Diabetologia (1999) 42: 978±986

Morphological evidence for the existence of nitric oxide andcarbon monoxide pathways in the rat islets of Langerhans:An immunocytochemical and confocal microscopical studyP.Alm1, P.Ekström2, R. Henningsson3, I. Lundquist3

1 Department of Pathology, University of Lund, Sweden2 Department of Zoology, University of Lund, Sweden3 Department of Pharmacology, University of Lund, Sweden

Ó Springer-Verlag 1999

Abstract

Aims/hypothesis. To map the cellular location of in-ducible and constitutive nitric oxide synthase andhaem oxygenase in rat islets to clarify the morpholog-ical background to putative nitric oxide and carbonmonoxide pathways.Methods. Immunocytochemistry and confocal mi-croscopy.Results. After treatment with endotoxin, immunore-activity for inducible nitric oxide synthase was ex-pressed in a large number of islet cells, most of whichwere insulin-immunoreactive beta cells and in singleglucagon-immunoreactive and pancreatic polypep-tide-immunoreactive cells. Somatostatin-immunore-active cells lacked immunoreactivity for inducible ni-tric oxide synthase. In untreated rats, immunoreactiv-ity for constitutive nitric oxide synthase occurred inthe majority of insulin-immunoreactive and gluca-gon-immunoreactive cells, in most pancreaticpolypeptide-immunoreactive and somatostatin-im-munoreactive cells and in islet nerves. Similarly, im-munoreactivity for constitutive haem oxygenase wasdetected in all four types of islet cells. Endotoxin

treatment did not change the pattern of immunoreac-tivity for constitutive and inducible haem oxygenase.After treatment with alloxan, insulin-immunoreactiv-ity was observed only in single islet cells, being almostdevoid of immunoreactivity for constitutive nitric ox-ide synthase and haem oxygenase.Conclusion/interpretation. In vivo endotoxin-inducedexpression of inducible nitric oxide synthase in insu-lin-producing and in scattered glucagon-producingand pancreatic polypeptide-producing cells strength-ens previous suggestions of a pathophysiological rolefor inducible nitric oxide synthase in the develop-ment of insulin-dependent diabetes mellitus. Thepresence of constitutive nitric oxide synthase andhaem oxygenase in all four types of islet cells, togeth-er with recent functional data of ours support rolesfor nitric oxide and carbon monoxide as intracellular,paracrine or neurocrine modulators of islet hormonesecretion. [Diabetologia (1999) 42: 978±986]

Keywords Pancreatic islets, nitric oxide synthase,haem oxygenase, imunocytochemistry, confocal mi-croscopy.

Received: 19 November 1998 and in revised form: 22 March1999

Corresponding author: P. Alm, MD, PhD, Department of Pa-thology, University Hospital, S-22 185 Lund, SwedenAbbreviations: CO, Carbon monoxide; HO, haem oxygenase;HO-1, inducible haem oxygenase; HO-2, constitutive haem ox-ygenase; NO, nitric oxide; NOS, nitric oxide synthase; GLUC,

glucagon; IG, immunoglobulins; INS, insulin; IR, immunore-active; LPS, lipopolysaccharide (endotoxin); iNOS, induciblenitric oxide synthase; cNOS, constitutive nitric oxide synthase;eNOS, endothelial nitric oxide synthase; nNOS, neuronal ni-tric oxide synthase; PP, pancreatic polypeptide; SOM, soma-tostatin; ZnPP, zinc protoporphyrin; FITC, fluorescein isothio-cyanate conjugated.

Nitric oxide (NO) is a free radical gas that conveysbiological information in a way greatly differingfrom that of the classical transmitters. In the nervoussystem NO does not act on conventional receptorsbut through effects on various regulatory processes,intracellularly or in the membrane [1]. The formationof NO is catalysed by the enzyme nitric oxide syn-thase (NOS), in a reaction in which l-arginine andoxygen are converted to NO and citrulline. Thereare two major types of NOS enzymes; one inducibleisoform iNOS, originally described in macrophagesand also shown to be expressed in a variety of mam-malian tissues among which are the islets of Langer-hans [2, 3], and constitutive isoforms (cNOS) presentin neurons (nNOS) and endothelial cells (eNOS) [1].We and others [4±8] have shown previously that thepancreatic islets contain a constitutive NOS as deter-mined by histochemical, immunocytochemical andbiochemical methods. Islet iNOS has been implicatedas an important factor in the pathogenesis of Type I(insulin-dependent) diabetes mellitus [2, 3], whereasislet cNOS has been suggested to be involved in thephysiological regulation of insulin and glucagon se-cretion [4±15]. Thus, both cNOS and iNOS seem tobe of great physiological and pathophysiological im-portance in the pancreatic islets. Constitutive nitricoxide synthase was localised by the use of an antiser-um to the neuronal isoform of cNOS.

There are diverging results as to the cellular loca-tion of the NOS isoforms within the islets, i. e. whe-ther only the insulin producing beta cells or, in addi-tion, other endocrine cell types such as glucagon-pro-ducing, somatostatin-producing, and pancreaticpolypeptide (PP)-producing cells also contain NOSactivity [4±8, 16±18]. Further, in this context, atten-tion has also been drawn to another gaseous mole-cule, carbon monoxide (CO), since a number of re-cent studies have shown that CO may serve as a neu-ronal messenger molecule similar to NO [19±21].Carbon monoxide is produced by the action of haemoxygenase (HO), at which haem from haemoglobinis degraded to CO and biliverdin [19±22]. The lattercompound can then be converted to bilirubin, whichis an important antioxidant, the reaction being cataly-sed by the enzyme biliverdin reductase. Similar toNOS, HO consists of at least two isoenzymes, an in-ducible (HO-1), and a constitutively expressed iso-form (HO-2) [19±21]. Expression of HO-1 is inducedby various stress factors, e.g. fever, starvation, oxida-tive injury. A cytokine-induced expression of a pro-tein (presumably HO-1) has previously been ob-served in islet tissue, and suggested to be a protectivemechanism against oxidative stress [2, 3, 23±25]. Con-stitutive haem oxygenase is highly expressed in ner-vous tissue, in which CO may have a transmitter-likefunction [19, 20] and recent findings of ours suggesta role for CO in the regulation of the release of islethormones in rats [26].

To further clarify the morphological backgroundto the putative NO and CO functional pathways inthe islets of Langerhans, the aim of this study was tomap the cellular location of iNOS, cNOS, HO-1 andHO-2 in islets of normal and alloxan diabetic rats bymeans of combined immunocytochemical and confo-cal microscopical methods.

Materials and methods

Tissue handling. Female Sprague-Dawley rats (300±400 g bodyweight, aged about 3±4 months) were purchased from B&KUniversal, Stockholm, Sweden. The animals had free accessto water and standard pellets and were used in different exper-imental groups consisting of four to six animals. One groupconsisted of rats that received no treatment. One group ofrats was given LPS (lipopolysaccharide endotoxin from salmo-nella typhimurium, Sigma, St Louis, Mo., USA; 10 mg/kg i. p.,dissolved in saline) and used after 6 h, at which time there is ahigh expression of iNOS [34]. One group was treated with al-loxan (Sigma; 60 mg/kg i. v., dissolved in saline with the addi-tion of a drop of 0.1 N acetic acid to acidify the solution) andkilled after 5 days. Plasma glucose was determined by a glu-cose oxidase method [18, 27] to ensure that the animals had be-come diabetic. The concentrations of plasma glucose in freelyfed diabetic animals were 15.1±38.7 mmol/l (total range) com-pared with 8.9±11.7 mmol/l in normal rats. The rats were an-aesthesized with ketamine (100 mg/kg intramuscular; Ketalar,Parke Davis, Barcelona, Spain) and xylazin (15 mg/kg intra-muscular; Rompun, Bayer, Leverkusen, Germany) and perfus-ed transcardially through the ascending aorta, first with 100 mlof ice-cold calcium-free Krebs buffer (containing 0.5 g/l sodi-um nitrite and 10.000 iU/l of heparin), and then with 300 ml ofan ice-cold, freshly prepared solution of 4 % formaldehyde inphosphate buffered saline (PBS, 0.1 mol/l, pH 7.4). The pan-creatic glands were then rapidly dissected out and dividedinto pieces, which were fixed in the same fixative for 4 hours.After this they were rinsed in ice-cold 15 % sucrose in PBS(three rinses during 48 h). The tissue specimens were frozenin isopentane at ±40 °C and then stored at ±70 °C. Principles oflaboratory animal care (NIH publication No 85±23 1985)were followed and the experimental design was also approvedby the animal ethics committee of the University of Lund,Lund, Sweden.

Immunocytochemistry. Cryostat section were cut at a thicknessof 8 mm and thaw-mounted onto chrom alum-coated glassslides and air dried for 30 min to 1 h. To show iNOS, nNOS,HO-1 and HO-2, sections were pre-incubated in PBS with0.2 % Triton X-100 for about 2 h, and then incubated for2 days in the presence of rabbit antisera to iNOS, nNOS, HO-1 or HO-2. The antiserum to iNOS (1:500) was generated inrabbits against a 25 amino acid peptide of a cloned inducibleNOS from a murine macrophage cell line [28, 29]. The antiserato nNOS were generated in rabbits against a 15 amino acid se-quence (nNOS-15, 1:1280) [30], or a 21 amino acid sequence(nNOS-21, peptide 58; 1:2000) [31] from the C-terminal partof a cloned rat cerebellar NOS [32]. The antiserum to HO-1(1:500; code OSA 100, StressGen Biotechnol, Victoria, Cana-da) was generated in rabbits against rat liver HO-1. The rabbitHO-2 antiserum (1:1000, code OSA-200; StressGen) was gen-erated in rabbits against rat testes HO-2. After rinsing in PBS(three rinses during 10 min), the sections were incubated for90 min with fluorescein isothiocyanate conjugated (FITC)

P. Alm et al.: Islet nitric oxide synthase and haem oxygenase 979

swine anti-rabbit immunoglobulins (IG) (1:80; Dakopatts,Stockholm, Sweden) or Texas red-conjugated affinity purifiedF(ab¢)2 fragments of donkey anti-rabbit IG (1:80; code711±076±132; Jackson Immuno Research, West Grove, Pa.,USA). After rinsing, the sections were mounted in PBS/glycer-ol with p-phenylenediamine to prevent fluorescence fading[33].

To show two antigens simultaneously [34], sections were in-cubated overnight with iNOS, nNOS, or HO-2 antisera (seeabove), rinsed and then incubated overnight with antisera gen-erated in guinea-pigs to insulin (1:16 000), glucagon (1:4000)and pancreatic polypeptide (1:500) (the latter antisera pur-chased from Linco Res, St Louis, Mo., USA), and a mousemonoclonal antiserum to somatostatin (1:5 of a prediluted an-tiserum; cat no 8330±0496, Biogenesis, Poole, England). Afterrinsing, the sections were incubated for 90 min with FITCgoat anti-guinea-pig IG or goat anti-mouse IG (1:80; Sigma,St Louis, Mo., USA), rinsed and then incubated with Texasred conjugated affinity purified F(ab¢)2 fragments of donkeyanti-rabbit IG (1:80; see above). The sections were rinsed andmounted as described above. An Olympus 3 ´ 50 fluorescencemicroscope (LRI Instrument AB, Lund, Sweden) equippedwith epi-illumination and appropriate filter settings for TexasRed-immunofluorescence and FITC-immunofluorescencewas used for the examinations of the sections [35].

The primary and secondary antisera were diluted in PBS. Incontrol experiments no immunoreactivity could be detected insections incubated in the absence of the primary antisera orwith nNOS-15, HO-2, glucagon, somatostatin or pancreaticpolypeptide antisera absorbed with excess of the correspond-ing immunizing antigen (100 mg/ml). No absorption controlscould be done with the iNOS or nNOS-21 antisera as antigenicsubstances were not available. The characteristics of the iNOSand the nNOS antisera have been presented previously[29±31]. In control experiments iNOS-immunoreactivity wasonly observed in LPS-induced tissues (macrophages in lungand liver), in which no nNOS-immunoreactivity could beseen. As cross reactions to antigens sharing similar amino acidsequences cannot be completely excluded the structures shownare referred to as iNOS-, nNOS-, HO-1-, HO-2-, insulin-(INS-),glucagon-(GLUC-), pancreatic polypeptide-(PP-), or soma-tostatin-(SOM-) immunoreactive (IR).

Confocal microscopy. To evaluate whether two immunoreac-tivities were colocalized within the same cellular structures,sections were analysed in a confocal laser scanning microscope(Multiprobe 2001 TM CLSM; Molecular Dynamics) equippedwith an Ar/Kr laser and an inverted Nikon Diaphot TMD mi-croscope as described elsewhere [35].

Results

No iNOS-immunoreactivity could be detected in is-lets of untreated animals. After LPS treatment,iNOS-immunoreactivity was expressed in a largenumber of islet cells, which were diffusely spreadover the islets (Fig.1A, D, G, J). Double immuno-staining showed that these cells were also INS-IR,which was further verified by confocal microscopy(Fig.1C).

Moreover, single iNOS-IR cells also displayedGLUC-immunoreactivity and PP-immunoreactivity(Fig.1F, I) but in most of the GLUC-IR and PP-IR

cells iNOS-immunoreactivity was lacking. No iNOS-IR cells expressed SOM-immunoreactivity (Fig.1L).In comparison, treatment with LPS did not seem tochange the nNOS-immunolabelling pattern withboth the nNOS antisera used.

Constitutive NOS expressed as nNOS-immunore-activity could be detected in the cytoplasm of most is-let cells of untreated animals (Fig.2A, D, G). Thenumber and distribution patterns of nNOS-IR cellswere similar with the two NOS antisera used, al-though the intensity of the NOS-immunofluores-cence was weaker with the nNOS-21 than with thenNOS-15 antiserum. Vessels of capillary size, be-tween the trabecula of islet cells (Fig.2G) and be-tween exocrine acini, were accompanied by varicoseNOS-IR nerve terminals, which were also foundaround arteries of various sizes. No nNOS-immu-noreactivity could be detected in endothelial cells ofthe islet microvasculature.

Double immunolabelling showed that nNOS-im-munoreactivity was in most INS-IR cells (Fig.2A±C).In the periphery of the islets there were, however,several nNOS-IR cells which lacked INS-immunore-activity. Most GLUC-IR cells, which were locatedalong the periphery of the islets, were also nNOS-IR(Fig.2D±F), although single GLUC-IR cells were dis-covered which lacked nNOS-immunoreactivity. Pan-creatic polypeptide-IR and some SOM-IR cells,which were also located in the periphery of the islets,also expressed nNOS-immunoreactivity (data notshown), which in the SOM-IR cells were as far as tothe ends of their long and gracile dendritic processes.

In the cytoplasm of almost all islet cells HO-2 im-munoreactivity could be detected (Fig.3 A, D, G,L). Double immunolabelling in combination withconfocal microscopy revealed that most INS-IR cellsalso showed HO-2 immunoreactivity, although therewere HO-2 IR cell in the periphery of the islets thatlacked INS-immunoreactivity (Fig.3A±C). Further,along the periphery of the islets there was a broadring of GLUC-IR, which also were HO-2 IR (Fig.3D±F) and dispersed PP-cells and SOM-IR cells withextended processes. These also expressed HO-2 im-munoreactivity (Fig.3G±K, L±N). No HO-1 immu-noreactivity could be detected in any type of isletcells.

After treatment with alloxan almost no specificnNOS-immunoreactivity and HO-2 immunoreactivi-ty was discovered in most of the damaged beta cellsand INS-immunoreactivity was only seen in single is-let cells, which were vacuolized and enlarged. In com-parison, nNOS-immunoreactivity and HO-2 immu-noreactivity was well preserved only in non beta cells(GLUC-IR cells and others) (data not shown).

P. Alm et al.: Islet nitric oxide synthase and haem oxygenase980

P. Alm et al.: Islet nitric oxide synthase and haem oxygenase 981

Fig. 1 A±L. Confocal microscopy of rat islets of Langerhans af-ter treatment with LPS. Left panel: red fluorescence in A,D,Gand J indicating expression of iNOS-immunoreactivity (Texasred immunofluorescence). Middle panel: green fluorescence(FITC-immunofluorescence) shows immunoreactivities for in-sulin (B), glucagon (E), pancreatic polypeptide (H) and soma-

tostatin (K). Right panel: overlay picture of A + B ( = C),D + E ( = F), G + H ( = I) and J + K ( = L). Cells showing yel-lowish fluorescence (arrowheads) indicate colocalization ofiNOS/insulin (C), iNOS/glucagon (F), and iNOS/pancreaticpolypeptide (I). Bars with numerals indicate lengths (mm)

Discussion

A characteristic of Type I diabetes is a local inflam-matory reaction in the pancreatic islets that are un-dergoing autoimmune destruction [2, 3]. Nitric oxidehas been proposed as a possible mediator in the dam-age process to the insulin producing beta cells andthere is ample in vitro evidence that IL-1 and othercytokines are able to induce iNOS expression in islettissue [2, 3]. The pancreatic islet consists, however,of a heterogeneous cell population, making it difficultto localize the cellular source of iNOS expression andNO production. It was recently shown that rat islets

exposed to cytokine in vitro expressed iNOS in theirinsulin cells whereas the glucagon cells seemed unaf-fected [7]. No data on the possible existence of iNOSin somatostatin cells or PP-cells have so far appearedin the literature.

Lipopolysaccharide (endotoxin) is known to stim-ulate cytokine production [2, 3]. It is important, how-ever, to note that apart from cytokines, other factorsalso have been suggested to serve as direct or indirectmediators of effects of LPS. Thus, it is known thatLPS may cause the synthesis of reactive oxygen spe-cies such as superoxide and hydrogen peroxide, andthat NO can combine with superoxide to form the po-tent oxidizing agent peroxynitrite [2, 3]. Hence, ourimmunocytochemical data cannot be extrapolated toanswer questions on the intimate mechanisms of im-mune destruction of the islet beta cells. Our resultsshow that after treatment with LPS in vivo iNOS isexpressed in most INS-IR cells and also, although toa much lesser extent, in scattered GLUC-IR and PP-IR cells. No iNOS was detected in cells immunostain-ed for somatostatin. Our in vivo data of iNOS expres-sion in INS-IR cells, agree with findings of previousstudies of islets exposed to cytokine in vitro [2, 3, 7].The observation that iNOS expression can be elicitedin GLUC-IR cells and PP-IR cells has not been de-scribed previously. This may be explained by differ-ences between the in vitro and the in vivo situationand the possible induction by LPS of unknowniNOS stimulatory factors. On the other hand, sincethe fraction of iNOS positive cells among theGLUC-IR cell and PP-IR cells is much smaller thanamong the INS-IR cells, it is not inconceivable thatrefined techniques such as confocal microscopy arerequired to observe these iNOS positive GLUC-IRcell and PP-IR cells. It is difficult to explain whySOM-IR cells are not influenced by LPS. It hasbeen shown [36, 37] that cells producing somatostatinin contrast to those producing insulin, glucagon andPP are not members of the group of endocrine cellsbelonging to the amine precursor uptake and decar-boxylation series. Whether the ability of endocrinecells to store amine is coupled to that of expressingiNOS remains to be explained. If NO finally turnsout, however, to be of pathophysiological importancein the development of Type I diabetes, it is notablethat not only insulin cells but also GLUC-IR cellsand PP-IR cells are able to express iNOS. Whetherthe number of these non-beta cells observed express-ing iNOS is, however, enough to actually contributeto damaging the beta cells remains to be explained.It is also tempting to speculate that NO derived bythe action of iNOS in the glucagon cells is at leastpartly responsible for the increased glucagon secre-tion in the diabetic condition. Indirectly, such amechanism might also explain why there is an in-creased somatostatin response to glucagon in diabe-tes.

P. Alm et al.: Islet nitric oxide synthase and haem oxygenase982

Fig. 2 A±G. Confocal microscopy of islets of Langerhans ofuntreated rats. Left panel: red fluorescence in A and D (TexasRed immunoflurescence) indicates nNOS-immunoreactivity.Middle panel: green fluorescence (FITC-immunofluores-cence) shows immunoreactivities for insulin (B) and glucagon(E). Right panel: overlay picture of A + B ( = C), and D + E( = F). Yellowish fluorescent cells indicate colocalization ofnNOS/insulin (C) and nNOS/glucagon (F). Bars with numeralsindicate lengths (mm). G. Rat islet of Langerhans with nNOS-IR varicose nerve terminals (arrowheads) running along ves-sels of capillary size between endocrine cells. FITC-immuno-fluorescence. Bar = 100 mm

P. Alm et al.: Islet nitric oxide synthase and haem oxygenase 983

Fig. 3 A±L. Confocal microscopy of islets of Langerhans of un-treated rats. Left panel: red fluorescence in A,D,G and J dis-plays expression of HO-2 immunoreactivity (Texas red immu-nofluorescence). Middle panel: green fluorescence (FITC-im-munofluorescence) shows immunoreactivities for insulin (B),glucagon (E), pancreatic polypeptide (H) and somatostatin

(K). Right panel: overlay picture of A + B ( = C), D + E( = F), G + H ( = I) and J + K ( = L). Cells showing yellowishfluorescence indicate colocalization of HO-2/insulin (C), HO-2/glucagon (F), HO-2/pancreatic polypeptide (I), and HO-2/somatostatin (L). Bars with numerals indicate lengths (mm)

Our data show that all four endocrine cell types,i. e. those containing insulin, glucagon, somatostatinand PP showed immunoreactivity for two differentantisera of the neuronal isoform of constitutiveNOS. As previously discussed it has been known forseveral years [2, 3] that rat pancreatic islets are ableto express the inducible NOS isoform after treatmentin vitro with different cytokines. The possibility of aconstitutive NOS isoform being present has, howev-er, been a matter of debate. We and others [4, 5, 7, 8,16, 38] have previously observed cNOS activity in ratand mouse islet cells by both histochemical (NAD-PH-diaphorase activity) and immunocytochemicalmethods. In a recent study, nNOS immunoreactivitywas shown in rat islets with a weak intensity [38] thatwas apparently lower than the findings in this study.This is possibly due to differences in methodologyand nNOS antisera used but also to differences inbrightness of the fluorophores of the secondary anti-bodies [39]. In comparison, other studies carried outwith other NOS antisera as well as with the NAD-PH-diaphorase method were either unable to showcNOS activity in rat islet cells or reported that onlycells containing somatostatin or islet nerves or bothwere positive for NOS [40, 41, 42]. The reasons forthese discrepancies are not known. The existence ofdifferent local isoforms, differences between antiserain recognizing epitopes or differences in methodolo-gy are possible explanations. Further, the recent ob-servation that a constitutive eNOS is localized to glu-cagon and somatostatin (but not to insulin cells) in ratislets suggests the possibility that both eNOS andnNOS are localized in the same cell type [5, 18]. Inthe present study using two antisera of differentsources directed against neuronal NOS, we observedthat nNOS-immunoreactivity was located in all fourtypes of endocrine cells. Moreover, confocal micros-copy disclosed coinciding profiles between cells ex-pressing nNOS-immunoreactivity and immunoreac-tivity for insulin, glucagon, pancreatic polypeptideand somatostatin, respectively. Taken together withthe results from previous studies [4±8, 16] this datastrongly suggest that a constitutive nNOS resides inall these four cell types and in nervous structures aswell. Moreover, results of most functional studies onthe influence of NOS-inhibitors and NO donors oninsulin and glucagon secretion favour NO as an im-portant modulator of the secretory processes of thesehormones [4±6, 8±11, 13±15, 43]. Although some ear-ly studies [8, 10] and a recent one (using islets fromnewborn rats) [18] suggested that NO had a positiveeffect on glucose-stimulated and l-arginine stimulat-ed insulin release, we [4±6, 9, 14, 15, 44±46] and oth-ers [13, 43, 47] have repeatedly shown that NO isstrongly inhibitory to insulin secretion induced bythese secretagogues. The inhibitory effect of NO oninsulin release induced by nutrients is probably dueto the formation of S-nitrosothiols [48], which impair

important regulatory thiol groups. Thiol groups havelong been shown to be essential for stimulus-secre-tion coupling induced by glucose [49, 50]. In contrast,NO is a positive modulator of glucagon secretion,probably acting by stimulating the cyclic GMP system[5, 6, 44±46].

It has previously been shown that cytotoxicity me-diated by cytokine can induce expression of HO-1 incultured islets [23±25]. That we did not observe anyimmunocytochemical evidence for HO-1 expressionin rats islets after injection with endotoxin does notexclude that HO-1 could be detected by other moresensitive methods but not by immunocytochemistrywhich might be too insensitive in the present situa-tion. We have very recently obtained evidence forthe presence of mechanisms mediated by HO-2 andCO in the release of insulin and glucagon from rat is-lets [26]. Thus, islet tissue was found to produce largeamounts of CO [26]. As this CO production wasstrongly suppressed by the HO-inhibitor zinc proto-porphyrin-IX (ZnPP-IX), and since both insulin andglucagon secretion from intact islets could be sup-pressed by ZnPP-IX and stimulated by the HO-sub-strate haemin, we concluded that CO should be re-cognized as a putative physiologic stimulator of insu-lin and glucagon release [26]. The data in this studystrongly suggest that HO-2 resides in all four typesof endocrine cells in rat pancreatic islets and confocalmicroscopy showed coinciding profiles between cellsexpressing HO-2 immunoreactivity and immunoreac-tivities for insulin and glucagon as well as for PP andsomatostatin. Hence, the CO-pathway could be offunctional significance as an intracellular modulatorsystem, not only for the release of insulin and gluca-gon [26] but also for the secretion of PP and soma-tostatin. Thus, from the most recent data and thosefrom this study it seems likely that NO as well as COformed within the islets of Langerhans are capableof acting both within their cells of origin and also asparacrine, neurocrine or even as endocrine media-tors.

In conclusion, we have shown the existence of amorphological substrate for a putative functionalrole of iNOS, nNOS and HO-2 as important regulato-ry enzymes in the physiology and pathophysiology ofhormone secretion from the islets of Langerhans.

Acknowledgements. The technical help of L. Thuresson andthe secretarial help of E. Björkbom is gratefully acknow-ledged. This study was supported by the Swedish Medical Re-search Council (12X-11205, 14X-4286), the foundations ofCrafoord, Magnus Bergvall, Albert Påhlsson, Thelma Zo�gaand �ke Wiberg, the Swedish Diabetes Association and theMedical Faculty, University of Lund, Lund, Sweden. The gen-erous supply of iNOS and nNOS-21 antisera by V. Riveros-Moreno, Wellcome Research Laboratories, Beckenham,England is gratefully appreciated.

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