The role of leucine-enkephalin on insulin and glucagon secretion from pancreatic tissue fragments of normal and diabetic rats

Leucine-enkephalin (Leu-Enk) has been shown to be present in endocrine cells of the rat pancreas and may play a role in the modulation of hormone secretion from the islets of Langerhans. Since little is known about the effect of Leu-Enk on insulin and glucagon secretion, it was the aim of this study to determine the role of Leu-Enk on insulin and glucagon secretion from the isolated pancreatic tissue fragments of normal and diabetic rats. Pancreatic tissue fragments of normal and streptozotocin-induced diabetic rats were incubated for 1 h with different concentrations of Leu-Enk (10(-12)-10(-6)M) alone or in combination with either atropine or yohimbine or naloxone. After the incubation period the supernatant was assayed for insulin and glucagon using radioimmunoassay techniques. Leu-Enk (10(-12 )-10(-6)M) evoked large and significant increases in insulin secretion from the pancreas of normal rats. This Leu-Enk-evoked insulin release was significantly (p < 0.05) blocked by atropine, naloxone and yohimbine (all at 10(-6)M). In the same way, Leu-Enk at concentrations of 10(-12)M and 10(-9)M induced significant (p < 0.05) increases in glucagon release from the pancreas of normal rats. Atropine, yohimbine but not naloxone significantly (p < 0.05) inhibited Leu-Enk-evoked glucagon release from normal rat pancreas. In contrast, Leu-Enk failed to significantly stimulate insulin and glucagon secretion from the pancreas of diabetic rats. In conclusion, Leu-Enk stimulates insulin and glucagon secretion from the pancreas of normal rat through the cholinergic, alpha-2 adrenergic and opioid receptor pathways.     

Distribution of vasoactive intestinal polypeptide, neuropeptide-Y and substance P and their effects on insulin secretion from the in vitro pancreas of normal and diabetic rats

This study examined the pattern of distribution of vasoactive intestinal polypeptide (VIP), neuropeptide-Y (NPY) and substance P (SP) in the pancreas of diabetic rat to determine whether there are changes in the number and pattern of distribution of these neuropeptides after the onset of diabetes. Moreover, the effect of VIP, NPY and SP on insulin secretion from the pancreas of normal and diabetic rats was also examined. Diabetes mellitus (DM) was induced by a single dose of streptozotocin (STZ) given intraperitoneally (i.p.) (60 mg kg body weight(-1)). Four weeks after the induction of DM, diabetic (n = 6) and normal (n = 6) rats were anesthetized with chloral hydrate and their pancreases removed and processed for immunohistochemistry and insulin secretion. The number of insulin-positive cells in the islets of Langerhans was reduced while that of VIP and NPY increased significantly after the onset of diabetes. The pattern of distribution of VIP, NPY and SP in the nerves innervating the pancreas was similar in both normal and diabetic rats. VIP-evoked large and significant (P < 0.02) increases in insulin secretion from the pancreas of normal and diabetic rats. NPY also induced a marked (P < 0.005) increase in insulin release from pancreatic tissue fragments of normal rat. Stimulation of pancreatic tissue fragments of diabetic rat with NPY resulted in a slight but not significant increase in insulin release. SP induced a large and significant (P < 0.005) increase in insulin secretion from the pancreas of normal rat but inhibited insulin secretion significantly (P < 0.03) from isolated pancreas of diabetic rat. In summary, VIP and NPY can stimulate insulin secretion from the pancreas after the onset of diabetes. The stimulatory effect of SP on insulin secretion is reversed to inhibitory in diabetic rats.     

GABA in the endocrine pancreas: cellular localization and function in normal and diabetic rats

Gamma amino butyric acid (GABA) and its related enzymes have been demonstrated in pancreatic beta cells of normal rat. Antibodies against GABA-synthesizing enzymes have been implicated in the pathogenesis of Type I diabetes. In spite of the importance of GABA in the aetiology of diabetes mellitus, detailed morphological data on the pattern of distribution of GABA in the pancreas of normal and diabetic rats are lacking. Diabetes mellitus (DM) was induced by a single dose of streptozotocin (STZ) given intraperitoneally (60 mg kg body weight(-1)). Four weeks after the induction of DM, normal (n = 6) and diabetic (n = 6) rats were anesthetized with chloral hydrate and their pancreata were removed and processed for the localization and effect of GABA on insulin secretion using immunohistochemistry and radioimmunoassay techniques. The number of GABA-like immunoreactive (GABA-LIR) cells in the pancreatic islets of STZ-diabetic rats decreased significantly (P<0.0001) when compared to non-diabetic control rats. The pattern and percentage distribution of GABA in the islet of Langerhans of normal and diabetic rat was similar to that of insulin. GABA induced a significant (P<0.0007) increase in insulin secretion from the pancreas of normal rats. In diabetic pancreas, GABA evoked a higher but not significant (P<0.1) increase in insulin secretion. These findings showed that the number of GABA-LIR cells is reduced significantly in diabetes. Moreover, GABA is a strong secretagogue of insulin from the pancreas of normal rat.     

Insulin restores neuronal nitric oxide synthase expression in streptozotocin-induced diabetic rats

Nitric oxide (NO) is known to play an important role in the pathophysiology of insulin-dependent diabetic mellitus (IDDM). In an attempt to investigate the relation between insulin and NO in IDDM, the present study employed male Wistar rats to induce IDDM by intravenous injection of streptozotocin (STZ). Four groups of rats were used; untreated normal control group, insulin treated STZ group, vehicle-treated STZ control, and one group of age-matched rats which were orally supplied with glucose to increase plasma glucose (glucose-challenged rats). Changes of the activity and gene expression of neuronal nitric oxide synthase (nNOS) were examined in cerebellum and kidney of these groups. The activity of nNOS in cerebellum, determined by conversion of [3H] L-arginine to [3H] L-citrulline, in STZ-induced diabetic rats was markedly lower than normal rats. Insulin treatment reversed the nNOS activity. Similar reversion by insulin treatment was also obtained in the gene expression of nNOS. However, the activity and gene expression of nNOS in glucose-challenged rats were not different from those in normal rats. The role of hyperglycemia can thus be ruled out. These findings indicated that an impairment of nNOS in the brain of rats with IDDM is mainly due to the absence of insulin.     

Supplementation of L-arginine improves hypertension and lipid metabolism but not insulin resistance in diabetic rats

Nitric oxide (NO) plays an important role in glucose and lipid metabolism. We previously reported that NO synthesis inhibitors, such as NG-nitro-L-arginine methyl ester (L-NAME), deteriorate insulin sensitivity and lipid metabolism, while the addition of L-arginine reverses this deterioration. L-arginine is a precursor of NO, and is used as a supplement in the US. In the present study, we evaluated whether the administration of L-arginine alone improves insulin resistance and serum lipid levels in insulin-resistant and hypertriglycemic rat models. Diabetic rats were divided into 3 groups: the control (Cont) group (standard diet), the L-NAME group (diet containing L-NAME), and the Arg group (diet containing L-arginine). After 4 weeks of breeding, urinary NOx, glucose infusion rate (GIR), glucose and lipid tolerance tests were performed. Urinary NOx levels were significantly lower in the L-NAME group than in the Cont group. The GIR in the L-NAME group was significantly lower than that in the Cont group, suggesting increased insulin resistance. However, the administration of L-arginine did not influence insulin resistance in the Arg group. Oral lipid administration significantly increased plasma triglyceride levels in the L-NAME group and plasma triglyceride levels were significantly lower in the Arg group than in the Cont group. The area under the curve of plasma triglyceride levels after oral lipid administration was larger in the L-NAME group than in the Cont group. The administration of L-NAME increased insulin resistance and decreased lipid metabolism. L-arginine significantly increased urinary NO secretion but did not improve insulin resistance, although it did improve lipid metabolism. These findings suggest that supplementation of L-arginine cannot improve insulin resistance in diabetic rats probably due to increased insulin secretion by L-arginine.     

Combined administration of sodium chloro-2-propionate and insulin on the secretion of glucagon by the pancreas in the diabetic rat

This work was designed to study the effects of sodium 2-chloropropionate (2CP) alone or combined with insulin, in vitro, on glucagon secretion from pancreas isolated from rats, made diabetic by streptozotocin (66 mg/kg i.p.). The pancreata were perfused with a physiological solution containing 2.8 mM glucose (0.5 g/l) and glucagon secretion was stimulated by an arginine infusion (5 mM) for 30 min. When 2CP (1 mM) and/or insulin (4 IU/l) were applied, they were infused from the start of the organ perfusion. In the presence of glucose alone, a marked decrease in glucagon output was observed in diabetic rat pancreas. The arginine perfusion induced a biphasic glucagon secretion both in normal and diabetic rat pancreas; this response was however clearly reduced in diabetic rat pancreas. In diabetic rat pancreas, the infusion of either 2CP or insulin had no effect on glucagon output in presence of glucose alone, nor did it modify the response to arginine. In contrast, the combined infusion of insulin and 2CP induced different effects depending on the conditions: whereas in presence of glucose alone it restored a glucagon output close to that recorded in normal rat pancreas, it did not modify the response to arginine.     

Effect of electrical field stimulation on insulin and glucagon secretion from the pancreas of normal and diabetic rats.

The effect of electrical field stimulation (EFS) on insulin (INS) and glucagon (GLU) secretion from normal and diabetic rat pancreas is poorly understood. In our study, EFS (5-20Hz, 50 V amplitude and 1.0 ms pulse width), when applied alone, resulted in a significant (p<0.05) increase in INS secretion from the pancreas of both normal and diabetic rats. Atropine (10(-5) M) did not inhibit the EFS (5 Hz)-evoked INS secretion in normal pancreas and failed to alter the effect of EFS (10-20 Hz) on INS secretion from the pancreas of both normal and diabetic rats. Propranolol (Prop) inhibited INS secretion to below basal level in the presence of EFS (5 Hz) but not at EFS (10- 20 Hz). Tetrodotoxin (TTX) also significantly (p = 0.002) inhibited INS secretion from normal pancreas in the presence of EFS (5-20 Hz). The decrease in insulin secretion observed when pancreatic tissue fragments were incubated in Prop and TTX in the presence of EFS was reversed by yohimbine (10(-5) M). In contrast, TTX did not significantly modify INS secretion from diabetic pancreas in the presence of EFS. EFS (5-20 Hz) significantly (p<0.05) increased GLU release from normal and diabetic rat pancreas when applied alone. Neither atropine, Prop nor TTX significantly modified GLU release from the pancreas of either normal or diabetic rats. This suggests that GLU secretion may be controlled through a different pathway. The EFS-evoked INS and GLU secretion is probably executed via different mechanisms. These mechanisms include 1) activation of cholinergic nerves by EFS; 2) EFS of alpha- and beta-adrenergic nerves; 3) activation of non-adrenergic non-cholinergic pathway by EFS; 4) EFS-induced depolarization and subsequent action potential in pancreatic endocrine cells and 5) electroporosity caused by EFS-induced membrane permeability. All of these effects may be summative. In conclusion, EFS (5-20 Hz), when applied alone, can evoke significant increases in INS and GLU secretion from the pancreas of both normal and diabetic rats. Insulin secretion is controlled via alpha-2 adrenergic (inhibition) and beta-adrenergic (stimulation) receptors. Glucagon secretion is enhanced by alpha2 adrenergic stimulation.     

The influence of nitric oxide on basal and cholecystokinin-8-induced proliferation and apoptosis in the rat pancreas

Nitric oxide (NO) is formed by different cell types in the pancreas. In this study, inhibition of endogenous nitric oxide by N(omega)-nitro-L-arginine (L-NNA) reduced the urinary excretion of NO(2)/NO(3) and raised serum L-arginine and the NO donator S-nitroso-N-acetylpenicillamine (SNAP) increased the urinary excretion of NO(2)/NO(3). The peptide cholecystokinin-8 (CCK-8) has a strong influence on exocrine pancreatic proliferation. Rat pancreas was excised and studied with regard to tissue weight, protein and DNA contents after 3 days of treatment with saline, L-NNA or SNAP given separately or combined with CCK-8. Further, proliferation of different pancreatic cells was studied with [3H]-thymidine incorporation and apoptotic activity was studied by analysing caspase-3 activity and histone-associated DNA fragments. The effects of L-NNA indicate that endogenous nitric oxide formation has a tonic inhibition on apoptosis in the pancreas during both basal condition and growth stimulation by CCK-8. In CCK-induced hyperplasia, NO inhibits the proliferation of acinar cells but stimulates ductal cells. Endogenous NO may regulate the balance between proliferation and apoptosis and in a situation of growth stimulation by CCK-8, it has a tonic inhibition on both mitogenesis and apoptosis thus slowing down the acinar cell turnover in the pancreas.     

Insulin-induced impairment via peroxynitrite production of endothelium-dependent relaxation and sarco/endoplasmic reticulum Ca(2+)-ATPase function in aortas from diabetic rats

We designed this study to determine whether a high insulin level and a diabetic state need to exist together to cause an impairment of endothelium-dependent relaxation. In diabetic rat aortas organ-cultured with insulin [vs both control rat aortas cultured with insulin and diabetic rat aortas cultured in serum-free medium]: (1) the relaxation responses to both acetylcholine (endothelium-dependent relaxation) and Angeli's salt (nitric oxide donor) were significantly weaker, (2) acetylcholine-stimulated nitric oxide production was significantly smaller, (3) superoxide and nitric oxide production into the culture medium was greater, and (4) the levels of both nitrotyrosine and tyrosine-nitrated sarco/endoplasmic reticulum calcium ATPase (SERCA) protein were greater. The insulin-induced effects were prevented by cotreatment with either a superoxide scavenger or a peroxynitrite scavenger. After preincubation with an irreversible SERCA inhibitor, the relaxation induced by the nitric oxide donor was significantly impaired in control aortas cultured with or without insulin and in diabetic aortas cultured without insulin, but not in diabetic aortas cultured with insulin. These results suggest that the coexistence of a high insulin level and an established diabetic state may lead to an excessive generation of peroxynitrite, and that this may in turn trigger an impairment of endothelium-dependent relaxation via a decrease in SERCA function.     

Effect of L-arginine-nitric oxide system on the metabolism of essential fatty acids in chemical-induced diabetes mellitus

Several studies have shown that the activities of delta-6-desaturase and delta-5-desaturase are depressed in experimental diabetes and in humans with insulin- and non-insulin-dependent diabetes mellitus (type I and type II diabetes mellitus respectively). Furthermore, treatment with insulin is known to correct the defects in desaturases in rats and humans with diabetes, especially in type I. In a recent study, we demonstrated that L-arginine and nitric oxide can prevent alloxan-induced beta cell damage, and the severity of diabetes, and restore the antioxidant status to near normal levels. But, no information is available as to the relationship between L-arginine-nitric oxide system and the metabolism of essential fatty acids in diabetes mellitus. In the present study, it was noted that the plasma levels of saturated fatty acids: stearic and palmitic were increased where as unsaturated fatty acids such as oleic, linoleic, gamma-linolenic and eicosapentaenoic acids (OA, LA, GLA and EPA respectively) were decreased in alloxan-induced diabetic rats. In the liver phospholipid (PL) fraction, GLA, DGLA (dihomo-GLA) and alpha-linolenic acid (ALA) were decreased in the alloxan-treated group; in the muscle PL fraction, LA, GLA and DGLA were low, whereas an increase in the saturated fatty acid content was noted. L-arginine (the precursor of nitric oxide) and sodium nitroprusside (a nitric oxide donor) treatment of alloxan-induced diabetic rats enhanced the levels of LA, GLA and DGLA. Further, nitric oxide synthase inhibitor, L-NMMA, prevented this beneficial action of L-arginine-nitric oxide system on essential fatty acid metabolism. The abnormalities in the essential fatty acid metabolism observed also reverted to normalcy following control of diabetes with insulin. These results indicate that nitric oxide can enhance the activities of delta-6- and delta-5 desaturases, which are depressed in diabetes, and suggests that there is a close interaction between L-arginine-nitric oxide system and the metabolism of essential fatty acids.     

Distribution of NPY and SP and their effects on glucagon secretion from the in vitro normal and diabetic pancreatic tissues

Neuropeptides modulate the function of classic neurotransmitters in the regulation of body function. The role of neuropeptides in the regulation of endocrine secretion from the pancreas of diabetic rat is poorly understood. This study examined the pattern of distribution of neuropeptide-Y (NPY) and substance P (SP) in normal and diabetic rat pancreases. In addition to this, the effect of NPY and SP on glucagon secretion was also examined in the pancreases of normal and diabetic rats. Four weeks after the induction of diabetes, the pancreaseses of normal and diabetic rats were removed and processed for immunohistochemistry and glucagon secretion. The pattern of distribution of glucagon in the pancreas of diabetic rat was conspicuously deranged after the onset of diabetes. The pattern of distribution of NPY and SP was, however, similar in the pancreases of both normal and diabetic rats. Stimulation of normal rat pancreatic tissue with NPY (10(-12) and 10(-9) M) evoked large and significant (P < 0.001) increases in glucagon secretion compared to basal. In contrast to this, NPY inhibited glucagon secretion from the pancreas of diabetic rat. Treatment of pancreatic tissue fragments of normal rat with 10(-9) M SP resulted in significant (P < 0.03) increases in glucagon secretion. SP inhibited glucagon secretion from diabetic rat pancreas. In conclusion, NPY and SP stimulated glucagon secretion from the pancreas of normal rat. In contrast, NPY and SP inhibited glucagon secretion from diabetic rat pancreas.     

The effect of L-arginine/nitric oxide pathway on salivary amylase secretion in conscious rats

In a recent study we have demonstrated the presence of nitric oxide synthase immunoreactive neurons and also perivascular, periacinar and periductal nerve fibres in feline submandibular salivary gland. The role of nitric oxide (NO) in salivary vasoregulation has been suggested by other data too, but the effect of NO on salivary amylase secretion has not been investigated yet. Under ether anaesthesia a catheter was introduced into the oesophagus for salivary juice collections, and a cannula was inserted into the jugular vein for infusions. After postanaesthesia recovery, submaximal carbachol infusion was given as a background to obtain steady secretion because of the low basal secretory rate. Then different groups of animals received NO synthase inhibitor NOLA (N^G-nitro-L-arginine), L-arginine, D-arginine or NO donor SIN-1 (3-morpholinosydnonimine). Volume and amylase activity were determined in mixed saliva samples collected for 30 min. Carbachol background infusion alone induced an elevated, sustained salivary secretion. NOLA (30 mg/kg) increased both volume and amylase output (P < 0.001). This effect was prevented by L-arginine but not by D-arginine. SIN-1 did not change either volume or amylase secretion. The present results suggest that the L-arginine/NO pathway has a modulatory effect on salivary fluid and amylase secretion, which is probably not related to its effect on salivary blood flow. NO may block certain presently unidentified secretagogue mechanisms and/or may relax myoepithelial cells.     

Impaired glucose-stimulated insulin secretion in the GK rat is associated with abnormalities in islet nitric oxide production

We investigated implications of nitric oxide (NO) derived from islet neuronal constitutive NO synthase (ncNOS) and inducible NOS (iNOS) on insulin secretory mechanisms in the mildly diabetic GK rat. Islets from GK rats and Wistar controls were analysed for ncNOS and iNOS by HPLC, immunoblotting and immunocytochemistry in relation to insulin secretion stimulated by glucose or l-arginine in vitro and in vivo. No obvious difference in ncNOS fluorescence in GK vs control islets was seen but freshly isolated GK islets displayed a marked iNOS expression and activity. After incubation at low glucose GK islets showed an abnormal increase in both iNOS and ncNOS activities. At high glucose the impaired glucose-stimulated insulin release was associated with an increased iNOS expression and activity and NOS inhibition dose-dependently amplified insulin secretion in both GK and control islets. This effect by NOS inhibition was also evident in depolarized islets at low glucose, where forskolin had a further amplifying effect in GK but not in control islets. NOS inhibition increased basal insulin release in perfused GK pancreata and amplified insulin release after glucose stimulation in both GK and control pancreata, almost abrogating the nadir separating first and second phase in controls. A defective insulin response to l-arginine was seen in GK rats in vitro and in vivo, being partially restored by NOS inhibition. The results suggest that increased islet NOS activities might contribute to the defective insulin response to glucose and l-arginine in the GK rat. Excessive iNOS expression and activity might be deleterious for the beta-cells over time.     

Role of nitric oxide synthase isoforms in glucose-stimulated insulin release

The role of islet constitutive nitric oxide synthase (cNOS) in insulin-releasing mechanisms is controversial. By measuring enzyme activities and protein expression of NOS isoforms [i.e., cNOS and inducible NOS (iNOS)] in islets of Langerhans cells in relation to insulin secretion, we show that glucose dose-dependently stimulates islet activities of both cNOS and iNOS, that cNOS-derived nitric oxide (NO) strongly inhibits glucose-stimulated insulin release, and that short-term hyperglycemia in mice induces islet iNOS activity. Moreover, addition of NO gas or an NO donor inhibited glucose-stimulated insulin release, and different NOS inhibitors effected a potentiation. These effects were evident also in K+-depolarized islets in the presence of the ATP-sensitive K+ channel opener diazoxide. Furthermore, our results emphasize the necessity of measuring islet NOS activity when using NOS inhibitors, because certain concentrations of certain NOS inhibitors might unexpectedly stimulate islet NO production. This is shown by the observation that 0.5 mmol/l of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) stimulated cNOS activity in parallel with an inhibition of the first phase of glucose-stimulated insulin release in perifused rats islets, whereas 5.0 mmol/l of L-NMMA markedly suppressed cNOS activity concomitant with a great potentiation of the insulin secretory response. The data strongly suggest, but do not definitely prove, that glucose indeed has the ability to stimulate both cNOS and iNOS in the islets and that NO might serve as a negative feedback inhibitor of glucose-stimulated insulin release. The results also suggest that hyperglycemia-evoked islet NOS activity might be one of multiple factors involved in the impairment of glucose-stimulated insulin release in type II diabetes mellitus.     

Involvement of nitric oxide in the regulation of regional hemodynamics in streptozotocin-diabetic rats

In experimental and human diabetes mellitus, evidence for an impaired function of the vascular endothelium has been found and has been suggested to contribute to the development of vascular complications in this disease. The aim of the study was to evaluate possible regional hemodynamic in vivo differences between healthy and diabetic rats which would involve nitric oxide (NO). Central hemodynamics and regional blood flow (RBF) were studied using radioactive microspheres in early streptozotocin (STZ)-diabetic rats and compared to findings in healthy control animals. This method provides a possibility to study the total blood flow and vascular resistance (VR) in several different organs simultaneously. L-NAME iv induced widespread vasoconstriction to a similar extent in both groups. In the masseter muscle of both groups, acetylcholine 2 microg/kg per min, induced a RBF increase, which was abolished by pretreatment with L-NAME, suggesting NO as a mediator of vasodilation. In the heart muscle of both groups, acetylcholine alone was without effect while the combined infusion of acetylcholine and L-arginine induced an L-NAME-sensitive increase in RBF. The vasodilation induced by high-dose acetylcholine (10 microg/kg per min) in the kidney was more pronounced in the STZ-diabetic rats. The results indicate no reduction in basal vasodilating NO-tone in the circulation of early diabetic rats. The sensitivity to vasodilating effects of acetylcholine at the level of small resistance arterioles vary between tissues but was not impaired in the diabetic rats. In the heart muscle the availability of L-arginine was found to limit the vasodilatory effect of acetylcholine in both healthy and diabetic rats. In conclusion, the results indicate a normal action of NO in the investigated tissues of the early STZ-diabetic rat.     

Nitric oxide-cyclic GMP system potentiates glucose-induced rise in cytosolic Ca2+ concentration in rat pancreatic beta-cells.

Involvement of nitric oxide (NO) in the regulation of insulin secretion from pancreatic beta-cells was investigated by measuring cytosolic Ca2+ concentration ([Ca2+]i) in isolated rat pancreatic beta-cells. At 7.0 mM glucose, L-arginine (0.1 mM) elevated [Ca2+]i in about 50% of the beta-cells examined. The response was partially inhibited by an NO synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMA; 0.1 mM), suggesting that part of the response was mediated by the production of NO from L-arginine. D-Arginine at higher concentrations (3 or 10 mM) also increased [Ca2+]i at 7.0 mM glucose; however, the response was not affected by L-NMA (0.1 mM). Similar [Ca2+]i elevation was produced by NO (10 nM) and sodium nitroprusside (SNP; 10 microM) at 7.0 mM glucose. The SNP-induced increase in [Ca2+]i was abolished by nicardipine (1 microM), suggesting that the [Ca2+]i response is mediated by Ca2+ influx through L-type voltage-operated Ca2+ channels. In the presence of oxyhemoglobin (1 microM), the [Ca2+]i elevation induced by NO (10 nM) was abolished. Neither degradation products of NO, NO2- nor NO3-, caused any changes in [Ca2+]i. 8-Bromo-cyclic GMP (8-Br-cGMP; 3 mM) and atrial natriuretic peptide (0.1 microM) elevated [Ca2+]i at 7.0 mM glucose. We conclude that NO, which is produced from L-arginine in pancreatic islets, facilitates glucose-induced [Ca2+]i increase via the elevation of cGMP in rat pancreatic beta-cells. NO-cGMP system may physiologically regulate insulin secretion from pancreatic beta-cells.     

<Emphasis Type="SmallCaps">l</Emphasis>-Arginine Uptake in Normal and Diabetic Rat Retina and Retinal Pigment Epithelium

Diabetes-induced increase in oxidative stress is postulated as playing a significant role in the development of retinopathy. The retinal pigment epithelium (RPE) which forms part of the retinal blood barrier has been reported to be affected in diabetes. Besides functioning as a neurotransmitter, the radical nitric oxide (NO) can act as a cytotoxic agent. NO is synthesized by nitric oxide synthase (NOS) that oxidizes arginine to citrulline producing NO. Given that intracellular concentration of arginine depends mainly on its transport, we studied arginine transport in RPE and retina from normal and streptozotocin-induced diabetic rats. Retina and RPE take up arginine by a saturable system with an apparent K_M of 70–80 μM. Tissue incubation in the presence of insulin or high glucose concentrations significantly increased arginine transport in RPE but not in retina from control rats. Similarly, arginine uptake was enhanced in RPE, but not in the retina from streptozotocin-induced diabetic rats. However, NO content was two-fold higher in diabetic retina and RPE compared to that in the control rats. Such findings may suggest that diabetes induced an increase in NO levels in RPE, which may have brought about alterations in its functioning and in turn manifestations of diabetic retinopathy. Special issue article in honor of Dr. Ricardo Tapia.     

The role of arginine vasopressin in diabetes-associated increase in glucagon secretion

The purpose of this study was to investigate the role of arginine vasopressin (AVP) on glucagon secretion in both normal and diabetic rats. Diabetes was induced by intravenous administration of 50 mg/kg streptozotocin, 14 days before pancreatic perfusion. Diabetic rats were maintained on insulin replacement therapy until approximately 48 h before the perfusion experiments. Both glucagon and AVP were determined in the effluent of the perfused pancreas using RIA. Both normal and diabetic rats had similar basal glucagon secretion. AVP (3-30 pM) increased glucagon secretion from both normal and diabetic rats in a concentration-dependent manner. However, diabetic subjects were more sensitive to AVP administration than normal subjects with regard to glucagon secretion. By comparison of the areas under the curves, AVP-induced glucagon secretion in diabetic rats was approximately 2-fold that of the normal rats. In addition, immunoreactive AVP was detected in the effluent of the perfused pancreas, and diabetic rats had 70% higher AVP concentrations in the pancreatic effluent than normal rats. We conclude that AVP is secreted from the pancreas and diabetic rats can secrete more AVP from the pancreas than normal rats. Consequently, AVP may have a greater impact on glucagon secretion in diabetic subjects than normal ones. AVP might play an important role in the hypersecretion of glucagon in diabetic subjects.     

L-arginine stimulates an endogenous ADP-ribosyltransferase

An ubiquitous biochemical pathway known to synthesize nitric oxide (NO) from L-arginine has been identified in many cell types. Recent studies indicate that besides activating soluble guanylate cyclase NO is likely to have effects unrelated to the known signal transduction pathway. Activation of the soluble NO synthase stimulates an endogenous ADP-ribosylation of a predominant 39 kDa protein, known to be activated by NO releasing agents. This is demonstrated using the cytosolic fraction of rat cerebellum and HL-60 cells. The ADP-ribosylation is suppressed by the known NO synthase inhibitors N-nitro-L-arginine and N-methyl-L-arginine. These observations indicate that NO derived from its physiological precursor L-arginine activates an endogenous ADP-ribosyltransferase.     

Influence of nitric oxide synthase inhibitors on the vasopressin-induced pituitary-adrenocortical activity and hypothalamic catecholamine levels.

In the present study the role of endogenous nitric oxide (NO) in the vasopressin-induced ACTH and corticosterone secretion was investigated in conscious rats. Vasopressin (AVP 5 microg/kg i.p.) considerably augmented ACTH and corticosterone secretion. L-arginine (120 and 300 mg/kg i.p.) did not significantly alter the AVP-induced secretion of those hormones. Nitric oxide synthase (NOS) blockers N(omega)-nitro-L-arginine (L-NNA) and its methyl ester (L-NAME) given i.p. 15 min before AVP markedly increased the AVP-induced ACTH secretion. L-NNA (2 mg/kg) more potently and significantly increased the AVP-induced ACTH secretion, whereas L-NAME elicited a weaker and not significant effect. Both those NOS antagonists intensified significantly and to a similar extent the AVP-induced corticosterone secretion. L-arginine (120 mg/kg i.p.) reversed the L-NNA-induced rise in the AVP-stimulated ACTH secretion and substantially diminished the accompanying corticosterone secretion. Neither vasopressin alone nor in combination with L-arginine and L-NAME evoked any significant alterations in the hypothalamic noradrenaline and dopamine levels. L-NNA (2 and 10 mg/kg i.p.) elicited a dose dependent and significant decrease in the hypothalamic noradrenaline level. The hypothalamic dopamine level was not significantly altered by any treatment. These results indicate that in conscious rats endogenous NO has an inhibitory influence on the AVP-induced increase in ACTH and corticosterone secretion. L-NNA is significantly more potent than L-NAME in increasing the AVP-induced ACTH secretion. This may be connected with a considerable increase by L-NNA of hypothalamic noradrenergic system activation which stimulates the pituitary-adrenal axis in addition to specific inhibition of NOS.