Pharmacological characterization of the novel helodermin/VIP receptor present in human SUP-T1 lymphoma cell membranes

[Acetyl-His1]VIP stimulated adenylate cyclase with higher potency than VIP in membranes from human SUP-T1 lymphoblasts and was used as an efficient radioiodinated ligand with low non-specific binding to evaluate the relationship between receptor occupancy and adenylate cyclase activation and the possible interference of peptide T (an epitope derived from HIV envelope protein gp120). Various peptides inhibited [125I-acetyl-His1]VIP binding and activated the enzyme, their order of potency being: helodermin greater than [acetyl-His1]VIP greater than VIP = PHI = [Phe1]VIP greater than [D-Phe2]VIP = [D-Ala4]VIP = [D-Phe4]PHI greater than or equal to [D-Phe4]VIP greater than [D-His1]VIP giving further support for the existence of a novel subtype of helodermin/VIP receptors. [D-Ala1]peptide T and VIP-(10-28) did not recognize the binding site and did not inhibit, even at high concentration, VIP - or VIP analogue - stimulated adenylate cyclase activities.     

A new type of functional VIP receptor has an affinity for helodermin in human SUP-T1 lymphoblasts

A new type of VIP receptor was characterized in human SUP-T1 lymphoblasts. The order of potency of unlabeled peptides, in the presence of [125I]helodermin, was: helodermin(1-35)-NH2 = helodermin(1-27)-NH2 greater than helospectin greater than VIP = PHI greater than [D-Ser2]VIP greater than [D-Asp3]VIP greater than [D-His1]VIP greater than or equal to [D-Ala4]VIP greater than or equal to secretin = GRF. This specificity was distinct from that of all VIP receptors described so far in that: (i) the affinity for helodermin (Kd = 3 nM) was higher than that of VIP (Kd = 15 nM) and PHI (Kd = 20 nM); and (ii) position 4 played an important role in ligand binding. The labeled sites were likely to be functional receptors as adenylate cyclase in crude lymphoblastic membranes (200-10,000 x g pellets) was stimulated by peptides, in the presence of GTP, with the following order of potency: helodermin(1-35)-NH2 greater than helodermin(1-27)-NH2 greater than helospectin = VIP = PHI.     

Cerebral Vascular Adenylate Cyclase: Evidence for Coupling to Receptors for Vasoactive Intestinal Peptide and Parathyroid Hormone

We have studied the responsiveness of vascular adenylate cyclase to vasoactive intestinal peptide (VIP) and parathyroid hormone (PTH) using preparations of cerebral microvessels and arteries. Cerebral microvessels obtained from rats, guinea-pigs, cattle, and pigs all responded potently to bovine (b) PTH-(1-34), whereas considerable between-species variability was observed in the responsiveness to VIP. The homologous peptide to VIP, PHI (porcine heptacosapeptide), stimulated adenylate cyclase in both rat microvessels and a broken-cell preparation of bovine arteries. The ED50 values for activation of bovine arterial adenylate cyclase by VIP, PHI, and bPTH-(1-34) were 6.9 nM, 10 nM, and 100 nM, respectively, with the following order of efficacy: VIP = PHI greater than bPTH-(1-34). The other related peptides, hpGRF (human pancreatic growth hormone releasing factor), secretin, and glucagon, and the fragment VIP-(10-28) were inactive. The PTH antagonist, [Nle8, Nle18, Tyr34]bPTH-(3-34) amide, inhibited bPTH-(1-34) activation of vascular adenylate cyclase but did not affect activation by VIP using either microvessels or arteries. VIP or PHI demonstrated an additive effect with bPTH-(1-34) on vascular adenylate cyclase activity. However, the effects of VIP and PHI were nonadditive with each other. These data suggest that VIP and bPTH-(1-34) activate cerebral vascular adenylate cyclase by interacting with pharmacologically distinct receptors, whereas PHI and VIP likely interact with a common receptor.     

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.     

Comparison of helodermin, VIP and PHI in pancreatic secretion and blood flow in dogs

Helodermin, VIP and PHI, which share a high degree of homology with secretin, have been identified in the gut but their physiological role is unknown. In this study 3 series of tests were carried out to determine the actions of helodermin, VIP and PHI on pancreatic secretion in 6 conscious dogs and amylase release from the dispersed canine pancreatic acini and to correlate the alterations in pancreatic secretory and circulatory effects in 24 anesthetized dogs. Helodermin, VIP and PHI infused i.v. in graded doses (12.5-200 pmol/kg.h) resulted in a dose-dependent increase in pancreatic HCO3 secretion reaching, respectively, 100%, 7% and 2% of secretin maximum. When combined with constant dose infusion of CCK-8 (100 pmol/kg.h), helodermin but not VIP or PHI augmented dose-dependently the HCO3 secretion. When added in various concentrations (10(-10)-10(-5)M) to the incubation medium of dispersed pancreatic acini only helodermin but not VIP or PHI increased dose-dependently amylase release reaching about 50% of CCK-8 maximum. In anesthetized dogs, the pancreatic blood flow (PBF) measured by electromagnetic blood flowmetry showed an immediate and dose-dependent increase following the injections of various doses of helodermin, VIP, PHI and secretin, the peak blood flow preceding by about 1 min the peak secretory stimulation. This study shows that helodermin resembles secretin in its potent pancreatic HCO3 stimulation but differs from VIP or PHI which are poor secretagogues but potent vasodilators. We conclude that if tested peptides are released in the gut, helodermin, like secretin, may be involved in the hormonal stimulation of exocrine pancreas, whereas VIP and PHI may serve mainly as vasodilators in the pancreatic circulation.     

Molecular Characteristics and Peptide Specificity of Vasoactive Intestinal Peptide Receptors from Rat Cerebral Cortex

Vasoactive intestinal peptide (VIP) receptors have been identified in CNS by their chemical specificity and molecular size. Using synaptosomes isolated from rat cerebral cortex, it was shown that central VIP receptors discriminated among natural and synthetic VIP-related peptides, because half-maximal inhibition of [125I]VIP binding to synaptosomes was obtained for 0.6 nM VIP, 9 nM peptide histidine isoleucineamide (PHI), 50 nM VIP 2-28, 70 nM secretin, 100 nM rat growth hormone-releasing factor (GRF), and 350 nM human GRF. Other peptides of the VIP family, such as glucagon and gastric inhibitory polypeptide, did not interact with cortical VIP receptors. The molecular components of VIP receptors in rat cerebral cortex were identified after [125I]VIP cross-linking to synaptosomes using the cross-linker dithiobis(succinimidyl propionate). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of synaptosomal proteins revealed two major [125I]VIP-protein complexes of Mr 49,000 and 18,000. The labeling of the Mr 49,000 component was specific, because it was abolished by native VIP, whereas the labeling of the Mr 18,000 component was not. Natural VIP agonists reduced the labeling of the Mr 49,000 component with the following order of potency: VIP greater than PHI greater than secretin approximately equal to rat GRF. In contrast, glucagon and octapeptide of cholecystokinin were without effect, a result indicating its peptide specificity. Densitometric scanning of autoradiographs showed that the labeling of the Mr 49,000 component was inhibited by low VIP concentrations between 10(-10) and 10(-6) M (IC50 = 0.8 nM), a result indicating the component's high affinity for VIP.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Occurrence of VIP and peptide HM in human pancreas and their influence on pancreatic endocrine secretion in man

By immunohistochemistry it was found that VIP- and peptide HI/peptide HM (PHI/PHM)-like immunoreactivity occurred in autonomic neurons in the human pancreas. Antisera against both VIP and PHI/PHM reacted with neuronal cells in local ganglia and these ganglia also contained PHI/PHM- and VIP-immunoreactive fibre plexuses. VIP- and PHI/PHM-positive fibres were also seen close to the Langerhans' islets. In addition, PHI/PHM- but not VIP-like immunoreactivity was observed in the endocrine cells often located in the periphery of the islets. The nature of these PHI/PHM-positive cells remains to be established. I.v. infusion of VIP at constant rates of 300 and 900 pmol/kg X h for 30 min in 6 healthy volunteers resulted in plateau values of 102 +/- 26 and 291 +/- 25 pM, respectively. These levels of VIP which are above those found in the circulation under physiological conditions stimulated secretion of insulin, C-peptide and pancreatic glucagon dose-dependently. On the contrary prolonged (60 min) infusion of PHM in doses resulting in plasma levels up to 1340 +/- 405 pM had no effect on pancreatic hormone secretion. These findings suggest that VIP is a likely neurotransmitter in the control of endocrine pancreatic secretion while PHM has a less prominent role, if any.     

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.     

Helodermin stimulates glucagon secretion in the mouse

Helodermin is structurally similar to VIP (vasoactive intestinal peptide) and PHI (peptide histidine isoleucine). Since VIP and PHI both stimulate insulin and glucagon secretion, we investigated the effects of helodermin on insulin and glucagon secretion in the mouse, both in the basal state and during administration of glucose and the cholinergic agonist carbachol. After intravenous injection at dose levels between 0.5 and 8.0 nmol/kg, helodermin markedly enhanced basal plasma glucagon levels, for example at 8 nmol/kg from 139 +/- 14 to 421 +/- 86 pg/ml (p less than 0.001) after 6 minutes, without affecting basal plasma insulin levels. Together with glucose (2.8 mmol/kg), helodermin (2 and 8 nmol/kg) augmented plasma glucagon levels but had no effect on plasma insulin levels. When injected together with the cholinergic agonist carbachol (0.16 mumol/kg), helodermin markedly potentiated the increase in plasma glucagon levels (more than three-fold; p less than 0.001), again without affecting the plasma insulin levels. Combined alpha- and beta-adrenoceptor blockade (yohimbine + L-propranolol) reduced the augmenting effect of helodermin on glucagon secretion by approximately 60%. It is concluded helodermin stimulates glucagon secretion in the mouse by an effect that is partially antagonized by combined alpha- and beta-adrenoceptor antagonism.     

Molecular characteristics and evidence for internalization of vasoactive-intestinal-peptide (VIP) receptors in the tumoral rat-pancreatic acinar cell line AR 4-2 J

1. Vasoactive intestinal peptide (VIP) receptors were investigated in the tumoral acinar cell line AR 4-2 J derived from rat pancreas [125I]Iodo-VIP binding to cell membranes showed the following IC50 values for unlabeled peptides: VIP, 0.3 nM; peptide His-IleNH2, 2 nM; helodermin, 30 nM; secretin, 100 nM. After incubation with 20 nM dexamethasone, the binding capacity increased twofold but affinities were unchanged. External [125I]iodo-VIP binding to intact cells reached steady state after 5 min at 37 degrees C, while the sequestration-internalization of the [125I]iodo-VIP-receptor complex (tested by cold acid washing) increased progressively, reaching 75% of total binding after 1 h. This phenomenon was blocked at 4 degrees C. Further data with dexamethasone, tunicamycin, cycloheximide, low temperature, and/or phenylarsine oxide, suggested a half-life of 2 days for VIP receptors and the necessity of N-glycosylation for proper translocation. 2. For chemical [125I]iodo-VIP cross-linking bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone gave the best yield when compared with five other bifunctional reagents. In membranes, the main specifically cross-linked peptide had Mr 66,000 under nonreducing conditions, and migrated with lower velocity (-5%) under reducing conditions. Cross-linking was suppressed by VIP, peptide His-IleNH2 and helodermin (competitively) and also by GTP. In intact cells, the Mr of [125I]iodo-VIP-cross-linked peptides depended on the mode of cell solubilization. After direct solubilization, the major cross-linked radioactivity migrated as a smear of Mr 130,000-180,000 but an Mr-66,000 peptide was also detectable. In contrast, the solubilization of cross-linked cells detached by mild trypsinisation gave mainly the Mr-66,000 labeled peptide. This suggests that most VIP receptors in intact, attached cells were in a high-Mr complex and that mild cell treatment was sufficient to disrupt this complex.     

Presence of highly selective receptors for PACAP (pituitary adenylate cyclase activating peptide) in membranes from the rat pancreatic acinar cell line AR 4-2J

We characterized highly selective receptors for PACAP, the pituitary adenylate cyclase activating peptide, in the tumoral acinar cell line AR 4-2J derived from the rat pancreas. PACAP, a novel hypothalamic peptide related to vasoactive intestinal peptide (VIP), was tested as the full natural 38-residue peptide (PACAP-38) and as an N-terminal amidated 27-residue derivative (PACAP-27). The binding sites showed considerable affinity for [125I]PACAP-27 (Kd = 0.4 nM) and PACAP-38, while their affinity for VIP and the parent peptide helodermin was 1000-fold lower. These receptors were coupled to adenylate cyclase, the potency of PACAP-38 and PACAP-27 (Kact = 0.2 nM) being much higher than that of VIP (Kact = 100 nM) and helodermin (Kact = 30 nM). Chemical cross-linking of [125I]PACAP-27 followed by SDS-PAGE and autoradiography revealed a specifically cross-linked peptide with an Mr of 68,000 (including 3000 for one PACAP-27 molecule).     

Specific labelling by [125I]helodermin of high-affinity VIP receptors in rat liver membranes

Helodermin, a newly isolated peptide from Gila Monster venom, is structurally related to VIP and secretin. When used as radioligand, [125I]helodermin bound rapidly and reversibly to crude rat liver membranes, the dissociation being accelerated by GTP. Competition binding curves of [125I]helodermin and [125I]VIP with unlabelled peptides showed the following order of decreasing affinity: VIP greater than helodermin greater than secretin greater than hpGRF(1-29)-NH2. The shape of binding curves and of concurrent adenylate cyclase activation is compatible with the specific labelling, by [125I]helodermin, of a class of high-affinity VIP receptors that is capable to stimulate adenylate cyclase.     

Role of PAC(1) receptor in adrenal catecholamine secretion induced by PACAP and VIP in vivo

The present study was conducted to investigate the functional implication of the pituitary adenylate cyclase-activating polypeptide (PACAP) type I (PAC(1)) receptor in the adrenal catecholamine (CA) secretion induced by either PACAP-27 or vasoactive intestinal polypeptide (VIP) in anesthetized dogs. PACAP-27, VIP, and their respective antagonists were locally infused to the left adrenal gland via the left adrenolumbar artery. Plasma CA concentrations in adrenal venous and aortic blood were determined by means of a high-performance liquid chromatograph coupled with an electrochemical detector. Adrenal venous blood flow was measured by gravimetry. The administration of PACAP-27 (50 ng) resulted in a significant increase in adrenal CA output. VIP (5 microg) also increased the basal CA secretion to an extent comparable to that observed with PACAP-27. In the presence of PACAP partial sequence 6--27 [PACAP-(6--27); a PAC(1) receptor antagonist] at the doses of 7.5 and 15 microg, the CA response to PACAP-27 was attenuated by approximately 50 and approximately 95%, respectively. Although the CA secretagogue effect of VIP was blocked by approximately 85% in the presence of PACAP-(6--27) (15 microg), it remained unaffected by VIP partial sequence 10--28 [VIP-(10--28); a VIP receptor antagonist] at the dose of 15 microg. Furthermore, the CA response to PACAP-27 did not change in the presence of the same dose of VIP--(10--28). The results indicate that PACAP-(6--27) diminished, in a dose-dependent manner, the increase in adrenal CA secretion induced by PACAP-27. The results also indicate that the CA response to either PACAP-27 or VIP was selectively inhibited by PACAP-(6--27) but not by VIP-(10--28). It is concluded that PAC(1) receptor is primarily involved in the CA secretion induced by both PACAP-27 and VIP in the canine adrenal medulla in vivo.     

Functional expression of VIP receptors in normal, immortalized and transformed mammary epithelial cells.

The effect of VIP and its related peptides on cAMP production has been characterized: 1) in long term culture of normal human mammary epithelial cells (HMEC); 2) in immortalized and transformed ST cell lines established from normal HMEC after genomic insertion of the large T oncogene of SV40; 3) in the spontaneously immortalized HC-11 cells, a clone isolated from the mouse mammary epithelial cells COMMA-1D, described to exhibit normal morphogenesis in vivo and functional differentiation in vitro. Basal cAMP levels were increased 1.5- to 8.7-fold in mammary epithelial cells (p less than 0.001-0.05), with a potency EC50 = 0.02-0.6 nM VIP. The pharmacological specificity of the VIP receptors coupled to cAMP generation was established according to the following potency sequence: VIP greater than PACAP-38 greater than helodermin greater than PHM, PHV greater than helospectin 1 much greater than hpGRF, secretin in HMEC, VIP greater than PACAP-38 greater than helodermin greater than helospectin 1, PHM, PHV greater than hpGRF greater than secretin in S1T3 cells, and VIP, PHI, helodermin greater than PHV greater than rhGRF greater than secretin in HC-11 cells. Our data demonstrate the presence of functional, highly sensitive and specific VIP receptors in normal, immortalized and transformed mammary epithelial cells, suggesting a regulatory role for this neuropeptide on the growth, differentiation and function in normal and neoplastic breast tissue.     

Vasoactive intestinal polypeptide carboxy-terminal fragment, VIP(22-28), and other fragments of VIP, in the central nervous system of the rat

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).     

Rat and guinea pig pancreatic acini possess both VIP(1) and VIP(2) receptors, which mediate enzyme secretion

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.     

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.     

Functional receptors for vasoactive intestinal polypeptide in cultured astroglia from neonatal rat brain

The effects of vasoactive intestinal polypeptide (VIP) were assessed on astroglia cultured from rat CNS. In these cultures VIP (500 nM) promoted the hydrolysis of [3H]glycogen newly synthesized from [3H]glucose. This effect on [3H]glycogen levels was also observed with the structurally related peptide PHI-27 and with other substances which had been demonstrated to promote glycogenolysis in rodent CNS in vitro such as: norepinephrine (NE), serotonin, histamine, adenosine, K+ and dibutyryl cyclic-AMP (dbcAMP). Furthermore, VIP (500 nM) and PHI 27 (500 nM), when applied to astroglial cultures in serum-free medium, displayed marked effects on the morphological appearance of the cell population: they converted the flat cells present in the cultures into cells with typical astrocytic morphology. As previously reported, this effect on the cellular morphology of the cultures was also observed, under identical experimental conditions, after NE and dbcAMP application. These studies demonstrate that cultured rat neonatal astroglia possess receptors for VIP, and suggest that a cyclic AMP accumulation may mediate both the metabolic and morphologic components of this response.     

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.