Heme Oxygenase and Carbon Monoxide: Regulatory ... - Diabetes

Heme Oxygenase and Carbon Monoxide: Regulatory Roles in Islet Hormone Release A Biochemical, Immunohistochemical, and Confocal Microscopic Study Ragnar Henningsson, Per Alm, Peter Ekström, and Ingmar Lundquist

Carbon monoxide (CO) has been suggested as a novel messenger molecule in the brain. We now report on the cellular localization and hormone secretory function of a CO-producing constitutive heme oxygenase (HO-2) in mouse islets. Islet homogenates produced large amounts of CO which were suppressed dose-dependently by the HO inhibitor zincprotoporphyrin-IX (ZnPP-IX). We also show, for the first time, that glucose markedly stimulates the HO activity (CO production) in intact islets. A further potentiation was induced by the HO substrate hemin. Western blot showed that islet tissue expressed HO-2, and confocal microscopy revealed that HO-2 resided in insulin, glucagon, somatostatin, and pancreatic polypeptide cells. ZnPP-IX dose-dependently inhibited, whereas hemin enhanced, both insulin and glucagon secretion from glucose-stimulated islets. Stimulation or inhibition of CO production was accompanied by corresponding changes in islet cGMP levels. Exogenously applied CO stimulated insulin and glucagon release from isolated islets, whereas exogenous nitric oxide (NO) inhibited insulin and stimulated glucagon release. Islets stimulated by glucose or Larginine displayed a marked increase in their NO-synthase (NOS) activity. Such an increase was suppressed by hemin, conceivably because NOS activity was inhibited by hemin-derived CO. Consequently, hemin enhanced L-arginine–induced insulin secretion. Insulin release stimulated by either hemin-derived CO or exogenous CO was strongly inhibited by the guanylate cyclase inhibitor ODQ, but it was unaffected by ZnPP-IX. Glucagon release induced by CO (but not by hemin) was inhibited by ODQ and partly inhibited by ZnPP-IX. We propose that the islets of Langerhans are equipped with a heme oxygenase–carbon monoxide pathway, which constitutes a novel regulatory system of physiological importance for the stimulation of insulin and glucagon release. This pathway is stimulated by glucose, is at least partly dependent on the cGMP system, and displays interaction with islet NOS activity. Diabetes 48:66–76, 1999

From the Departments of Pharmacology (R.H., I.L.), Pathology (P.A.), and Zoology (P.E.), University of Lund, Lund, Sweden. Address correspondence and reprint requests to Dr. Ragnar Henningsson, Department of Pharmacology, Sölvegatan 10, S-223 62 Lund, Sweden. E-mail: [email protected]. Received for publication 24 March 1998 and accepted in revised form 28 September 1998. cNOS, constitutive NO synthase; FITC, fluoroescein isothiocyanate; HO, heme oxygenase; HO-1, inducible heme oxygenase; HO-2, constitutive heme oxygenase; IR, immunoreactive; L-NMMA, NG-monomethyl-L -arginine; NOS, NO synthase; ODQ, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one; PBS, phosphatebuffered saline; PP-IX, protoporphyrin-IX; ZnPP-IX, zincprotoporphyrin-IX. 66

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itric oxide (NO) is generally accepted as an important signaling molecule in many organ systems (1,2). We and others (3–7) previously showed morphological and biochemical evidence for the occurrence of a constitutive NO-synthase (cNOS) activity in the islets of Langerhans, and NO has been found to inhibit the release of insulin but to stimulate that of glucagon (3,5,7). Another two-atom and “poisonous” gas molecule, carbon monoxide (CO), seems to share many similarities with NO (1,2). CO, like NO, is a smooth-muscle relaxant (8–11). It has been suggested as a neuroendocrine modulator in the hypothalamus (12), and it may play a crucial role in brain neuronal signaling (13,14). Activation of the cGMP system is thought to be the most important signaling mechanism exerted by CO and NO (1,2,8–17). This activation is accomplished through NO or CO binding to the heme prosthetic group of guanylate cyclase (16). As CO is much less reactive than NO (for instance, CO is stable to oxygen), it might be capable of exerting its effects during longer time periods and over longer distances than NO. CO can be produced from the -methene bridge of heme (18,19) under the influence of the heme oxygenase (HO) enzyme (19–21), which occurs in two major isoforms (15,22,23): HO-1 (inducible) and HO-2 (constitutive). Heme degradation involves NADPH and three molecules of oxygen (18,23) and results in equimolar formation of CO, Fe2+, and biliverdin. The latter is subsequently transformed to bilirubin, which is postulated as an important antioxidant of possible physiological importance (24). The aim of the present investigation was to study the possible occurrence, morphological localization, and putative function of a constitutive HO enzyme in mouse islets of Langerhans. In a recent report (25), we provided immunocytochemical and biochemical evidence for the occurrence of a CO-producing HO-2 activity in the pancreatic islets of the rat. Functional studies with isolated rat islets also showed that glucose-induced insulin release was modestly inhibited by zincprotoporphyrin-IX (ZnPP-IX), a recognized inhibitor of HO activity (15), and potentiated by hemin, a natural substrate for the HO enzyme. In this article, we show the results of a more detailed morphological, biochemical, and functional study on the different characteristics of a CO-producing HO-2 activity in the pancreatic islets of another species, the mouse. In addition to showing some basal characteristics—which we found to be largely in accordance DIABETES, VOL. 48, JANUARY 1999

R. HENNINGSSON AND ASSOCIATES

with our observations in rat islets—we have now performed a detailed mapping, using immunocytochemistry and confocal microscopy, of all four islet endocrine cell types to localize the HO-2 enzyme. Furthermore, we have also extended our functional studies to involve a comparative analysis of the effects of CO and NO on islet hormone secretion and of the effects of different secretagogues on the activities of HO-2 and NO synthase (NOS) in intact islets, as well as an analysis of the putative implication of the guanylate cyclase–cGMP system in the action of CO on the hormone secretory processes. RESEARCH DESIGN AND METHODS Chemicals. The radioimmunoassay kits for insulin and glucagon determination were obtained from Diagnostika (Falkenberg, Sweden) and Euro-Diagnostica (Malmö, Sweden), respectively. Collagenase (CLS 4) was obtained from Worthington Biochemicals (Freehold, NJ). Hemin-HCl, -NADPH, NG-monomethylL -arginine ( L-NMMA) and 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) were purchased from Sigma Chemical (St. Louis, MO), ZnPP-IX and protoporphyrin-IX (PP-IX) were from Aldrich Chemical (Milwaukee, WI). Hemin-HCl and ZnPP-IX were dissolved in 0.1 mol/l NaOH, followed by titration with 0.1 mol/l HCl. Bovine serum albumin was from ICN Biomedicals (High Wycombe, U.K.). All other chemicals were from Merck AG (Darmstadt, Germany). Animals. Female mice of the NMRI strain (B&K, Sollentuna, Sweden) weighing 25–30 g were used in all studies. They were fed a standard pellet diet (B&K) and tap water ad libitum. The animals were killed by cervical dislocation. Preparation of pancreatic islets was performed by retrograde injection of a collagenase solution via the bile-pancreatic duct (26). Immunocytochemistry. The pancreatic glands were divided into pieces and immersion-fixed in an ice-cold, freshly prepared solution of 4% formaldehyde in phosphate-buffered saline (PBS; 0.1 mol/l, pH 7.4) for 4 h. After fixation, the specimens were rinsed in ice-cold 15% sucrose in PBS (three rinses in 48 h) and then frozen in isopentane at –40°C and stored at –70°C . Cryostat sections were then cut at a thickness of 8 µm, thaw-mounted onto chrom-alum–coated glass slides, and air dried for 30 min to 1 h. For the demonstration of constitutive (HO-2) and inducible (HO-1) heme oxygenase, sections were pre-incubated in PBS with 0.2% Triton X-100 for about 2 h and then incubated for 2 days in the presence of antisera raised in rabbits to rat HO-2 (1:1,000) or rat HO-1 (1:500) (both antisera purchased from StressGen Biotechnol, Victoria, Canada). After being rinsed in PBS (three rinses in 10 min), the sections were incubated for 90 min with Texas Red conjugated affinity purified F(ab)2 fragments of donkey anti-rabbit immunoglobulins (1:80; Jackson ImmunoResearch, West Grove, PA). After the rinsing, the sections were mounted in PBS/glycerol with p-phenylenediamine to prevent fluorescence fading (27). For the simultaneous demonstration of two antigens (28), sections were incubated overnight with rabbit HO-2 antiserum (see above), rinsed, and then incubated overnight with antisera raised in guinea pigs to insulin (1:1,600), glucagon (1:4,000), or pancreatic polypeptide (1:500) (the antisera were purchased from Linco, St. Louis, MO). Further, after incubation with the rabbit HO-2 antiserum, some sections were incubated with a rat monoclonal antiserum to somatostatin (1:500; MAB354; Chemicon, Temecula, CA). After rinsing, the sections were incubated for 90 min with fluoroescein isothiocyanate (FITC)-conjugated goat anti-guinea pig or goat anti-rat immunoglobulin G (1:80; Sigma), rinsed, and then incubated with Texas Red conjugated donkey anti-rabbit immunoglobulin G (see above). The sections were rinsed and mounted as described above. An Olympus 3 50 fluorescence microscope equipped with epi-illumination and the appropriate filter settings for Texas Red– and FITC-immunofluorescence was used for examinations of the sections (29). The primary and secondary antisera were diluted in PBS. In control experiments, no immunoreactivity could be detected in sections incubated with antisera absorbed with excess of the corresponding immunizing antigen (100 µg/ml). Because cross-reactions to antigens sharing similar amino acid sequences cannot be completely excluded, the structures demonstrated are referred to as HO-1–immunoreactive (IR), HO-2–IR, insulin-IR, glucagon-IR, pancreatic polypeptide–IR, and somatostatin-IR. Confocal microscopy. To evaluate whether two immunoreactivities were colocalized within the same cellular structures, sections were analyzed in a confocal laser scanning microscope (Multiprobe 2001 TM CLSM; Molecular Dynamics) equipped with an Ar/Kr laser and an inverted Nikon Diaphot TMD microscope, as described elsewhere (29). Western blot analysis. Approximately 200 islets and an equivalent amount of exocrine tissue were hand-picked in Hanks’ buffer under a stereomicroscope and sonicated on ice (3 10 s). The protein content was determined by using DIABETES, VOL. 48, JANUARY 1999

BCA Protein Assay Reagent (Pierce, Rockford, IL). Homogenate samples representing 5 or 10 µg of total protein from islet tissue or 10 µg from exocrine tissue were solubilized and boiled for 5 min in reducing buffer (2% SDS, 100 mmol/l Tris, pH 6.8, 100 mmol/l dithiothreitol, 0.3 mmol/l bromophenolblue, and 10% glycerol). The samples were run on 4–20% SDS-polyacrylamide gradient gels (Bio-Rad, Richmond, CA). After electrophoresis, proteins were transferred to nitrocellulose filters by electrotransfer (semi-dry transfer cell, Bio-Rad). The membranes were incubated in 2.5% milk powder and 1% bovine serum albumin for 60 min at room temperature to block nonspecific binding sites. Immunoblotting with rabbit anti-rat HO-1 and HO-2 antibodies (1:2,000) was performed for 16 h at room temperature. The membranes were then incubated with alkalinephosphatase conjugated antibodies (goat anti-rabbit 1:500) (DAKO, Glostrup, Denmark) for 60 min. Antibody binding to HO-1 and HO-2 was detected by nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate color reaction. Measurement of HO activity. CO production was determined with a sensitive gas chromatographic method, essentially as previously described (25,30). Islets were isolated and hand-picked under a stereomicroscope at room temperature. Then, when CO production in islet homogenates was directly assayed, they were thoroughly washed, collected in ice-cold phosphate buffer (0.1 mol/l, pH 7.4; ~1,000 islets in 350 µl buffer), and immediately frozen at –20°C until the day of assay. When intact islets were studied, they were first pre-incubated and incubated (300 islets in 1.5 ml buffer)—as described below for batch incubation experiments—and then thoroughly washed, collected in ice-cold phosphate buffer, and frozen as above. On the day of assay, the islets were sonicated on ice. Then methemalbumin (30 µl), -NADPH (100 µl; 4 mg dissolved in 1 ml phosphate buffer [0.1 mol/l]), and hemoglobin (2 mg) were added together with phosphate buffer up to a final volume of 1 ml. Methemalbumin solution was prepared by dissolving 25 mg hemin, 82.5 mg NaCl, and 12.1 mg Tris base in 5 ml of 0.1 mol/l NaOH, followed by the addition of 5 ml albumin solution (20 g/l) and 5 ml distilled water. The homogenate was then incubated in a water bath at 37°C while protected from light. Aliquots (330 µl) were taken after 3, 6, and 12 min of incubation, which was terminated by placing the tubes on ice. The samples were then injected into reaction tubes containing ferricyanide–citric acid (100 µl). Nitrogen was used as a carrier gas and to purge the reaction vessels for 4 min before the samples were injected into them. After a reaction time of 4 min, the liberated CO was brought to a nickel catalyst, mixed with H2 , and then further as methane to the detector. The standard of 99.9% CO was used. The amount of CO produced was calculated from the area under the curve. Protein was determined according to Bradford (31) on samples from the original homogenate. Hormone secretion. Freshly isolated islets (26) were pre-incubated for 30 min at 37°C in Krebs Ringer bicarbonate buffer, pH 7.4, supplemented with 10 mmol/l HEPES, 0.1% bovine serum albumin, and 1 mmol/l glucose. Each incubation vial contained 10 islets in 1.0 ml buffer solution and, unless otherwise stated, was gassed with 95% O2 / 5% CO2 to obtain constant pH and oxygenation. After pre-incubation, the buffer was changed to a medium containing 1, 7, or 20 mmol/l of glucose together with different test agents, and the islets were then incubated for 60 min. Aliquots of the medium were then removed and frozen for subsequent assays of insulin and glucagon (32–34). Effects of exogenously administered NO and CO. One hundred milliliters of the incubation buffer was purged of O 2 by helium and saturated with either NO or CO. Control buffer was saturated with helium. The solubility of CO (2.3 ml/100 ml H 2O) is very close to the solubility of NO (4.6 ml/100 ml H 2O). Islets were then incubated in these media as described above, except that the incubation vials were gassed with air instead of 95% O2 /5% CO2. Assay of islet NOS. As described above for batch incubation experiments, islets were isolated, handpicked, pre-incubated, and incubated (200 islets in 1.5 ml Krebs-Ringer buffer) as for hormone secretion. Then they were thoroughly washed, collected in ice-cold buffer (840 µl) containing 20 mmol/l HEPES, 0.5 mmol/l EDTA, and 1 mmol/l D,L-dithiothreitol, pH 7.2, and immediately frozen at –20°C. On the day of assay, the islets were sonicated on ice, and the buffer solution containing the islet homogenate was supplemented so that it also contained 0.45 mmol/l CaCl2, 2 mmol/l NADPH, 25 U calmodulin, and 0.2 mmol/l L-arginine in a total volume of 1 ml. The buffer composition is essentially the same as previously described for assay of NOS in brain tissue using radiolabeled L-arginine (35). The homogenate was then incubated at 37°C under constant air bubbling, 1.0 ml/min, for 3 h. It was ascertained that under these conditions the reaction velocity was linear for at least 6 h. Aliquots of the incubated homogenate (200 µl) were then passed through a 1-ml Amprep CBA cationexchange column for high-performance liquid chromatography analysis. The amount of L-citrulline formed was then measured in a Hitachi F 1000 fluorescence spectrophotometer (Merck), as previously described (7). NO and citrulline are produced in equimolar concentrations. The methodology was described in detail earlier (7,36), the only difference being that the incubation was now performed at 37°C instead of at room temperature. Protein was determined according to Bradford (31) on samples from the original homogenate. 67

HO-2, CO, AND ISLET HORMONE RELEASE

Measurement of islet cGMP. Incubation of isolated islets was stopped by removal of the buffer and the addition of 0.5 ml of ice-cold 10% trichloroacetic acid, followed by immediate freezing in a –70°C ethanol bath (37). Before assay, 0.5 ml of H2O was added, and the samples were sonicated for 3 5 s followed by centrifugation at 1,100g for 15 min. The supernatants were collected and extracted with 4 2 ml of water-saturated diethyl ether. The aqueous phase was removed and freeze dried, using a Lyovac GT 2 freeze dryer. The residue was then dissolved in 450 µl of 50 mmol/l Na-acetate buffer (pH 6.2). The amount of cGMP was quantified with a [125I]-cGMP radioimmunoassay kit (RIANEN, Du Pont, Boston, MA). Statistical analysis. Statistical significance between sets of data was assessed using the unpaired Student’s t test or where applicable analysis of variance followed by Tukey-Kramer’s multiple comparisons test. Results are expressed as means ± SE.

RESULTS

Formation of CO. Figure 1A shows a gas chromatogram from a single experiment illustrating the CO production by two samples taken from the same homogenate and incubated with (b) and without (a) addition of the HO inhibitor ZnPP-IX (100 µmol/l), and a control (c) without any islet tissue present. The production of CO was linear for the first 6 min of reaction, after which its velocity tended to slow down (data not shown). The reaction time was therefore set to 6 min in further experiments. Figure 1B shows the CO production in islet homogenates from a series of experiments in the absence and presence of different concentrations of ZnPP-IX. The CO production was dose-dependently inhibited by ZnPP-IX: 40% suppression at 10 µmol/l and 75% suppression at 100 µmol/l. In comparison, the HO activity was not influenced by PP-IX itself at a concentration of 100 µmol/l (Fig. 1B). CO production in islet homogenates prepared from intact islets first incubated for 60 min in the presence of either low (1 mmol/l) or high (20 mmol/l) glucose ± hemin is illustrated in Fig. 1C. CO production almost doubled when islets were incubated with 20 mmol/l glucose compared to incubation with 1 mmol/l, and it increased even further (~30%) when 100 µmol/l hemin was added to the incubation medium at 20 mmol/l glucose. Western blot analysis. As shown in Fig. 2, there was an exclusive expression of HO-2 in endocrine pancreas; no expression could be detected in exocrine pancreas. In contrast, expression of HO-1 could not be detected in either islets or exocrine tissue. Immunocytochemistry and confocal microscopy. HO-2 immunoreactivity could be detected in the cytoplasm of almost all islet cells (Fig. 3A, D, G, and J). Double immunolabeling in combination with confocal microscopy showed that most insulin-IR cells also displayed HO-2 immunoreactivity (Fig. 3B and C), although there were HO-2–IR cells in the periphery of the islets that lacked insulin immunoreactivity. Further, along the periphery of the islets was a broad ring of glucagon-IR cells—which also were HO-2–IR (Fig. 3E and F)—dispersed pancreatic polypeptide–IR cells (Fig. 3H and I), and somatostatin-IR cells (Fig. 3K and L) with extended processes, which also expressed HO-2 immunoreactivity. In comparison, HO-1 immunoreactivity could not be demonstrated in any islet cells (data not shown). Effects of inhibition or stimulation of CO production on islet hormone secretion. The effect of inhibition of HO-2 activity was investigated in the first set of experiments. At a low concentration of glucose (1 mmol/l), the secretion of insulin was not affected by ZnPP-IX (G1 in Fig. 4A). In contrast, glucagon secretion, which is increased at low concentrations of glucose, was dose-dependently inhibited by ZnPP68

FIG. 1. A: CO production within mouse islet homogenates estimated by gas chromatography. Chromatograms of two samples from one homogenate incubated in the absence (a) or presence (b) of the HO inhibitor ZnPP-IX (100 µmol/l) and of a control sample (c) without tissue present are shown. B: HO activity directly assayed in homogenates of freshly isolated islets expressed as CO formation (pmol · mg–1 protein · min–1) with and without addition of ZnPP-IX (10 and 100 µmol/l) or PP-IX (100 µmol/l). Data are means ± SE for 4–8 observations. *P < 0.05, **P < 0.01 for probability level of random difference. C: HO activity expressed as CO formation (pmol · mg–1 protein · min–1) within islet homogenates prepared after incubation of intact islets either at low glucose (1 mmol/l) or at high glucose (20 mmol/l) ± the HO substrate hemin (100 µmol/l). Data are means ± SE for 3–5 observations.

IX (G1 in Fig. 4B). At a maximal insulin-stimulatory concentration of glucose (20 mmol/l), the secretions of insulin and glucagon were inhibited by ZnPP-IX in a dose-dependent way, with the effect being much stronger on glucagon than on DIABETES, VOL. 48, JANUARY 1999


FIG. 2. Western blots. Lanes 1–3 show results from incubation with HO-1 antibody. Lanes 4–6 show results from incubation with HO-2 antibody. Lane 1, islet tissue (10 µg); lane 2, islet tissue (5 µg); lane 3, exocrine tissue (10 µg); lane 4, islet tissue (10 µg); lane 5, islet tissue (5 µg); lane 6, exocrine tissue (10 µg).

insulin secretion (G20 in Fig. 4A and B). PP-IX (100 µmol/l) itself had no effects on either insulin or glucagon secretion (Fig. 4A and B). The increase of insulin release at high glucose over that of low glucose, i.e., 20 mmol/l glucose versus 1 mmol/l glucose, was 25.1 ± 0.95 for the controls; 23.1 ± 1.71 for 10–6 mmol/l ZnPP-IX; 18.9 ± 0.98 for 10–5 mmol/l ZnPP-IX; and 16.4 ± 1.03 for 10–4 mmol/l ZnPP-IX. The effects of increased CO production from hemin (a natural substrate for the HO-2 enzyme) on insulin and glucagon secretion are shown in Fig. 4C and D. The secretions of insulin and glucagon were dose-dependently affected in the presence of glucose at a maximal insulin-stimulatory concentration (20 mmol/l). At a low glucagon-”stimulatory” glucose concentration (1 mmol/l), only glucagon secretion was stimulated by hemin. Furthermore, insulin as well as glucagon secretion stimulated by hemin was completely antagonized by ZnPP-IX (100 µmol/l) (Fig. 4C and D). The insulin increase ratio for 20 mmol/l versus 1 mmol/l of glucose was 10.9 ± 1.58 for the controls; 12.9 ± 1.61 for 10–6 mmol/l hemin; 16.8 ± 1.09 for 10–5 mmol/l hemin; and 36.4 ± 1.36 for 10–4 mmol/l hemin. Influence of glucose, ZnPP-IX, and exogenous CO on islet cGMP content and hormone secretion. Table 1 shows the islet levels of cGMP in relation to the release of insulin and glucagon in the presence of glucose, ZnPP-IX, or exogenous CO in the islet incubation medium. Islet cGMP levels did increase in response to a high glucose concentration (20 mmol/l) compared with a low glucose concentration (1 mmol/l). The increase in islet cGMP at high glucose was accompanied by a great increase in insulin release and a modest decrease in glucagon release (Table 1). At 20 mmol/l glucose, addition of ZnPP-IX and the use of a helium-saturated incubation medium with an atmosphere gassed with air significantly diminished the levels of islet cGMP in parallel with a decreased secretion of insulin. ZnPP-IX, but not helium saturation, also induced a suppression of glucagon secretion. Incubation at 20 mmol/l glucose in a medium saturated with exogenous CO markedly increased islet cGMP DIABETES, VOL. 48, JANUARY 1999

levels in parallel with a greatly enhanced insulin and glucagon release in comparison to incubation in the heliumsaturated control medium (Table 1). Comparative effects of exogenous CO and NO on islet hormone release. The comparative effects of exogenously applied CO and NO on insulin and glucagon secretion were studied at a maximal stimulatory concentration of glucose (20 mmol/l) (Fig. 5A and B). Exogenous CO stimulated, whereas exogenous NO inhibited, glucose-induced insulin release (Fig. 5A). Both gases stimulated glucagon secretion (Fig. 5B). Interaction of endogenously produced CO and NO on islet hormone release. Possible interactions between endogenously produced CO and NO were studied through different pharmacological manipulations. The direct effects on insulin and glucagon secretion induced by the amino acid L-arginine, inhibition of NOS by the selective NOS inhibitor L-NMMA, CO formed from hemin, and inhibition of HO-2 by ZnPP-IX at a basal glucose concentration (7 mmol/l) are shown in Fig. 6A and B. The presence of L-arginine or hemin stimulated both insulin and glucagon secretion. Inhibition of basal NOS activity by L-NMMA had no effect on either insulin or glucagon secretion, whereas inhibition of basal HO-2 activity by ZnPP-IX decreased the secretion of both hormones. Effects and interactions of endogenously manipulated CO and NO production on hormone secretion stimulated by L-arginine are illustrated in Fig. 7A and B. L-Arginine–induced insulin secretion was potentiated by inhibiting NO formation through the addition of L-NMMA, whereas glucagon secretion was suppressed. Inhibition of CO production by addition of ZnPP-IX did not influence L-arginine–stimulated insulin secretion, but it weakly inhibited glucagon secretion. Stimulation of HO-2 activity by hemin strongly enhanced Larginine–induced insulin secretion. This increase was totally suppressed by addition of ZnPP-IX. L-arginine–induced glucagon secretion was not significantly affected by hemin or hemin plus ZnPP-IX (Fig. 7B). To test our assumption that L-arginine and glucose could increase islet NOS activity, we directly measured islet NO production. Fig. 7C shows the production of NO in islet homogenates prepared from intact islets incubated for 60 min at 7 mmol/l glucose ± 10 mmol/l L-arginine or 20 mmol/l glucose ± 100 µmol/l hemin. L-Arginine in the incubation medium almost doubled NO production, whereas hemin markedly suppressed it. Furthermore, it should be noted that islet NOS activity was increased almost threefold in islets incubated in the presence of a high glucose concentration compared with a low glucose concentration. Influence of the selective guanylate cyclase inhibitor ODQ and the HO inhibitor ZnPP-IX on CO- and heminstimulated islet hormone secretion. In the last series of experiments, we studied the effects of ODQ—a recognized inhibitor of soluble guanylate cyclase (38,39)—and the HO inhibitor ZnPP-IX on insulin and glucagon release stimulated by CO or hemin. In the presence of 20 mmol/l glucose, ODQ (10 µmol/l) modestly suppressed insulin but did not affect glucagon release. As expected, ZnPP-IX (100 µmol/l) modestly suppressed the release of both hormones (Fig. 8). Further, in Fig. 8A it can be seen that CO-stimulated insulin release was not affected by ZnPP-IX, whereas it was almost abolished in the presence of the guanylate cyclase inhibitor ODQ. In addition, hemin-induced insulin release was markedly suppressed by ODQ. CO-stimulated (but not hemin69


FIG. 3. Immunofluorescence and confocal micrographs of islets of mouse pancreas. The four different sections were double immunolabeled for HO-2 and insulin (A–C), HO-2 and glucagon (D–F), HO-2 and pancreatic polypeptide (G–I), or HO-2 and somatostatin (J–L). Texas Red immunofluorescence in A, D, G, and J expresses HO-2 immunoreactivity. Clear green FITC-immunofluorescence in B, E, H, and K shows immunoreactivities for insulin (B), glucagon (E), pancreatic polypeptide (H), and somatostatin (K). Greenish-yellowish fluorescence in C, F, I, and L indicates cellular co-localization of immunoreactivities for HO-2 and the respective islet hormones. C shows HO-2 and insulin in most (but not all: see arrow heads) cells. In scattered cells, co-localization of HO-2 and glucagon (F), HO-2 and pancreatic polypeptide (I), or HO-2 and somatostatin (L) was observed. Caliber bars with length in micrometers are indicated in each micrograph. 70

DIABETES, VOL. 48, JANUARY 1999


FIG. 4. A and B: Effects of different concentrations of ZnPP-IX ( ) or 100 µmol/l PP-IX ( ) on insulin (A) and glucagon (B) release from isolated islets in the presence of 1 mmol/l (G1) or 20 mmol/l (G20) glucose. C and D: Effects of different concentrations of hemin ( ) or 100 µmol/l ZnPP-IX ( ) on insulin (C) and glucagon (D) release from isolated islets in the presence of 1 mmol/l (G1) or 20 mmol/l (G20) glucose. , controls incubated in the absence of ZnPP-IX or hemin. Means ± SE are shown for 10–20 batches of islets. Where no standard errors are denoted, they were smaller than the symbol. Each batch (incubation vial) consisted of 10 islets in 1 ml incubation medium. Incubation time was 60 min. The results are from 8–9 separate experiments run on different days. *P < 0.05, **P < 0.01, ***P < 0.001 for probability level of random difference versus controls. xxx P < 0.001 for probability level of random difference regarding hemin (100 µmol/l) vs. hemin + ZnPP-IX.

stimulated) glucagon release was also suppressed in the presence of ODQ (Fig. 8B). CO-stimulated glucagon secretion was modestly decreased in the presence of ZnPP-IX. However, it should be noted that CO was still able to increase significantly ZnPP-IX–inhibited glucagon release (Fig. 8B). DISCUSSION

Biochemistry and immunocytochemistry. The present study indicates the presence of a HO–CO pathway in the islets of Langerhans of the mouse. We recently suggested that such a pathway also occurs in the rat (25). In the present study, we observed that mouse islet homogenates were capable of producing large amounts of CO, which was

blocked in a dose-dependent way by ZnPP-IX, a widely used inhibitor of HO (15). Furthermore, we can show, for the first time, that intact islets incubated in 20 mmol/l glucose indeed produced more CO than did islets incubated at low, basal glucose. Moreover, this glucose-stimulated CO production in intact islets could be further stimulated by incubation of the islets in the presence of the HO substrate hemin. Hence, a glucose-induced stimulation of HO-2 activity apparently operates in intact islets. In fact, this observation strongly suggests that a hitherto unknown HO–CO pathway is implicated in glucoseinduced insulin release and thus might be of both physiological and pathophysiological importance for the insulin secretory process. With regard to the localization of the HO

TABLE 1 Effects of glucose and ZnPP-IX in a normal incubation medium and of CO (saturated CO medium) compared with a helium-saturated control medium on the islet content of cGMP and the release of insulin and glucagon from isolated islets

Normal medium 1.0 mmol/l glucose 20 mmol/l glucose 20 mmol/l glucose + 0.1 mmol/l ZnPP-IX Helium-saturated medium 20 mmol/l glucose CO-saturated medium 20 mmol/l glucose

Islet cGMP (amol/islet)

Insulin secretion (ng · islet–1 · h)

Glucagon secretion (pg · islet–1 · h)

276 ± 42 ‡ 896 ± 47 612 ± 47‡

0.19 ± 0.018‡ 5.16 ± 0.26 3.75 ± 0.24†

36.2 ± 2.66* 26.5 ± 1.64 20.0 ± 1.06†

338 ± 23‡

3.21 ± 0.24‡

20.9 ± 2.64

920 ± 95

4.48 ± 0.27§

34.1 ± 2.46§

Data are means ± SE for 5–12 observations. The amount of hormone secreted is expressed as ng · islet –1 · h (insulin) and pg · islet –1 · h (glucagon). *P < 0.05, †P < 0.01, ‡P < 0.001 for probability level of random difference vs. 20 mmol/l glucose. §P < 0.01, P < 0.001 for probability level of random difference for CO-saturated vs. helium-saturated medium at 20 mmol/l glucose. DIABETES, VOL. 48, JANUARY 1999

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FIG. 5. Effects of exogenous NO and CO on insulin (A) and glucagon (B) release from isolated islets in the presence of 20 mmol/l glucose. Incubation media were first purged of O2 by helium and then saturated by NO or CO. Controls ( ) were helium-saturated. Means ± SE are shown for 8–10 batches of islets. Incubation time was 60 min. The results are from four separate experiments run on different days. *P < 0.05, ***P < 0.001 for probability level of random difference versus controls.

FIG. 6. Effect of the NOS inhibitor L-NMMA (5 mmol/l), ZnPP-IX (100 µmol/l), L-arginine (10 mmol/l) and hemin (100 µmol/l) on insulin (A) and glucagon (B) release from isolated islets in the presence of 7 mmol/l glucose. , basal controls. Means ± SE are shown for 8–12 batches of islets. Incubation time was 60 min. The results are from three separate experiments run on different days. *P < 0.05, **P < 0.01, ***P < 0.001 for probability level of random difference versus basal controls.

enzyme, immunoblots showed expression of HO-2 in islet tissue but not in pancreatic exocrine tissue. Expression of HO1 could not be detected. The biochemical results were further supported by the immunocytochemical findings, which showed that most islet cells displayed HO-2 but not HO-1 immunoreactivity. The present observation that large amounts of constitutive HO-2 are expressed in mouse islets raises the question of whether the presence of HO-2, in addition to its role as a putative regulator of islet hormone release (by virtue of its CO-producing property, found in the present study), can serve as a protective mechanism against free radicals, such as NO, through its generation of bilirubin, which is regarded as a powerful antioxidant (24,40). Several studies (41–43) previously showed that HO-1 might serve as such a protective mechanism. Moreover, the HO substrate hemin was previously shown to be able to act as an NO scavenger (41). This is important because recent observations suggested that an exaggerated production of free radicals within the endocrine pancreas might be deleterious to islet -cells and thus tentatively be implicated in the pathogenesis of type 1 diabetes (42). In this context, it should be noted, however, that human islets have been shown to be more resistant to various free radical–producing -cell toxins than mouse and rat

islets, although both human and rat islets transplanted to nude mice expressed a similar HO content (43). The immunocytochemical and confocal microscopic findings revealed that HO-2 immunoreactivity was widely distributed throughout the islets of Langerhans, residing in insulin-, glucagon-, somatostatin-, and pancreatic polypeptide–producing cells. Since CO is a gas, it could easily penetrate plasma membranes and be transported from one cell to another, in which it could exert its effects. This is one of the major properties distinguishing the new gas neuromodulators CO and NO from the classic receptor-regulating transmitters. This concept applies especially to CO, which is much more inert than NO, and thus can travel “long” distances (16). Hence, if the HO2 activity in the insulin cells is specifically stimulated, the CO produced could influence not only surrounding insulin cells but also, e.g., glucagon cells and vice versa. Functional studies. The present data concerning changes in islet function in the presence of various agents known to affect HO-2-activity strongly support the idea of a HO–CO pathway, which could be of regulatory importance for the secretion of insulin and glucagon. Thus, hemin (15) dose-dependently enhanced not only glucose-induced insulin release but also the secretion of glucagon. Moreover, ZnPP-IX, a selective HO

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FIG. 7. Secretion of insulin (A) and glucagon (B) from L-arginine–stimulated (10 mmol/l) isolated islets in the presence of L-NMMA (5 mmol/l), ZnPP-IX (100 µmol/l), hemin (100 µmol/l), and hemin + ZnPPIX. Glucose concentration was 7 mmol/l. Dotted lines show basal insulin (A) and glucagon (B) release as measured in accompanying experiments (shown as in Fig. 6). Means ± SE are shown for 8–12 batches of islets. Incubation time was 60 min. The results are from four separate experiments run on different days. *P < 0.05, ***P < 0.001 for probability level of random difference versus L-arginine controls. xxxP < 0.001 as indicated in A. C: Islet NOS activity in islets incubated at 7 mmol/l (G7) or 20 mmol/l (G20) glucose in the absence and presence of L-arginine (10 mmol/l) or hemin (100 µmol/l). Glucose concentration was 7 mmol/l for L-arginine and 20 mmol/l for hemin. Islet NOS activity is expressed as NO production (pmol · min–1 · mg–1 protein). Groups of 200 islets per 1.5 ml medium were incubated for 60 min. Data are means ± SE for 4–5 batches of islets.. **P < 0.01 vs. corresponding G7 or G20 control; P < 0.001 for probability level of random difference as indicated in the figure.

inhibitor (15,44), dose-dependently suppressed glucoseinduced insulin release as well as the secretion of glucagon. Furthermore, it should be noted that ZnPP-IX completely abolished the potentiating effect of hemin on both insulin and glucagon release, thus providing good evidence that the stimulatory action of hemin was exerted through enhanced HO-2 activity with a subsequent increase in CO production. Moreover, we DIABETES, VOL. 48, JANUARY 1999

FIG. 8. Effects of ZnPP-IX (100 µmol/l), CO (1 µmol/l), the guanylate cyclase inhibitor ODQ (10 µmol/l) and hemin (100 µmol/l), alone or in different combinations, on the secretion of insulin (A) and glucagon (B) in the presence of 20 mmol/l glucose. Means ± SE are shown for 6–25 batches of islets in each group. Incubation time was 60 min. The results are from four separate experiments run on different days. P < 0.05, P < 0.001 for probability level of random difference versus control. **P < 0.01, ***P < 0.001 for probability level of random difference as indicated in the figure.

directly showed here that incubation of intact islets with hemin could potentiate the glucose-induced CO production. The inhibition of glucose-induced insulin release by ZnPP-IX was accompanied by a concomitant suppression of glucagon release. Thus, it could be argued that the decrease in insulin release might be a secondary consequence of decreased glucagon release. Although such an influence cannot be totally excluded, it should be noted that the ZnPP-IX–induced suppression of glucagon release either at 1 mmol/l glucose, at 10 mmol/l L-arginine, or after direct stimulation of CO itself was not accompanied by any changes in insulin release. Likewise, the marked inhibitory action of ZnPP-IX on insulin release induced by L-arginine + hemin was not reflected in any changes of glucagon release. The observation that ZnPP-IX could suppress glucagon (but not insulin) release at both low and high glucose concentrations might indicate that the 73


glucagon secretory machinery is more sensitive to and/or more dependent on islet CO production than that of insulin. The importance of HO-2 as a putative modulator of secretory processes is underlined by very recent data showing that both hemin and ZnPP-IX displayed regulatory effects (antagonizing each other) on the secretion of corticotropin-releasing hormone and oxytocin from rat hypothalamus (12,45) and on hormone secretion from isolated rat islets (25). Endogenous manipulations of islet CO production by hemin and ZnPP-IX thus provided good evidence for a regulatory role of HO-2 in islet hormone release. These data were further strengthened by our observation that CO itself, dissolved in the incubation medium, markedly enhanced both insulin and glucagon secretion. Our data also showed that CO markedly enhanced the islet cGMP levels, a property that has been shown to be an important mechanism of action of CO in many other biological systems (8–11,16,17). In accordance with this, we also found that ZnPP-IX suppressed the islet levels of cGMP. In contrast to the stimulating effects of CO on both insulin and glucagon secretion, another messenger gas, NO, was found to greatly inhibit glucose-induced insulin secretion but to stimulate glucagon secretion. It should be noted that endogenously produced NO is also known to raise cGMP levels in many organs (1,2,16,17), yet it inhibits glucoseinduced insulin release (7,37). We observed in this study that pure NO gas could inhibit insulin secretion stimulated by glucose. The most likely explanation can be found in certain biochemical differences between NO and CO. NO-induced stimulation of islet cGMP levels (37,46,47) is probably overshadowed by other NO effects within the -cell, because NO is a superreactive molecule, reacting almost momentarily with, e.g., catalytically important sulfhydryl groups in enzymes and important membrane constituents (48). It should also be noted that very high levels of NO derived from inducible NOS during long-term incubations of islets or -cells in the presence of inducible NOS–stimulating agents, such as interleukin-1 , have multiple effects on -cells, ranging from aconitase inhibition to DNA damage (49). We previously suggested (3,7,37) that the inhibiting effects of cNOS-derived NO on insulin secretion might be due to the formation of S-nitrosothiols, thereby impairing important thiol groups, which are known to be essential for glucoseinduced insulin secretion (50,51). Hence, the stimulating effect of NO on the cGMP system in the -cell is likely to be strongly counteracted by the formation of S-nitrosothiols, which negatively modulate the stimulus-secretion coupling of glucose-induced insulin release. In this context, it is worth noting that the functional role of the constitutive NO-producing enzyme (cNOS) in the islets of Langerhans is still an unsettled topic (3–7,37,46,47). Recent investigations in our laboratory, both in vivo and in vitro, suggest that NO is a negative modulator of insulin secretion but a positive modulator of glucagon secretion (3,5,7,37). With regard to the mechanism of action of CO and NO on glucagon secretion, we find it conceivable that at least part of the stimulating effect of CO, as well as of NO, on the glucagon secretory process might be mediated through activation of the cGMP system. It should be remarked that comparisons with CO-saturated or NO-saturated incubation media must be performed with a helium-saturated control, because a hypoxic condition itself is known to affect the 74

rate of islet hormone secretion in the isolated perfused pancreas (52). This was verified in our isolated islets with regard to glucose-stimulated insulin release, which was reduced by ~40% when islets were incubated in a helium-saturated medium with an atmosphere of air. Glucagon release, however, was apparently unaffected (Table 1), suggesting that the reported increase in glucagon secretion during hypoxic conditions (52) requires an intact pancreas. Finally, we studied whether the effects of CO and NO on islet hormone release would reveal any interrelationship and whether the guanylate cyclase inhibitor ODQ could influence CO-induced hormone release. We first observed that hemin potentiated L-arginine–stimulated insulin secretion. This observation suggested a possible interaction between the heme-CO and the L-arginine–NO systems. CO produced from heme is known to inhibit NOS activity in other tissues (53); hence, it seemed conceivable that CO might suppress L-arginine–induced NOS activation and thus the subsequent formation of NO in the insulin-producing -cells, which in turn could lead to a decrease of the NOinduced negative influence on the insulin secretory mechanisms (3,5,7,37). Such an assumption was indeed verified in the present study, where we could show, for the first time, by directly measuring NOS activity after incubation of intact islets that both L-arginine and glucose markedly increased islet NO production and that hemin-derived CO was a potent inhibitor of glucose-induced NOS activity. Moreover, both hemin-derived CO and L-arginine–derived NO stimulated glucagon release, but the combination of hemin and L-arginine did not induce a further increase in the amount of glucagon secreted than was induced by each of them alone (Figs. 6 and 7), again indicating a possible CO-induced inhibition of NOS activity. The question as to whether the stimulatory effects of CO on insulin and glucagon could be mediated by direct activation of guanylate cyclase was addressed by using ODQ (38,39). ODQ is a novel guanylate cyclase inhibitor, which reportedly is a specific inhibitor of soluble guanylate cyclase without effects on particulate guanylate cyclase and adenylate cyclase (38,39). Direct stimulation by CO of both insulin and glucagon release was indeed strongly inhibited by ODQ, which suggests that the guanylate cyclase–cGMP–protein kinase G system is an important, if not the sole, mediator of CO-induced islet hormone secretion. With regard to insulin secretion, these data were further supported by our observation that the potentiation of glucose-stimulated insulin release induced by hemin, the CO-producing HO substrate, was markedly inhibited by ODQ. Likewise, the suppression of glucose-stimulated insulin release induced by the potent HO inhibitor ZnPP-IX was totally abolished by direct guanylate cyclase stimulation through exogenous CO. Hence, these data, in addition to our observation that glucose itself can increase islet CO production, seem to be in favor of a COmediated activation of the guanylate cyclase–cGMP–protein kinase G system being an important messenger in the stimulation of insulin secretory processes induced by glucose. However, as seen in Table 1, there was a rather poor correlation between the glucose-stimulated increase in insulin release and the increase in islet cGMP content during the different experimental conditions. Moreover, ODQ had only a modest inhibitory effect on glucose-induced insulin release. Hence, although future experiments might reveal novel DIABETES, VOL. 48, JANUARY 1999


mechanisms of action of CO, the accumulated data up to now suggest that the CO-induced inhibition of islet NOS activity is at least as important as the cGMP system as a target for CO to positively modulate the transduction signaling in glucose-stimulated insulin release. With regard to glucagon release, the picture is also complex. ODQ totally abolished glucagon release induced by exogenous CO. However, in contrast to our observation with insulin release, the ZnPPIX–induced suppression of glucagon secretion was only partially reversed by addition of exogenous CO. Moreover, hemin-induced glucagon secretion was not at all affected by ODQ. Hence, evidently, the guanylate cyclase–cGMP–protein kinase G system is not the sole messenger operating in CO-induced glucagon release. In summary, from the present data we propose that the endocrine pancreas has an HO–CO pathway of great importance for the regulation of insulin and glucagon release. The intimate mechanisms of action of CO on islet hormone release will await further studies, although we believe that increased activity of the cGMP system might be a possible transduction signal in this respect. Moreover, our data also suggest the occurrence of interactive regulatory mechanisms between the HO–CO pathway and the NOS–NO pathway in islet hormone release. ACKNOWLEDGMENTS

This study was supported by the Swedish Medical Research Council (14X-4286 and 11205), the Swedish Diabetes Association, and the foundations of Magnus Bergvall, Crafoord, Albert Påhlsson, Åke Wiberg, and Thelma Zoega. The skillful technical assistance of Maj-Britt Johansson, Elsy Ling, Britt-Marie Nilsson, and Lillemor Thuresson is gratefully acknowledged. REFERENCES 1. Moncada S, Palmer RMJ, Higgs EA: Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev 43:109–142, 1991 2. Bordeaux A: Nitric oxide: an ubiquitous messenger. Fund Clin Pharmacol 7:401–411, 1993 3. Panagiotidis G, Alm P, Lundquist I: Inhibition of islet nitric oxide synthase increases arginine-induced insulin release. Eur J Pharmacol 229:277–278, 1992 4. Laychock SG, Modica ME, Cavanaugh CT: L-arginine stimulates cyclic guanosine 3 ,5 -monophosphate formation in rat islets of Langerhans and RIN m5F insulinoma cells: evidence for L-arginine:nitric oxide synthase. Endocrinol ogy 129:3043–3052, 1991 5. Panagiotidis G, Åkesson B, Alm P, Lundquist I: The nitric oxide system in the endocrine pancreas induces differential effects on the secretion of insulin and glucagon. Endocrine 2:787–792, 1994 6. Schmidt HHHW, Warner TD, Ishii K, Sheng H, Murad F: Insulin secretion from pancreatic B-cells caused by L-arginine-derived nitrogen oxides. Science 255:721–723, 1992 7. Salehi A, Carlberg M, Henningsson R, Lundquist I: Islet constitutive nitric oxide synthase: biochemical determination and regulatory function. Am J Physiol 270:C1634–C1641, 1996 8. Christodoulides N, Durante W, Kroll MH, Schafer AI: Vascular smooth muscle cell heme oxygenases generate guanylyl cyclase-stimulatory carbon monoxide. Circulation 91:2306–2309, 1995 9. Lefer DJ, Xin-liang M, Lefer AM: A comparison of vascular biological actions of carbon monoxide and nitric oxide. Meth Find Exp Clin Pharmacol 15:617–622, 1993 10. Suematsu M, Kashiwagi S, Sano T: Carbon monoxide as an endogenous modulator of hepatic vascular perfusion. Biochem Biophys Res Comm 205:1333–1337, 1994 11. Utz J, Ullrich V: Carbon monoxide relaxes ileal smooth muscle through activation of guanylate cyclase. Biochem Pharmacol 8:1195–1201, 1991 12. Pozzoli G, Mancuso C, Mirtella A, Preziosi P, Grossman AB, Navarra P: Carbon monoxide as a novel endocrine modulator: inhibition of stimulated corticotropin-releasing hormone from acute rat hypothalamic explants. DIABETES, VOL. 48, JANUARY 1999

Endocrinology 135:2314–2317, 1994 13. Verma A, Hirsch DJ, Glatt CE, Ronett GV, Snyder SH: Carbon monoxide: a putative neural messenger? Science 259:381–384, 1993 14. Baringa M: Carbon monoxide: killer to brain messenger in one step. Science 259:309, 1993 15. Marks GS: Heme oxygenase: the physiological role of one of its metabolites, carbon monoxide and interactions with zinc protoporphyrin, cobalt protoporphyrin and other metalloporphyrins. Cell Mol Biol 40:863–870, 1994 16. Marks GS, Brien JF, Nakatsu K, McLaughlin BE: Does carbon monoxide have a physiological function? Trends Pharmacol Sci 11:185–188, 1991 17. Schmidt HHHW: NO•,CO and •OH endogenous soluble guanylyl cyclase-activating factors. FEBS Lett 307:102–107, 1992 18. Rodgers PA, Vreman HJ, Dennery PA, Stevenson DK: Sources of carbon monoxide (CO) in biological systems and applications of CO detection technologies. Sem in Perinatol 18:2–10, 1994 19. Vreman JH, Stevenson DK: Heme oxygenase activity as measured by carbon monoxide production. Anal Biochem 168:31–38, 1988 20. Grundemar L, Johansson M-B, Ekelund M, Högestätt ED: Haem oxygenase activity in blood vessel homogenates as measured by carbon monoxide production. Acta Physiol Scand 153:203–204, 1995 21. Schacter BA: Heme catabolism by heme oxygenase: physiology, regulation and mechanism of action. Sem in Hematol 25:349–369, 1988 22. Braggins PE, Trakshel GM, Kutty RK, Maines MD: Characterization of two heme oxygenase isoforms in rat spleen: comparison with hematin-induced and constitutive isoforms of the liver. Biochem Biophys Res Comm 141:528–533, 1986 23. Maines MD: Heme oxygenase: function, multiplicity, regulatory mechanisms, and clinical applications. FASEB J 2:2557–2568, 1988 24. Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN: Bilirubin is an antioxidant of possible physiological importance. Science 235:1043–1046, 1987 25. Henningsson R, Alm P, Lundquist I: Occurrence and putative hormone regulatory function of a constitutive heme oxygenase in rat pancreatic islets. Am J Physiol 273:C703–C709, 1997 26. Gotoh M, Maki T, Kiyoizumi T, Satomi S, Monaco AP: An improved method for isolation of mouse pancreatic islets. Transplantation 40:437–438, 1985 27. Johnson GD, Araujo GM: A simple method of reducing the fading of immunofluorescence during microscopy. J Immunol Methods 43:349–350, 1981 28. Wessendorf MW, Elde RP: Characterization of an immunofluorescence technique for the demonstration of coexisting neurotransmitters within nerve fibers and terminals. J Histochem Cytochem 33:984–994, 1985 29. Ny L, Alm P, Larsson B, Ekström P, Andersson K-E: The nitric oxide pathway in cat esophagus; localization of nitric oxide synthase and functional aspects. Am J Physiol 268:G59–G70, 1995 30. Cavallin-Ståhl E, Jönsson GI, Lundh B: A new method for determination of microsomal heme oxygenase (EC 1.14.99.3) based on quantification of carbon monoxide formation. Scand J Clin Lab Invest 38:69–76, 1978 31. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 84:309–312, 1978 32. Ahrén B, Lundquist I: Glucagon immunoreactivity in plasma from normal and dystrophic mice. Diabetologia 22:258–263, 1982 33. Heding L: A simplified insulin radioimmunoassay method. In Labelled Proteins in Tracer Studies. Donato L, Milhaud G, Sirchis J, Eds. Brussels, Euratom, 1966, p. 345–350 34. Panagiotidis G, Salehi A, Westermark P, Lundquist I: Homologous islet amyloid polypeptide: effects on plasma levels of glucagon, insulin and glucose in the mouse. Diabetes Res Clin Pract 18:167–171, 1992 35. Bredt DS, Snyder SH: Nitric oxide mediates glutamate-linked enhancement of cGMP levels in cerebellum. Proc Natl Acad Sci U S A 86:9030–9033, 1989 36. Carlberg M: Assay of neuronal nitric oxide synthase by HPLC determination of citrulline. J Neurosci Methods 52:165–167, 1994 37. Panagiotidis G, Åkesson B, Rydell EL, Lundquist I: Influence of nitric oxide synthase inhibition, nitric oxide and hydroperoxide on insulin release induced by various secretagogues. Br J Pharmacol 114:289–296, 1995 38. Cellek S, Kasakov L, Moncada S: Inhibition of nitrergic relaxation by a selective inhibitor of the soluble guanylate cyclase. Br J Pharmacol 118:137–140, 1996 39. Brunner F, Stessel H, Kukovetz WR: Novel guanylyl cyclase inhibitor, ODQ reveals role of nitric oxide, but not of cyclic GMP in endothelin-1 secretion. FEBS Lett 376:262–266, 1995 40. Willis D, Moore AR, Frederick R, Willoughby DA: Heme oxygenase: a novel target for the modulation of the inflammatory response. Nat Med 2:87–90, 1996 41. Welsh N, Sandler S: Protective action by hemin against interleukin-1 induced inhibition of rat pancreatic islet function. Mol Cell Endocrinol. 103:109–114, 1994 75


42. Eizirik DL, Flodström M, Karlsen AE, Welsh N: The harmony of the spheres: inducible nitric oxide synthase and related genes in pancreatic beta cells. Dia betologia 39:875–890, 1996 43. Welsh N, Margulis B, Borg LAH, Wiklund HJ, Saldeen J, Flodström M, Mello MA, Andersson A, Pipeleers DG, Hellerström C, Eizirik DL: Differences in the expression of heat-shock proteins and antioxidant enzymes between human and rodent pancreatic islets: implications for the pathogenesis of insulindependent diabetes mellitus. Mol Med 1:806–820, 1995 44. Ny L, Andersson K-E, Grundemar L: Inhibition by zincprotoporphyrin-IX of receptor-mediated relaxation of the rat aorta in a manner distinct from inhibition of haem oxygenase. Br J Pharmacol 115:186–190, 1995 45. Kostoglou-Athanassiou I, Forsling ML, Navarra P, Grossman AB: Oxytocin release is inhibited by the generation of carbon monoxide from the rat hypothalamus: further evidence for carbon monoxide as a neuromodulator. Mol Brain Res 42:301–306, 1996 46. Jones PM, Persaud SJ, Bjaaland T, Pearson JD, Howell SL: Nitric oxide is not involved in the initiation of insulin secretion from rat islets of Langerhans. Dia betologia 35:1020–1027, 1992 47. Green IC, Delaney CA, Cunningham JM, Karmiris V, Southern C: Interleukin-

76

1 effects on cyclic GMP and cyclic AMP in cultured rat islets of Langerhans: arginine-dependence and relationship to insulin secretion. Diabetologia 36:9–16, 1993 48. Stamler JS, Simon DI, Osborne JA, Mullins ME, Jaraki O, Michel T, Singel DJ, Loscalzo J: Nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds. Proc Natl Acad Sci U S A 89:444–448, 1992 49. Eizirik DL, Pavlovic D: Is there a role for nitric oxide in -cell dysfunction and damage in IDDM? Diabetes Metab Rev 13:293–307, 1997 50. Ammon HPT, Mark M: Thiols and pancreatic -cell function: a review. Cell Biochem Funct 3:157–171, 1985 51. Hellman B, Idahl L-Å, Lernmark Å, Sehlin J, Täljedal IB: Membrane sulfhydryl groups and pancreatic beta cell recognition of insulin secretagogues. Excerpt Med Int Congr Ser 312:65–78, 1974 52. Narimiya M, Yamada H, Matsuba I, Ikeda Y, Tanese T, Abe M: The effect of hypoxia on insulin and glucagon secretion in the perfused pancreas of the rat. Endocrinology 111:1010–1014, 1982 53. Ingi T, Cheng J, Ronett GV: Carbon monoxide: an endogenous modulator of the nitric oxide-cyclic GMP signalling system. Neuron 16:835–842, 1996


Author Queries (please see Q in margin and underlined text) Q1: For sentence beginning “Such an increase…” do edits at end of sentence accurately clarify what is meant? Q2: Does IgG stand for immunoglobulin G? If not, please correct usage in the sentence beginning “After rinsing, the sections were incubated…” Q3: Please clarify the end of the sentence beginning “In control experiments, no immunoreactivity…” It sounds like the antisera were absorbed by the excess of the corresponding immunizing antigen. Is this what is meant? Q4: Please clarify the sentence beginning “After a reaction time of 4 min…” Some words seem to be missing near the end. The CO was further mixed? Do you mean that CO and H2 were mixed until methane was formed or detected by the detector? Q5: What does

XXX

denote in Figure 7B?

For Ref. 6, are you referring to the journal Science, which is published in Washington, D.C.? Original reference had Science Wash DC. The word Wash is not a part of the journal’s title, is it? Please add the closing page number for Ref. 14 or indicate if it is an abstract, letter, or one-page article. For Ref. 18, Stevenson did not have initials. Please check that DK, to match the Stevenson initials in Ref. 19, is also correct for ref. 18.