Stimulus-secretion coupling of arginine-induced insulin release. Functional response of islets to L-arginine and L-ornithine.

L-Arginine and L-ornithine stimulate insulin release from pancreatic islets exposed to D-glucose. This coincides with an increased outflow of 86Rb and 45Ca from prelabelled islets and an increased net uptake of 45Ca by the islets. In the presence of D-glucose, L-lysine stimulates insulin secretion to the same extent as L-arginine or L-ornithine, but the hormonal release is not further enhanced by combinations of these cationic amino acids. L-Arginine or L-ornithine failed to enhance insulin release evoked by either L-leucine or 2-ketoisocaproate. The inhibitor of ornithine decarboxylase D,L-alpha-difluoromethyl ornithine failed to affect the metabolism and insulinotropic action of D-glucose in pancreatic islets, and only caused a partial inhibition of the secretory response to either L-arginine or L-ornithine. The latter amino acids inhibited modestly but significantly D-glucose utilization and oxidation by pancreatic islets. These and complementary findings suggest that the secretory response to L-arginine and L-ornithine is not attributable to any major change in the overall oxidative catabolism of nutrients, but involves mainly a biophysical component, such as the depolarization of the plasma membrane by these cationic amino acids.

The activation of adenylate cyclase by pituitary adenylate cyclase activating polypeptide (PACAP) via helodermin-preferring VIP receptors in human SUP-T1 lymphoblastic membranes.

Competition binding curves, using [125I-acetyl-His1]PACAP-27 as radioligand and dose-effect curves of adenylate cyclase activation in human SUP-T1 lymphoblastic membranes showed that PACAP-27 and PACAP-38 stimulate the enzyme through a single class of helodermin-preferring VIP receptors with the following order of potency: helodermin = [acetyl-His1]PACAP-27 greater than PACAP-38 greater than PACAP-27 greater than VIP. PACAP (6-27) (Ki 0.5-0.8 microM) and [Des-His1, Asn3]PACAP-27 (Ki 1-2 microM) acted as competitive antagonists. Using a series of 13 PACAP-27 analogues and fragments and three VIP analogues, we identified positions 1, 2, 3, 9 and 13 in PACAP-27 as being of importance for high-affinity binding. Thus, we added further evidence for considering that the present helodermin-preferring VIP receptors, when compared to a majority of VIP receptors and PACAP receptors, exhibit an original specificity pattern.

Pituitary adenylate cyclase-activating polypeptide receptors of types I and II and glucagon-like peptide-I receptors are expressed in the rat medullary carcinoma of the thyroid cell line 6/23.

The expression of the messenger RNAs coding for glucagon-like peptide-I (GLP-I) receptor, VIP receptor, and pituitary adenylate cyclase-activating polypeptide (PACAP) receptor as well as the expression of the receptor proteins were demonstrated in the rat medullary carcinoma of thyroid cell line 6/23 by the following experiments: 1) RNA extraction, reverse transcriptase, and polymerase chain reaction with specific primers; 2) binding of the radiolabeled ligands [125I]GLP-I-(7-36)-NH2, [125I]PACAP-(1-27), and [125I]VIP and inhibition by, respectively, unlabeled GLP-I-(7-36)-NH2, PACAP-(1-27), and VIP; and 3) study of adenylate cyclase activation by the peptides and selective inhibition of the VIP/PACAP response by the antagonist [D-Phe2]VIP. Besides the highly selective GLP-I receptor, PACAP receptors of types I and II were present on the cell line and coupled to adenylate cyclase. PACAP stimulated the adenylate cyclase through type I and II receptors, whereas VIP interacted with type II receptors only. Messenger RNA analysis indicated that at least three splice variants of the PACAP type I receptor may be expressed in 6/23 cells.

Multiple effects of leucine on glucagon, insulin, and somatostatin secretion from the perfused rat pancreas.

The effects of increasing concentrations of leucine (0.2, 2.0, and 15.0 mmol/liter) on glucagon secretion from the perfused rat pancreas were examined at various glucose levels (0, 3.3, or 8.3 mmol/liter) and in the absence or presence of either arginine (5.0 mmol/liter) or glutamine (10.0 mmol/liter). At a low glucose concentration (3.3 mmol/liter), leucine caused a dose-related biphasic increase in glucagon output in the absence of arginine, but only a transient increase in the presence of the latter amino acid. These positive responses were markedly reduced and, on occasion, abolished at a high glucose concentration (8.3 mmol/liter). Moreover, at a low glucose concentration (3.3 mmol/liter) and in the presence of arginine, the highest concentration of leucine (15.0 mmol/liter) provoked a sustained and reversible inhibition of glucagon release. Likewise, leucine (15.0 mmol/liter) reversibly inhibited glucagon secretion evoked by glutamine in the absence of glucose. Thus, leucine exerted a dual effect on the secretion of glucagon, the inhibitory effect of leucine prevailing at a high concentration of the branched chain amino acid and when glucagon secretion was already stimulated by arginine or glutamine. At a physiological concentration (0.2 mmol/liter), however, leucine was a positive stimulus for glucagon release, especially in the absence of another amino acid. Concomitantly, leucine was always a positive stimulus for both insulin and somatostatin secretion. The intimate mechanisms involved in the dual effect of leucine on glucagon secretion remain to be elucidated.

The two forms of the pituitary adenylate cyclase activating polypeptide (PACAP (1-27) and PACAP (1-38)) interact with distinct receptors on rat pancreatic AR 4-2J cell membranes.

The existence of specific receptors for the two PACAPs (Pituitary Adenylate Cyclase Activating Peptides of 27 and 38 amino acids) was previously demonstrated on membranes from the pancreatic acinar cell line AR 4-2J (Buscail et al., FEBS Lett. 202, 77-81, 1990) by [125I]PACAP-27 binding. Here we demonstrate, by comparing Scatchard analysis of saturation curves and competition binding curves obtained with [125I]PACAP-27 and [125I]PACAP-38 as radioligands, the coexistence of two classes of receptors: 1/PACAP-A receptors that recognize PACAP-27 and PACAP-38 with the same high affinity (Kd 0.3 nM) and 2/PACAP-B receptors that recognize PACAP-38 with a high affinity (Kd 0.3 nM) and PACAP-27 with a lower affinity (Kd 30 nM). These two receptors are coupled to adenylate cyclase but can be clearly distinguished by the ability of PACAP(6-27) to specifically inhibit PACAP-27 adenylate cyclase activation.

Presence of helodermin-like peptides of the VIP-secretin family in mammalian salivary glands and saliva.

Helodermin is a biologically active peptide isolated from the venom of the Gila monster lizard (Heloderma suspectum) whose structure is related to that of vasoactive intestinal peptide and secretin. Using a specific radioimmunoassay based on antisera prepared by immunizing rabbits with natural helodermin, we demonstrated the presence of helodermin-like material in mammalian salivary glands, including parotid, submaxillary and sublingual glands from rat and dog, and parotid and submaxillary glands from man. All helodermin-like materials had an apparent molecular mass of 4-12 kDa. Dog saliva, collected after pilocarpine stimulation, revealed similar immunoreactivity with a major component around 6 kDa.

Failure to pentagastrin administration to restore postprandial acid secretion from Heidenhain pouches after antrectomy in dogs.

In four dogs with Heidenhain pouche, acid outputs from the pouch were measured in response to feeding boiled liver with or without simultaneous administration of pentagastrin. Antrectomy was performed and the experiments were repeated. Antrectomy decreased the acid response to feeding by 90 percent. The preantrectomy response was restored by infusing pentagastrin but near maximal doses were needed and the maximal response to feeding plus pentagastrin could not be restored. These results indicate that the decrease of the acid secretory response to feeding from Heidenhain pouches after antrectomy cannot be explained fully through suppression of endogenous gastrin release. Antrectomy probably interferes with the intestinal phase of gastric secretion.

Chromogranin A(210-301) is the major form of pancreastatin-like material in human gut extracts and endocrine tumors.

A radioimmunoassay of human pancreastatin was developed using a rabbit antiserum that selectively recognized the C-terminal amidated end of the peptide, and it was used for the identification of the molecular forms of pancreastatin in human gut (stomach, duodenum, small intestine, colon) and endocrine tumor extracts (liver metastasis of a gastrinoma and a medullary carcinoma of thyroid, one nonsecreting pancreatic tumor, one recurrence of a gut carcinoid, one vipoma and one insulinoma). In all gut extracts, a gel filtration chromatography revealed the presence of three peaks of pancreastatin-like immunoreactivity. The predominant form eluted with an apparent molecular weight higher than that of pancreastatin. This form was also predominant in the endocrine tumors analyzed, except in the insulinoma, where a lower molecular weight form predominated. The high molecular form was further purified from a liver metastasis of a gastrinoma. The pancreastatin-like immunoreactivity eluted in all the chromatographical systems (reverse-phase, ion exchange) as a single peak that was finally purified to homogeneity and sequenced. The sequence of the first 29 N-terminal amino acids was obtained unambiguously and corresponded to the sequence 210-238 of chromogranin A. Considering the selectivity of the assay used for peptide identification, this major form was identified as the fragment 210-301 of chromogranin A. It is likely that the predominant form of pancreastatin in human gut extracts and noninsular tumors is a 92 amino acid peptide.

Identification of a new chromogranin B fragment (314-365) in endocrine tumors.

A rabbit antiserum was raised against the fragment (350-365) of human chromogranin B corresponding to the C-terminal end of a putative proteolytic fragment generated by the cleavage of a dibasic doublet located in position 366-367 of the precursor. A radioimmunoassay was developed. Chromatographic analysis of 10 endocrine tumor extracts (one liver metastasis of a gastrinoma, one liver metastasis of a medullary carcinoma of the thyroid, one VIPoma, one insulinoma, one nonsecreting pancreatic endocrine tumor, one local recurrence of a gut carcinoid, two pituitary gonadotropinoma, and two non-secreting pituitary adenomas) revealed the presence of two forms of immunoreactive material. The most abundant form had an apparent molecular weight of 4500 and was purified to homogeneity by successive reverse-phase HPLC chromatographies and partially sequenced. The N-terminal sequence of the peptide, established by automated Edman degradation, was A-S-E-E-E-P-E-Y-G-E-E-I-K-G-Y-P-V-Q and corresponded to the 314-332 sequence of human chromogranin B. Taking into account the specificity of the antiserum used for peptide identification, we deduced that the purified peptide was chromogranin B(314-365) and represented a new form generated by limited proteolysis of chromogranin B.

Expression of pituitary adenylate cyclase activating polypeptide (PACAP) receptors in human glial cell tumors.

Twenty-three human gliomas were analyzed: 13 astroglial neoplasms including three grade II, four grade III, and six grade IV tumors; seven ependymomas; and three oligodendrogliomas. A crude membrane fraction was prepared within 30 min after surgical removal of the tumors and was immediately tested for the presence of pituitary adenylate cyclase activating polypeptide (PACAP) receptors. PACAP stimulated adenylate cyclase activity in 23 tumors, but a specific binding of [125I-acetyl-His1]PACAP-27 was detected in only 16 tumors. In all cases, PACAP-27 and -38 were equipotent (Kd or Kact of 1-3 nM) and were 100- to 1000-fold more potent than VIP. PACAP stimulated threefold the adenylate cyclase activity in the presence of GTP. The results were compatible with an interaction of PACAP with a highly selective type I PACAP receptor and not with a high-affinity VIP/PACAP type II receptor. The presence of PACAP receptors on glial neoplasic opens the possibility of a control of the tumor growth by this family of peptides.

Effect of cimetidine, ranitidine and omeprazole on postprandial gastrin and somatostatin release in conscious dogs.

During a first series of experiments, the gastrin responses to a meal were measured and compared to the responses seen after administration of cimetidine (2.5 mg/kg/h) or omeprazole (2 mg/kg). During a second series of experiments the effects of cimetidine (2.5 mg/kg/h), ranitidine (0.5 mg/kg/h) and omeprazole (2 mg/kg) on post-prandial gastrin and somatostatin release were determined in experiments during which the intragastric pH was maintained close to 6.4. During a third series of experiments, the effects of cimetidine (2.5 mg/kg/h) and omeprazole (2 mg/kg) on basal gastrin and somatostatin release were estimated. Postprandial gastrin release was increased by cimetidine and by omeprazole. When acidification of the gastric content was prevented by intragastric titration, postprandial gastrin release was increased by about 100%. No further increase was observed when the animals were concomitantly treated with cimetidine, ranitidine or omeprazole. Intragastric titration did not alter postprandial somatostatin release. Concomitant administration of H2 blockers decreased the somatostatin response to the meal, while concomitant administration of omeprazole did not alter this release. No significant changes were observed in basal gastrin or somatostatin levels after administration of cimetidine or omeprazole.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of vagotomy and atropine on plasma somatostatin response to a meal in conscious dogs.

In 4 conscious dogs with gastric fistulas the somatostatin responses to a meal were measured and compared to the responses seen after i.v. infusion of atropine sulfate (20 and 50 micrograms.kg-1.h-1) or cimetidine (8 mg.kg-1.h-1). The experiments were repeated after truncal vagotomy. The somatostatin responses to bombesin (0.5 micrograms.kg-1.h-1) were also measured before and after vagotomy. Vagotomy decreased basal and postprandial somatostatin levels and reduced the somatostatin responses to feeding during the first 30-min period following the ingestion of the meal but not during subsequent periods. Bombesin-induced somatostatin release was increased after vagotomy. Atropine decreased the somatostatin responses to the meal before and after vagotomy. Cimetidine had no significant effect. These studies suggest that, in conscious dogs, somatostatin released into the circulation is partly under vagal control and that, as for gastrin release, vagal pathways for stimulation and inhibition are present. Our studies also suggest that cholinergic mechanisms are involved in the control of postprandial somatostatin release.

Presence of calcitonin gene-related peptide receptors coupled to adenylate cyclase in human gliomas.

Eleven surgical samples of gliomas (1 of grade II, 3 of grade III and 7 of grade IV) were analyzed. Calcitonin gene-related peptide (CGRP) receptors were identified by 125I-alpha h-CGRP binding in 9 cases and the presence of a CGRP-stimulated adenylate cyclase in all the 11 cases. Tracer binding was inhibited by unlabelled alpha h-CGRP (Kd of 0.3 nM), by (8-37) alpha h-CGRP (Kd of 30 nM), by (12-37) alpha h-CGRP (Kd of 3.000 nM) but not by human calcitonin. The mean density of CGRP receptors (120 fmol/mg membrane protein) was comparable to that of beta-adrenergic receptors. CGRP stimulated 1.4 to 4.7-fold (mean 2.7) the adenylate cyclase activity with a K(act) of 2.0 nM. The CGRP fragments had no intrinsic activity but inhibited the CGRP effect. The (8-37)CGRP fragment had a Ki of 30 nM. Thus, at variance with previous reports on rat and human brain membranes, that showed the presence of CGRP receptors not coupled to adenylate cyclase, we observed in human gliomas the presence of CGRP receptors that, when occupied, stimulated efficiently the adenylate cyclase activity.

Effect of met-enkephalin on acid secretion from gastric fistulas and Heidenhain pouches in dogs stimulated by pentagastrin, pentagastrin plus bethanechol, or a meal.

In dogs with denervated Heidenhain pouches and gastric cannulas the authors studied the action of met-enkephalin on acid secretion stimulated by a liquid meal, pentagastrin or pentagastrin + bethanechol. Serum gastrin levels were determined during the feeding experiments. Meal-stimulated and pentagastrin-stimulated acid secretion from the gastric fistula were inhibited by met-enkephalin. A rise in pentagastrin-induced acid secretion was observed in the denervated stomach. No significant changes were observed in the postprandial acid response from the pouch to the meal, nor from the main stomach or the pouch during stimulation with pentagastrin + bethanechol. Gastrin release was decreased by met-enkephalin during the first hour following feeding and increased during the second hour. Our data indicate that met-enkephalin can either inhibit or stimulate acid secretion in dogs. The inhibition occurs only in the innervated fundic mucosa, and might be explained by a decrease in gastrin release and by a decrease in vagal tone.

Increase in gastrin and somatostatin cell numbers in the antrum of dogs after small bowel resection.

A proximal 50 p. 100 resection of the small bowel was performed in six dogs while six control animals underwent a simple intestinal transsection and reanastomosis. The pre- and postoperative serum gastrin levels were measured in both groups before and after a standard test meal. The stomach was resected in both groups 6 to 8 weeks after the operation. A morphometric method using specific antigastrin and antisomatostatin antibodies was used to estimate the density and the total number of antral gastrin and somatostatin cells. No variation in basal serum gastrin was found after surgery. A 50 p. 100, though not significant, increase in the postprandial gastrin response was observed after intestinal resection whereas a significant (P less than 0.01) 50 p. 100 increase in the gastrin cell density was found in the antral mucosa of these dogs. At the same time, a remarkable hyperplasia of the antral somatostatin cell population was observed in the animals submitted to small bowel resection.

[Interactions between secretin and somatostatin on acid secretion and gastric emptying in dogs]. / Interactions entre la sécrétine et la somatostatine sur la sécrétion acide et la vidange gastrique chez le chien.

Secretin and somatostatin are two peptides released by H+ ions. The fact that blood levels of these peptides increase during the postprandial period makes them the most probable candidates for gastric acid secretion retrocontrol . The aim of our study was to determine whether a potentiation of inhibitory effects exists when the two peptides are administered simultaneously. Seven dogs were provided with a gastric fistula. Gastric acid secretion was stimulated by intragastric infusion of a bactopeptone solution or by pentagastrin (1 microgram X kg-1 X h-1). Acid outputs and gastric emptying rates were measured at regular intervals. The tests were repeated during intravenous infusion of secretin (0.125 micrograms X kg-1 X h-1), somatostatin (0.2 micrograms X kg-1 X h-1) or both peptides together at the same doses. Both secretin and somatostatin inhibited acid secretion and gastric emptying. The inhibition was not greater when both peptides were administered together. Under the conditions used, no potentiation or additive effects between the two peptides were observed on gastric acid secretion or gastric emptying.

[Basal concentrations and postprandial integrated flows of gastrin in patients with atrophic gastritis or duodenal ulcer. Limits of diagnostic usefulness]. / Concentrations basales et débits intégrés post-prandiaux de gastrine chez des malades atteints de gastrite atrophique ou d'ulcère duodénal. Limites à l'utilité diagnostique.

Maximal acid outputs were determined during intravenous pentagastrin tests (6 micrograms/kg/h) in 119 male subjects: 17 controls, 74 patients with duodenal ulcer and 28 with atrophic gastritis. Basal and postprandial serum gastrin levels were also determined in order to estimate the integrated gastrin response to the meal. In patients with atrophic gastritis the maximal acid output was decreased (p less than 0.01) and the integrated gastric response was increased (p less than 0.01) but the basal gastrin levels in these patients did not differ from that of controls. An integrated gastrin response greater than 2.5 ng/ml/100 min was observed in 89 p. 100 of patients with atrophic gastritis. An integrated gastrin response smaller than 2.5 ng/ml/100 min was observed in 76 p. 100 of controls. The maximal acid output was smaller than 20 mmol/l in all patients with atrophic gastritis but was greater than this value in all controls. In duodenal ulcer patients, the measured parameters were not significantly different from control values. The measure of the integrated gastrin response which reflects the presence of an antral endocrine hyperactivity may be useful to detect patients with atrophic gastritis, but this test is less sensitive and less specific than the determination of the maximal acid output.

[Physiological control and pharmacological inhibition of digestive secretions]. / Contrôle physiologique et inhibition pharmacologique des sécrétions digestives.

The pharmacological treatment of alimentary tract fistulas was for a long time limited to the use of atropine and related substances. More recently H2 blockers, pirenzepine, omeprazole and loperamide have been used. It is likely that in the coming years specific and long acting derivatives of somatostatin and enkephalins inhibiting digestive secretions will be synthetized.