived hyperpolarizing factors and vas-cular cytochrome P450 metabolites of arachidonic acid in the regulation of tone.Circ Res1999;84:484^488.

11. Furchgott RF, ZawadzkiJV.The obligatory role of endothelial cells in the relaxationof arterial smooth muscle by acetylcholine. Nature 1980;288:373^376.

12. Furchgott RF. Research leading to nitric oxide: the importance of accidental ¢nd-ings. In Gryglewski RJ and Minuz P eds. Proceedings of 59th NATO ASI Course:Nitric Oxide^Basic Research and Clinical Application. Erice, September 7^17,2000.

13. Murad F. Cyclic guanosine monophosphate as a mediator of vasodilation. J CliniInvesti 1986;78:1^5.

14. Gryglewski RJ, Palmer RM, Moncada S. Superoxide anion is involved in the break-down of endothelium-derived vascular relaxing factor. Nature 1986;320:454^456.

15. Vergnani L, Hatrik S, Ricci F, PassaroA, Manzoli N, Zuliani G, BrovkovychV, FellinR, Malinski T. E¡ect of native and oxidized low-density lipoprotein on endothelialnitric oxide and superoxide production: key role of L-arginine availability. Circula-tion 2000;101:1261^1266.

16. Abu-Soud HM,WangJ, Rousseau DL, FukutoJM, Ignarro LJ, Stuehr DJ. Neuronalnitric oxide synthase self-inactivates by forming a ferrous-nitrosyl complex duringaerobic catalysis. J Biolog Chem1995;270:22997^23006.

17. Abu-Soud HM, Rousseau DL, Stuehr DJ. Nitric oxide binding to the heme of neu-ronal nitric-oxide synthase links its activity to changes in oxygen tension. J BiologiChemi1996;271:32515^32518.

18. Gryglewski RJ, Moncada S, Palmer RM. 1986. Bioassay of prostacyclin andendothelium-derived relaxing factor (EDRF) from porcine aortic endothelial cells.BrJ Pharmacol 1986;87:685^694.

19. SwierkoszTA, MitchellJA,WarnerTD, Botting RM,VaneJR. Co-induction of nitricoxide synthase and cyclo-oxygenase: interactions between nitric oxide and prosta-noids. BrJ Pharmacol1995;114:l335^1342.

20. Salvemini D, Settle SL, Masferrer JL, Seibert K, Currie MG, Needleman P.Regulation of prostaglandin production by nitric oxide; an in vivo analysis. Br JPharmacol1995;114:1171^1178.

21. Salvemini D, Currie MG, MollaceV. Nitric oxide-mediated cyclooxygenase activa-tion. A key event in the antiplatelet e¡ects of nitrovasodilators. J Clini Investi1996;97:2562^2568.

22. Salvemini D.; Regulation of cyclooxygenase enzymes by nitric oxide. Cell Mol LifeSci 1997;53:576^582.

22a. Gryglewski RJ, Chlopicki S, UraczW, Marcinkiewicz E. Signi¢cance of endothelialprostocyclin and nitric oxide in peripheral and pulmonary circulation. Med SciMonit 2001;7:1^16.

23. Ziemianin B, Olszanecki R, UraczW, Marcinkiewicz E, Gryglewski RJ.Thienopyri-dines: e¡ects on cultured endothelial cells. J Physiol Pharmacol1999;50:597^604.

288 R. J. Gryglewski et al.

Feta

l Ped

iatr

Pat

hol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

Lib

rary

Utr

echt

on

09/1

1/13

For

pers

onal

use

onl

y.

24. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2 ‡ indicators withgreatly improved £uorescence properties. J Biolog Chem1985;260:3440^3450.

25. Malinski T,Taha Z. Nitric oxide release from a single cell measured in situ by a por-phyrinic-based microsensor. Nature 1992;358:676^678.

26. Jia L, Furchgott RF. Inhibition by sulfhydryl compounds of vascular relaxationinduced by nitric oxide and endothelium-derived relaxing factor. J Pharmacol ExpTher 1993;267:371^378.

27. Ja¡e EA, Armellino D, Lam G, Cordon-Cardo C, Murray HW, Evans RL. IFN-gamma and IFN-alpha induce the expression and synthesis of Leu-13 antigen bycultured human endothelial cells. J Immunol 1989;143:3961^3966.

28. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidi-nium thiocyanate-phenol-chloroform extraction. Anal Biochem1987;162:156^159.

29. Gryglewski RJ, Korbut R, Ocetkiewicz A, StachuraJ. In vivo method for quantita-tion for anti-platelet potency of drugs. Naunyn-Schmiedebergs Arch Pharmacol1978;302:25^30.

30. Gryglewski RJ, Korbut R, Swies J., Uracz W. E¡ect of ticlopidine on streptokinase-induced thrombolysis in rats.W|ener KlinischeWochenschrift 1999;111:98^102.

31. Gryglewski RJ, Uracz W, Swies J. Unusual e¡ects of aspirin on ticlopidine inducedthrombolysis.Thorax 2000;50 suppl 2:S17^S19.

32. Mackiewicz ZGRJ, Swies J, Dabros W, Uracz W. Streptokinase-induced changes inthe structure of platelets. Ex vivo study. Acta Med Lituanica 1996;2:3^8.

33. Chlopicki S, BartusJB, Gryglewski RJ. Biphasic response to lipopolysaccharide fromE-coli in the isolated-ventilated blood-perfused rat lung. J Physiol Pharmacol1999;50:551^565.

34. Uhlig S, Heiny O. Measuring the weight of the isolated perfused rat lung duringnegative pressure ventilation. J PharmacolToxi Meth 1995;33:147^152.

35. Mugge A, PetersonT, Harrison DG. Release of nitrogen oxides from cultured bovineaortic endothelial cells is not impaired by calcium channel antagonists. Circulation1991;83:1404^1409.

36. Ferreira SH. A bradykinin-potentiating factor (BPF) present in the venom of Bo-thropsJaracca. BrJ Pharmacol1965;24:163^169.

37. Bakhle YS, Reynard AM,Vane JR. Metabolism of the angiotensins in isolated per-fused tissues. Nature1969;222:956^959.

38. Yusuf S, Sleight P, PogueJ, BoschJ, Davies R, Dagenais G. E¡ects of an angiotensin-converting-enzyme inhibitor, ramipril, on cardiovascular events in high-risk pa-tients. The Heart Outcomes Prevention Evaluation Study Investigators [see com-ments] [published errata appear in N Eng J Med 2000 Mar 9;342(10):748 and 2000May 4;342(180:1376]. N EnglJ med 2000;342:145^153.

39. Wagener FA, da SilvaJL, FarleyT, deW|tteT, Kappas A, Abraham NG. Di¡erentiale¡ects of heme oxygenase isoforms on heme mediation of endothelial intracellularadhesion molecule 1 expression. J Pharmacol ExpTher 1999;291:416^423.

40. Johnson RA, Lavesa M, Askari B, Abraham NG, Nasjletti A. A heme oxygenaseproduct, presumably carbon-monoxide, mediates a vasodepressor function in rats.Hypertension 1995;25:166^169.

41. Yamasaki S, Sawada S, Komatsu S, KawaharaT,TsudaY, SatoT,Toratani A, KonoY,Higaki T, Imamura H, Tada Y, Akamatsu N, Tamagaki T, Tsuji H, Nakagawa M.

Bradykinin and Endothelium 289

Feta

l Ped

iatr

Pat

hol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsity

Lib

rary

Utr

echt

on

09/1

1/13

For

pers

onal

use

onl

y.

E¡ects of bradykinin on prostaglandin I(2) synthesis in human vascular endothelialcells. Hypertension 2000;36:201^207.

42. Gryglewski RJ,Wolkow PP, UraczW, Janowska E, Bartus JB, Balbatun O, Patton S,Brovkovych V, Malinski T. Protective role of pulmonary nitric oxide in the acutephase of endotoxemia in rats. Circulation Research 1998;82:819^827.

43. Kanwar S, Kubes P. Nitric oxide is an antiadhesive molecule for leukocytes. NewHoriz.1995;3:93^104.

44. Provost P, Merhi Y. Endogenous nitric oxide release modulates mural plateletthrombosis and neutrophil-endothelium interactions under low and high shearconditions.Thromb Res 1997;85:315^326.

45. VaneJR.The Croonian Lecture,1993. The endothelium: maestro of the blood circu-lation. PhilosTrans R Soc Lond B Biol Sci 1994;343:225^246.

46. Radomski MW, Moncada S.The biological and pharmacological role of nitric oxidein platelet function. Adv Exp Med Biol 1993;344:251^264.

47. Kuehl FA, Jr., HumesJL, Egan RW, Ham EA, Beveridge GC,Van Arman CG. Roleof prostaglandin endoperoxide PGG2 in in£ammatory processes. Nature1977;265:170^173.

290 R. J. Gryglewski et al.

Feta

l Ped

iatr

Pat

hol D

ownl

oade

d fr

om in

form

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re.c

om b

y U

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rary

Utr

echt

on

09/1

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For

pers

onal

use

onl

y.