Vagal modulation of arginine- and glucagon-induced pancreatic somatostatin secretion

In order to elucidate the role of the vagus nerve in the regulation of pancreatic somatostatin secretion, the effect of electrical stimulation of the vagus on the isolated perfused rat pancreas was studied. Somatostatin release induced by 19 mM arginine in the presence of 11 mM glucose or 10(-6)M glucagon in the presence of 5.5 mM glucose was suppressed by vagal stimulation. This suppressive effect on somatostatin was eliminated in the presence of 10(-5)M atropine plus glucagon, while somatostatin release was significantly enhanced in the presence of atropine plus arginine. We conclude that pancreatic somatostatin secretion may be regulated not only by a cholinergic inhibitory neuron but also by a stimulatory non-cholinergic neuron.     

Somatostatin release from isolated perfused rat stomach.

Gastric somatostatin release from the isolated rat stomach was studied using a perfusion technique. Somatostatin released from the isolated perfused rat stomach was found to be identical in molecular size and immunoreactively with synthetic somatostatin. Infusion of glucagon (10^?7 M) caused biphasic increase of gastric somatostatin release. Gastric somatostatin release was also stimulated by infusion of theophylline (10^?3 M) and dibutyryl cyclic AMP (10^?3 M). These results indicate the possible involvement of adenylate cyclase-cyclic AMP system in the regulatory mechanism of gastric somatostatin release.     

Effect of somatostatin on glucose-induced 45Ca uptake in the pancreatic islets.

This study was designed in an attempt to elucidate a mechanism of somatostatin inhibition of glucose-induced Ca+ uptake by rat pancreatic islets. Rat pancreatic islets were perifused with Krebs-Ringer bicarbonate (KRB) buffer containing 16.7 mM of glucose with somatostatin (2 micrograms/ml) or/and diltiazem HCl (2 x 10(-5) M). Somatostatin inhibited preferentially the early phase of glucose-induced insulin release, whereas diltiazem HCl inhibited the late one. And the concomitant presence of the submaximal concentration of somatostatin (2 micrograms/ml) and diltiazem HCl (2 x 10(-5 M) provided the completely additive inhibition of glucose-induced insulin release. Rat pancreatic islets were incubated with KRB buffer supplemented with 16.7 mM of glucose and 45CaCl2 (10 muCi/ml) for 5--60 min and the biphasic 45Ca uptake by pancreatic islets was obtained. Somatostatin (500 ng/ml-4 micrograms/ml) gave the suppressive effect on the early phase of glucose-induced 45Ca uptake, but the higher concentration (2 micrograms/ml) of somatostatin did not impair the late phase of 45Ca uptake by pancreatic islets. On the other hand, diltiazem HCl did suppress the late phase of glucose-induced 45Ca uptake dose-dependently, but did not suppress the early phase (2 x 10(-5) M). These data indicate that somatostatin suppresses the early phase of glucose-induced Ca2+ uptake preferentially to the late one and has a different action mechanism from Ca antagonist on glucose-induced insulin release.     

Somatostatin and gastrin release into the gastric lumen in rats

Somatostatin and gastrin release into the gastric lumen was investigated in anaesthetized, vagally intact rats. The stomach was perfused at a flow rate of 0.5 mL.min-1. During perfusion with 0.1 M HCl or buffers of varying pH the somatostatin ans gastrin concentrations in the perfusate were less than 10 pg.mL -1 and approximately 30 pg.mL-1, respectively. Peptone caused a gastrin concentrations in the perfusate were less than 10 pg.mL-1 and approximately 30 pg.mL-1, respectively. Peptone caused a slight pH-independent increase in somatostatin release; gastrin release was unchanged despite an increase in serum gastrin from a basal of 15 +/- 4 to 155 +/- 34 pg.mL-1 during peptone stimulation. intravenous infusion of carbachol (1 microgram.kg-1.min-1) strongly stimulated luminal somatostatin and gastrin release (from 5 +/- 1 to 192 +/- 52 pg.mL-1 and from 27 +/- 5 to 198 +/- 41 pg.mL-1, respectively) during perfusion with 0.1 M HCl. Phosphate buffer perfusion at pH 7.5 abolished the cholinergic-mediated somatostatin release but the gastrin response was unaffected. It is suggested that changes of luminal hormone concentrations in the rat stomach do not reflect the secretory activity of the endocrine cells in the gastric mucosa.     

The interaction of glucagon, gastric inhibitory peptide and somatostatin with cyclic AMP production systems present in rat gastric glands

The effects of glucagon, gastric inhibitory peptide (GIP) and somatostatin on the generation of cyclic AMP have been studied under basal and histamine- or secretin-stimulated conditions in tubular gastric glands isolated by means of EDTA from the rat fundus and antrum. Four types of cell could be identified by electron microscopy; namely, parietal, mucous, peptic and some endocrine cells with a good morphological preservation of the cellular topography as seen in the intact mucosa. Immunoreactive somatostatin was found in antral glands (210 +/- 16 ng/g cell, wet wt., n = 9) as well as in fundic glands, but in smaller concentration (50 +/- 8 ng/g cell, wet wt., n = 9). (1) In rat fundic glands, glucagon, in supraphysiologic doses (3 . 10(-9) -5 . 10(-7) M), raised cyclic AMP levels 46 times above the basal. At maximally effective doses, combination of glucagon plus histamine was not additive whereas glucagon and secretin stimulations resulted in an additive response. Somatostatin (10(-10) -10(-7) M) inhibited both glucagon- and histamine-induced cyclic AMP production, whereas cimetidine specifically blocked the histaminergic stimulation. (2) In the same conditions, 10(-6)M glucagon produced a marginal effect (4-fold increase) in rat antrum, whereas GIP (10(-9) -10(-6)M) was unable to induce a significant rise of cyclic AMP production in either fundic or antral glands, or to prevent cyclic AMP production stimulated by histamine. (3) The present data do not support the view that circulating glucagon or GIP may regulate gastric secretion directly by a cyclic AMP-dependent mechanism in rat gastric glands and raise the possibility that gastric somatostatin may be the final mediator of the inhibitory actions of these hormones on acid secretion. (4) It is proposed that pancreatic glucagon acts through a receptor-cyclic AMP system which is specific for the bioactive peptide enteroglucagon ('oxyntomodulin'), probably in rat parietal cells.     

The effect of somatostatin on baseline and stimulated acid secretion and vascular histamine release from the totally isolated vascularly perfused rat stomach

The effect of somatostatin-(1-14) (S1-14) on the gastrin- and histamine-induced acid secretion and gastrin-evoked vascular histamine release was studied in isolated vascularly perfused rat stomachs being continuously perfused by a gassed buffer containing 10% ovine erythrocytes and 50 microM isobutyl methylxanthine (IMX). Concentrations of gastrin (520 pM) and histamine, (0.5 microM) were chosen to give acid secretion in the same range (61.5 +/- 7.0 and 49.4 +/- 9.4 mumol/60 min). S1-14 induced a concentration-dependent decrease in acid secretion stimulated by both gastrin and histamine. Even at the lowest concentration examined (0.1 nM) somatostatin gave a significant inhibition of both gastrin- and histamine-stimulated acid secretion. The inhibitory effect was, however, most marked for gastrin-stimulated acid secretion (P less than 0.05 at 1 nM concentration of S1-14). Gastrin gave an immediate and marked vascular histamine release which was inhibited by somatostatin in the higher concentrations (1.0 and 5.0 nM). Somatostatin at the lowest concentration tested (0.1 nM) did not inhibit the gastrin-induced vascular histamine release although it did inhibit acid secretion. Furthermore, baseline histamine release was not affected by somatostatin. This study suggests that somatostatin inhibits acid secretion both via a direct effect of the parietal cell and by inhibiting gastrin-induced histamine release. Baseline histamine release is regulated by a mechanism not sensitive to somatostatin.     

Effect of gastrointestinal hormones on choleresis from the isolated perfused rat liver

Using isolated perfused rat liver, the direct effect of secretin, glucagon, caerulein, insulin and somatostatin on choleresis was investigated. When the liver was perfused in the absence of sodium taurocholate, the bile volumes were: control, 0.33 +/- 0.01 (mean +/- S.E.M.) ml/10 g liver per 50 min; secretin 0.05 U/ml, 0.39 +/- 0.01 (P less than 0.01); glucagon 10(-10) M, 0.44 +/- 0.02 (P less than 0.01); caerulein 10(-8) M, 0.34 +/- 0.03 (n.s.); insulin 1 mU/ml, 0.35 +/- 0.02 (n.s.); glucagon plus somatostatin 10(-7) M, 0.46 +/- 0.03 (n.s. vs. glucagon alone), respectively. When 10(-5) M sodium taurocholate was present in the perfusate, the bile volumes were: control, 0.61 +/- 0.03; secretin, 0.63 +/- 0.01 (n.s.); glucagon, 0.70 +/- 0.01 (P less than 0.05); caerulein, 0.55 +/- 0.01 (n.s.); insulin, 0.62 +/- 0.04 (n.s.); somatostatin, 0.59 +/- 0.01 (n.s.); respectively. Glucagon increased glucose output and cyclic AMP in the effluent from the liver neither of which were suppressed by somatostatin. Secretin increased cyclic AMP but not glucose output. These results indicate that glucagon has the most potent action on bile acid-independent canalicular bile, that caerulein and insulin do not act on canalicular bile production directly and that somatostatin does not directly suppress canalicular bile production nor hepatic glucose output produced by glucagon in rats.     

Interaction of somatostatin and calcium in regulating insulin release from isolated pancreatic islets of rats.

The effect of synthetic somatostatin on insulin release was studied in vitro by using isolated islets of rats. Somatostatin, with concentrations from 10 ng/ml to 10μg/ml, inhibited insulin release induced by 16.7 mM glucose. Insulin release elicited by 10 μg/ml glucagon or 2 mM dibutyryl cyclic AMP was likewise inhibited by 100ng/ml somatostatin. By raising the calcium concentration of the incubation medium to 6 mM, glucose-induced insulin release was fully restored even in the presence of somatostatin.However, the same maneuver only partially counteracted the somatostatin inhibition of dibutyryl cyclic AMP-induced insulin release. These results suggest the involvement of calcium mobilization process in the inhibitory action of somatostatin.     

Pulses of somatostatin release are slightly delayed compared with insulin and antisynchronous to glucagon

It was early proposed that somatostatin-producing delta-cells in pancreatic islets have local inhibitory effects on the release of insulin and glucagon. Recent observations that pulses of insulin and glucagon are antisynchronous make it important to examine the temporal characteristics of glucose-induced somatostatin release. Analysis of 30 s fractions from the perfused rat pancreas indicated that increase of glucose from 3 to 20 mmol/l results in initial suppression of somatostatin release followed by regular 4-5 min pulses. During continued exposure to 20 mmol/l glucose, the pulses of somatostatin overlapped those of insulin with a delay of 30 s. Somatostatin and glucagon pulses were coupled in antisynchronous fashion (phase shift 2.4+/-0.2 min), supporting the idea that the delta-cells have a local inhibitory effect on glucagon release. It was possible to remove the pulses of somatostatin and glucagon with maintenance of the insulin rhythmicity by addition of 1 micromol/l of the P2Y(1) receptor antagonist MRS 2179.     

Glucose regulation of arginine-induced pancreatic somatostatin release from the isolated perfused rat pancreas

The effects of glucose alone, combinations of glucose with arginine or tolbutamide and either arginine or tolbutamide alone, on somatostatin, insulin, and glucagon secretion were investigated using the isolated perfused rat pancreas. When glucose alone was raised in graded increments at 15-min intervals from an initial concentration of 0 mM to a maximum of 16.7 mM, somatostatin as well as insulin in the perfusate increased with the glucose, while glucagon decreased. The similarity of the glucose stimulated somatostatin and insulin release was especially evident when the perfusate glucose was increased from an initial dose of 4.4 mM rather than 0 mM to 8.8 mM or 16.7 mM. In addition, glucose at concentrations varying from 4.4 mM to 11 mM dose-dependently enhanced arginine-induced somatostatin and insulin release and suppressed glucagon release dose-dependently as before. Arginine in the absence of glucose was not capable of stimulating somatostatin secretion whereas tolbutamide, in contrast, was capable of stimulating somatostatin secretion even in the absence of glucose.     

Failure of chronic hyperinsulinemia to suppress pancreatic glucagon in vivo in the rat.

To determine the effects of chronic hyperinsulinemia on glucagon release, rats were made hyperinsulinemic for 14 days by supplementation of drinking water with sucrose (10%; sucrose-fed) to increase endogenous release or by implantation of osmotic minipumps (subcutaneous, s.c.; or intraperitoneal, i.p.) to deliver exogenous insulin (6 U/day). Both s.c. and i.p. rats also had sucrose in the drinking water to prevent hypoglycemia. Plasma insulin levels were significantly elevated in sucrose-fed, s.c., and i.p. rats. However, glucose levels were significantly elevated in sucrose-fed rats only. Surprisingly, plasma glucagon concentrations were elevated in i.p. and s.c. rats and were not suppressed in sucrose-fed rats. Inverse relationships were found between the plasma levels of insulin and glucose (n = 65; r = -0.42, p less than 0.0001) and between glucose and glucagon (n = 73; r = -0.46, p less than 0.0001). However, unexpectedly, a positive correlation between insulin and glucagon (n = 65; r = 0.47, p less than 0.0001) was established. As suppression of plasma glucagon levels below basal was not observed in any of the hyperinsulinemic or hyperglycemic rats, we wished to establish further whether pancreatic glucagon release could be suppressed below basal levels in the rat by another means. Thus, high doses of somatostatin (50-100 micrograms.kg-1.min-1) were infused for 45 min into normal rats without or with a concomitant hyperinsulinemic, hyperglycemic glucose clamp. Somatostatin fully suppressed insulin, but although plasma glucagon levels were decreased by somatostatin infusion relative to saline-infused animals, there was still no suppression below basal levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin stimulates somatostatin and inhibits glucagon secretion from the perfused chicken pancreas-duodenum.

The isolated perfused chicken pancreas-duodenum was used to study the secretion of somatostatin and glucagon. With perfusate glucose at 50 mg/dl, bovine insulin was infused at a concentration of 20, 000 μU/ml, resulting in a rapid increase of somatostatin secretion, with peak concentrations seen at 5 minutes. This was accompanied by suppression of glucagon secretion. These data suggest that there may be a paracrine negative feedback loop between B and D cells.     

Mechanism of action of growth-hormone-releasing hormone in stimulating insulin secretion in vitro from isolated rat islets and dispersed islet cells

Human growth-hormone-releasing hormone [(1-44)NH2] (hGHRH) was a potent stimulus for insulin release from rat islets of Langerhans in vitro; the optimum concentration used was 10(-11) M. The dose response curves for hGHRH effects on insulin secretion were notably different in intact islets of Langerhans compared to cultured dispersed islet cells. Pancreatic islets responded to a very low hGHRH concentration (10(-12) M), but at a higher hGHRH concentration (10(-9) M) no stimulation of insulin release was observed. When somatostatin antiserum was included in the incubation medium, hGHRH (10(-9) M) stimulated insulin release from intact islets. In cultured dispersed islet cells, which are principally insulin-secreting B cells, hGHRH directly and potently stimulated insulin release even at a concentration of 10(-9) M. Addition of somatostatin (10(-7), 10(-8) M) significantly reduced the hGHRH-induced insulin-secretory responses of dispersed islet cells. hGHRH (10(-11)-10(-9) M) raised islet cAMP levels; individually, hGHRH and theophylline exerted positive effects on insulin release, their combined effect was greater than that caused by either one. We conclude that hGHRH directly affects insulin secretion in vitro by a cAMP-dependent mechanism, and that the difference in responses of intact islets versus islet cells to increasing concentrations of hGHRH may be related to hGHRH-induced release of somatostatin in intact rat islets.     

Lipoproteins in a nonrecirculating perfusate of rat liver

Rat livers were perfused in a nonrecirculating system for 30-40 min with Krebs-Ringer bicarbonate-0.1% glucose solution gassed with 95% O(2)-5% CO(2) at 37 degrees C at a flow rate of 3 ml/g/min. The livers appeared normal as judged by O(2) uptake, bile flow, transaminase release, and net protein output (2.5 mg/g/hr). The perfusate was concentrated by ultrafiltration using Amicon PM-10 or PM-30 membranes. The concentrated perfusate was subjected to sequential ultracentrifugation at solution densities of 1.006, 1.04, 1.06, and 1.21, and the top fractions were analyzed for protein and lipid. The net release of protein in the four density classes, suitably corrected, averaged 39, 10, 5, and 20 micro g/g/hr. The lipid composition of the perfusate lipoprotein fractions differed from that of serum mainly in the high percentage of free cholesterol, reflecting the lack of exposure to lecithin:cholesterol acyltransferase. When rat serum was fractionated in the same way, most of the lipoprotein in the d 1.006-1.06 range had a density greater than 1.04. It was concluded from these experiments that the liver secretes very low density lipoprotein (VLDL), high density lipoprotein (HDL), and a modified form of VLDL containing less lipid. Comparison of secretion rates and serum lipoprotein levels leads to the conclusion that the latter are largely determined by catabolic rates. When labeled amino acids were present, the perfusate HDL had a higher specific activity than VLDL. Addition of carrier whole serum did not alter recovery of labeled lipoproteins, but when these were isolated from Golgi membranes after a 40-min perfusion, more than twice as much label was recovered in HDL, suggesting the presence of precursors within the Golgi. The main advantages of the nonrecirculation perfusion technique are the avoidance of catabolic reactions, simplicity, and complete control over the composition of the perfusing medium.     

The effects of somatostatin on the arachidonate cascade of platelets

Somatostatin (10(-9) M) significantly elevated the synthesis of thromboxane B2 in rat platelets. The transformation of arachidonic acid to active lipoxygenase metabolites was suppressed by somatostatin (10(-9) and 10(-8) M). The ratio of the lipoxygenase/cyclooxygenase products was significantly reduced by the polypeptide (10(-9) and 10(-8) M) in rat platelets. Higher concentrations (10(-7), 10(-6) and 10(-5) M) of somatostatin did not modify the lipoxygenase pathway of the platelets. The synthesis of the vasoconstrictor - proaggregatory cyclooxygenase products was stimulated by the polypeptide (10(-9) and 10(-8) M), while the formation of vasodilatator - antiaggregatory cyclooxygenase metabolites was induced by higher concentrations of somatostatin (10(-7) and 10(-6) M). Somatostatin might act on the deacylation process of phospholipids, reducing the free arachidonic acid substrate level, resulting in a lower lipoxygenation rate in the platelets, which could be responsible for the increased formation of thromboxane. The contradictory results reported by others concerning the action of somatostatin on the platelet function might be explained by our results that the effect of somatostatin depends on the applied dose.     

The effect of gastrin-releasing peptide on the endocrine pancreas

The 27-amino acid peptide gastrin releasing peptide (GRP-(1–27)) was infused at 4 dose levels (0.01, 0.1, 1.0, and 10 nM) into the arterial line of the isolated perfused porcine pancreas. Infusions were performed at 3 different perfusate glucose levels (3.5, 5.0, and 8.0 mM) and at two levels of amino acids (5 and 15 mM). GRP-(1–27) stimulated insulin and pancreatic polypeptide secretion and inhibited somatostatin secretion in a dose-dependent manner. Glucagon secretion was unaffected by infusion of GRP under all circumstances. The effect of GRP-(1–27) on insulin secretion was enhanced with increasing perfusate glucose levels, whereas the effects upon somatostatin and pancreatic polypeptide secretion were independent of perfusate glucose levels. The responses to GRP were unaffected by elevation of the concentration of amino acids in the perfusate. The effects of GRP were unaffected by atropine at 10⁻⁶ M. The localization of GRP within the porcine pancreas, its release during electrical stimulation of the vagus nerve, and its potent effects upon pancreatic endocrine secretion make it conceiveable that the peptide participates in parasympathetic regulation of pancreatic endocrine secretion.     

Effect of starvation of gastric somatostatin release

The present study is an investigation of the effects of 16 and 48 hours starvation on gastric somatostatin release using the isolated perfused rat stomach. Before sacrifice the body weights and blood glucose levels of fasted rats were significantly lower than fed rats. In the presence of 4.4 mM glucose, basal somatostatin concentrations in the stomach perfusate of fasted rats were also significantly lower. Gastric somatostatin release was stimulated in all three groups similarly by 5 × 10^?8 M glucagon when the decrease in basal levels is considered. These results suggest that gastric somatostatin as well as pancreatic somatostatin contributes to nutrient homeostasis and that nutrient homeostasis influences somatostatin levels in turn.     

Somatostatin potentiates cholinergic neurotransmission in ferret trachea

We studied the effect of somatostatin on contractile responses to electrical field stimulation (EFS) in isolated ferret tracheal segments. Somatostatin (up to 10(-5) M) did not change resting tension, but it potentiated the contractile response to EFS dose dependently, with a maximum effect at 10(-6) M. Thus, at a concentration of 10(-6) M, somatostatin significantly decreased the mean log of EFS frequency producing 50% of maximum contraction from a control value of 0.52 +/- 0.07 to 0.24 +/- 0.06 (SE) Hz (P less than 0.01). The potentiating effect of somatostatin (10(-6) M) was not inhibited by hexamethonium, indomethacin, BW755C, pyrilamine, methysergide, or D,Pro2,D,Trp7,9-SP, but it was inhibited by atropine or by the somatostatin antagonist cyclo[7-aminoheptanoyl-Phe-D-Trp-Lys-Thr(Bzl)]. In contrast to EFS-induced contraction, contractions produced by acetylcholine (10(-9) to 10(-3) M) were not affected by somatostatin at a concentration of 10(-6) M. These results suggest that somatostatin potentiates contractions produced by EFS via presynaptic cholinergic mechanisms and probably through a specific somatostatin receptor.     

Assessment of the role of Ca2+ mobilization from intracellular pool(s), using dantrolene, in the glycogenolytic action of alpha-adrenergic stimulation in perfused rat liver

To identify the role of Ca2+ mobilization from intracellular pool(s) in the action of alpha-adrenergic agonist, the effects of dantrolene on phenylephrine-induced glycogenolysis were investigated in perfused rat liver. Dantrolene (5 X 10(-5) M) inhibited both glycogenolysis and 45Ca efflux induced by 5 X 10(-7) M phenylephrine. The inhibition by dantrolene was observed in the presence and absence of perfusate calcium. In contrast, dantrolene did not inhibit glycogenolysis induced by glucagon. To confirm the specificity of dantrolene action on calcium release in liver, experiments were also carried out using isolated hepatocytes. Dantrolene did not affect phenylephrine-induced production of inositol 1,4,5-trisphosphate. The compound did inhibit a rise in cytoplasmic Ca2+ concentration induced by phenylephrine both in the presence and absence of extracellular Ca2+. Thus, these results suggest that calcium release from an intracellular pool is essential for the initiation of alpha-adrenergic stimulation of glycogenolysis in the perfused rat liver.     

Effect of glucagon antiserum on somatostatin,glucagon and insulin release from the isolated perfused rat pancreas

In order to elucidate the effect of glucagon antiserum on the endocrine pancreas, the release of somatostatin, glucagon, and insulin from the isolated perfused rat pancreas was studied following the infusion of arginine both with and without pretreatment by glucagon antiserum. Various concentrations of arginine in the presence of 5.5 mM glucose stimulated both somatostatin and glucagon secretion. However, the responses of somatostatin and glucagon were different at different doses of arginine. The infusion of glucagon antiserum strongly stimulated basal secretion in the perfusate total glucagon (free + antibody bound glucagon) and also enhanced its response to arginine, but free glucagon was undetectable in the perfusate during the infusion. On the other hand, the glucagon antiserum had no significant effect on either insulin or somatostatin secretion. Moreover, electron microscopic study revealed degrannulation and vacuolization in the cytoplasm of the A cells after exposure to glucagon antiserum, suggesting a hypersecretion of glucagon, but no significant change was found in the B cells or the D cells. We conclude that in a single pass perfusion system glucagon antiserum does not affect somatostatin or insulin secretion, although it enhances glucagon secretion.