Molecular cloning and in vitro properties of the recombinant rabbit secretin receptor

The rabbit secretin receptor cDNA was cloned from rabbit pancreas using combined polymerase chain reaction (PCR)/rapid amplification of cDNA ends (PCR/RACE) approaches. The rabbit cDNA encoded 445 amino acids and had 80 and 85% homology with rat- and human receptor, respectively, in terms of nucleic and amino acid sequences. Several regions where the rabbit receptor sequence diverged from the rat/human receptor sequences were observed in the putative extracellular domains of the receptor. A cDNA coding for a similar sequence with a 76 bp deletion after the 5th transmembrane domain was also found; it probably encoded an inactive protein. The whole rabbit secretin receptor cDNA was subcloned in expression vector pCR3.1, then stably and transiently transfected in Chinese hamster ovary (CHO) cells. The pharmacological properties of the rat and rabbit secretin receptor studies were compared by radiolabeled secretin binding, binding inhibition, and adenylate cyclase activation (using secretin analogs and fragments). Porcine secretin was equipotent with rabbit secretin on the rabbit secretin receptor, but fivefold more potent than rabbit secretin on the rat receptor. This was due to the serine → arginine residue replacement, in position 16 of rabbit secretin. Amino terminal modified secretin analogs (secretin(2–27), [E³]secretin, [N³]secretin) and VIP were less potent than secretin on both secretin receptors, but more potent on the rabbit than on the rat receptor. The carboxy-terminally truncated fragment (1–26) had the same reduced potency on rat and rabbit receptors. Thus, the rabbit secretin receptor had original properties, different from those of the rat receptor.     

Secretin Stimulates Cyclic AMP Formation in the Rat Brain

The effects of secretin on cyclic AMP levels in the rat brain were determined. Incubation of rat brain frontal cortex slices with secretin or the structurally related peptides peptide histidine leucine (PHI) or vasoactive intestinal polypeptide (VIP) in the presence of 10 mM theophylline resulted in a dose-dependent increase in the cyclic AMP levels. The half-maximal increase in cyclic AMP occurred using a 1 microM dose of secretin or a 2 microM dose of PHI or VIP. Preincubation of slices with secretin-(5-27) produced a dose-dependent inhibition of the secretin but not VIP- or PHI-stimulated increase in the cyclic AMP content. Also, in receptor binding studies, secretin-(5-27) produced a dose-dependent inhibition (Ki = 400 nM) of 125I-secretin but not of 125I-VIP binding to rat brain membranes. Guanyl-5'-yl imidodiphosphate decreased the affinity of radiolabelled secretin binding as a result of an increased rate of dissociation of bound 125I-secretin. These data suggest that secretin receptors in the rat brain may be coupled to adenylate cyclase in a stimulatory manner and that secretin-(5-27) may function as a central secretin receptor antagonist.     

Pharmacology and molecular identification of secretin receptors in rat gastric glands

The structure of the secretin receptor in purified plasma membranes isolated from the antral and fundic parts of the rat gastric mucosa was probed, using the cross linking reagent dithiobis succinimidyl propionate (DSP) and HPLC-purified [125I] secretin. [125I] secretin binding sites were preferentially located in rat antrum and displayed the pharmacological properties expected for specific secretin receptors: secretin greater than helodermin greater than rhGRF greater than rPHI. SDS gel electrophoresis of the solubilized receptor allowed identification of two radiolabeled peptides of 62 and 33 KDa connected by disulfide bonds. According to the sensitivity of the 62 KDa component to low doses of secretin and to GTP, it constitutes the membrane domain involved in the physiological regulation of adenylate cyclase by secretin in rat gastric glands.     

Molecular cloning and expression of a cDNA encoding the secretin receptor.

Secretin is a 27 amino acid peptide which stimulates the secretion of bicarbonate, enzymes and potassium ion from the pancreas. A complementary DNA encoding the rat secretin receptor was isolated from a CDM8 expression library of NG108-15 cell line. The secretin receptor expressed in COS cells could specifically bind the iodinated secretin with high and low affinities. Co-expression of the secretin receptor with the alpha-subunit of rat Gs protein increased the concentration of the high affinity receptor in the membrane fraction of the transfected COS cells. Secretin could stimulate accumulation of cAMP in COS cells expressing the cloned secretin receptor. The nucleotide sequence analysis of the cDNA has revealed that the secretin receptor consists of 449 amino acids with a calculated Mr of 48,696. The secretin receptor contains seven putative transmembrane segments, and belongs to a family of the G protein-coupled receptor. However, the amino acid sequence of the secretin receptor has no significant similarity with that of other G protein-coupled receptors. A 2.5 kb mRNA coding for the secretin receptor could be detected in NG108-15 cells, and rat heart, stomach and pancreatic tissue.     

Different actions of secretin and Gly-extended secretin predict secretin receptor subtypes

Only one secretin receptor has been cloned and its properties characterized in native and transfected cells. To test the hypothesis that stimulatory and inhibitory effects of secretin are mediated by different secretin receptor subtypes, pancreatic and gastric secretory responses to secretin and secretin-Gly were determined in rats. Pancreatic fluid secretion was increased equipotently by secretin and secretin-Gly, but secretin was markedly more potent for inhibition of basal and gastrin-induced acid secretion. In Chinese hamster ovary cells stably transfected with the rat secretin receptor, secretin and secretin-Gly equipotently displaced (125)I-labeled secretin (IC(50) values 5.3 +/- 0.5 and 6.4 +/- 0.6 nM, respectively). Secretin, but not secretin-Gly, caused release of somatostatin from rat gastric mucosal D cells. Thus the equipotent actions of secretin and secretin-Gly on pancreatic secretion appear to result from equal binding and activation of the pancreatic secretin receptor. Conversely, secretin more potently inhibited gastric acid secretion in vivo, and only secretin released somatostatin from D cells in vitro. These results support the existence of a secretin receptor subtype mediating inhibition of gastric acid secretion that is distinct from the previously characterized pancreatic secretin receptor.     

Heart receptors for VIP, PHI and secretin are able to activate adenylate cyclase and to mediate inotropic and chronotropic effects. Species variations and physiopathology

We have assessed the presence of VIP/PHI/secretin receptors in heart by: (1) testing the ability of the corresponding peptides to activate adenylate cyclase in cardiac membranes from rat, dog, Cynomolgus monkey and man, and (2) examining the ability of the same peptides to exert inotropic and chronotropic effects on heart preparations from rat and Cynomolgus monkey in vitro. Based on their affinity for natural peptides and synthetic analogs, two types of VIP/PHI/secretin receptors were characterized: the relatively nonspecific "secretin/VIP receptor" of rat heart (that is "secretin-preferring" only in that secretin was more efficient than VIP in stimulating adenylate cyclase), and the "VIP/PHI-preferring" receptor of man, monkey and dog heart. Four physiopathological situations affecting secretin/VIP receptors in rat heart were explored: In male rats from the Okamoto strain and the Lyon strain, two strains presenting spontaneous hypertension, heart membranes exhibited a markedly decreased response of adenylate cyclase to secretin/VIP, with lesser alterations in the responses to isoproterenol and glucagon. This impairment developed in parallel with the occurrence of hypertension and was reproduced in normotensive rats submitted to chronic isoproterenol treatment (but not in Goldblatt hypertensive rats). These findings are consistent with a hyperactivity of norepinephrine pathways in spontaneously hypertensive rats, leading to a reduced number of cardiac post-junctional secretin/VIP receptors bound to adenylate cyclase. Heart membranes from genetically obese (fa/fa) Zucker rats also exhibited severely decreased responses to secretin/VIP with lesser alterations in the responses to glucagon and isoproterenol. These anomalies were specific for the heart, and developed in concomitance with obesity. The first anomaly could not be corrected by severe food restriction. Secretin stimulation of heart adenylate cyclase was also selectively altered in streptozotocin-diabetic rats. Thus, two types of diabetic cardiomyopathy were characterized by a severe local alteration of secretin/VIP receptors coupled to adenylate cyclase. Hypothyroidism, provoked in rat by thyroidectomy or propylthiouracil treatment, again induced a marked decrease in secretin-stimulated cardiac adenylate cyclase activity. In rat papillary muscle electrically stimulated in vitro, secretin exerted a positive inotropic effect. This effect was reduced in obese (fa/fa) Zucker rats. In rat right atrium, secretin also exerted a positive chronotropic effects.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rat pancreas adenylate cyclase. VI. Role of the enzyme in secretin stimulated enzyme secretion.

1. The responsiveness of adenylate cyclase and enzyme secretin for secretin and the C-terminal octapeptide of pancreozymin has been investigated in particulate fractions of the pancreas of five different species. 2. The adenylate cyclase is sensitive to the C-terminal octapeptide of pancreozymin in all species investigated. 3. The enzyme is much more sensitive to secretin in rat and cat than in mouse and rabbit, whereas with guinea pig intermediate values are obtained. 4. The enzyme secretion is stimulated by secretin in pancreatic fragments of rat and cat, but not in those of mouse and rabbit. 5. These results suggest that in species where secretin stimulated enzyme secretion, it does so by stimulating the adenylate cyclase system.     

Evidence that helodermin, a newly extracted peptide from Gila monster venom, is a member of the secretin/VIP/PHI family of peptides with an original pattern of biological properties

Helodermin, a newly isolated peptide from the venom of Gila monster (Heloderma suspectum) was shown to stimulate the adenylate cyclase activity of rat pancreatic membranes as efficiently as secretin and VIP. It also increased cyclic AMP levels and inhibited [125I]VIP binding in rat pancreatic acini. Finally, helodermin activated adenylate cyclase in membranes from rat heart, rat brain, and human heart, showing properties analogous yet distinct from those of secretin, VIP and PHI.     

Dog secretin: sequence and biologic activity

Secretin was purified from the first 50 cm of proximal intestine of a single dog. The purification sequence included extraction in acid-ethanol, differential precipitation with acetone, Sephadex gel filtration and three successive HPLC steps. Dog secretin has the following sequence: HSDGTFTSELSRLRESARLQRLLQGLV. The underlined amino acid represents a substitution of Glu for Asp at position 15 from the NH2-terminus of pig secretin. Dog secretin is equal in potency to pig secretin in stimulating pancreatic ductal flow in a rat bioassay system.     

促胰液素对血管灌流大鼠离体胃运动的抑制作用

本工作采用血管灌流大鼠离体胃制备,探讨促胰液素对胃运动的影响。结果表明:(1)促胰液素能明显抑制胃窦自发和五肽胃泌素兴奋的胃运动;(2)抗促胰液素血清可完全取消促胰液素的抑制胃窦运动作用;(3)抗生长抑素血清和消炎痛都能阻断促胰液素的抑制胃窦运动作用。上述结果提示,促胰液素的抑制作用除通过直接作用于促胰液素受体外,还可能部分通过胃窦局部生长抑素和前列腺素介导来抑制胃的运动。     

Evidence for a cyclic AMP system highly sensitive to secretin in gastric glands isolated from the rat fundus and antrum

The effects of secretin and vasointestinal peptide (VIP) on the production of cyclic AMP have been studied in gastric glands isolated by means of EDTA from rat fundic and antral mucosa. (1) In gastric fundus, secretin and VIP caused a time- and temperature-dependent stimulation of cyclic AMP production that was maximal when the test agents were incubated for 60 min at 20 degrees C in the presence of 0.5 mM 3-isobutyl-1-methylxanthine as a phosphodiesterase inhibitor. The dose-response curve was monophasic for both peptides, the production of cyclic AMP being sensitive to 10(-10) M secretin and to 5 . 10(-8) M VIP. Half-maximal stimulation was obtained with 2.9 10(-9) M secretin or 2 . 10(-7) M VIP and the maximal stimulation represented a 21-fold and a 19-fold increase above control for secretin and VIP, respectively. Histamine also stimulated cyclic AMP production, with a Km of about 5 . 10(-4) M. No additive effect on cyclic AMP production was oberved when secretin and VIP were simultaneously added at maximally active concentrations, while an additive effect was observed when secretin and histamine were added together. (2) In gastric antrum, the characteristics of the secretin- and VIP-stimulated cyclic AMP production were similar to those observed in gastric fundus. Histamine nevertheless failed to stimulate the formation of cyclic AMP in antral mucosa. (3) These data demonstrate the existence of a cyclic AMP system highly sensitive to secretin in gastric glands isolated from the rat fundus and antrum and suggest that VIP operates through this system. (4) It is proposed that the pepsinogen- and/or mucous-secreting cells are implicated in the regulation of cyclic AMP production by secretin in gastric glands of the rat.     

Interaction of Gila monster venom with secretin receptors in rat pancreatic membranes

The stimulatory effect of Gila monster venom on adenylate cyclase activity in rat pancreatic membranes was compared to that of porcine secretin and porcine VIP. The maximal effect exerted by the venom was identical to that of VIP but significantly lower than that of secretin. The effect of Gila monster venom could, however, be attributed to its interaction with secretin receptors rather than with VIP receptors, at variance with its previously described action on guinea pig pancreatic acini. Adenylate cyclase activation by both Gila monster venom and secretin in rat pancreatic membranes was, indeed: (1) dose-dependently inhibited by two secretin fragments secretin-(4-27) and secretin-(7-27), and (2) more severely depressed than VIP stimulation, after pretreating pancreatic membranes with dithiothreitol (DTT).     

Interaction of GRF with VIP receptors and stimulation of adenylate cyclase in rat and human intestinal epithelial membranes. Comparison with PHI and secretin

GRF (10(-8) - 10(-5) M) is shown to inhibit competitively the binding of [125I]VIP to human and rat intestinal epithelial membranes. The affinity of GRF for VIP receptor is 700-800-times lower than that of VIP in both species. The order of affinity of different peptides is VIP greater than PHI greater than secretin greater than GRF in rat, and VIP greater than GRF greater than PHI greater than secretin in man. The important species specificity of VIP receptors in recognizing PHI and secretin does not occur in the case of GRF. GRF stimulates adenylate cyclase through its interaction with VIP receptors in rat and human membranes. However, while GRF behaves as a VIP agonist in human tissue, it is a partial agonist/antagonist of VIP in the rat.     

cAMP and in vitro inotropic actions of secretin and VIP in rat papillary muscle.

Secretin and VIP stimulate cardiac adenylyl cyclase activity and exert a positive inotropic action in several mammalian species. This study examined positive inotropic activity and cAMP levels in rat papillary muscle. Isoproterenol and secretin increased contractions by 150+/-31% and 129+/-27%, respectively. VIP increased contraction by 30+/-21% only at 10 microM. Isoproterenol significantly increased cAMP levels by 82%, whereas increases by secretin (58%) and VIP (56%) were not significant. These results are consistent with reports that secretin and VIP stimulate cardiac adenylyl cyclase in the rat, but suggest that cAMP tissue levels cannot totally explain the positive inotropic responses to secretin and VIP.     

Expression and motor effects of secretin in small and large intestine of the rat

Immunocytochemistry and in situ hybridization revealed abundant secretin expressing cells on duodenal villi with a gradual decrease throughout the small intestines of the rat. They were absent in pancreas, stomach and colon. Secretin caused relaxation of rat intestinal longitudinal muscle in vitro. Studies on colon revealed that the secretin-evoked response was unaffected by apamin, tetrodotoxin, L-NAME, VIP or PACAP pretreatment; secretin itself caused desensitization. Addition of VIP or PACAP when the secretin-evoked relaxation was maximal evoked a further relaxation suggesting the presence of distinct receptors. Secretin causes relaxation via activation of secretin receptors located on the smooth muscle and not via any of the related VIP/PACAP receptors.     

Secretin induces rapid increases in inositol trisphosphate, cytosolic Ca2+ and diacylglycerol as well as cyclic AMP in rat pancreatic acini.

Previous studies have shown that the dose-response relationship for secretin-stimulated cyclic AMP accumulation is different from that for secretin-stimulated enzyme secretion in the rat exocrine pancreas. Here we show that secretin concentrations of 10(-10) M and higher stimulated a rise in cyclic AMP levels, with maximum effect on cyclic AMP accumulation being achieved already with 10(-8) M-secretin. However, at this concentration of secretin, enzyme secretion rates were approximately half-maximal. Unexpectedly, at concentrations of secretin greater than 10(-8) M there was evidence suggestive of phosphatidylinositol bisphosphate hydrolysis with rapid increases in inositol trisphosphate, cytosolic free calcium and diacylglycerol content of rat pancreatic acini. Furthermore, there was a dose-response relationship among secretin concentration (in the range 10(-8) M-2 X 10(-6) M), increases in inositol trisphosphate and increases in cytosolic free calcium ([Ca2+]i). Contrary to what has been previously believed, these results clearly indicate that in rat pancreatic acini secretin not only stimulates cyclic AMP accumulation but also raises inositol trisphosphate, [Ca2+]i and diacylglycerol. Thus, two second messenger systems may play a role in the regulation of secretin-induced amylase release.     

Secretin receptors in the rat kidney: adenylate cyclase activation and renal effects

In previous studies it has been demonstrated that pharmacological administration of secretin can alter urine output. Whether the effect is due to a direct action on kidney was investigated by examining the effect of secretin on renal output, and determining whether there were secretin receptors and a secretin sensitive adenylate cyclase in the kidney. Secretin had an antidiuretic action on kidney when administered intravenously to anesthetized hydrated rats. In addition, binding sites for (125I)-secretin, and a secretin sensitive adenylate cyclase were identified in rat kidney. Binding was saturable and reversable and was half maximally inhibited by 1 X 10(-7) M synthetic porcine secretin. Autoradiographic studies revealed a high density of secretin binding sites in the outer medulla of the kidney, a region that is composed mainly of the thick ascending limb of the loop of Henle, and is also the major site of action for the antidiuretic hormone, vasopressin. The data indicate that a functional secretin receptor system exists in kidney which may have a physiological role in regulating urine output.     

The effect of various secretagogues on accumulation of cyclic AMP and secretion of α-amylase from rat exocrine pancreas

The relationship between accumulation of cyclic AMP and the secretion of α-amylase was investigated in the rat pancreas $\text{in vitro}$. Theophylline and secretin induced an increase in tissue cyclic AMP levels, however, only secretin stimulated secretion of α-amylase. Pancreozymin caused a release of α-amylase and had a biphasic effect on nucleotide levels — stimulation followed by inhibition. Carbachol, which induced a secretory response in the rat pancreas, reduced tissue levels of the cyclic nucleotide.     

The secretin precursor gene. Structure of the coding region and expression in the brain

Secretin is a 27-amino acid gastrointestinal hormone that stimulates the secretion of bicarbonate-rich pancreatic fluid. We isolated and analyzed the coding region of the gene for the rat secretin precursor. The entire coding region spans 692 base pairs and is divided into four regions corresponding to the signal peptide and NH2-terminal peptide, the secretin peptide and processing signal sequences, a part of the COOH-terminal peptide, and the remainder of the COOH-terminal peptide, which are interrupted by three short introns (81, 105, and 104 base pairs). The organization is similar to those of the genes for other members of the secretin family, glucagon and VIP/PHI-27 precursors, supporting the assumption that the genes for the secretin family peptide precursors originated from a common ancestral gene. We also demonstrated that the secretin precursor gene is widely expressed in the brain and in the hypophysis. The regional expression pattern of the secretin precursor gene in the brain is quite different from those of the glucagon and VIP/PHI-27 precursor genes. The secretin precursor gene is highly expressed in the medulla oblongata and pons of the brain and the hypophysis, the expression levels of which are comparable to those in the duodenum. The secretin precursor mRNA in the brain and the hypophysis has the same coding sequence as that in the duodenum, indicating that secretin in the brain and the hypophysis is produced from the same secretin precursor protein as that in the duodenum. This is the first evidence to be reported that the secretin precursor gene is definitely expressed in the brain.