Receptor binding of pancreatic spasmolytic polypeptide in intestinal mucosal cells and membranes

Rat intestinal mucosal cells contain receptors for pancreatic spasmolytic polypeptide (PSP). The binding of 125I-PSP was rapid, saturable, reversible and specific. PSP competed with 125I-PSP for binding to the receptors and 10(-7) M of PSP half-maximally inhibited 125I-PSP binding. The normalized PSP dose-response graphs in intact cells and crude membranes were superimposable. Scatchard plots of PSP binding to membranes were curvilinear, indicating multiple classes of binding sites, negative cooperative interaction between sites or a combination of both. PSP increased the rate of dissociation of the 125I-PSP-receptor complex compared to the rate observed by dilution only, thus giving evidence that negative cooperative interaction may occur between PSP binding sites. The half-life of the fast dissociating complex was about 1.5 min and that of the slow dissociating complex 38 min. These values were independent of the receptor occupancy. The increased rate of dissociation at high receptor occupancy stemmed from a shift in the ratio of the pool sizes of fast and slow dissociating receptor complexes.     

Immunohistochemical localization of pancreatic spasmolytic polypeptide (PSP) in the pig

Summary Pancreatic spasmolytic polypeptide (PSP) is a peptide that is isolated from the porcine pancreas and that affects intestinal motility and growth of intestinal tumour cells in vitro. The peptide was recently demonstrated to be present in large amounts in pancreatic juice. The cellular origin of the peptide, however, is largely unclarified and the localization was therefore studied of PSP in pigs using immunohistochemistry. Positive immunoreactions were seen in the pancreas, the stomach, the duodenum, the jejunum and the ileum. In the pancreas, the PSP immunoreaction was seen in all acinar cells; no immunoreaction was seen in the endocrine islets. In the stomach, it was localized to the mucous cells of the glands in the cardiac gland region, the corpus and the pylorus. In the duodenum a strong immunoreaction was present in Brunner's glands and in the cells of their excretory ducts. In the jejunum and ileum, PSP immunoreactivity was seen in some of the cells in the epithelium of the crypts of Lieberkühn. A peptide chromatographically identical to highly purified PSP was identified in pancreas and stomach extracts. Thus epithelial cells in all parts of the stomach and small intestine contribute to the supply of PSP to the gut lumen.     

Immunohistochemical localization of pancreatic spasmolytic polypeptide (PSP) in the pig.

Pancreatic spasmolytic polypeptide (PSP) is a peptide that is isolated from the porcine pancreas and that affects intestinal motility and growth of intestinal tumour cells in vitro. The peptide was recently demonstrated to be present in large amounts in pancreatic juice. The cellular origin of the peptide, however, is largely unclarified and the localization was therefore studied of PSP in pigs using immunohistochemistry. Positive immunoreactions were seen in the pancreas, the stomach, the duodenum, the jejunum and the ileum. In the pancreas, the PSP immunoreaction was seen in all acinar cells; no immunoreaction was seen in the endocrine islets. In the stomach, it was localized to the mucous cells of the glands in the cardiac gland region, the corpus and the pylorus. In the duodenum a strong immunoreaction was present in Brunner's glands and in the cells of their excretory ducts. In the jejunum and ileum, PSP immunoreactivity was seen in some of the cells in the epithelium of the crypts of Lieberkühn. A peptide chromatographically identical to highly purified PSP was identified in pancreas and stomach extracts. Thus epithelial cells in all parts of the stomach and small intestine contribute to the supply of PSP to the gut lumen.     

Domoic acid inhibits adenylate cyclase activity in rat brain membranes

Adenylate cyclase activity measured by the formation of cyclic AMP in rat brain membranes was inhibited by a shellfish toxin, domoic acid (DOM). The inhibition of enzyme was dependent on DOM concentration, but about 50% of enzyme activity was resistant to DOM-induced inhibition. Rat brain supernatant resulting from 105,000×g centrifugation for 60 min, stimulated adenylate cyclase activity in membranes. Domoic acid abolished the supernatant-stimulated adenylate cyclase activity. The brain supernatant contains factors which modulate adenylate cyclase activity in membranes. The stimulatory factors include calcium, calmodulin, and GTP. In view of these findings, we examined the role of calcium and calmodulin in DOM-induced inhibition of adenylate cyclase in brain membranes. Calcium stimulated adenylate cyclase activity in membranes, and further addition of calmodulin potentiated calcium-stimulated enzyme activity in a concentration dependent manner. Calmodulin also stimulated adenylate cyclase activity, but further addition of calcium did not potentiate calmodulin-stimulated enzyme activity. These results show that the rat brain membranes contain endogenous calcium and calmodulin which stimulate adenylate cyclase activity. However, calmodulin appears to be present in membranes in sub-optimal concentration for adenylate cyclase activation, whereas calcium is present at saturating concentration. Adenylate cyclase activity diminished as DOM concentration was increased, reaching a nadir at about 1 mM. Addition of calcium restored DOM-inhibited adenylate cyclase activity to the control level. Similarly, EGTA also inhibited adenylate cyclase activity in brain membranes in a concentration dependent manner, and addition of calcium restored EGTA-inhibited enzyme activity to above control level. The fact that EGTA is a specific chelator of calcium, and that DOM mimicked adenylate cyclase inhibition by EGTA, indicate that calcium mediates DOM-induced inhibition of adenylate cyclase activity in brain membranes. While DOM completely abolished the supernatant-, and Gpp (NH)p-stimulated adenylate cyclase activity, it partly blocked calmodulin-, and forskolin-stimulated adenylate cyclase activity in brain membranes. These results indicate that DOM may interact with guanine nucleotide-binding (G) protein and/or the catalytic subunit of adenylate cyclase to produce inhibition of enzyme in rat brain membranes.     

Characterization of a vasoactive intestinal peptide-sensitive adenylate cyclase in rat intestinal epithelial cell membranes

A vasoactive intestinal peptide-sensitive adenylate cyclase in intestinal epithelial cell membranes was characterized. Stimulation of adenylate cyclase activity was a function of vasoactive intestinal peptide concentration over a range of 1 · 10⁻¹⁰−1 · 10⁻⁷ M and was increased six-times by a maximally stimulating concentration of vasoactive intestinal peptide. Half-maximal stimulation was observed with 4.1 ± 0.7 nM vasoactive intestinal peptide. Fluoride ion stimulated adenylate cyclase activity to a higher extent than did vasoactive intestinal peptide. Under standard assay conditions, basal, vasoactive inteetinal peptide- and fluoride-stimulated adenylate cyclase activities were proportional to time of incubation up to 15 min and to membrane concentration up to 60 μg protein per assay. The vasoactive intestinal peptide-sensitive enzyme required 5–10 mM Mg²⁺ and was inhibited by 1 · 10⁻⁵ M Ca²⁺. At sufficiently high concentrations, both ATP (3 mM) and Mg²⁺ (40 mM) inhibited the enzyme.Secretin also stimulated the adenylate cyclase activity from intestinal epithelial cell membranes but its effectiveness was 1/1000 that of vasoactive intestinal peptide. Prostaglandins E₁ and E₂ at 1 · 10⁻⁵ M induced a two-fold increase of cyclic AMP production. Vasoactive intestinal peptide was the most potent stimulator of adenylate cyclase activity, suggesting an important physiological role of this peptide in the cyclic AMP-dependent regulation of the intestinal epithelial cell function.     

Galanin inhibits adenylate cyclase of rat brain membranes.

The ubiquitous neuropeptide, galanin, strongly inhibits adenylate cyclase in rat brain membranes. While basal enzyme activity was not altered, galanin from 10(-11) M to 5 x 10(-7) M decreased forskolin- and VIP-stimulated adenylate cyclase with a half-maximal effect being elicited by 0.7 nM neuropeptide and a maximal 80% inhibition of the enzyme activity. The galanin fragments (2-29) and (1-15) dose-dependently inhibited the forskolin-stimulated adenylate cyclase, while the fragments (3-29) and (10-29) were found inactive. These results indicate that the regulatory action of galanin in the central nervous system involves the coupling of galanin receptors to the inhibition of the adenylate cyclase system.     

Vasoactive intestinal polypeptide (VIP) stimulates adenylate cyclase in selected areas of rat brain

VIP stimulated adenylate cyclase activity in homogenates of some areas of rat brain that are rich in this peptide, e.g., cerebral cortex, hypothalamus and hippocampus, as well as in cerebellar cortex, where VIP content is low. No stimulation occurred in caudate nucleus or brainstem. The enzyme stimulation was inhibited by Ca²⁺, but unaffected by guanine nucleotides. Synthetic fragments of VIP (VIP_6?28 & VIP_14–28) neither stimulated cyclase activity nor inhibited VIP-induced stimulation.     

Vasoactive intestinal polypeptide and glucagon: stimulation of adenylate cyclase activity via distinct receptors in liver and fat cell membranes

Vasoactive intestinal polypeptide (VIP), a peptide hormone that is chemically and biologically related to glucagon and secretin, stimulates the activity of adenylate cyclase in liver and fat cell membranes. Effects of combinations of VIP with glucagon and secretin at concentrations that maximally activate adenylate cyclase suggest that in adipose tissue, the three hormones act on the same enzyme, whereas in liver, VIP and secretin activate a common enzyme that is distinct from that responding to glucagon. Studies with radioiodinated derivatives of VIP and glucagon indicate that these hormones interact with separate receptors. Secretin, which gives a maximal stimulation of adenylate cyclase activity virtually identical to that elicited by VIP, inhibits the binding of the latter to its receptor. However, the apparent affinity of secretin for adenylate cyclase and for the VIP receptor is about two order of magnitude lower than that of VIP. It is suggested that VIP and secretin may activate adenylate cyclase via a common receptor.     

Pancreatic spasmolytic polypeptide (PSP): II. Radioimmunological determination of PSP in porcine tissues, plasma and pancreatic juice

A radioimmunoassay (RIA) for the determination of porcine Pancreatic Spasmolytic Polypeptide (PSP) has been developed. The antisera raised in rabbits were sensitive to 5 pgequiv. of PSP in a volume of 100 microliter. Immunoreactive PSP (IR-PSP) has been determined in extracts from 22 porcine organs. Pancreas was found to be the only organ containing substantial amounts of IR-PSP (0.1 mgequiv. IR-PSP/g wet weight). The fasting porcine plasma level of IR-PSP was about 10 ngequiv./ml, corresponding to 850 pM. The concentration of IR-PSP in porcine pancreatic juice varied from 0.2 mugequiv./ml in the fasting state to 46-116 mugequiv./ml after stimulation with pancreozymin or secretin. A linear correlation was found between the exocrine secretion of IR-PSP and total protein.     

Pituitary adenylate cyclase activating polypeptide (PACAP) in the rat mammary gland

Pituitary adenylate cyclase activating polypeptide (PACAP), a member of the vasoactive intestinal polypeptide (VIP) family of peptides, is present in the brain and in neuronal elements of a number of peripheral organs. Since no information on PACAP in the mammary gland exists, we have investigated, by radioimmunoassay and immunohistochemistry, the occurrence and distribution of PACAP immunoreactivity in the mammary gland of lactating and non-lactating rats. A specific monoclonal mouse anti-PACAP antibody'has been used to show that the peptide is located in nerve fibres associated with bundles of circular and longitudinal smooth muscle surrounding the lactiferous duct of the nipple. PACAP-immunoreactive nerve fibres and nerve bundles are present in the subepidermal connective tissue of the nipple and in the mammary parenchyma, some of the fibres being in close contact with blood vessels. Occasionally, a few delicate varicose fibres are associated with secretory alveoli and lactiferous ducts. The majority of PACAP-positive nerve fibres are, however, located in the glabrous skin of the nipple and the hairy skin adjacent to the nipple forming a subepithelial plexus from which delicate varicose nerve fibres enter the overlying epithelium. Double immunostaining for PACAP and a marker for sensory neurons, calcitonin gene-related peptide, has disclosed that the two peptides are almost completely co-localized. A minor population of the PACAP-immunoreactive nerve fibres shows co-existence with VIP. Although no obvious changes at the immunohistochemical level could be observed during pregnancy or lactation, elevated concentrations of immunoreactive PACAP-38 in mammary extracts have been found during lactation. Our data suggest that PACAP is involved in the nervous control of mammary gland function, probably in the transmission of suckling stimuli.     

Demonstration of specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat astrocytes

The high and low affinity binding sites for PACAP were identified in rat astrocytes using [125I]PACAP27 as the labeled ligand. Scatchard analysis of displacement of the bound tracer by unlabeled PACAP27 indicated the existence of two classes of binding sites, with the dissociation constant (Kd) = 1.22 +/- 0.4 nM, the binding maximal capacity (Bmax) = 821 +/- 218 fmols/mg protein for the high affinity binding site, and Kd = 0.59 +/- 0.06 microM, Bmax = 563 +/- 12 pmols/mg protein for the low affinity binding site, respectively. The specificity of [125I]PACAP27 binding was tested using PACAP38 and peptides structurally related to PACAP, such as VIP, GHRF, PHI, secretin and glucagon. PACAP38 completely displaced the binding of [125I]PACAP27 and Scatchard analysis also indicated the presence of two classes of binding sites with similar Kd and Bmax to those for PACAP27. VIP and GHRF competed with [125I]PACAP27, but to a much lesser extent than unlabeled PACAP27 in binding. Other peptides tested did not displace the binding of [125I]PACAP27 at 10(-6) M.     

Dimethylnitrosamine inhibits the glucagon-stimulated adenylate cyclase activity of rat liver plasma membranes and decreases plasma membrane fluidity

The effect of the hepatocarcinogen dimethylnitrosamine on rat liver plasma membrane adenylate cyclase activity and lipid fluidity was assessed. Glucagon-stimulated adenylate cyclase activity exhibited a complex response to increasing concentrations of dimethylnitrosamine, whereas fluoride-stimulated adenylate cyclase activity was progressively inhibited. Maximal inhibitory effects were observed at a concentration of 15 mM in both cases. The activity of detergent-solubilized adenylate cyclase was unaffected by dimethylnitrosamine. ESR analysis using a fatty acid spin probe showed that dimethylnitrosamine produced a marked, dose-dependent reduction in the fluidity of the plasma membrane with a maximal effect occurring at 20 mM. Dimethylnitrosamine also elevated the temperature at which the lipid phase separation occurred in rat liver plasma membranes, from 28 degrees C to 31 degrees C. The non-carcinogenic but structurally similar compound, dimethylamine hydrochloride neither inhibited adenylate cyclase nor decreased plasma membrane fluidity. It is suggested that the decrease in membrane fluidity, induced by dimethylnitrosamine, via its effects on membrane fluidity, could influence plasma membrane function and cellular regulation.     

Effect of pituitary adenylate cyclase activating polypeptide on rat pancreatic exocrine secretion.

A novel neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), which has been isolated from ovine hypothalami, shows 68% homology with vasoactive intestinal peptide (VIP). Since VIP stimulates amylase secretion from the pancreas, we investigated the effect of PACAP and VIP on rat pancreatic exocrine secretion after intravenous injections of PACAP-27, PACAP-38, or VIP at doses of 2.5, 5 or 10 nmol/kg. Results showed: 1) Bolus injection of PACAP stimulated pancreatic amylase and protein secretions in a dose-dependent manner; and 2) Stimulation of amylase secretion with 10 nmol/kg of PACAP-27 was greater than that induced with the same dose of VIP or PACAP-38 (p less than 0.05).     

Guanosine diphosphate binding,metabolism and regulation of follitropin-sensitive adenylate cyclase activity in Sertoli cell membranes

Nucleotides such as GTP and GDP appear to be involved in signal transduction via G protein modulation of adenylate cyclase activity. Studies on direct binding of [³H]GDP to membranes prepared from cultured immature rat Sertoli cells indicated that this process was reversible, approached steady state within 10 min, had a K_a of 4.5 ·10⁶M⁻¹ and was specific for guanine nucleotides. The non-hydrolyzable analog, guanosine 5′-O-[3-thio]triphosphate (GPPP[S]), was most effective as an inhibitor of [³H]GDP binding (ED₅₀ = 4.8·10⁻⁸M), whereas guanosine 5′-O-[2-thio]diphosphate (Gpp[S]) was less potent (ED₅₀ = 3.4·10⁻⁷M). Release of bound GDP was enhanced by follitropin (FSH) in the presence of Gppp[S], although not by FSH alone. Sertoli cell membranes possess guanine nucleotide hydrolase activity, where 95% of added nucleotide was rapidly degraded to guanosine. Binding kinetics were significantly influenced by nucleotide metabolism, which was prevented by controlling the Mg²⁺ concentration with EDTA and including App[NH]p to reduce nonspecific hydrolysis. Kinetic studies indicated that Gpp[S] inhibited (P < 0.05) Gppp[S]-stimulated adenylate cyclase activity (K_i = 1.8·10⁻⁷M), whereas basal activity remained unaffected. Addition of Gpp[S] to pre-activated enzyme (FSH plus GTP) resulted in a time-dependent decay of adenylate cyclase activity with a K_off value of 6 ± 1·min⁻¹. Using a two-stage pre-inculbation technique, adenylate cyclase activity was demonstrated to be sensitive to the nucleotide bound. When FSH was included, catalytic activity was not altered by the order of pre-incubation with the nucleotides. This suggested that the exchange of bound Gpp[S] for Gppp[S] was enhance by FSH. Activation and attenuation of FSH-sensitive adenylate cyclase activity is dependent on a nucleotide exchange mechanism which is driven by (1) the higher affinity of G for GTP than GDP, (2) enhanced release of GD when FSH is present and (3) GTP hydrolysis coupled to rapid metabolism of guanine nucleotides.     

Specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat cultured astrocytes: molecular identification and interaction with vasoactive intestinal peptide (VIP)

Molecular identification of the binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) and the effect of vasoactive intestinal peptide (VIP) on the specific binding sites for PACAP in rat cultured astrocyte membrane preparations were investigated. Affinity cross-linking of astrocyte membrane preparations with [125I]PACAP27 showed the presence of a 60 kDa radiolabeled ligand-receptor complex. The labeling of this band was completely abolished in the presence of 10(-8) M or higher concentrations of unlabeled PACAP27. The molecular weight of this binding protein was estimated to be 57 kDa assuming an equimolar interaction of ligand and receptor in the 60 kDa complex. The labeling of [125I]PACAP27 binding to this binding protein was partly reduced by the addition of 10(-6) M VIP, but not by 10(-8) M. In the binding assay, VIP displaced the specific binding of [125I]PACAP27 at 10(-7) M or a greater concentration. Displacement of [125I]PACAP27 binding by unlabeled PACAP27 was analyzed in the presence or absence of 10(-6) M VIP. VIP at 10(-6) M reduced the maximal binding capacity (Bmax) of the high affinity binding site for PACAP27 by about 50% but did not alter the Bmax of the low affinity binding site. The dissociation constants (Kd) for both the high and low affinity binding sites were unaltered. These results indicate that PACAP binds to a 57 kDa membrane protein with high affinity and that VIP, at much higher concentrations, binds to this same binding site, suggesting that VIP mimics the biological action of PACAP in astrocytes at high concentrations.     

Presence of vasoactive intestinal peptide receptors coupled to adenylate cyclase in rat lung membranes

(1) The binding of 125I-labelled vasoactive intestinal peptide (VIP) to a particulate fraction from rat lung was rapid, temperature dependent, saturable and specific. This process was also reversible and 125I-labelled VIP dissociation was accelerated by guanine triphosphate nucleotides. The curves describing the inhibition of tracer binding by peptides of the VIP-secretin family suggested the presence of at least two classes of VIP receptor: a "high-affinity' type with decreasing affinity for VIP in the order: VIP = [Val5]secretin greater than [Ala4, Val5]secretin; and a "low-affinity type' with decreasing affinity for VIP in the order: VIP greater than [Val5]secretin greater than [Ala4, Val5]secretin = secretin greater than [Ala4]secretin. (2) VIP and related peptides stimulated the adenylate cyclase activity of the same lung membrane preparation more efficiently than beta-adrenergic agonists and prostaglandins E1 and E2. The dose-effect curves of stimulation of adenylate cyclase by VIP and parent peptides were also compatible with the existence of two classes of VIP receptor, the relative peptide potencies being identical with their ability to compete with 125I-labelled VIP for binding.     

Calcitonin gene-related peptide stimulates adenylate cyclase activity in trout gill cell membranes

The physiological significance of calcitonin gene-related peptide (CGRP) was investigated by assessing the CGRP stimulated adenylate cyclase activity in various tissues of trout. The highest enzyme concentration was found in gill and stomach membranes. The maximal activity (190% of the basal value) was observed for a concentration of 53.3 nM CGRP I or II. In the presence of 58 nM sCT, the maximal enzyme activity represented 120% of the basal value. No additive effect was observed; this suggests that both CGRP and sCT activities are mediated through the same receptor. The present data are in favour of a role for this neuropeptide operating in branchial cell functions such as calcium transfer from the external to the internal milieu.     

Thyrotropin binding to and adenylate cyclase activity of porcine thyroid plasma membranes.

Plasma membranes have been purified from porcine thyroid gland homogenate by discontinuous sucrose gradient centrifugation. The preparations contained specific binding sites for thyrotropin but not for luteinizing hormone or the beta subunits of thyrotropin and luteinizing hormone. Optimum conditions of 125I-labeled thyrotropin binding were pH 6.0-6.5 and 37 degrees C. Thyrotropin binding was reduced by divalent (Ca2+, Mg2+) and monovalent cations (Na+, K+, Li+), 50% inhibition being obtained at 10 mM and 50 mM respectively. Displacement curves of 125I-labeled bovine or porcine thyrotropin by the unlabeled hormone from three species was in the order of increasing concentrations (bovine greater than porcine greater than human) which is the order of decreasing biological activity of these hormone preparations in the assay in vivo in the mouse. The validity of the results was established by controlling that porcine membranes bound the native and the 125I-labeled hormones with equal affinity. A single type of high-affinity (Kd = 0.28 nM) binding sites was detected for bovine and porcine thyrotropins. In contrast, porcine plasma membranes bound human thyrotropin with a lower affinity (Kd = 70 nM). A good correlation was found at equilibrium and in the conditions of the cyclase assay, between receptor occupancy and adenylate cyclase activation for the three hormones.     

Potentiation by guanine nucleotides of the VIP-induced adenylate cyclase stimulation in intestinal epithelial cell membranes

The GTP analog 5′-quanylyl-imidodiphosphate Gpp(NH) p potentiated the action of VIP on adenylate cyclase from intestinal epithelial cell membranes. The other nucleotides tested were also active on adenylate cyclase with the following order of potency GTP>GDP>GMP>ITP>UTP=CTP. Guanine nucleotides act by increasing the Vmax of the enzyme activity and by decreasing the Km of enzyme activation by VIP. Activation of the peptide-induced adenylate cyclase activity by Gpp (NH) p was inhibited by GTP and the other nucleotides with the same order and range of potency than those observed for their intrinsic stimulatory effect on adenylate cyclase. These data demonstrate the potent and specific action of quanine nucleotides on the VIP-sensitive adenylate cyclase.