Vagally mediated, nonparacrine effects of cholecystokinin-8s on rat pancreatic exocrine secretion

Cholecystokinin (CCK) has been proposed to act in a vagally dependent manner to increase pancreatic exocrine secretion via actions exclusively at peripheral vagal afferent fibers. Recent evidence, however, suggests the CCK-8s may also affect brain stem structures directly. We used an in vivo preparation with the aims of 1) investigating whether the actions of intraduodenal casein perfusion to increase pancreatic protein secretion also involved direct actions of CCK at the level of the brain stem and, if so, 2) determining whether, in the absence of vagal afferent inputs, CCK-8s applied to the dorsal vagal complex (DVC) can also modulate pancreatic exocrine secretion (PES). Sprague-Dawley rats (250-400 g) were anesthetized and the common bile-pancreatic duct was cannulated to collect PES. Both vagal deafferentation and pretreatment with the CCK-A antagonist lorglumide on the floor of the fourth ventricle decreased the casein-induced increase in PES output. CCK-8s microinjection (450 pmol) in the DVC significantly increased PES; the increase was larger when CCK-8s was injected in the left side of the DVC. Protein secretion returned to baseline levels within 30 min. Microinjection of CCK-8s increased PES (although to a lower extent) also in rats that underwent complete vagal deafferentation. These data indicate that, as well as activating peripheral vagal afferents, CCK-8s increases pancreatic exocrine secretion via an action in the DVC. Our data suggest that the CCK-8s-induced increases in PES are due mainly to a paracrine effect of CCK; however, a relevant portion of the effects of CCK is due also to an effect of the peptide on brain stem vagal circuits.     

Suppression of ANP secretion by somatostatin through somatostatin receptor type 2

Somatostatin is a cyclic-14 amino acid peptide which mainly distributed in digestive system and brain. Somatostatin receptor (SSTR) is a G-protein coupled receptor and all five SSTR subtypes are expressed in cardiomyocytes. The aim of this study was to investigate the effect of somatostatin on atrial natriuretic peptide (ANP) secretion and its signaling pathway. Somatostatin (0.01 and 0.1 nM) decreased ANP secretion in isolated beating rat atrium in a dose-dependent manner. But atrial contractility and translocation of extracellular fluid were not changed. Somatostatin-induced decrease in ANP secretion was significantly attenuated by the pretreatment with CYN 154806 (SSTR type 2 antagonist; 0.1 μM), but not by BIM 23056 (SSTR type 5 antagonist; 0.1 μM) and urantide (urotensin II receptor antagonist; 0.1 μM). When pretreated with an agonist for SSTR type 2 (Seglitide, 0.1 nM) and SSTR type 5 (L 817818, 0.1 nM), only Seglitide reduced ANP secretion similar to that of somatostatin. The suppressive effect of somatostatin on ANP secretion was attenuated by the pretreatment with an inhibitor for adenylyl cyclase (MDL-12330A, 5 μM) or protein kinase A (KT 5720, 0.1 μM). In diabetic rat atria, the suppressive effect of somatostatin on ANP secretion and concentration was attenuated. Real time-PCR and western blot shows the decreased level of SSTR type 2 mRNA and protein in diabetic rat atria. These data suggest that somatostatin decreased ANP secretion through SSTR type 2 and an attenuation of suppressive effect of somatostatin on ANP secretion in diabetic rat atria is due to a down-regulation of SSTR type 2.     

Involvement of prostacyclin/IP receptors in decreased acid response of damaged stomachs — Mediation by somatostatin/SST2 receptors

AimsWe examined the effect of a prostacyclin (PGI₂) analog iloprost on histamine-induced acid secretion and investigated how endogenous PGI₂ mediated the decreased secretory response in the damaged stomach after exposure to taurocholate (TC).Main methodsMale C57BL/6 mice, both wild-type (WT) and IP receptor knockout (IP-KO) animals, were used after 18 h of fasting. Under urethane anesthesia, the abdomen was incised, and an acute fistula was provided in the stomach.Key findingsAcid secretion in WT and IP-KO mice was similarly and dose-dependently increased by histamine. Iloprost decreased the histamine-stimulated secretion in WT but not IP-KO mice. The inhibitory effect of iloprost in WT mice was totally abrogated by the prior administration of CYN154806, a selective somatostatin SST2 receptor antagonist. On the other hand, the acid secretion in WT mice was decreased after exposure of the stomach to 20 mM TC for 20 min, with an increase in mucosal PGI₂ content, but the decrease was significantly less marked in IP-KO mice. The decreased acid response to TC in WT mice was totally prevented by the prior administration of CYN154806 as well as indomethacin. Somatostatin contents in the stomach were reduced after the administration of iloprost or the mucosal exposure to TC, while the blood levels increased.SignificanceSomatostatin/SST2 receptors are involved in the decreased acid response of the damaged stomach, in addition to PGI₂/IP receptors. It is assumed that PGI₂ releases somatostatin from D cells, which in turn decreases acid secretion via the activation of SST2 receptors.     

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.     

Effect of somatostatin on exocrine pancreatic secretion stimulated by pancreozymin-secretin or by a test meal in the dog

In 4 dogs with chronic duodenal and gastric fistulae, exocrine pancreatic function was assessed by cannulating the pancreatic duct and collecting the duodenal contents. Both methods were applied in each animal. Pancreatic secretion was stimulated by infusion of 2 CHR units of pancreozymin and secretin or by administration of a liquid test meal, injected into the stomach through the gastric fistula. During both experiments 3.5 microgram/kg somatostatin was given as bolus injection followed by an infusion of 3.5 microgram/kg/h. Somatostatin caused a significant reduction in protein and amylase output and in the bicarbonate concentration during stimulation with pancreozymin-secretin. Volume and bicarbonate slightly decreased but not to a significant extent. Duodenal volume and the duodenal activities of trypsin and amylase were significantly reduced during test meal stimulation and somatostatin infusion. Somatostatin is a potent inhibitor of exocrine pancreatic function mainly influencing enzyme secretion.     

Pancreatic polypeptide, microinjected into the dorsal vagal complex, potentiates glucose-stimulated insulin secretion in the rat

Specific binding sites for circulating pancreatic polypeptide (PP) have been found within the dorsal vagal complex (DVC) in the caudal medulla oblongata. Therefore, the effects of rat PP on pancreatic hormone secretion upon its microinjection into the DVC in halothane-anesthetized rats at doses of 0.4–40 pmol were investigated. At this range of doses, the changes in plasma concentrations of insulin, glucagon and glucose over basal levels did not differ from those after vehicle microinjection. In a separate series of experiments, vehicle and PP at doses of 0.4 and 4 pmol were microinjected into the right DVC 40 min after the continuous infusion of -glucose had been started. In animals receiving continuous infusion of -glucose, PP microinjected into the DVC (4 pmol), resulted in markedly higher insulin levels at corresponding time points compared to those with vehicle microinjected into the DVC. These data indicate, for the first time, that microinjection of PP into the DVC may potentiate glucose-stimulated insulin secretion in halothane-anesthetized rats.     

Somatostatin receptors (sst2) regulate cGMP production in rat retina

The present study investigated the effect of somatostatin in the regulation of cGMP levels in rat retina and the mechanisms involved in this process. Isolated rat retinas were treated alone or in the presence of somatostatin (0.01-10 microM), BIM23014 (sst2 agonist, 0.01-10 microM), L-796,778 (sst3 agonist, 10 microM), somatostatin (0.1 microM) in combination with CYN154806 (sst2 antagonist, 1 microM), N(G)-methyl-L-arginine acetate salt (NMMA, inhibitor of the nitric oxide synthase (NOS), 250 microM), orthovanadate (inhibitor of tyrosine phosphatase, SHP-1, 1 microM), and arginine alone (250 microM). cGMP levels were quantified by ELISA. Immunohistochemistry studies were performed for the detection of cGMP and nNOS, while Western blot analysis was employed for the detection of SHP-1. Somatostatin increased cGMP levels in a concentration-dependent manner. This increase was inhibited by CYN154806. BIM23014 increased cGMP levels only at the concentration of 10 microM, while L-796,778 had no effect. NMMA blocked completely the somatostatin stimulated increase of cGMP levels and nNOS was detected in rat retina. cGMP immunoreactivity was observed primarily in bipolar cells only of nitroprusside-treated retinas. SHP-1 inhibition by orthovanadate reduced the somatostatin effect in a statistically significant manner. These results suggest that a SRIF/SHP-1/NO/cGMP mechanism underlies the actions of somatostatin in the retina and in its influence of retinal circuitry.     

Somatostatin stimulates intestinal NHE8 expression via p38 MAPK pathway

Diarrhea is a common manifestation of gastrointestinal disorders. Diarrhea-induced losses of fluid and electrolyte could lead to dehydration and electrolyte imbalances, resulting in significant morbidity and mortality, especially in children living in developing countries. Somatostatin, a peptide hormone secreted by D-cells, plays an important role in regulating motility and intestinal Na(+) absorption. Although octreotide, a somatostatin analog, is used to treat diarrhea, its mechanisms of action are unclear. Here we showed that octreotide increased brush-border membrane Na(+)/H(+) exchanger 8 (NHE8) expression in the small intestine to the exclusion of other NHEs that participate in Na(+) absorption. The same effect also occurred in human intestinal cells (Caco-2). We found that the increase of NHE8 expression by somatostatin required p38 mitogen-activated protein kinase (MAPK) activation. Furthermore, the somatostatin receptor SSTR2 antagonist CYN154806 could abolish somatostatin-induced NHE8 expression and p38 MAPK phosphorylation. Thus our data provided the first concrete evidence indicating that somatostatin stimulates intestinal Na(+) absorption by increasing intestinal NHE8 expression through the SSTR2-p38 MAPK pathway.     

Thyrotropin-releasing hormone in the dorsal vagal complex stimulates pancreatic blood flow in rats

Central administration of thyrotropin-releasing hormone (TRH) enhanced pancreatic blood flow in animal models. TRH nerve fibers and receptors are localized in the dorsal vagal complex (DVC), and retrograde tracing techniques have shown that pancreatic vagal nerves arise from the DVC. However, nothing is known about the central sites of action for TRH to elicit the stimulation of pancreatic blood flow. Effect of microinjection of a TRH analog into the DVC on pancreatic blood flow was investigated in urethane-anesthetized rats. After measuring basal flow, a stable TRH analog (RX-77368) was microinjected into the DVC and pancreatic blood flow response was observed for 120 min by laser Doppler flowmetry. Vagotomy of the several portions, or pretreatment with atoropine methyl nitrate or N(G)-nitro-l-arginine-methyl ester was performed. Microinjection of RX-77368 (0.1-10 ng) into the left or right DVC dose-dependently increased pancreatic blood flow. The stimulation of pancreatic blood flow by RX-77368 microinjection was eliminated by the same side of cervical vagotomy as the microinjection site or subdiaphragmatic vagotomy, but not by the other side of cervical vagotomy. The TRH-induced stimulation of pancreatic blood flow was abolished by atropine or N(G)-nitro-l-arginine-methyl ester. These results suggest that TRH acts in the DVC to stimulate pancreatic blood flow through vagal-cholinergic and nitric oxide dependent pathways, indicating that neuropeptides may act in the specific brain nuclei to regulate pancreatic function.     

Antinociceptive effect of somatostatin microinjected into caudate putamen.

The effects of somatostatin microinjected bilaterally and unilaterally (left or right) at a dose of 10, 50 and 100 ng into the caudate putamen of male Wistar rats on nociception (analgesy-meter test) were studied. Somatostatin injected into caudate putamen resulted in analgesia. Bilateral microinjections of somatostatin significantly increased the pain threshold in a dose-dependent manner, i.e. somatostatin exerted antinociceptive effect. The pain threshold after left-side microinjections was significantly higher than that after injections into right-side. These findings suggest antinociceptive and asymmetric effects of somatostatin on pain in the caudate putamen.     

Cholecystokinin-58 and cholecystokinin-8 produce similar but not identical activations of myenteric plexus and dorsal vagal complex

The enteric nervous system (ENS: myenteric and submucosal plexuses) of the gastrointestinal tract may have a role in the reduction of food intake by cholecystokinin (CCK). Exogenous cholecystokinin-8 (CCK-8) activates the myenteric plexus and the feeding control areas of the dorsal vagal complex (DVC) of the brainstem. An increasing number of reports, however, have shown that CCK-58 is the sole or the major circulating form of CCK in rat, human and dog, and that it is qualitatively different from CCK-8 in evoking various gastrointestinal physiological responses (e.g., contraction of the gallbladder and exocrine pancreatic secretion). In the current report, we compared the abilities of exogenous CCK-58 to activate the myenteric plexus and the dorsal vagal complex with those of exogenous CCK-8 by quantifying Fos-like immunoreactivity (Fos-LI; a marker for neuronal activation). We report that CCK-58 (1, 3, and 5 nmol/kg) increased Fos-LI in the myenteric plexus (p<0.001) and in the DVC (p<0.001) compared to the saline vehicle. The highest dose of CCK-58 increased Fos-LI more than an equimolar dose of CCK-8 in the myenteric plexus and the area postrema. Thus, CCK-8 and CCK-58 produce the same qualitative pattern of activation of central and peripheral neurons, but do not provoke identical quantitative patterns at higher doses. The different patterns produced by the two peptides at higher doses, in areas open to the circulation (myenteric plexus and area postrema) may reflect endocrine actions not observed at lower doses.     

Effects of naloxone on basal and vagus nerve-induced secretions of GRP, gastrin, and somatostatin from the isolated perfused rat stomach

The effects of naloxone, an opiate antagonist, on basal and vagus nerve-induced secretions of GRP, gastrin, and somatostatin were examined using the isolated perfused rat stomach prepared with vagal innervation. Naloxone (10(-6) M) significantly inhibited basal somatostatin secretion in the presence and absence of atropine and of hexamethonium, whereas basal GRP and gastrin secretion was not affected by naloxone. Electrical stimulation (10 Hz, lms duration, 10V) of the distal end of the subdiaphragmatic vagal trunks elicited a significant increase in both GRP and gastrin but a decrease in somatostatin. Naloxone (10(-6) M) failed to affect these responses in the presence or absence of atropine. On the other hand, when hexamethonium was infused, naloxone significantly inhibited both the GRP and gastrin responses to electrical vagal stimulation. Somatostatin secretion was unchanged by vagal stimulation during the infusion of hexamethonium with or without naloxone. These findings suggest that basal somatostatin secretion is under the control of an opiate neuron and that opioid peptides might be involved in vagal regulation of GRP and gastrin secretion.     

Central action of somatostatin analog, SMS 201-995, to stimulate gastric acid secretion in rats

The effects of intracisternal and intravenous injections of the somatostatin analog, SMS 201-995, on gastric acid secretion were investigated in rats with pylorus ligation or gastric cannula. Intracisternal injection of SMS 201-995 induced a dose-related (0.1-0.3 microgram) and long-lasting stimulation of gastric acid output with a peak response at 3 h postinjection in conscious, pylorus-ligated rats. Intracisternal SMS 201-995 increased histamine levels in the portal blood, whereas plasma gastrin levels were not modified. Atropine, cimetidine and adrenalectomy abolished the stimulatory effect of intracisternal SMS 201-995 (0.3 microgram). SMS 201-995 (0.03 microgram), microinjected unilaterally into the dorsal vagal complex, increased gastric acid output in urethane anesthetized rats. SMS 201-995, injected intravenously at 0.5 microgram, did not alter gastric secretion, whereas higher doses (5-20 micrograms) resulted in a dose-related inhibition of gastric acid secretion in conscious pylorus-ligated rats. These data indicate that SMS 201-995, a selective ligand for somatostatin-1 receptor subtype, induces a centrally mediated stimulatory effect on gastric acid secretion in rats. The central action involves the parasympathetic system, muscarinic and H2 receptors as well as adrenal-dependent pathways.     

Prevention of pancreatic reactions by bolus somatostatin administration in patients undergoing endoscopic retrograde cholangio-pancreatography and endoscopic sphincterotomy

Mild pancreatitis is a common complication of endoscopic retrograde cholangio-pancreatography (ERCP) and endoscopic sphincterotomy. Knowing that a bolus injection of natural somatostatin (SRIF) dramatically reduces pancreatic secretion, a study was conducted in 33 subjects undergoing invasive diagnostic procedures. A placebo (n = 16) or SRIF (n = 17; 4 micrograms/kg) were injected before cannulation. Enzymatic rise was observed in 16 (94%) subjects receiving placebo and in 8 (50%) injected previously with SRIF. In the former group 65% reported abdominal pain whereas only 19% had this complaint in the SRIF series. Results suggest that a bolus injection of SRIF may attenuate pancreatic irritation caused by diagnostic procedures or sphincterotomy.     

Physiological significance of gastrointestinal somatostatin

Somatostatin participates in the regulation of nutrient entry from the intestinal tract into the circulation. Thus, dietary fats and proteins may elicit significant increases of gastropancreatic somatostatin. Regulation of postprandial somatostatin secretion may occur via neural, hormonal and humoral factors. This peptide, in pharmacological doses, inhibits virtually all gastrointestinal exo- and endocrine functions as well as local motor activity. Neutralization of endogenous circulating somatostatin with specific antiserum is followed by increases in GH and enteroglucagon, augmenting also the postprandial rise of gastrin, insulin and pancreatic polypeptide. Somatostatin deficiency can be observed in obese subjects with hyperinsulinism. Concomitant elevation of insulin and gastrin levels can be antagonized by exogenous somatostatin. These findings confirm the importance of somatostatin as a peripheral regulator in experimental and human biology.     

Lack of effect of somatostatin on the stimulation of hepatic glycogenolysis by epinephrine in isolated canine hepatocytes

The effects of somatostatin on epinephrine's ability to stimulate glucose output have been examined in hepatocytes isolated from dogs fasted overnight. Half-maximal stimulation of phosphorylase a activity and glucose output occurred at an epinephrine concentration of approx. 5 X 10(-9) M. Somatostatin at 10, 100 or 1000 ng/ml had no effect on the ability of a maximal (1 X 10(-7) M) and a submaximal (1 X 10(-8) M) dose of epinephrine to activate phosphorylase at 2 min, or to stimulate glucose output over 20 min. Since the doses of somatostatin used in the present study are up to 50-fold higher than the blood concentrations commonly found when somatostatin is used in vivo to inhibit pancreatic hormone secretion, it seems unlikely that use of somatostatin in this way would affect stimulation of hepatic glycogenolysis by epinephrine in vivo.     

Binding of somatostatin to guinea-pig pancreatic membranes: regulation by ions

Somatostatin receptors were characterized on guinea-pig pancreatic acini membranes using 125I-[Tyr11] somatostatin 14 as a radioligand. In 0.1 mM Ca2+ buffer the binding was saturable and slowly reversible, exhibiting a single class of high affinity binding sites (KD = 0.15 +/- 0.03 nM) with a maximal binding capacity (B max) of 178 +/- 18 fmol/mg protein. In 30 nM) free Ca2+ buffer, the binding was highly reversible. Affinity and B max were decreased by about 2-fold. Ca2+ exhibited an EC50 of 2.4 +/- 0.9 microM to potentiate the binding of somatostatin. Na+, but not K+, inhibited the binding: Bmax was decreased with no change in affinity. Somatostatin analogs inhibited the binding of 125I-[Tyr11] somatostatin 14. The relative potencies were: somatostatin 14 greater than somatostatin 28 = [Nle8]somatostatin 28 greater than [D Tryp8, D Cys14]somatostatin 14.     

Differential mechanism and site of action of CCK on the pancreatic secretion and growth in rats

Recent studies demonstrated that cholecystokinin (CCK) at physiological levels stimulates pancreatic enzyme secretion via a capsaicin-sensitive afferent vagal pathway. This study examined whether chemical ablation of afferent vagal fibers influences pancreatic growth and secretion in rats. Bilateral subdiaphragmatic vagal trunks were exposed, and capsaicin solution was applied. Pancreatic wet weight and pancreatic secretion and growth in response to endogenous and exogenous CCK were examined 7 days after capsaicin treatment. Perivagal application of capsaicin increased plasma CCK levels and significantly increased pancreatic wet weight compared with those in the control rats. Oral administration of CCK-1 receptor antagonist loxiglumide prevented the increase in pancreatic wet weight after capsaicin treatment. In addition, continuous intraduodenal infusion of trypsin prevented the increase in plasma CCK levels and pancreatic wet weight after capsaicin treatment. There were no significant differences in the expression levels of CCK-1 receptor mRNA and protein in the pancreas in capsaicin-treated and control rats. Intraduodenal administration of camostat or intravenous infusion of CCK-8 stimulated pancreatic secretion in control rats but not in capsaicin-treated rats. In contrast, repeated oral administrations of camostat or intraperitoneal injections of CCK-8 significantly increased pancreatic wet weight in both capsaicin-treated and control rats. Present results suggest that perivagal application of capsaicin stimulates pancreatic growth via an increase in endogenous CCK and that exogenous and endogenous CCK stimulate pancreatic growth not via vagal afferent fibers but directly in rats.     

Long-term effect of somatostatin 14 on mouse stomach, antrum, intestine and exocrine pancreas

Mice were injected 3 times a day for 12 days with 8 micrograms/kg of somatostatin 14 which caused a hypoplasia of parietal and goblet cells, a hypotrophy and hypofunctionality of pancreatic acinar cells with a decrease in lipase and chymotrypsin activities, a decrease in the secretory fuction of the Brunner gland and in the number of dark granules of G cells. Neither villous and microvillous areas nor brush border hydrolase activities were affected. The number of peptic cells and Paneth cells increase as the level of pepsin and lysozyme. Mice were injected 4 times per hour with 2 micrograms/kg of somatostatin. 2 h after the first injection of somatostatin and 90 min after a single injection of tritiated thymidine, fundic, antral, jejunal and ileal labelling indexes strongly decrease (maximal effect in ileum). The inhibitory effect of somatostatin on the digestive epithelial cell proliferation compared to its long-term action only directed on specific cell types evokes probable compensatory mechanisms induced to maintain the equilibrium of the digestive epithelia.