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1.
A peptide that cross reacted with N-terminal, but not C-terminal, antisera to vasoactive intestinal peptide (VIP) was isolated from extracts of intestine from the dogfish Scyliorhinus canicula. Microsequence analysis gave the structure His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Ser-Arg-Ile-Arg-Lys-Gln-Met-Ala-Val-Lys - Lys-Tyr-Ile-Asn-Ser-Leu-Leu-Ala-NH2. C-terminal amidation was determined by HPLC analysis of phenylthiocarbamyl amino acid derivatives after carboxypeptidase Y digestion. The peptide differs at five positions from the porcine octacosapeptide. Dogfish VIP was equipotent with its porcine counterpart in inhibiting binding of 125I-labelled VIP to guinea pig dispersed pancreatic acini, and in stimulating amylase secretion by the same preparation. The data indicate a strong conservation of VIP during evolution and permit identification of residues crucial for bioactivity.  相似文献   

2.
The effect of vasoactive intestinal peptide (VIP) on the cAMP system of the thyroid and on the secretion of T4 and T3 from the follicular cells and calcitonin and somatostatin from the C-cells was studied in perfused dog thyroid lobes. Activation of the cAMP system was evaluated by measurements of the amount of cAMP released into the perfusion medium. T4, T3, calcitonin and somatostatin were measured by radioimmunoassays. 3 X 10(-6) M VIP induced increases in cAMP release and T4 and T3 secretion from the thyroid while there were no significant alterations in calcitonin and somatostatin release (n = 4). In experiments employing both of the two isolated thyroid lobes 100 microU/ml TSH gave considerably higher increases in T4 and T3 secretion than 10(-6) M VIP (n = 4). The effect of 10(-9) M VIP on T4 and T3 secretion was similar to that of 10(-6) M VIP (n = 4). 10(-10) M VIP induced a small but statistically significant increase in T4 and T3 secretion in two experiments while no effect was observed in two dogs. This high sensitivity of the follicular cells to VIP and the demonstration by others of VIP containing nerves in the thyroid suggest that VIP-ergic nerves may be involved in the regulation of thyroid hormone secretion.  相似文献   

3.
Using isolated submucosal glands from feline trachea, we examined the effect of vasoactive intestinal peptide (VIP) on mucus glycoprotein secretion and glandular contraction by measuring released radiolabeled glycoconjugates and induced tension, respectively. VIP (10(-10) to 10(-6) M) produced a dose-dependent increase in [3H]glycoconjugate release of up to 300% of controls, which was inhibited by VIP antiserum and not inhibited by atropine, propranolol, or phentolamine. VIP at a low concentration (10(-9) M), which did not produce any significant increases over controls, produced a 2.4- to 5-fold augmentation of the glycoconjugate release induced by 10(-9) to 10(-7) M methacholine (MCh). Atropine or VIP antiserum abolished the augmentation. VIP did not produce any alteration in isoproterenol- or phenylephrine-evoked glycoconjugate secretion. VIP (up to 10(-5) M) did not produce any alteration in the tension, even when the gland had contracted with MCh, or any augmentation of contraction induced by MCh (10(-9) to 10(-7) M). These results indicate that VIP induces mucus glycoprotein release from secretory cells and also that it potentiates the secretion induced by cholinergic stimulation.  相似文献   

4.
The physiological role of VIP in the liver is controversial. VIP receptors are present, but their function in the metabolic regulation is uncertain. The interaction of porcine VIP with isolated cells from pig liver was studied with respect to receptor-binding, degradation and glycogenolytic action. In this model, VIP and liver showed homology of animal species. 1. Receptor-binding was heterogenous with Kd values of 10(-9) mol/l and 4 X 10(-8) mol/l, and a total amount of binding sites of 7 X 10(-11) mol per 10(9) cells. The peptide specificity showed that porcine and chicken VIP were equally potent in inhibiting receptor-bound 125I-VIP; secretin was about 30 times less potent; glucagon and somatostatin were ineffective. 2. Receptor-bound 125I-VIP was degraded since about 70% was released as radioactivity not reacting with VIP-antiserum. 3. Glucose-release was not stimulated by VIP (10(-6) mol/l) whereas the rate was increased two-fold by glucagon (10(-6) mol/l). In conclusion, VIP receptors in pig liver cells are different from other tissues regarding peptide specificity. It is suggested that receptor-binding mediates degradation of VIP by pig liver rather than metabolic effects.  相似文献   

5.
The possible existence in rat brain tissues of shorter peptides related to VIP has been examined. VIP and PHI both contain paired basic amino acid residues at which posttranslational cleavage of these peptides might occur. Antiserum to VIP(22-28) was raised in rabbits. The antiserum was carboxy-terminus directed, showing cross-reactivity with all tested peptides containing the VIP carboxy-terminus sequences. Chromatographic analysis of rat brain extracts demonstrated that recovered VIP(22-28) immunoreactivity [VIP(22-28)-ir] was heterogeneous, consisting of a major fraction [60-70% of total VIP(22-28)-ir] which eluted as authentic VIP(1-28) on gel filtration and on reversed phase high performance liquid chromatography (HPLC) columns. A second fraction (30-35% of total VIP(22-28)-ir] eluted from gel filtration columns in the position of VIP(22-28). HPLC analysis of this fraction from extracts of rat cortex, hippocampus, and midbrain indicated that it was heterogeneous. One component corresponded to authentic VIP(22-28). The other two components have not been identified; one appears to be a VIP fragment intermediate in size between VIP(1-28) and VIP(22-28).  相似文献   

6.
The endothelium and its interaction with smooth muscle play a central role in the local control of the pulmonary vasculature, and endothelial dysfunction is thought to contribute to pulmonary hypertension and chronic obstructive pulmonary disease. Vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide, relaxes the rat pulmonary artery, but there is controversy as to whether or not this action of VIP depends on the endothelium. The aim of this study, therefore, was to investigate the role of the endothelium and nitric oxide (NO), the major endothelium-derived relaxing factor, in the dilator action of VIP on the rat isolated pulmonary artery. Pulmonary artery preparations pre-contracted by the alpha(1)-adrenoceptor agonist L-phenylephrine were relaxed by VIP (0.003-1 microM) and acetylcholine (0.003-10 microM) in a concentration-dependent manner. Mechanical removal of the endothelium reduced the maximal response to VIP by about 50% and practically abolished the response to acetylcholine. Inhibition of NO synthesis by N(omega)-nitro-L-arginine methyl ester (0.5 mM) had a similar effect, abolishing the vasorelaxation caused by acetylcholine and attenuating the vasorelaxation caused by VIP by about 50%. From these data it is concluded that the relaxant action of VIP on the rat isolated pulmonary artery depends in part on the presence of the endothelium and that this part is mediated by endothelial NO.  相似文献   

7.
Vasoactive intestinal peptide (VIP) is secreted from many cancer lines and VIP binding was observed in many tumors. We have shown before that VIP antagonists are potent inhibitors of neoplastic growth of neuroblastoma, lung and breast cancer cells in vitro. Here, the cultured colon cancer cell line HCT-15 that exhibited VIP receptor expression was treated with the VIP hybrid antagonist neurotensin(6-11)VIP(7-28). The antineoplastic activity was assessed by thymidine incorporation. Neurotensin(6-11)VIP(7-28) efficiently inhibited cancer growth with a maximal effect at nanomolar concentrations. Once the inhibitory properties of the VIP antagonist on colon cancer cells were established, the in vivo curative effects were analyzed. Sprague-Dawley rats were injected with azoxymethane (AOM) (15 mg/kg/week) for 2 weeks, providing artificial induction of colon tumors. The rats were then allocated into four experimental groups: (1) receiving no treatment; (2) receiving treatment with saline; (3, 4) receiving treatment with 10 or 20 microg of neurotensin(6-11)VIP(7-28), respectively. After 10 weeks of daily injections, rats were sacrificed and tumors assessed for stage, volume, location, differentiation and lymphocytic infiltrate. Embedded mucosa was assessed for dysplastic crypts. Results showed that the antagonist treatment reduced the tumor volume, staging, lymphocyte infiltrate and the number of dysplastic crypts. Thus, neurotensin(6-11)VIP(7-28) could serve as an effective cancer treatment and a preventing agent.  相似文献   

8.
Vasoactive intestinal polypeptide (VIP) is released into the portal circulation by a meal stimulus, but is rapidly cleared from plasma. Although it is known to bind to receptors on liver cells, the role of the liver in the clearance of VIP is not clearly defined. We therefore studied the disappearance of VIP in recirculating and in single pass isolated perfused rat liver (IPRL) preparations. Disappearance of added VIP was rapid in recirculating IPRL experiments with a half life of ca. 30 min. In single-pass steady-state studies in which livers were perfused at 16 ml/min for 30 min, clearance of VIP was complete (16 ml/min) at concentrations of 500 fmol/ml, but clearance fell to 3 and 1 ml/min at perfusate concentrations of 8 and 40 pmol/ml respectively. Further experiments to evaluate whether VIP was disappearing in perfusate itself demonstrated substantial metabolism of VIP in perfusate which had previously been circulated through a liver for 90 min. The products of metabolism were identical to those found in the IPRL. We conclude that VIP is rapidly cleared as it passes through the isolated perfused rat liver model with a significant proportion of clearance attributable to release of a peptidase from the liver into the perfusate.  相似文献   

9.
By immunohistochemistry galanin-like immunoreactivity and vasoactive intestinal polypeptide (VIP)-like immunoreactivity were found in nerve cell bodies mostly in the submucous plexus and in nerve fibres in the mucosa, submucosa and muscularis including the myenteric plexus of the porcine ileum and were found to co-exist in most of these structures. Using isolated, perfused porcine ileum we studied the release of galanin and VIP in response to electrical stimulation of the mixed periarterial nerves or to intraarterial infusions of different neuroactive agents. Nerve stimulation (4-10 Hz) inhibited the basal release of galanin and VIP from the ileum (to 69 +/- 6 and 62 +/- 6% of basal release). After infusion of the alpha-adrenergic blocker, phentolamine, (10(-6) M) electrical stimulation increased the release of both galanin and VIP (to 140 +/- 12 and 133 +/- 13% of basal output). This increase was abolished by atropine (10(-6) M) and by hexamethonium (3.10(-5) M). Infusion of norepinephrine (10(-6) M) inhibited, whereas acetylcholine (10(-6) M) stimulated the release of both peptides. The effect of the latter was abolished by atropine. The inhibitory effect of nerve stimulation was not influenced by atropine. Our results suggest that the galanin- and VIP-producing intrinsic neurons receive inhibitory signals by noradrenergic nerve fibers and stimulatory signals mediated by cholinergic nerves, possibly via a cholinergic interneuron.  相似文献   

10.
Intramuscular injection of synthetic VIP (200 micrograms) resulted in a rapid increase in plasma prolactin (PRL) concentrations in normal women, which was accompanied by the 4- to 7-fold increase in plasma VIP levels. Mean (+/- SE) peak values of plasma PRL obtained 15 min after the injection of VIP were higher than those of saline control (28.1 +/- 6.7 ng/ml vs. 11.4 +/- 1.6 ng/ml, p less than 0.05). Plasma growth hormone (GH) and cortisol levels were not affected by VIP in normal subjects. VIP injection raised plasma PRL levels (greater than 120% of the basal value) in all of 5 patients with prolactinoma. In 3 of 8 acromegalic patients, plasma GH was increased (greater than 150% of the basal value) by VIP injection. In the in vitro experiments, VIP (10(-8), 10(-7) and 10(-6) M) stimulated PRL release in a dose-related manner from the superfused pituitary adenoma cells obtained from two patients with prolactinoma. VIP-induced GH release from the superfused pituitary adenoma cells was also shown in 5 out of 6 acromegalic patients. VIP concentrations in the CSF were increased in most patients with hyperprolactinemia and a few cases with acromegaly. These findings indicate that VIP may play a role in regulating PRL secretion in man and may affect GH secretion from pituitary adenoma in acromegaly.  相似文献   

11.
The human colon adenocarcinoma cell line HT-29 in culture exhibits a cyclic AMP production system highly sensitive to vasoactive intestinal peptide (VIP), making HT-29 cells a unique cultured cell system for studying the mechanism of VIP action [Laburthe, Rousset, Boissard, Chevalier, Zweibaum & Rosselin (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2772-2775]. The quantitative characteristics of VIP receptors in HT-29 cells and their structural requirement and molecular size were studied. 125I-labeled VIP bound in a time-dependent manner to HT-29 cell homogenates. At equilibrium (60 min incubation at 30 degrees C), unlabelled VIP in the 0.01-10 nM concentration range competed with 125I-VIP for binding to cell homogenates. Scatchard analysis of binding data gave a straight line, indicating that VIP bound to a single population of sites with a KD of 0.12 +/- 0.02 nM and a capacity of 120 +/- 9 fmol/mg of protein. The structural requirement of these receptors was studied with peptides structurally related to VIP, either natural or synthetic. Several peptides inhibited 125I-VIP binding to HT-29 cell homogenates with the following order of potency, which is typical of the human VIP receptor: VIP (IC50 = 0.1 nM) greater than VIP-(2-28)-peptide (IC50 = 13 nM) greater than human growth hormone releasing factor (IC50 = 56 nM) greater than peptide histidine isoleucine amide (IC50 = 80 nM) greater than secretin (IC50 greater than 10 000 nM). To characterize the molecular component(s) of the VIP receptor in HT-29 cells, 125I-VIP was covalently bound to cell homogenates by using the cross-linker dithiobis(succinimidyl propionate). Sodium dodecyl sulphate/polyacrylamide-gel autoradiographic studies of affinity-labelled cell homogenates revealed two major bands, corresponding to 125I-VIP-protein complexes of Mr 66 000 and 16 000. The labelling of the Mr-66 000 component was specific, since it was abolished by native VIP, whereas that of the Mr-16 000 component was not. Densitometric scanning of autoradiographs indicated that the labelling of the Mr-66 000 complex was inhibited by low VIP concentrations in the 0.1-10 nM range (IC50 = 0.6 nM), but was unaffected by 1 microM-glucagon or octapeptide of cholecystokinin. It was also decreased by VIP-(2-28)-peptide with a potency 1% that of VIP. Assuming that one molecule of 125I-VIP bound per molecule of protein, one protein of Mr 63 000 was identified as a component of the VIP receptor in HT-29 cells.  相似文献   

12.
Vasoactive intestinal polypeptide (VIP) is now considered to be a prolactin-releasing factor (PRF). The aim of this study was to determine the VIP concentration in peripheral blood in patients with prolactin-secreting adenoma compared to healthy subjects. We also examined the effect of bromocriptine administration on the plasma VIP concentration in patients with prolactinoma. Nine patients with prolactinoma (6 women and 3 men, aged 27-50) and 7 healthy control subjects (4 women and 3 men, aged 26-40) were examined. Blood samples for prolactin and VIP were collected at 06:00, 12:00, 18:00, 24:00. In prolactinoma blood was taken before and after bromocriptine administration. Serum prolactin concentration was determined by the radioimmunoassay. VIP concentration was measured by a specific radioimmunoassay Kit-INCSTAR Corp. (Minnesota, USA). Statistical significance was calculated using the analysis of variance. A single 5 mg oral dose of bromocriptine decreased the mean prolactin concentration during the first 24 hours of treatment. Plasma VIP concentration was higher in prolactinoma patients compared to healthy subjects. There was no change in plasma VIP level after bromocriptine administration. In conclusion: in patients with prolactin secreting adenoma the plasma VIP concentration is increased.  相似文献   

13.
Porcine VIP was synthesized from three segments. The segments, VIP(1-6), VIP(7-13), and VIP(14-28), were synthesized via the Repetitive Excess Mixed Anhydride (REMA) method. The low solubility of the C-terminal segment was greatly improved by a temporary substitution of Asn28 by a beta-t-butyl aspartic acid ester. The segments VIP(1-6) and VIP(7-13) were purified by HPLC and coupled via the mixed anhydride method. The product was purified by gel filtration. VIP was synthesized from VIP(1-13) and VIP(14-28) by the same procedure. After deprotection, Met17-sulfoxide reduction, and purification by ion-exchange chromatography, the product was found to have the expected amino acid composition and biological potency. A HPLC purified sample was compared with several commercial preparations of varying purity.  相似文献   

14.
In order to develop potent shortened analogues of vasoactive intestinal peptide (VIP), the structure-activity relationship of C-terminally truncated analogues of VIP was investigated by examining the binding activity to rat lung VIP receptors and relaxation of smooth muscle in isolated mouse stomach. VIP(1-27) showed VIP receptor binding activity comparable to that of VIP but the activity of VIP(1-26) was reduced to one-third of VIP. The receptor binding activity of VIP(1-26) to VIP(1-23) was reduced in proportion to the decrease in amino acid residues. There was a significant correlation between the number of amino acid residues and VIP receptor binding activities of VIP and its C-terminally truncated analogues. VIP(1-22) and VIP(1-21) exhibited little binding activity even at high concentrations, suggesting the requisite of 23 amino acid residues as the minimal essential sequence for the conservation of VIP receptor binding activity. The chemical modification of VIP(1-23) generated a potent analogue, [Arg(15, 20, 21), Leu(17)]-VIP(1-23), that displayed a 22-fold higher receptor binding activity and 1.6-fold more potent relaxation of mouse stomach than VIP(1-23) did. In conclusion, it was shown that [Arg(15, 20, 21), Leu(17)]-VIP(1-23) could be a relatively potent and stable agonist of VIP receptors. The present study has provided further insight into the structure-activity relationship of VIP to generate novel shortened VIP analogues having a high affinity to VIP receptors and potent pharmacological activity.  相似文献   

15.
In HGT-1 cells incubated at 20 degrees C for 15 min with 1 mM 3-isobutyl-1-methylxanthine (IBMX), histamine (10(-4)M) increased basal cAMP levels from 2.12 +/- 0.14 to 22.9 +/- 2 pmol per 10(6) cells, with a potency of 6.4 X 10(-6)M. IBMX was added in order to inhibit cAMP degradation by low and high Km cAMP-phosphodiesterases (cAMP-PDE). The use of specific H1, H2 agonists or antagonists indicated that the histamine effect was due to an interaction with typical H2 -receptors that are involved in gastric acid secretion. Cyclic AMP levels were also increased (10-fold) by vasoactive intestinal peptide VIP (3 X 10(-11) - 10(-8)M). Porcine peptide having N-terminal histidine and C-terminal isoleucine amide (PHI) and secretin were respectively 80 and 3600 times less potent than VIP and did not produce additive effect when tested in combinations with VIP. This observation indicates that these two peptides, structurally related to VIP, are acting through the recognition sites for VIP. Combination of VIP and histamine results in additive stimulation on intact cells as well as on membrane-bound adenylate cyclase, suggesting the existence of two cell populations bearing respectively the two sets of receptors. Two other human cancer cell lines originating from nongastric tumors (HT-29 and HL-60) possess only VIP or histamine receptors, respectively, indicating the gastric cellular originality of the HGT-1 cells. It is concluded that HGT-1 cells possess both VIP and histamine H2 receptors with similar pharmacological properties to those characterized in normal human fundic glands (1,2). Therefore, this cell line can be a good model to study drugs used therapeutically during the treatment of patients for gastric ulcer or cancer.  相似文献   

16.
Pancreatic acini from most species possess vasoactive intestinal peptide (VIP) receptors. Recently, two subtypes of VIP receptors, VIP(1)-R and VIP(2)-R, were cloned. Which subtype exists on pancreatic acini or mediates secretion is unclear. To address this, we examined pancreatic acini from both rat and guinea pig. VIP(1)-R and VIP(2)-R mRNA were identified in dispersed acini from both species by Northern blot analysis and in rat by Southern blot analysis. With the use of the VIP(2)-R-selective ligand Ro-25-1553 in both species, inhibition of binding of (125)I-labeled VIP to acini showed a biphasic pattern with a high-affinity component (10%) and a second representing 90%. The VIP(1)-R-selective ligand, [Lys(15),Arg(16),Leu(27)]VIP-(1-7)-GRF-(8-27), gave a monophasic pattern. Binding of Ro-25-1553 was better fit by a two-site model. In both rat and guinea pig acini, the dose-response curve of Ro-25-1553 for stimulation of enzyme secretion was biphasic, with a high-affinity component of 10-15% of the maximal secretion and a low-affinity component accounting for 85-90%. At low concentrations (10 nM) of Ro-25-1553 and [Lys(15),Arg(16), Leu(27)]VIP-(1-7)-GRF(8-27), which only occupy VIP receptors, a 4-fold and a 56-fold increase in cAMP occurred, respectively. These results show that both VIP(1)-R and VIP(2)-R subtypes exist on pancreatic acini of rat and guinea pig, their activation stimulates enzyme secretion by a cAMP-mediated mechanism, and the effects of VIP are mediated 90% by activation of VIP(1)-R and 10% by VIP(2)-R. Because VIP has a high affinity for both VIP-R subtypes, its effect on pancreatic acini is mediated by two receptor subtypes, which will need to be considered in future studies of the action of VIP in the pancreas.  相似文献   

17.
The purpose of this study was to determine whether association of vasoactive intestinal peptide with sterically stabilized liposomes (VIP on SSL) amplifies DNA synthesis evoked by the peptide in cultured chemically transformed hamster oral keratinocytes (HCPC-1) and, if so, whether this response in mediated, in part, by SSL-induced inactivation of neutral endopeptidase 24.11 (NEP; EC 3.4.24.11) and angiotensin I-converting enzyme (ACE; EC 3.4.15.1), two ectoenzymes that modulate HCPC-1 cell growth, in these cells. We found that VIP (10(-9)-10(-6) M) alone elicited a modest, albeit significant, concentration-dependent increase in DNA synthesis in HCPC-1 cells that was maximal after 48-72-h incubation (p < 0.05). VIP on SSL potentiated DNA synthesis in these cells relative to VIP alone. The magnitude of VIP on SSL-induced responses was 1.2-1.6-fold higher than that of VIP alone with maximal effects observed at 10(-9) M and 10(-6) M after 72- and 48-h incubation, respectively. Empty SSL had no significant effects on DNA synthesis. Empty SSL and VIP on SSL had no significant effects on NEP 24.11 and ACE activity in HCPC-1 cells. Collectively, these data indicate that association of VIP with SSL potentiates DNA synthesis in cultured oral keratinocytes relative to VIP alone and that this response is not related to non-specific effects of SSL.  相似文献   

18.
The protected heptapeptide derivative t-butyloxycarbonyl- -threonyl-β-benzyl- -aspartyl- -asparaginyl-O-benzyl- -tyrosyl- -threonyl-nitro- -arginyl- - leucine methyl ester was prepared by stepwise chain lengthening. The protecting groups on the side chains of arginine, tyrosine, and aspartic acid residues were removed by hydrogenolysis and the partially deprotected heptapeptide ester converted to the hydrazide, an intermediate in the synthesis of the (porcine) vasoactive intestinal peptide (VIP).After the removal of the tert-butyloxycarbonyl group, the heptapeptide ester was exposed to the action of trypsin which split off its C-terminal residue, -leucine methyl ester. The hexapeptide was then exposed to chymotrypsin, which cleaved it into an acidic, and a basic fragment. The former was, under the conditions used, indistinguishable on paper chromatography and paper electrophoresis from the tetrapeptide threonyl-aspartyl-asparaginyl-tyrosine which had previously been isolated from natural VIP, of which it comprises the sequence 7–10. Similarly, the basic fragment was indistinguishable from threonyl-arginine, the sequence 11–12 of VIP. This intestinal peptide increases visceral blood flow and reduces blood pressure in the dog, and also causes relaxation of different smooth muscle preparations, e.g., the trachea of guinea pigs. The principal aim of the present synthesis is to provide independent evidence for the sequence of (porcine) VIP.  相似文献   

19.
The 19 amino acid carboxyl terminus fragment of vasoactive intestinal peptide (VIP), VIP(10-28), inhibits [125I]VIP binding in intact HT29 colonic adenocarcinoma cells and in membranes from these cells. However, VIP(10-28) alone has no effect on adenylate cyclase activity (membranes) or cyclic AMP synthesis (intact cells) in HT29 cells although VIP receptor agonists are markedly stimulatory. The indicated antagonist character of VIP(10-28) was confirmed by rightward parallel shifts of VIP dose response curves in the presence of VIP(10-28) in adenylate cyclase and cyclic AMP synthesis experiments. The equilibrium dissociation constant values for VIP(10-28) from these experiments agree with values from inhibition binding studies. The lack of effect of VIP(10-28) on forskolin dose response curves in HT29 adenylate cyclase assays indicates the specificity of the VIP(10-28) antagonism, thus suggesting that VIP(10-28) may be a useful tool in studying VIP receptor regulation and other aspects of the mechanisms of VIP action. The potential regulatory role of a proteolytically generated fragment of VIP acting antagonistically at VIP receptors is discussed.  相似文献   

20.
HT 29, a cell line derived from a human colonic adenocarcinoma, is highly responsive to the vasoactive intestinal peptide (VIP) as shown by a more than 100-fold intracellular cAMP increase (Ka = 0.3 nM), the stimulations of protein kinase A (Ka = 0.1 nM) and the low-Km cAMP phosphodiesterase (Ka = 40 nM). Remarkably, adenylate cyclase, cAMP-dependent kinase and cAMP-specific phosphodiesterase are activated in a sequential manner. Binding studies with [125I]-labeled VIP indicate a high affinity site with a Kd value (0.5 nM) close to the activation constant value (Ka) of the three enzymes. The molecular structure of the VIP receptor was studied by immunological and chemical approaches. A monoclonal antibody (mAb 109-10-16) which partially decreased the binding of VIP to its receptor allowed the characterization of Mr = 53,000 and Mr = 48-49,000 polypeptides. More precise identification of protein components of the VIP receptor resulted from covalent cross-linking on intact HT 29 cells by four bifunctional reagents: dithiobis-(succinimidyl propionate) and its non-cleavable analog disuccinimidyl suberate, the photoactivable azido phenyl glyoxal and dimethylpimelimidate. Analysis by SDS-polyacrylamide gel electrophoresis demonstrated a major band of Mr = 67,000 regardless of which cross-linker was used. The same band and an Mr = 49,000 species were found in experiments using a crude membrane fraction of HT 29 cells. Assuming one molecule of VIP (Mr = 3326) linked per polypeptide, these observations suggest that an Mr = 64,000 species belongs to the VIP specific plasma membrane receptor. This protein contains an Mr = 20,000 N-linked sialic acid rich oligosaccharidic moiety.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

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