Receptors for vasoactive intestinal peptide and secretin on guinea pig pancreatic acini

We investigated the abilities of VIP and secretin to occupy receptors and to increase cellular cyclic AMP using dispersed acini from guinea pig pancreas. The dose-inhibition curve for inhibition of binding of 125I-VIP by VIP was broad with detectable inhibition at 0.1 nM VIP, half-maximal inhibition at 2 nM VIP and complete inhibition at 10 microM VIP. Secretin also inhibited binding of 125I-VIP was compatible with two VIP-preferring receptors with one class having a high affinity for VIP (Kd 1.1 nM) and a low affinity for secretin (Kd 5 microM) and the other class having an intermediate affinity for VIP (Kd 470 nM). The dose inhibition curve for inhibition of binding of 125I-secretin by secretin was not broad. Half-maximal inhibition occurred with 7 nM secretin or with 10 microM VIP. Computer analysis was compatible with a single secretin-preferring receptor with a high affinity for secretin (Kd 7 nM) and a low affinity for VIP (Kd 5.9 microM). Comparison of the ability of VIP to increase cyclic AMP with or without the secretin-receptor antagonist, secretin-5-27, demonstrated only occupation of the high affinity VIP-preferring or high affinity secretin-preferring receptors increase cyclic AMP. Our results demonstrate that, in contrast to previous reports, guinea pig pancreatic acini possess 3 classes of receptors that interact with VIP and secretin. The low affinity receptor seen with 125I-VIP is not the same as the secretin-preferring receptor and does not increase cellular cyclic AMP.     

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.     

D-Phe4]peptide histidine-isoleucinamide ([D-Phe4]PHI), a highly selective vasoactive-intestinal-peptide (VIP) agonist, discriminates VIP-preferring from secretin-preferring receptors in rat pancreatic membranes

The capacity of vasoactive intestinal peptide (VIP), peptide histidine-isoleucinamide (PHI), secretin, and a series of analogs to discriminate between VIP-preferring and secretin-preferring receptors that coexist in rat pancreatic plasma membranes was evaluated by their ability to inhibit [125I]iodo-VIP and [125I]iodo-secretin binding and to activate adenylate cyclase. VIP, the VIP analogs [D-His1]VIP, [D-Ser2]VIP, [D-Asp3]VIP and [D-Ala4]VIP, PHI, [D-Phe4]PHI, and secretin inhibited the binding of both ligands in a concentration range of 10(-11) M to 10(-5) M and with a selectivity factor varying from 18,000 to 0.1. The only exception was [D-Phe4]PHI that inhibited 125I-VIP binding only, with an IC50 of 7 nM, and with no inhibition of 125I-secretin binding at 10 microM. The peptides tested stimulated adenylate cyclase in the same membranes and the slope of the dose-effect curves indicated that all peptides, except [D-Phe4]PHI, interacted with at least two classes of receptors: VIP-preferring and secretin-preferring receptors. By contrast, the dose-effect curve of [D-Phe4]PHI activation of adenylate cyclase was monophasic and competitively modified by [D-Phe2]VIP (a VIP antagonist) but not by secretin(7-27) (a secretin antagonist), indicating an interaction with VIP-preferring receptors only. Thus, [D-Phe4]PHI appears to be a highly selective tool to characterize these receptors.     

The effects of VIP on cyclic AMP and glycogen levels in vertebrate retina

The effects of VIP and related-peptides (PHI, secretin, glucagon) on cyclic AMP formation were investigated in intact pieces of rabbit retina. VIP and PHI increased cyclic AMP levels with EC50 of 160 nM and 300 nM respectively. At 5 microM the peptides increased cyclic AMP 46 fold (VIP) and 38 fold (PHI). Secretin was much less potent and glucagon was totally inactive. VIP was also tested for its effects on glycogen levels under similar experimental conditions. In contrast to its pronounced glycogenolytic action in mouse cerebral cortical slices, VIP at 1 microM decreased only moderately (38.3%) 3H-glycogen newly synthesized from 3H-glucose by pieces of rabbit retina. Furthermore a discrepancy between the efficacy of VIP in increasing cyclic AMP and in promoting glycogenolysis appears to exist. A similar dissociation between these two cellular events was also observed with other neuroactive substances. Thus the pronounced increase in cyclic AMP induced by dopamine and forskolin was accompanied by only a moderate decrease in 3H-glycogen levels. Conversely 50 mM potassium induced a 79.9% decrease in 3H-glycogen levels without any significant increase in cyclic AMP.     

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.     

GABAB Receptor-Mediated Enhancement of Vasoactive Intestinal Peptide-Stimulated Cyclic AMP Production in Slices of Rat Cerebral Cortex

Basal and vasoactive intestinal peptide (VIP)-stimulated accumulations of cyclic AMP were measured in slices of rat cerebral cortex. Neither gamma-aminobutyric acid (GABA) nor the selective GABAB receptor agonist (-)-baclofen stimulated basal cyclic AMP accumulation, whereas VIP caused a large dose-dependent increase in cyclic AMP levels. However, in the presence of 100 microM (-)-baclofen, the effects of VIP on cyclic AMP accumulation were significantly enhanced, with the responses to 1 microM and 10 microM VIP being approximately doubled. The enhancing effects of (-)-baclofen was dose related (1-1,000 microM), but an enhancing effect was not observed with 100 microM (+)-baclofen. In the presence of the GABA uptake inhibitor nipecotic acid (1 mM), GABA caused a similar dose-related enhancement of the VIP response. The ability of either GABA or (-)-baclofen to augment VIP-stimulated production of cyclic AMP was not mimicked by the GABAA, agonists isoguvacine and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and was not antagonized by the GABAA antagonist bicuculline. The putative GABAB antagonist 5-aminovaleric acid (1 mM) significantly reduced the effect of (-)-baclofen. The ability of (-)-baclofen to enhance VIP-stimulated accumulation of cyclic AMP was observed in slices of rat cerebral cortex, hippocampus, and hypothalamus. These results indicate that GABA and (-)-baclofen can enhance VIP-stimulated accumulation of cyclic AMP in rat brain slices via an interaction with specific GABAB receptors.     

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).     

Vasoactive Intestinal Polypeptide-Related Peptides Modulate Tyrosine Hydroxylase Gene Expression in PC12 Cells Through Multiple Adenylate Cyclase-Coupled Receptors

Abstract: We investigated the receptor mechanisms by which vasoactive intestinal polypeptide (VIP) and related peptides exert their effects on tyrosine hydroxylase (TH) gene expression. VIP, secretin, and peptide histidine isoleucine (PHI) each produced increases in TH gene expression, as measured by increases in TH mRNA levels and TH activity. The concentrations at which the effects of these peptides were maximal differed for TH activity and TH mRNA. Moreover, maximal increases in TH activity were 130-140% of control, whereas maximal increases in TH mRNA were 250% of control. The concentration dependence of the increases in TH mRNA in response to the three peptides was analyzed by fitting the data to nonlinear regression models that assume either one or two components to the response. The data for secretin fit best to a model that assumes a single component to the increase in TH mRNA levels. The data derived for PHI and VIP fit best to models that assumed two components to the TH mRNA response. These data suggested that there may be more than one receptor or signal transduction mechanism involved in the response to the various peptides. We examined whether the peptides exerted their effects through common or multiple second messenger systems. The ability of maximally active concentrations of these peptides to stimulate increases in TH mRNA was not additive, indicating that the peptides work through a common receptor or signal transduction pathway. Each peptide stimulated increases in protein kinase A (PKA) activity. Secretin and VIP were ineffective in increasing TH mRNA levels in a PKA-deficient mutant PC12 cell line (A 126-1B2). Moreover, the adenylate cyclase antagonist 2′,5′-dideoxyadenosine prevented the increase in TH mRNA produced by each peptide. Thus, each peptide requires an intact cyclic AMP second messenger pathway to produce changes in TH gene expression, suggesting that the complex pattern of response to VIP and PHI revealed by concentration-response analysis was due to the actions of these peptides at multiple receptors. To evaluate this possibility, we examined the effect of several peptide receptor antagonists on the increase in TH gene expression elicited by VIP, PHI, and secretin. The secretin antagonist secretin (5–27) (20 μM) had no significant effect on VIP or PHI stimulation of TH gene expression, but reduced the effect of secretin. The VIP antagonist VIP (10–28) (20 μM) reduced the effect of VIP on increasing TH mRNA, but had no significant effect on the response of TH mRNA to secretin or PHI. Interestingly, the VIP antagonist [Ac-Tyr¹,D-Phe²]-growth hormone releasing factor [GRF(1–29)] amide (20 μM) potentiated the effect of VIP on elevating TH mRNA levels, but had no effect on secretin-stimulated TH mRNA induction. To determine whether this response was mediated through the cyclic AMP pathway, we examined the effects of the VIP antagonist [Ac-Tyr¹,D-Phe²]-GRF(1–29) amide on VIP stimulation of PKA activity. Although the antagonist had no effect alone, it enhanced stimulation of PKA activity by VIP. Taken together, these findings indicate that VIP and secretin stimulate TH mRNA through different adenylate cyclase-linked receptors and that a second VIP receptor may modulate TH induction by inhibiting VIP stimulation of PKA.     

Homologous regulation of adenylate cyclase-coupled receptors for vasoactive intestinal peptide (VIP) on human mononuclear leucocytes

The effect of agonists on VIP receptor regulation has been investigated in mononuclear human blood leucocytes. VIP receptor number and affinity, as well as VIP-stimulated cyclic AMP accumulation were measured after pretreatment with VIP, PHM-27 or secretin. Pretreatment for 30 min with 0.1 μM VIP caused 28% (S.E.M. = 15) reduction in specific binding, and 52% (S.E.M. = 12) reduction in cyclic AMP accumulation, while 3 h of pretreatment caused 59% (S.E.M. = 10) and 68% (S.E.M. = 12) reduction. Only VIP concentrations at the nanomolar level and higher were shown to have any effect. B_max of the high-affinity receptor was reduced by 66% (S.E.M. = 8) after 30 min, and 95% (S.E.M. = 3) after 3 h of exposure to 0.1 μM VIP. No significant change was observed in receptor affinity, in B_max of the low-affinity receptor, in ED₅₀, or in ED₁₀₀ of VIP-stimulated cyclic AMP accumulation. Pretreatment with PHM-27 (0.1 μM, 3 h) caused 24% reduction in [¹²⁵I]VIP binding and 25% reduction in cyclic AMP accumulation, while no effect was detected after pretreatment with secretin (0.1 μM, 3 h).     

Stimulatory effect of vasoactive intestinal peptide (VIP) on cyclic AMP production in rat peritoneal macrophages.

Vasoactive intestinal peptide (VIP) stimulated cyclic AMP production in rat peritoneal macrophages. The stimulatory effect of VIP was dependent on time, temperature and cell concentration, and was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). At 15 degrees C, the response occurred in the 0.1-1000 nM range of VIP concentrations. Half maximal stimulation of cellular cyclic AMP (ED50) was obtained at 1.2 +/- 0.5 nM VIP, and maximal stimulation (about 3-fold basal level) was obtained between 100-1000 nM. The cyclic AMP system of rat peritoneal macrophages showed a high specificity for VIP. The order of potency observed in inducing cyclic AMP production was VIP greater than rGRF greater than hGRF greater than PHI greater than secretin. Glucagon, insulin, pancreastatin and octapeptide of cholecystokinin did not modify cyclic AMP levels at concentrations as high as 1 microM. The beta-adrenergic agonist isoproterenol increased the cyclic AMP production and show additive effect with VIP. Somatostatin inhibits the accumulation of cyclic AMP in the presence of both vasoactive intestinal peptide and isoproterenol. The finding of a VIP-stimulated cyclic AMP system in rat peritoneal macrophages, together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, strongly suggest that VIP may be involved in the regulation of macrophage function.     

Characterization of functional receptors for vasoactive intestinal peptide (VIP) in rat peritoneal macrophages.

Functional vasoactive intestinal peptide (VIP) receptors have been characterized in rat peritoneal macrophages. The binding depended on time, temperature and pH, and was reversible, saturable and specific. Scatchard analysis of binding data suggested the presence of two classes of binding sites: a class with high affinity (kd = 1.1 +/- 0.1 nM) and low capacity (11.1 +/- 1.5 fmol/10(6) cells), and a class with low affinity (kd = 71.6 +/- 10.2 nM) and high capacity (419.0 +/- 80.0 fmol/10(6) cells). Structural requirements of these receptors were studied with peptides structurally or not structurally related to VIP. Several peptides inhibited 125I-VIP binding to rat peritoneal macrophages with the following order of potency: VIP greater than rGRF greater than hGRF greater than PHI greater than secretin. Glucagon, insulin, somatostatin, pancreastatin and octapeptide of cholecystokinin (CCK 26-33) were ineffective. VIP induced an increase of cyclic AMP production. Half-maximal stimulation (ED50) was observed at 1.2 +/- 0.5 nM VIP, and maximal stimulation (3-fold above basal levels) was obtained between 0.1-1 microM. Properties of these binding sites strongly support the concept that VIP could behave as regulatory peptide on the macrophage function.     

Demonstration of [125I]VIP binding sites and effects of VIP on cAMP-formation in rat insulinoma (RINm5F and RIN14B) cells.

Vasoactive intestinal polypeptide (VIP)-immunoreactive nerves have been demonstrated in close association with the islets of Langerhans, and VIP has been shown to stimulate insulin and somatostatin secretion. Using [125I]VIP and membranes prepared from rat insulinoma (RIN) cells, i.e., the subclones m5F (m5F; mainly insulin-secreting) and 14B (14B; mainly somatostatin-secreting), it was found that VIP (10(-10)-10(-7) M) competitively inhibited the binding of [125I]VIP. A single class of high affinity binding sites with Kd values of 0.40 +/- 0.06 nM and 0.36 +/- 0.08 nM for m5F and 14B, respectively, with a corresponding number of binding sites (Bmax) of 163 +/- 20 and 254 +/- 51 fmol/mg protein was observed. The rank order of potency in inhibiting [125I]VIP binding was in both cell lines: VIP greater than helodermin greater than pituitary adenylate cyclase activating polypeptide 1-27 (PACAP27) greater than peptide histidine isoleucine (PHI) greater than secretin. VIP caused a dose-dependent increase in cAMP-formation in both m5F and 14B cell membranes with EC50 values of 3.0 and 3.5 nM, respectively, but VIP (1.10(-9)-3.10(-6) M) had no effect on insulin secretion (over 2 h) from the m5F cells. Thus, the data suggest that the VIP-receptors in these neoplastic rat cell lines, despite an apparent coupling to adenylate cyclase activity, seem to be functionally uncoupled to an effect on insulin secretion following an acute exposure to VIP.     

Phosphorylation of 3 particulate proteins in rat pancreatic acini in response to vasoactive intestinal peptide (VIP), secretin and cholecystokinin (CCK-8)

Rat pancreatic acini were preincubated with 0.4 mM 32Pi for 45 min at 37 degrees C, then exposed for 15 min to VIP, secretin or CCK-8. The incubation was terminated with a stop solution and a fraction rich in mitochondria and zymogen granules was separated from a microsome-rich fraction by differential centrifugation. After heating in the presence of SDS, beta-mercaptoethanol was added and the pattern of equivalent amounts of 32P-labelled proteins was examined by autoradiography of SDS-PAGE gels. VIP, secretin, and CCK-8 stimulated the phosphorylation of a Mr=33 K microsomal protein and that of two proteins of Mr=21 K and Mr=25 K mostly present in a fraction rich in mitochondria and zymogen granules. Stimulations were dose-dependent, the highest stimulant concentrations tested allowing 2- to 3-fold increases of phosphorylation over basal. When 1 nM CCK-8 was used simultaneously with 1 microM VIP, the cyclic AMP levels attained and the pattern of protein phosphorylation were similar to those obtained with VIP alone, and there was a potentiation of amylase secretion; when a supra-maximal 0.1 microM CCK-8 concentration was added, the VIP-induced elevation in cyclic AMP levels and the phosphorylation of the Mr=21 K and Mr=25 K proteins were partially antagonized, and no potentiation any more of secretion occurred. To conclude the in vitro phosphorylation of three particulate proteins (Mr=33 K, 25 K, and 21 K) was similarly increased in rat pancreatic acini in response to secretin and VIP (acting through cyclic AMP) and to CCK-8 (acting mostly through Ca2+).     

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.     

Binding of vasoactive intestinal peptide and its stimulation of adenylate cyclase through two classes of receptors in rat liver membranes effects of 12 secretin analogues and 2 secretin fragments

1. Vasoactive intestinal peptide (VIP) receptors were identified in crude rat hepatic membranes by ¹²⁵I-labelled VIP binding and by the ability of VIP to stimulate adenylate cyclase activity. The specificity of these receptors was evaluated by the capacity of secretin, synthetic secretin analogues, and secretin fragments to inhibit ¹²⁵I-labelled VIP binding and to stimulate adenylate cyclase. 2. The results were compatible with the existence of two classes of VIP binding sites that could be distinguised according to their affinity for VIP and their specificity. High-affinity sites were more specific for VIP as secretin was 175 times less potent than VIP for recognition of these sites while being only 33 times less potent than VIP for recognition of low-affinity sites. 3. Secretin analogues, monosubstituted in position 2, 3, 4, or 6 were less potent than secretin for adenylate cyclase stimulation as well as for the recognition of the two classes of receptors. [Val⁵]Secretin was more potent than secretin and appeared definitely more VIP-like than secretin; [Ala⁴, Val⁵]secretin were equipotent to secretin. 4. The fragment secretin (7–27) was unable to recognize VIP receptors and to stimulate adenylate cyclase. The substituted fragment [Gln[⁹,Asn¹⁵]secretin (5–27) recognized these receptors with weak potency but could not activate the enzyme.     

Functional and structural characterization of the secretin receptors in rat gastric glands: Desensitization and glycoprotein nature

We have documented and characterized the down-regulation of the¹²⁵I-secretin binding sites and the associated desensitization of the secretin receptor-cAMP system in rat gastric glands. Secretin induced a rapid decrease of the high-affinity¹²⁵I-secretin binding sites with t^1/2=30 min at 37°C. Half-maximal down-regulation and desensitization occurred at 10^–9 M secretin, a physiological concentration corresponding to the half-maximal activation of the secretin receptor. The Scatchard parameters of the low-affinity¹²⁵I-secretin binding sites were unaffected by the pretreatment. This desensitization is heterologous in view of the loss of responsiveness to the truncated glucagon-like peptide 1 (TGLP-1), and pharmacologically selective since the sectetin-related analogue VIP (10^–7 M) does not alter the secretin-induced cAMP generation in rat gastric glands. The glycoprotein nature of the secretin receptor has also been demonstrated using WGA-agarose affinity chromatography of the solubilized¹²⁵I-secretin receptor complex.     

Reduced peptide bond pseudopeptide analogues of secretin. A new class of secretin receptor antagonists.

The ability to assess the importance of secretin in various physiological processes is limited by the lack of specific potent antagonists. Recently, reduced peptide bond (psi) analogues of bombesin or substance P in which the -CONH- bond is replaced by -CH2NH- are reported to be receptor antagonists. To attempt to develop a new class of secretin receptor antagonists, we have adopted a similar strategy with secretin and sequentially altered the eight NH2-terminal peptide bonds, the biological active portion of secretin. In guinea pig pancreatic acini, secretin caused a 75-fold increase in cyclic AMP (cAMP). Secretin inhibited 125I-secretin binding with a half-maximal effect at 7 nM. Each of the psi analogues inhibited 125I-secretin binding. [psi 4,5]Secretin was the most potent, causing the half-maximal inhibition at 4 microM, and was 2-fold more potent than the [psi 1,2]secretin; 7-fold more than [psi 3,4]secretin, [psi 5,6]secretin, and [psi 8,9]secretin; 9-fold more than [psi 7,8]secretin; 13-fold more potent [psi 6,7]secretin, and 17-fold more than [psi 2,3]secretin. Secretin caused a half-maximal increase in cAMP at 1 nM. At concentrations up to 10 microM, [psi 2,3]secretin, [psi 4,5]secretin, and [psi 8,9]secretin did not alter cAMP whereas [psi 1,2]secretin and [psi 6,7]secretin caused a detectable increase in cAMP at 10 nM, [psi 7,8]secretin at 300 nM, [psi 5,6]secretin at 1 microM, and [psi 3,4]secretin at 10 microM. The [psi 4,5], [psi 2,3], and [psi 8,9] analogues of secretin each inhibited 1 nM secretin-stimulated cAMP as well as [psi 3,4]secretin, which functioned as a partial agonist. [psi 4,5]Secretin was the most potent, causing half-maximal inhibition at 3 microM whereas [psi 8,9]secretin was 6-fold less potent, and [psi 2,3]secretin and [psi 3,4]secretin were 17-fold less potent. [psi 4,5]Secretin inhibited secretin-stimulated cAMP and binding of 125I-secretin in a competitive manner. [psi 4,5]Secretin did not interact with cholecystokinin, bombesin, calcitonin gene-related peptide, or cholinergic receptors but did interact with receptors for vasoactive intestinal peptide, causing half-maximal inhibition at 72 microM and thus had a 18-fold higher affinity for secretin than vasoactive intestinal peptide receptors. These results indicate that reduced peptide bond analogues of the NH2 terminus of secretin represent a new class of secretin receptor antagonists. It is likely that in the future even more potent members of this class can be developed which may be useful to investigate the role of secretin in various physiological processes.     

Autoradiographic visualization of CNS receptors for vasoactive intestinal peptide

Receptors for VIP were characterized in the rat CNS. 125I-VIP bound with high affinity to rat brain slices. Binding was time dependent and specific. Pharmacology studies indicated that specific 125I-VIP binding was inhibited with high affinity by VIP and low affinity by secretin and PHI. Using in vitro autoradiographic techniques high grain densities were present in the dentate gyrus, pineal gland, supraoptic and suprachiasmatic nuclei, superficial gray layer of the superior colliculus and the area postrema. Moderate grain densities were present in the olfactory bulb and tubercle, cerebral cortex, nucleus accumbens, caudate putamen, interstitial nucleus of the stria terminalis, paraventricular thalamic nucleus, medial amygdaloid nucleus, subiculum and the medial geniculate nucleus. Grains were absent in the corpus callosum and controls treated with 1 microM unlabeled VIP. The discrete regional distribution of VIP receptors suggest that it may function as an important modulator of neural activity in the CNS.     

Stimulatory effects of Gila monster venom on rat pancreatic acini

The effects of Gila monster venom on dispersed rat pancreatic acini were compared with those of secretin and VIP. The efficacy of the venom in terms of amylase release was much higher (a 24-fold increase over basal secretion) than that of secretin (a 4-fold increase) and VIP (+ 40% only). On the other hand, cyclic AMP levels increased 12-fold with the venom, as compared to 18-fold with secretin and 16-fold with VIP. The venom, VIP and secretin all displaced 125I-VIP and the competition curve with the venom was steeper, suggesting that all VIP-recognizing receptors bound the venom with the same affinity. VIP receptors were, however, not responsible for the release of amylase provoked by the venom since VIP (and secretin) did not inhibit the secretory action of the venom. The venom exerted no effect on 45Ca efflux and its secretory effect did not depend on the presence of external calcium. Besides, the effect of CCK-8 on amylase release was additive with the effect of the venom. A first exposure to the venom induced a refractoriness to itself with respect to amylase release but not in terms of cyclic AMP increase. In conclusion, Gila monster venom may contain one component binding to VIP/secretin receptors with resulting cyclic AMP elevation. A second venom component may be responsible for the high secretory efficacy, without involving cyclic AMP or calcium efflux.     

High affinity peptide histidine isoleucine-preferring receptors in rat liver

Peptide Histidine Isoleucine (PHI) is generally considered a low affinity agonist for Vasoactive Intestinal Peptide (VIP) receptors. In this study, we investigated the presence and characteristics of [125I]PHI binding sites on rat liver membranes. Detergents at nonsolubilizing concentrations (1 mM CHAPS or 0.01% Tween-20) were included in the assay buffer to reduce adsorptive loss of PHI to acceptable levels and permit measurement of PHI-binding to receptors. Under these conditions, binding of PHI to liver membranes was time- and temperature-dependent, reversible and saturable. Unlabeled PHI was 9.7-fold more potent than VIP, and 357-fold more potent than secretin in displacing [125I]-PHI binding. Scatchard analysis suggested the presence of two classes of PHI receptors, with Kd 27 pM and 512 pM. The data from [125I]-PHI and [125I]-VIP binding studies suggested that one class of receptors was PHI-preferring, and the other, equally reactive with PHI and VIP. The concentration of immunoreactive PHI, measured by radioimmunoassay, in blood from the hepatic portal vein of anesthetized rats was 2-fold higher than that from the hepatic vein, suggesting uptake of circulating PHI by the liver.