Effects of nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester on duodenal alkaline secretory and ulcerogenic responses induced by mepirizole in rats

The inhibition of nitric oxide (NO) production by NO synthase inhibitors stimulates HCO ₃ ^– secretion in the rat duodenal mucosa. Therefore, we examined the effects of N^G-nitro-l-arginine methyl ester (l-NAME, the NO synthase inhibitor) and nitroprusside (the exogenous NO donor) on the duodenal HCO ₃ ^– and ulcerogenic responses in anesthetized rats. Animals were administered mepirizole (200 mg/kg, subcutaneously) for induction of duodenal ulcers, and gastric acid and duodenal HCO ₃ ^– secretions were measured with or without pretreatment withl-NAME (5 mg/kg, intravenously) or nitroprusside (4 mg/kg, intravenously). Mepirizole increased acid secretion, decreased the acid-induced duodenal HCO ₃ ^– secretion, and induced hemorrhagic lesions in the proximal duodenum. The inhibition of NO production byl-NAME potentiated the acid secretory response, increased the duodenal HCO ₃ ^– secretion, and prevented the duodenal lesions, and these changes were all antagonized by simultaneous administration ofl-arginine (200 mg/kg, intravenously) but notd-arginine. On the other hand, nitroprusside slightly reduced the acid response but further decreased the HCO ₃ ^– output, resulting in aggravation of duodenal lesions induced by mepirizole. These data suggest that the inhibition of endogenous NO production by the NO synthase inhibitorl-NAME increases duodenal HCO ₃ ^– secretion and protects the duodenal mucosa against acid injury.     

Inhibition of endogenous nitric oxide reduces basal mesenteric vascular tone but does not alter intraduodenal hydrochloric acid-induced intestinal hyperemia in rats

There are conflicting reports on the role of endogenous nitric oxide (NO) in the regulation of basal intestinal blood flow. The effect of inhibition of NO on intraduodenal hydrochloric acid (HCl) induced intestinal hyperemia remains to be confirmed. We investigated the effect of inhibition of endogenous NO on basal intestinal blood flow, HCl-induced intestinal hyperemia, and duodenal villous injury. Superior mesenteric artery blood flow in rats was measured by pulsed Doppler flowmetry and duodenal villous injury evaluated by histology. Intravenous N^G-nitro-l-arginine methyl ester (l-NAME), orl-arginine ord-arginine followed byl-NAME, was given to show inhibition, reversal of inhibition of endogenous NO synthase, and stereospecificity, respectively. An intraduodenal 2 ml/kg bolus or perfusion for 30 min of 0.1 N HCl was given 15 min afterl-NAME or vehicle. Mean arterial blood pressure was increased byl-NAME, which also significantly reduced intestinal blood flow under basal condition and after intraduodenal HCl. Basal mesenteric blood flow was not altered byl- ord-arginine. Thel-NAME-induced increase in blood pressure and decrease in basal blood flow was attenuated byl- but notd-arginine. The villous damage and the magnitude of the peak hyperemia was unchanged byl-NAME,l- ord-arginine. Inhibition of endogenous NO byl-NAME is suggested by the significant rise in blood pressure. The rise in blood pressure and reduction in blood flow are attenuated byl- but notd-arginine, indicating stereospecificity. Inhibition of endogenous NO reduces basal mesenteric vascular tone but does not alter intraduodenal HCl-induced intestinal hyperemia. The increase in blood flow after intraduodenal HCl predicts the absence of exacerbation of HCl-induced duodenal villous damage.     

Mechanism of bradykinin-induced cyclic GMP accumulation in bovine tracheal smooth muscle

Bradykinin (10^–8-10^–5M) caused a concentration-dependent increase in cyclic GMP (cGMP) production in bovine tracheal smooth muscle in the absence of epithelium. The effect was calcium-dependent and was inhibited by pyrogallol (10 M) and methylene blue (10 M). The inhibition of pyrogallol was reversed by superoxide dismutase (100 Usnowml). Nitric oxide (NO) synthase inhibitors, N ^G-methyl-l-arginine (10–100 M) and N ^G-nitro-l-arginine (10–100 M) reduced cGMP accumulation induced by bradykinin in a concentration-dependent fashion, and the inhibition was reversed by l-arginine. Immunohistochemistry with a specific antibody against neuronal NO synthase from rat cerebellum showed positive staining localized in some nerve fibers. Bradykinin-induced cGMP accumulation appears to be related to the release of NO, part of which is probably synthesized in nonadrenergic noncholinergic nerve in bovine trachea.Offprint requests to: Dr. Hong Sheng     

Cold restraint stress-induced gastric mucosal dysfunction role of nitric oxide

The objectives of this study were to determine the cold restraint stress-induced changes in gastric mucosal permeability and to assess whether nitric oxide synthesis inhibition affects gastric mucosal integrity after cold-restraint administration. Cold-restraint stress caused multiple gastric lesions in 90% of animals. The lesion index was found to be 3.87 ± 0.97 mm. Gastric mucosal permeability to the [⁵¹Cr]EDTA molecule was significantly elevated in the cold-restraint group compared to control. In order to evaluate the role of nitric oxide in cold restraint stress-induced gastropathy,l-arginine analogN ^G-nitro-l-arginie methyl ester (l-NAME) was given as a bolus (10 mg/kg, intravenously) and infused at a rate of 2 mg/ml/hr for 2 hr after cold-restraint administration.l-NAME greatly exacerbated gastric mucosal dysfunction associated with cold-restraint stress.d-NAME, the biologically inactive enantiomer, did not enhance mucosal dysfunction, whereasl-arginine, the substrate for nitric oxide, reversed the effect ofl-NAME. In an additional group of experiments, effects of cold-restraint stress andl-NAME on net transmucosal fluid flux as well as tissue myeloperoxidase activity (MPO) were assessed. Cold-restraint stress administration significantly reduced the absorptive capacity of stomach, whereasl-NAME treatment did not affect the stress-induced alterations on net fluid absorption. Furthermore,l-NAME treatment did not affect the cold restraint stress-induced changes in tissue MPO activity. Our results suggest that gastric barrier function is altered after cold-restraint stress and nitric oxide production is important in minimizing mucosal barrier dysfunction associated with cold-restraint stress administration. Our results also indicate thatl-NAME-induced alterations on mucosal permeability are not related to net transmucosal fluid flux and tissue neutrophils.Supported by a grant from the Turkish Scientific and Technical Research Council (TÜBITAK, TAG 1209).     

Inhibition of calcium-dependent nitric oxide synthase causes ileitis and leukocytosis in guinea pigs

As nitric oxide reduces gut epithelial permeability, we designed a study to determine if chronic nitric oxide synthase inhibition predisposes the gut to inflammation. Nitric oxide synthase (NOS) inhibitors were administered in the drinking waterad libitum, for seven days: aminoguanidine (10 µg/ml), a selective inhibitor of the inducible form of nitric oxide synthase; andN ^G-nitro-l-arginine methyl ester (l-NAME, 1, 10, and 100 µg/ml), which inhibits both the constitutive and inducible forms. Control animals drank tap water only or water withd-NAME, the inactive enantiomer. After one week, circulating leukocyte count and tissue myeloperoxidase activity were measured.l-NAME (100 µg/ml), but notd-NAME or aminoguanidine, caused a twofold increase in a circulating leukocyte numbers. This increase in leukocyte numbers was time- and dose-dependent, but the differential count was unaltered. Tissue myeloperoxidase (MPO) activity as an index of granulocyte infiltration was comparable in all groups in the stomach, jejunum, colon, liver, lung, kidney, heart, and skeletal muscle. However, ileal MPO activity was elevated threefold in thel-NAME- (100 µg/ml) treated group (P<0.05). Results in thed-NAME and aminoguanidine groups were similar to controls.l-NAME administration resulted in a reduction in NOS activity ([¹⁴C]citrulline formation) in the ileum but not jejunum, whereas cGMP levels were elevated in both ileum and jejunum. We conclude that chronic inhibition of the constitutive form of nitric oxide synthase predisposes the ileum to inflammation and leads to a progressive leukocytosis.     

Role ofl-arginine, a substrate for nitric oxide-synthase, in gastroprotection and ulcer healing

Nitric oxide (NO) synthesized froml-arginine interacts with prostaglandins (PG) and sensory neuropeptides in the regulation of mucosal integrity, but the role ofl-arginine, a substrate for NO-synthase, in gastroprotection and healing of chronic gastric ulcers has been little studied. In this study we compared the effects of intragastric (i.g.) and systemic (i.v.) administration ofl-arginine ord-arginine on gastric secretion and acute gastric lesions provoked in rats by i.g. application of 100% ethanol, acidified aspirin (ASA), or the exposure to 3.5h of water immersion and restraint stress (WRS). In addition, the effects ofl-arginine on ulcer healing and the formation of new vessels (angiogenesis) were determined, using monoclonal antibody (MAb E-9).l-arginine (10–200 mg/kg i.g.) failed to significantly affect gastric secretion but dose-dependently reduced the gastric lesions induced by 100% ethanol, ASA, and WRS, the doses inhibiting 50% of these lesions being 65, 94, and 72 mg/kg, respectively. This protection was accompanied by a significant rise in the gastric blood flow (GBF), whereasl-arginine given i.v. failed to affect the ethanol-lesions and the GBF.d-arginine or the NO-related amino acids—l-glutamine,l-citrulline, orl-ornithine—failed to significantly influence these lesions. Suppression of the generation of mucosal PG by indomethacin or capsaicin-denervation attenuated the protection and hyperemia induced byl-arginine. The inhibition of constitutive NO synthase byl-NNA had no significant effect on the protection afforded byl-arginine, but reduced the gastric hyperemia accompanying this protection.l-arginine (150 mg/kg per day, i.g.) accelerated the ulcer healing and increased GBF at the ulcer margin, and angiogenesis, whereas treatment with L-NNA had an opposite effect.l-arginine added to N^G-nitro-l-arginine (L-NNA) restored the ulcer healing, hyperemia, and angiogenesis. We conclude that: (1) the protective activity ofl-arginine involves gastric hyperemia mediated by NO and a mild irritant effect due to enhanced generation of endogenous PG, and (2) the ulcer healing properties ofl-arginine depend upon its hyperemic and angiogenic actions, possibly involving NO.     

Insulin increases cyclic nucleotide content in human vascular smooth muscle cells: a mechanism potentially involved in insulin-induced modulation of vascular tone

Summary It has been suggested that insulin exerts a vasodilating effect, but the mechanisms involved are not completely understood. Since cyclic nucleotides mediate the vasodilation induced by endogenous substances, such as prostacyclin and nitric oxide, we aimed to investigate the influence of insulin (concentration range 240–960 pmol/l) on both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) content in human vascular smooth muscle cells. Insulin dose-dependently increased both nucleotides (cAMP: from 0.7±0.1 to 2.6±0.4 pmol/10⁶ cells, p=0.0001; cGMP: from 1.3±0.2 to 3.4±0.7 pmol/10⁶ cells, p=0.033). This increase is receptor-mediated, since it was blunted when cells were preincubated with the tyrosine kinase inhibitor genistein. The effect of insulin remained significant (p=0.0001) when preincubation with the phosphodiesterase inhibitor theophylline prevented cyclic nucleotide catabolism. The increase of cGMP was blunted when the cells were preincubated with the guanylate cyclase inhibitor methylene blue, and with the nitric oxide-synthase inhibitor N^G-monomethyl-l-arginine. At all the concentrations tested, insulin potentiated the increase of cAMP induced by the stable prostacyclin analogue Iloprost (p=0.0001), whereas only at 1920 pmol/l did it potentiate the cGMP increase induced by glyceryltrinitrate (p=0.05). This study demonstrates that the vasodilating effects exerted by insulin may at least in part be attributable to an increase of both cGMP and cAMP via a receptor-mediated activation of adenylate and guanylate cyclases in human vascular smooth muscle cells and that the insulin effect on cGMP is mediated by nitric oxide.Abbreviations cAMP cyclic adenosine monophosphate - cGMP cyclic guanosine monophosphate - PDE phosphodiesterases - NO nitric oxide - hVSMC human vascular smooth muscle cells - l-NMMA N^G-monomethyl-l-arginine - GTN glyceryltrinitrate - BSA bovine serum albumin - NIDDM non-insulin-dependent diabetes mellitus - MEM minimal essential medium - RIA radioimmunoassay     

Mechanisms of arginine-induced increase in cytosolic calcium concentration in the beta-cell line NIT-1

Summary The effects of l-arginine and its analogues N^G-nitro-l-arginine, N^G-methyl-l-arginine, l-homoarginine and d-arginine on cytosolic calcium concentration were investigated to characterise the mechanisms of arginine-induced stimulation and to determine if nitric oxide production played a role in this stimulation. NIT-1 cells, a transgenic beta-cell line, were used for this purpose since they release insulin in response to typical beta-cell stimuli. Our data demonstrate that the arginine-induced increase in cytosolic calcium concentration was completely dependent on the influx of extracellular Ca^{2 +} via verapamil-sensitive voltage-activated Ca^{2 +} channels and that arginine stimulation requires the presence of a nutrient in order to cause an increase in cytosolic calcium concentration. The nutrient likely acted by closing the K ⁺ _ATP channels, since its effect could be inhibited by activation of these channels with diazoxide. l-arginine, as well as nitro-arginine and methyl-arginine which are not substrates for the production of nitric oxide, caused similar increases in cytosolic calcium concentration. Non-metabolisable arginine analogues homoarginine and d-arginine also caused increases in the cytosolic calcium concentration although not to the same extent. Insulin secretion was enhanced to the same extent by all analogues of arginine. It can be concluded that the arginine-induced increase in cytosolic calcium concentration in NIT-1 cells is attributable to an electrogenic effect following the transport of arginine leading to depolarisation of the plasma membrane potential, although metabolism of the amino acid itself may also partially contribute to the response. [Diabetologia (1997) 40: 374–382] Received: 16 October 1996 and in revised form: 24 December 1996     

Intragastric nicotine protects against 40% ethanol-induced gastric injury despite pretreatment with NG-Nitro-l-Arginine methyl ester or adrenal medullectomy in rats

We tested the hypotheses that the protective effect of intragastric nicotine against ethanol-induced gastric mucosal injury is dependent on endogenous nitric oxide or peripheral sympathoadrenal mechanisms. Rats were pretreated with N^G-nitro-l-arginine methyl ester (3 mg/kg subcutaneous, 1 h prior to study) to block endogenous nitric oxide synthesis or with adrenal medullectomy (three weeks prior to study) to ablate the effect of the adrenal medulla. At 1-h intervals, vehicle or nicotine (4 mg/kg) and 40% ethanol were then given intragastrically. The total lengths of the linear gastric corpus mucosal lesions were measured unbiasedly. The protective effect of intragastric nicotine was not modified by either pretreatment. We conclude that the mechanism mediating intragastric nicotine protection against 40% ethanol-induced gastric mucosal injury is independent of endogenous nitric oxide or the adrenal medulla.     

A Novel Method for Study of Gastric Mechanical Functions in Conscious Mice

A novel method has been developed for simultaneous study of gastric emptying, antral motility, and gastric muscle tone in conscious mice. Intragastric pressure was measured during infusion of an X-ray-opaque, viscous meal through a chronically implanted gastric fistula (0.25 ml/min). Compared with vehicle treatment, molsidomine (nitric oxide donor) and atropine (muscarinic receptor antagonist) treatment significantly reduced the area under the intragastric pressure curve (AUC) by 37 ± 4% and 35 ± 3%, respectively, (mean ± S.E.M.) whereas N ^G-nitro-l-arginine methyl ester (l-NAME; nitric oxide synthase inhibitor) significantly increased the AUC by 20 ± 3%. Atropine also significantly reduced the frequency and amplitude of stomach contraction-induced intragastric pressure waves while molsidomine only reduced the frequency. Gastric emptying, as assessed by X-ray imaging, was significantly delayed after l-NAME and atropine treatment. This methodology is the first to enable simultaneous assessment of gastric emptying, antral motility, and gastric tone in conscious mice and confirmed the important role of nitrergic and cholinergic innervation.     

Vascular action of the hypoglycaemic agent gliclazide in diabetic rabbits

Summary ATP-dependent potassium channel blockers used as hypoglycaemic agents may have effects on vascular disease in diabetes mellitus beyond their effect on blood glucose control. This study was designed to determine the effects of treatment with gliclazide on the isolated abdominal aorta of diabetic rabbits in which endothelium-dependent relaxation is impaired by a mechanism involving oxygen-derived free radicals. After induction of diabetes with alloxan, there was no effect of gliclazide (10 mg · kg⁻¹· day⁻¹ orally) on blood glucose or insulin levels over a 6 week period. Hence, this permitted an examination of the vascular effects of gliclazide in diabetic rabbits exclusive of metabolic effects. Acetylcholine- and nitric oxide-induced relaxation in aortae from rabbits treated with or without gliclazide were measured in the absence or presence of the nitric oxide synthase inhibitor, N^G-nitro-l-arginine (l-NAME). Diabetes was associated with significant impairment of acetylcholine-induced endothelium-dependent relaxation of the abdominal aorta which was not significant in diabetic rabbits treated with gliclazide in vivo. Aortae from diabetic rabbits studied in the presence of l-NAME showed an exaggerated contraction to acetylcholine which was prevented in rabbits treated with gliclazide. Gliclazide treatment did not affect the response to acetylcholine of normal rabbit aorta, and gliclazide when added in vitro had no effect on the response of diabetic rabbit aorta, suggesting that the effect of gliclazide was specific to the abnormality arising with diabetes and was not due to an acute effect of the drug. These data indicate that gliclazide, aside from either a direct antioxidant action or an effect on insulin or glucose levels, may ameliorate diabetic endothelial cell dysfunction. [Diabetolgia (1998) 41: 9–15] Received: 31 January 1997 and in final revised form: 25 August 1997     

Effects of submucosal administration of endothelin-3 on rat gastric mucosa

The present study was carried out to examine the effects of submucosal administration of endothelin on gastric mucosal integrity in rats. Injection of endothelin-3 into the submucosal space of the stomach induced gastric mucosal damage dose-dependently and site-specifically. The gastric injury was localized only at the injected site and the mucosal damage was associated with hemorrhage. Macroscopic and microscopic examinations revealed that mucosal injury had developed 15 min after endothelin application. Submucosal injection of either adrenalin or noradrenalin also induced gastric mucosal damage, but produced multiple gastric mucosal lesions; i.e., the macroscopic appearance of endothelin-induced gastric lesions differed from those produced by catecholamines. The endothelin-induced mucosal lesions were significantly inhibited by pretreatment with either atropine, pirenzepine, or ranitidine; or by vagotomy. In addition, N^G-nitro-l-arginine methyl ester (l-NAME), a nitric oxide synthase inhibitor, significantly enhanced the formation of gastric mucosal lesions. Thus, it appears that nitric oxide synthesis, possibly induced by endothelin, may play a role as an antiulcer mechanism in endothelin-induced gastric mucosal damage. Vagotomy and anti-cholinergic or antisecretory treatment significantly attenuated the severity of the mucosal lesions, suggesting that vagal cholinergic pathways and acid secretion may influence the development of the gastric mucosal damage induced by endothelin-3. These results suggest that endothelin-3 may play an important role in the development of gastric ulceration; the submucosal application of endothelin-3 in the gastric mucosa may be a useful experimental model for investigating acute gastric mucosal ulceration.     

Nitric oxide in ischaemic acute renal failure of streptozotocin diabetic rats

Summary Changes in nitric oxide (NO) levels were determined in ischaemic acute renal failure in streptozotocin-induced diabetes mellitus rats. Two weeks after streptozotocin administration and immediately after right nephrectomy, the left renal artery was occluded for 60 min. Similar procedures were carried out in non-diabetic rats. The nitrite (NO₂) + nitrate (NO₃) levels were measured in plasma and urine. The effects of chronic oral supplementation with l-arginine and an NO synthase inhibitor (N-omega-nitro-l-arginine) were also studied in both diabetic and non-diabetic rats before and after renal artery clamping. The rats with diabetic acute renal failure had a much lower creatinine clearance (90±22 l · min^–1 · 100 g body weight^–1, p<0.005), and higher fractional excretion of sodium (FENa)% (10.90±4.2, p<0.001) and protein excretion (2078±69 g/ml creatinine clearance, p<0.001) compared with the respective values in the non-diabetic groups (163±30; 1.46±86; 453.3±31). The plasma and urine NO₂ + NO₃ levels were significantly higher in the untreated diabetic rats compared with the untreated normal rats before ischaemia (p<0.001). The ischaemic acute renal failure in non-diabetic rats increased the plasma and urinary NO₂ + NO₃ excretion after ischaemia. The urinary excretion of these metabolites decreased significantly and their plasma levels remained unchanged in the ischaemic diabetic rats. The l-arginine administration resulted in a small but significantly higher creatinine clearance after clamping in the non-diabetic rats. The NO synthase inhibitor caused deterioration in renal function in all ischaemic and non-ischaemic groups. In summary, the greater vulnerability to ischaemia of the diabetic kidney seems to be associated with both impaired response to and impaired production of NO.Abbreviations IARF Ischaemic acute renal failure - STZ streptozotocin - NOSi nitric oxide synthase inhibitor - ARF acute renal failure - Ccr creatinine clearance - FENa% fractional excretion of sodium     

Autocrine growth inhibition of IL-1β-treated cultured human aortic smooth muscle cells: Possible role of nitric oxide

Summary This study was designed to investigate whether interleukin (IL)-1 would stimulate nitric oxide (NO) production in cultured human aortic vascular smooth muscle cells (VSMCs), and to determine the basic effect of the liberated NO on VSMC proliferation. NO production was estimated from nitrite concentration of culture medium in multi-well plates, determined by the Griess method. VSMCs were IL-1-pretreated in insert cups, and co-cultured with untreated VSMC in the wells.³H-thymidine (³H-Tdr) incorporation into the VSMC in wells was evaluated for VSMC proliferative activity. IL-1 stimulated NO production in VSMCs in a concentration-dependent manner. This effect was further enhanced by the addition of a membrane-permeable cyclic adenosine monophosphate derivative, dibutyryl cyclic AMP (db-cAMP), and was significantly reduced by concomitant use of an NO synthase inhibitor, N^G-nitro-l-arginine methyl ester (l-NAME). IL-1-pretreated VSMCs significantly inhibited³H-Tdr incorporation of the co-cultured VSMC. This inhibitory effect was significantly enhanced by the addition of db-cAMP, while this inhibition was significantly decreased by preincubation withl-NAME, and was abolished in thel-arginine-free medium. These results suggest that, in human VSMC, IL-1 stimulates NO production that is enhanced by intracellular cAMP accumulation, and that the liberated NO inhibits further VSMC proliferation in an autocrine fashion.     

Pharmacological manipulation of vascular endothelium function in non-diabetic and streptozotocin-diabetic rats: effects on nerve conduction,hypoxic resistance and endoneurial capillarization

Summary We examined the potential for some of the abnormalities of vascular endothelium found in diabetes mellitus to cause neuropathic changes. Non-diabetic rats were treated for 2 months with the cyclo-oxygenase inhibitor flurbiprofen (5 mg·kg⁻¹·day⁻¹) to reduce prostacyclin production, the nitric oxide synthase inhibitor N^G-nitro-L-arginine (5 or 25 mg·kg⁻¹·day⁻¹), or combined treatment. There were dose-dependent reductions in sciatic motor and saphenous sensory conduction velocity. The two inhibitors acted synergistically, thus, the 5–6% motor conduction deficits (p<0.01) produced by either flurbiprofen or N^G-nitro-l-arginine (5 mg·kg⁻¹·day⁻¹) increased to 17% (p<0.001) for combined treatment. With N^G-nitro-l-arginine (25 mg·kg⁻¹·day⁻¹) and flurbiprofen, motor and sensory conduction velocity were reduced by 23% (p<0.001) and 12% (p<0.001), respectively, matching the deficits following 2-month streptozotocin diabetes. N^G-nitro-l-arginine (25 mg·kg⁻¹·day⁻¹) and flurbiprofen together produced a 13% prolongation of the time taken for 80% hypoxic conduction failure in vitro (p<0.05) and a 10% reduction in sciatic capillary density. A second investigation tested an alternative hypothesis that overproduction of nitric oxide was responsible for vascular-related complications in diabetes, the prediction being that N^G-nitro-L-arginine (5 mg·kg⁻¹·day⁻¹) would prevent nerve dysfunction. However, rather than prophylaxis during 2-month streptozotocin diabetes, treatment exacerbated nerve abnormalities. Thus, N^G-nitro-L-arginine worsened (8%,p<0.001) the motor conduction deficit, there was an 11% increase in hypoxic conduction failure time (p<0.01) and an 11% reduction in endoneurial capillary density (p<0.01). We conclude that overproduction of nitric oxide is unlikely to be involved in the aetiology of experimental diabetic neuropathy. However, endothelial dysfunction resulting in impaired nitric oxide and prostacyclin synthesis could make a substantial contribution.     

Cytotoxicity of activated rat macrophages against syngeneic islet cells is arginine-dependent,correlates with citrulline and nitrite concentrations and is identical to lysis by the nitric oxide donor nitroprusside

Summary Lysis of rat islet cells by syngeneic activated macrophages in vitro can be completely inhibited by the nitric oxide-synthase-inhibitor N^G-methyl-l-arginine. This inhibition can be reversed by an excess of l-arginine. Time-dependent lysis of islet cells by activated macrophages is accompanied by increasing concentrations of nitrite and citrulline in the culture medium both of which are measures of nitric oxide formation derived from l-arginine. Lysis of isolated islet cells and disintegration of isolated whole islets is also obtained within 15 h by culture in the presence of the nitric oxide generating vasodilator sodium nitroprusside. We thus conclude that nitric oxide is extremely toxic for islet cells and that nitric oxide alone and in the absence of other macrophage-generated potentially toxic products can rapidly and completely kill islet cells.     

Vascular and metabolic effects of methacholine in relation to insulin action in muscle

Aims/hypothesis Methacholine (MC) is a nitric oxide vasodilator, but unlike other vasodilators, it potentiates insulin-mediated glucose uptake by muscle. The present study aimed to resolve whether this action was the result of a vascular effect of MC leading to increased muscle perfusion or a direct effect of MC on the myocytes. We hypothesise that vascular-mediated insulin-stimulated glucose uptake responses to MC occur at lower doses than direct myocyte MC-mediated increases in glucose uptake. Methods The vascular and metabolic effects of this vasodilator were examined in rats in vivo using a novel local infusion technique, and in the pump-perfused rat hindlimb under conditions of constant flow. Results Local infusion of low-dose MC (0.3 μmol/l) into the epigastric artery of one leg (test) in vivo markedly increased femoral blood flow and decreased vascular resistance, without effects in the contra-lateral leg. Capillary recruitment, but not glucose uptake, was increased in the test leg. All increases caused by MC were confined to the test leg and blocked by local infusion into the test leg of N-nitro-l-arginine methyl ester (l-NAME), but not by infusion of N-nitro-d-arginine methyl ester (d-NAME). In the constant-flow pump-perfused rat hindlimb, infusion of 0.6 μmol/l MC vasodilated the pre-constriction effected by 70 nmol/l noradrenaline or 300 nmol/l serotonin, and this was blocked by 10 μmol/l l-NAME. 2-Deoxyglucose in muscle was increased by 30 μmol/l MC (p<0.05), but was unaffected by 3 μmol/l MC. All increases in 2-deoxyglucose uptake by 30 μmol/l MC were blocked by 10 μmol/l l-NAME. Conclusions/interpretation MC has dose-dependent effects both on the vasculature and on muscle metabolism. At low dose (0.3–3 μmol/l), MC is a potent vasodilator in muscle, both in vivo and in vitro, without metabolic effects; at higher doses (≥30 μmol/l) MC has a direct metabolic effect leading to increased glucose uptake. Both the vascular and metabolic effects are sensitive to l-NAME. The low-dose enhancement of insulin action in vivo by MC, which has been reported previously, thus seems to be attributable to vascular effects.     

Nitric oxide modulates pancreatic edema formation in rat caerulein-induced pancreatitis

This study was designed to investigate the role of nitric oxide (NO) in the formation of pancreatic edema in caerulein-induced pancreatitis in rats. Pancreatitis was produced by two intraperitoneal injections of caerulein, and plasma amylase concentration, pancreatic edema index (pancreatic wet weight/body weight), and Evans blue extravasation (as a measure of vascular permeability) were evaluated 5h after the first injection. Four doses (1, 2.5, 5, and 10 mg/kg) of N^G-nitro-L-arginine (l-NNA), an NO synthase inhibitor, were subcutaneously administered at −0.5, 0.5, 1.5, 2.5, and 3.5h after the first injection of caerulein.l-NNA significantly lowered the edema index, the wet/dry weight ratio of the pancreas, and Evans blue extravasation in the rats with pancreatitis. The maximal effect was obtained byl-NNA at a dose of 2.5 mg/kg; this inhibited the increase in pancreatic edema formation from the control value by 60%–70%. Intraperitoneal injections (20 mg/kg, five times) ofl-arginine, a substrate for NO production, partly reversed thel-NNA-induced inhibition of pancreatic edema formation, butd-arginine, an enantiomer ofl-arginine, did not show any effect. Plasma amylase concentrations were not significantly affected by any dose of L-NNA, nor were they affected byl- ord-arginine. These findings strongly suggest that endogenous NO plays an important role in the formation of pancreatic edema in caerulein-induced pancreatitis in rats, probably by increasing vascular permeability and protein extravasation.     

Immediate and delayed effects of D-fructose upon insulin, somatostatin, and glucagon release by the perfused rat pancreas

Novel information was recently provided concerning the reciprocal effects of d-glucose and d-fructose upon their respective metabolism in rat pancreatic islets. In the light of such findings, this study aims at comparing the effects of d-glucose and d-fructose on insulin, somatostatin, and glucagon release from the isolated perfused rat pancreas. A rise in d-glucose concentration from 3.3 to 5.0 or 7.3 mM or the administration of d-fructose (17 and 40 mM) in the presence of 3.3 mM d-glucose stimulated insulin release in a concentration-related manner, but failed to affect somatostatin output. The secretion of glucagon was decreased in all cases. The secretory response to l-arginine (5 mM), 25 min after restoring the basal concentration of d-glucose, was more markedly affected, in terms of potentiation of insulin and somatostatin release and reduction of glucagon output, after prior administration of d-fructose than after a prior increase in d-glucose concentration. These findings argue against any major role for a paracrine regulation of hormonal release and, instead, are consistent with a causal link between metabolic and secretory events in the islet cells. Nevertheless, the present results emphasize differences in the response of distinct pancreatic endocrine cell types to the same or distinct hexoses.