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.     

Interaction of Gila monster venom with VIP receptors in intestinal epithelium of human. A comparison with rat

Gila monster venom (1-300 micrograms/ml) is shown to inhibit completely the binding of [125I]VIP to human and rat intestinal epithelial cell membranes. In both models, the venom inhibits [125I]VIP binding and stimulates adenylate cyclase with a maximal efficiency that is similar to that of VIP and a potency that is 10000-50000 times lower than that of the peptide, on a weight basis. At maximal doses, VIP and Gila monster venom do not exert an additive effect on adenylate cyclase, suggesting that the activation of the enzyme by the venom occurs through VIP receptors. As is the case for VIP, adenylate cyclase activation by Gila monster venom requires the presence of GTP in the incubation medium. Finally, no VIP-like immunoreactivity was detected in the venom using an antiserum raised against mammalian VIP. All these data suggest the presence in the venom of the Gila monster, of a new substance which behaves as a VIP agonist in human as well as rat intestine.     

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.     

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.     

Aldehydic peptides inhibiting renin

The metabolism of 20:4 (arachidonic acid) in alkenylacyl, alkylacyl and diacyl lipid classes in choline glycerophospholipids (CGP) and ethanolamine glycerophospholipids (EGP) in rabbit alveolar macrophages was examined. [3H]20:4 was very rapidly incorporated into diacyl glycerophosphocholine (GPC). After the removal of free 20:4, the radioactivity was gradually lost from diacyl GPC. Concomitantly, the radioactivities in alkylacyl GPC and alkenylacyl glycerophosphoethanolamine (GPE) were increased, indicating that 20:4 was mobilized from diacyl GPC to alkylacyl GPC and alkenylacyl GPE. The mobilization was considered to be a 20:4-specific event. The gradual accumulation of 20:4 in ether phospholipids leads to a high abundance of 20:4 in these lipids. These results suggest metabolic relationships between 20:4 and ether phospholipids, including platelet-activating factor (PAF).     

Importance of disulfide bonds in receptors for vasoactive intestinal peptide and secretin in rat pancreatic plasma membranes

(1) Vasoactive intestinal peptide (VIP), secretin, and C-terminal octapeptide of cholecystokinin (CCK-8) receptors were identified in rat pancreatic plasma membranes by the ability of these peptides to stimulate adenylate cyclase activity. The membrane preparation procedure was conducted through a series of steps including discontinuous sucrose density gradient fractionation. 5 mM β-mercaptoethanol was added stepwise. Membrane preparations obtained stepwise were preincubated for 10 min at 25°C in the presence of various concentrations of β-mercaptoethanol or dithiothreitol before assaying adenylate cyclase. The use of the reducing agents exerted no effect on p[NH]ppG-, NaF-, and CCK-8- stimulated activities. By contrast, stimulation of adenylate cyclase by low VIP concentrations was specifically altered when β-mercaptoethanol was used during tissue homogeneization at 5°C. (2) In addition, both VIP and secretin responses were highly sensitive towards a preincubation of 10 min at 25°C in the presence of dithiothreitol. (3) These results were likely to reflect alterations at the receptor level. ¹²⁵I-VIP binding was, indeed, reduced after dithiothreitol preincubation, low concentrations of the thiol reagent decreasing the apparent number of high-affinity VIP receptors and higher dithiothreitol concentrations reducing the affinity of VIP receptors.     

Vasoactive Intestinal Peptide (VIP) and peptide having N-terminal histidine and C-terminal isoleucine amide (PHI) stimulate adenylate cyclase activity in human heart membranes

The presence of receptors, recognized by Vasoactive Intestinal Peptide (VIP) and Peptide having N-terminal Histidine and C-terminal Isoleucine amide (PHI), was documented in membranes from human right auricle and left ventricular cardiac muscle by the ability of these peptides to stimulate adenylate cyclase. The capacity of VIP and PHI to activate the enzyme was comparable, in auricle as well as ventricle membranes, the affinity of the system being moderately higher for VIP than for PHI. In auricles, dose-effect curves appeared compatible with the coexistence of high-affinity and low-affinity VIP receptors. PHI could not, however, discriminate these subclasses of VIP receptors.     

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 secretin5–27 and its analogues with hormone receptors on pancreatic acini

The C-terminal tricosapeptide of secretin (S_5–27) and two analogues, one with asparagine replacing aspartic acid in position 15 (15-Asn-S_5–27) and one with lysine replacing aspartic acid in position 15 (15-Lys-S_5–27) were tested for their abilities to interact with hormone receptors on pancreatic acinar cells. In interacting with the receptors which prefer vasoactive intestinal peptide (vaso-active intestinal peptide-preferring receptors), the apparent affinity of 15-Asn-S_5–27 was equal to that of 15-Lys-S_5–27 and was greater than that of S_5–27. In interacting with secretin-preferring receptors, the apparent affinity of 15-Asn-S_5–27 was equal to that of S_5–27 and was greater than that of 15-Lys-S_5–27. In interacting with the secretin-preferring receptors each of the secretin fragments was approximately 2% as effective as secretin in causing an increase in cellular cyclic AMP. None of these fragments was able to cause a detectable increase in cyclic AMP mediated by the vasoactive intestinal peptide-preferring receptors. The dose vs. response curves for the action of secretin and vasoactive intestinal peptide on cellular cyclic AMP and on amylase secretion as well as the patetern of effects of secretin fragments on these actions indicated that the increase in amylase secretion caused by vasoactive intestinal peptide and secretin is mediated exclusively by the vasoactive intestinal peptide-preferring receptors. Furthermore, occupation of approximately 50% of the vasoactive intestinal peptide-preferring receptors is sufficient to cause maximal stimulation of amylase secretion.     

Secretin Receptors on Neuroblastoma Cell Membranes: Characterization of 125I-Labeled Secretin Binding and Association with Adenylate Cyclase

Secretin, a gut-brain peptide, elicited cyclic AMP production in a clone of neuroblastoma cells derived from the C1300 mouse tumor. Adenylate cyclase (EC 4.6.1.1) in plasma membranes from these cells was stimulated by secretin greater than vasoactive intestinal peptide greater than peptide histidine isoleucine amide, but not by the related peptides glucagon, gastric inhibitory polypeptide, or human growth hormone releasing factor. Hill coefficients for stimulation approximated one and the response to submaximal peptide concentrations was additive, as expected for hormones competing for a single receptor associated with the enzyme. Binding of 125I-labeled secretin to the neuroblastoma plasma membranes was saturable, time-dependent, and reversible. The KD determined from kinetic and equilibrium binding studies approximated 1 nM. The binding site displayed marked ligand specificity that paralleled that for stimulation of adenylate cyclase. The secretin receptor was regulated by guanine nucleotides, with guanosine 5'-(beta, gamma-imino)-triphosphate being the most potent to accelerate the rate of dissociation of bound secretin. These findings demonstrate the functional association of the secretin receptor with adenylate cyclase in neuronally derived cells.     

Specific decrease of secretin/VIP-stimulated adenylate cyclase in the heart from the Lyon strain of hypertensive rats

The cardiac adenylate cyclase activity of genetically hypertensive rats from the Lyon strain (LH) was compared to that of Lyon normotensive rats (LN) and that of low blood pressure Lyon rats (LL). The major finding was a 30-35% decrease of secretin- and VIP-stimulated adenylate cyclase activity in cardiac membranes of LH rats that was already obvious in 5 week-old prehypertensive animals: this alteration was apparently specific for the cardiac secretin/VIP-stimulated adenylate cyclase activity, the same activity in membranes from brain, anterior pituitary, and liver being similar in LH, LN and LL rats. It is tempting to conclude that a selective alteration of functional cardiac secretin/VIP receptors in LH rats reflects a local hyperactivity of the sympathetic adrenergic system.     

Primary structure of helodermin, a VIP-secretin-like peptide isolated from Gila monster venom

The inhibitory regulatory component of adenylate cyclase (Ni) was highly purified from rat brain synaptic membranes. A low Km GTPase activity was always associated with Ni through the purification, and the recovery of GTPase activity correlated well with that of Ni. Purified Ni was hardly ADP-ribosylated by islet-activating protein (IAP). A heat-labile factor in the fraction of the stimulative regulatory component (Ns) restored ADP-ribosylation and also activated the GTPase about 2-fold. NaF which was reported to interact with Ni markedly reduced GTPase activity. The purified Ni fraction inhibited adenylate cyclase only in the presence of a heat-stable factor found in the partially purified regulatory component. GTPase and inhibitory activities were weak in myelin which contained only a small amount of Ni. These findings support the view that GTPase activity is an intrinsic activity of Ni and some factors are necessary for the function of Ni.     

Do Secretin and Vasoactive Intestinal Peptide Have Independent Receptors on Striatal Neurons and Glial Cells in Primary Cultures?

Vasoactive intestinal peptide (VIP) and secretin are two related peptides that activate adenylate cyclase on membranes of striatal neurons and glial cells from embryonic mouse brain grown in primary culture. On the two cell types, the maximal activation that could be induced by secretin was only 40% above basal activity, which represented less than 15% of the maximal effect obtainable with VIP. From competition experiments performed on glial cells and the neuroblastoma X glioma hybrid, NG 108-15, a cell line known to possess both VIP and secretin sensitive-adenylate cyclase, we demonstrate that secretin does not activate VIP receptors. Furthermore, secretin has an apparent high affinity (EC50 10(-8) M) for its receptors on striatal neurons and NG 108-15 whereas an apparent low affinity (EC50 7 X 10(-6) M) was found on striatal glial cells. This suggests the existence of either two distinct secretin receptors or a desensitized form.     

Interactions Between Vasoactive Intestinal Peptide and Dopamine in the Rabbit Retina: Stimulation of a Common Adenylate Cyclase

Although 3,4-dihydroxyphenylethylamine (dopamine, DA) and vasoactive intestinal peptide (VIP) have been reported to stimulate adenylate cyclase activity in the rabbit retina, possible interactions between VIP-sensitive and DA-sensitive adenylate cyclase systems have not been previously investigated. To elucidate the interactions between these two putative transmitter-stimulated cyclase systems, the effects of VIP, DA, and VIP + DA on the conversion of [alpha-32P]ATP to [32P]cyclic AMP in rabbit retinal homogenates were measured. VIP stimulated adenylate cyclase activity in a biphasic manner, suggesting that two classes of VIP receptors may be involved in the induction of cyclic AMP formation. DA was less potent than VIP, and stimulated cyclase activity with a monophasic dose-response curve. When assayed together, these stimulations were partially nonadditive, implying the existence of a common adenylate cyclase pool that may be stimulated by both putative neurotransmitters. The dopaminergic antagonist (+)-butaclamol completely blocked dopaminergic stimulation, but had no significant effect on VIP-induced stimulation, indicating that VIP interacts with specific VIP receptor sites, which are distinct from the dopaminergic receptor sites. Furthermore, the specific D-2 dopaminergic receptor agonist LY141865 demonstrated no inhibitory effect on adenylate cyclase activity, suggesting that the interaction between the VIP- and DA-sensitive adenylate cyclase systems does not result from a D-2 receptor-mediated cyclase inhibition in the rabbit retina. Finally, at maximally effective concentrations, DA and VIP were less potent than fluoride or forskolin in the stimulation of cyclic AMP formation, suggesting that adenylate cyclase pools that are not sensitive to DA and VIP may also be present in this retina.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of HIS1 modifications on the ability of vasoactive intestinal peptide to stimulate adenylate cyclase from rat and human tissues

The importance of the N-terminal His residue of VIP for stimulating adenylate cyclase was appreciated by estimating the intrinsic activity and EC50 of four VIP analogues on membranes from rat lung, liver, brain, anterior pituitary, and pancreas, and on human heart membranes. In all tissue preparations tested except one, the order of efficacy (and often potency) was: VIP greater than (Ac-His1)VIP greater than (Phe1)VIP = (3-Me-His1)VIP greater than (D-His1)VIP. In rat heart membranes, the order of efficacy was somewhat different: VIP greater than (Ac-His1)VIP = (Phe1)VIP greater than (D-His1)VIP greater than (3-Me-His1)VIP. These data demonstrated the key role of His1 in VIP in activating adenylate cyclase. They suggest that a given VIP analogue might act as full agonist in tightly coupled adenylate cyclase systems (such as those of rat lung and liver membranes) whereas the same analogue could not promote full activity in poorly coupled systems (such as that present in rat brain synaptic membranes).     

In-vivo stimulation of rat pancreatic acinar cells by infusion of secretin

Summary Infusion of synthetic secretin in conscious unrestricted rats for periods up to 24 h was used to study the structural and functional adaptation of pancreatic acinar cells to this secretagogue. Initial dose-response studies established 16 clinical units (CU) per kg and h (corresponding to 4.64 ug x kg^-1 x h^-1) as optimal dose for persistent stimulation of enzyme discharge. Infusion of this dose led to a slow but progressive depletion of enzyme stores with minimal content by 12 h stimulation. As a result of persistent stimulation total protein synthesis in the acinar cells increased after a lag period of 3 h and reached maximal values 90% above controls by 6 and 12 h secretin infusion. No structural equivalent for pronounced fluid and bicarbonate secretion was observed for either acinar or duct cells over the entire dose range (1 to 64 CU x kg^-1 x h^-1) and infusion period (1–24 h), except an increased number of coated vesicles in duct cells.Discharge of enzymes from acinar cells was paralleled by a high frequency of exocytotic images at the luminal plasma membrane and was accompanied by the occurrence of membrane fragments in the luminal space, especially after 3 and 6 h secretin infusion. An increased number of lysosomal bodies at these time points especially in the vicinity of the Golgi complex was interpreted in relation to membrane recycling following massive exocytosis. This pattern of structural and functional adaptation of acinar cells following secretin infusion corresponds to previously described changes following caerulein and carbamylcholine stimulation.Supported by a grant from Deutsche Forschungsgemeinschaft (Ke 113/15-1)     

Effects of Vasoactive Intestinal Peptide and Other Peptides on Cyclic AMP Accumulation in Intact Pieces and Isolated Horizontal Cells of the Teleost Retina

The effects of vasoactive intestinal peptide (VIP) and several other peptides have been examined on cyclic AMP accumulation in intact pieces and isolated horizontal cells of the teleost (carp) retina. VIP was the most effective peptide examined, inducing a dose-related response, and an approximately fivefold increase in cyclic AMP production when used at a concentration of 10 microM. Porcine histidine isoleucine-containing peptide and secretin, peptides structurally related to VIP, also stimulated cyclic AMP accumulation, but at concentrations of 10 microM induced responses which were only approximately 40% and 10%, respectively, of the response observed with 10 microM VIP. In contrast, several other peptides, including glucagon, neurotensin, somatostatin, luteinizing hormone-releasing hormone, alpha-melanocyte-stimulating hormone, cholecystokinin octapeptide26-33, gastrin-releasing peptide, thyrotropin-releasing hormone, and VIP10-28 were totally inactive. The response to 10 microM VIP was not antagonized by several dopamine antagonists, indicating the presence of a population of specific VIP receptors coupled to adenylate cyclase, distinct from the population of dopamine receptors coupled to adenylate cyclase also known to be present in this tissue. Finally, experiments involving the use of fractions of isolated horizontal cells indicate that these neurons possess a population of VIP receptors coupled to cyclic AMP production which would appear to share a common pool of adenylate cyclase with a population of similarly coupled dopamine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of a bovine thymic peptide extract with vasoactive intestinal peptide (VIP) receptors

Bovine t hymic peptide extract (1–100 g/ml) is shown to completely inhibit the binding of [¹²⁵I]VIP to rat blood mononuclear cells, lymphoid cells of spleen, and liver plasma membranes. In the three models, the bovine thymic peptide extract inhibits [¹²⁵I]VIP binding with a potency that is 4000–7000 times lower than that of the native VIP, on a weight basis. In rat liver plasma membranes, the bovine thymic peptide extract stimulates adenylate cyclase with a maximal efficiency that is similar to that of VIP. At maximal doses, VIP and thymic peptide extract do not exert an additive effect on adenylate cyclase, suggesting that the activation of the enzyme by the bovine thymic peptide extract occurs through VIP receptors. Finally, no VIP-like immunoreactivity was detected in the thymic peptide extract using an antiserum raised against mammalian VIP. All these data suggest the presence in the bovine thymic peptide extract of a new substance which behaves as a VIP agonist in rat.     

Corticosteroid modulation of muscarinic receptors in rat myocardial membranes

Rat ventricular myocardial membanes contain muscarinic acetylcholine receptors which can be identified by binding of the muscarinic antagonist (-)-[³H]quinuclidinyl benzilate. Scatchard analysis of saturation binding data revealed binding to a single class of non-cooperative sites (0.693 pmol/mg protein) with high affinity (i.e. with an equilibrium dissociation constant of 0.24 nM). Competition binding curves of the agonist carbamylholine were shallow (with a Hill coefficient, n_H of 0.71) for membranes of untreated rats, suggesting the presence of two receptor subpopulations with different agonist affinity. These curves were steeper (n_H = 0.86) for adrenalectomized animals and more shallow (n_H = 0.62) for hydrocortisone-treated animals. In contrast, both treatments did not affect the total receptor number. This suggests that corticosteroids are required for the myocardial muscarinic receptors to adopt high agonist affinity. However, the inhibition of adenylate cyclase by muscarinic agonists disappeared after both corticosteroid treatment and adrenalectomy. But agonist receptor binding could still be modulated by guanine nucleotides. This indicates that both high and low affinity froms of muscarinic receptors induced by altered corticosteroid states retain functional coupling with the inhibitory nucleotide binding site, but are uncoupled from the adenylate cyclase catalytic subunit, C.