Temperature-dependent stimulation and inhibition of adenylate cyclase by Aplysia bag cell peptides

The bag cell peptides (alpha-, beta-, and gamma-BCP) are secreted by the neuroendocrine bag cells of Aplysia, and provide feedback modulation of bag cell excitability and cAMP levels. We report here that if 200-500 mM NaCl is included in the assay buffer, the BCPs alter adenylate cyclase activity in a manner consistent with their effects on cAMP levels in intact bag cells. Specifically, beta-BCP and the related peptide A from the atrial gland stimulate the enzyme, while the effects of alpha-BCP(1-7) and gamma-BCP are temperature-dependent, stimulating at 30 degrees C and inhibiting at 15 degrees C. Both stimulation and inhibition require GTP, suggesting mediation by Gs and Gi. The ionic requirements of stimulation and inhibition differ: Cl- is necessary to support stimulation, but not inhibition. Moreover, pertussis toxin blocks inhibition, but does not affect stimulation. These results suggest that the temperature-sensitive mechanism lies upstream from the G-proteins in the signal transduction pathway.     

Inhibition of adenylate cyclase of rat hepatic membranes by glycosaminoglycans

Glycosaminoglycans are long non-branched polysaccharides composed of repeating disaccharide units. In a previous in vitro study we have shown that such molecules are able to modulate substrate phosphorylation catalyzed by cAMP-dependent protein kinase. Here, we investigate the impact of glycosaminoglycans, such as heparan sulfate, dermatan sulfate, chondroitin 4- and 6-sulfate, keratan sulfate and hyaluronic acid upon adenylate cyclase, which directly regulates cAMP-dependent protein kinase activity via cAMP synthesis. In rat liver plasma membrane preparation we have determined forskolin- and guanosine-5'-beta, gamma-imidotriphosphate-induced cAMP formation catalyzed by adenylate cyclase in the presence of increasing concentrations of glycosaminoglycans. The results indicate that glycosaminoglycans strongly influence enzymic conversion of ATP into cAMP. The highest reduction of adenylate cyclase activity is observed in the presence of dermatan sulfate and heparan sulfate. Moreover, the inhibitory effect of these two glycosaminoglycans is higher when guanosine-5'-beta, gamma-imidotriphosphate, instead of forskolin, is used as stimulator of adenylate cyclase. Further characterization of enzyme inhibition mediated by dermatan sulfate shows that this molecule exerts an inhibitory effect of mixed type.     

Pharmacological characterization of metabotropic glutamate receptors linked to the inhibition of adenylate cyclase activity in rat striatal slices

The pharmacological profile of mGlu receptors negatively linked to adenylyl cyclase was characterized in adult rat striatal slices. Among the mGlu agonists tested, (+)-2-aminobicyclo-[3.1.0]-hexane-2,6-di carboxylate (LY354740), was the most potent inhibitor of forskolin-stimulated cAMP formation (EC50 = 11 +/- 2 nM). Inhibition of forskolin stimulation by the group III agonist L-2-amino-4-phosphono-butanoate (L-AP4) was biphasic, the two parts of the concentration curve having EC50 values of 6 +/- 1 microM and 260 +/- 4 microM, suggesting a sequential recruitment of mGlu4/8 and mGlu7. The effects of several new phenylglycine derivative antagonists were tested on the inhibition of forskolin cAMP response by (2S,1'S,2'S)-2-(carboxy-cyclopropyl)-glycine (L-CCG I) and L-AP4. At 500 microM, (RS)-alpha-methyl-3-carboxy-methyl-pheny lglycine was unable to antagonize the effect of L-CCG I or L-AP4 but (S)-alpha-methyl-3-carboxy-phenylalanine inhibited the effect of L-AP4 with a low potency. Finally, (RS)-alpha-methyl-4-tetrazolylphenylglyc ine and particularly (RS)-alpha-methyl-4-phosphonophenylglyci ne, appeared to be the most potent and selective antagonists of L-AP4 induced inhibition of forskolin-stimulated cAMP production in adult rat striatal slices.     

Effects of prostaglandins and catecholamines on rat spleen adenylate cyclase in vitro

The results reported here show some characteristics of adenylate cylase (EC 4.6.1.1) derived from homogenates of rat spleen, and describe the in vitro stimulation of this enzyme by prostaglandins, nucleotides, and F- under conditions where cyclic nucleotide degradative pathways are effectively inhibited. Particulate fractions from rat spleen homogenates contain high adenylate cyclase activities, and the highest specific activity is recovered in a particulate fraction prepared by low speed (1200 X g) centrifugation. Activity found in all particulate fractions is stimulated by fluoride, prostaglandins E1 and E2, catecholamines, and purine nucleotides. No stimulation is caused by prostaglandins F1 alpha and F2 alpha. Stimulation by prostaglandin E1 and E2 is augmented by GTP and other purine nucleotides, and stimulation by the combination of GTP and prostaglandin E1 is equal to that caused by optimal fluoride concentrations. Stimulation caused by L-isoproterenol is additive to that caused by GTP but is not increased by GTP.     

Activation by cholera toxin of adenylate cyclase solubilized from rat liver

Cholera toxin, or peptide A1 from the toxin, activates adenylate cyclase solubilized from rat liver with Lubrol PX, provided that cell sap, NAD+, ATP and thiol-group-containing compounds are present. The activation is abolished by antisera to whole toxin, but not to subunit B.     

Independent variation of beta-adrenergic receptor binding and catecholamine-stimulated adenylate cyclase activity in rat erythrocytes

The pharmacokinetics, following i.v. administration of (+)-propranolol (40 mg) have been compared to in vitro measurement of protein binding and biochemical parameters of liver function in six normal subjects and twenty patients with stable chronic liver disease. The clearance of (+)-propranolol decreased with evidence of increasing severity of impairment of liver function correlating significantly with a fall in serum albumin, a rise in bilirubin and a prolongation in prothrombin index. The clearance of (+)-propranolol correlated with and was numerically similar to the clearance of indocyanine green in normal subjects and also in patients with chronic liver disease. Protein binding was decreased in chronic liver disease, but this change was not related to changes in plasma proteins. In normal subjects and patients without ascites the volume of distribution increased with decreases in protein binding. Ascites was associated with a further increase in the volume of distribution. The considerable variation in half-life largely depends on changes in liver blood flow, the degree of protein binding and the plasma protein pool size.     

Loop diuretics act directly on adenylate cyclase in rat renal tubular basolateral membranes

We have reported previously that loop diuretics, especially azosemide and ethacrynic acid, may act not only on the AVP receptor site, but also on the post-AVP receptor site in rat renal tubular basolateral membranes. The purpose of this study was to examine whether loop diuretics (furosemide, azosemide, ethacrynic acid) affect the post-AVP receptor components, using GTP-gamma S, forskolin and cholera toxin as tools acting distal to the receptor. Adenylate cyclase activity stimulated by 10(-9)M AVP was inhibited more potently by azosemide and ethacrynic acid than by furosemide at the concentration of 10(-3) M. Azosemide and ethacrynic acid at concentrations above 10(-4) M also significantly decreased the enzyme activity that was stimulated by 10(-7) M GTP-gamma S and 10(-5)M forskolin, while significant inhibition by furosemide was observed only at 10(-3)M. In addition, the inhibitory effect of these loop diuretics on cholera toxin-stimulated enzyme activity was almost similar to the results observed in AVP-, GTP-gamma S- or forskolin-stimulated the enzyme activity. From these results, we conclude that loop diuretics, especially azosemide and ethacrynic acid, directly affect adenylate cyclase in part as well as the AVP receptor site.     

Hemodynamic actions of systemically injected pituitary adenylate cyclase activating polypeptide-27 in the rat

The aims of this study were (1) to characterize the hemodynamic mechanisms underlying the hypotensive effects of pituitary adenylate cyclase activating polypeptide-27 (PACAP-27 0.1-2.0 nmol/kg, i.v.) in pentobarbital-anesthetized rats, and (2) to determine the roles of the autonomic nervous system, adrenal catecholamines and endothelium-derived nitric oxide (NO) in the expression of PACAP-27-mediated effects on hemodynamic function. PACAP-27 produced dose-dependent decreases in mean arterial blood pressure and hindquarter and mesenteric vascular resistances in saline-treated rats. PACAP-27 also produced pronounced falls in mean arterial blood pressure in rats treated with the ganglion blocker, chlorisondamine (5 mg/kg, i.v.). The hypotensive and vasodilator actions of PACAP-27 were not attenuated by the beta-adrenoceptor antagonist, propranolol (1 mg/kg, i.v.), or the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME 50 micromol/kg, i.v.). PACAP-27 produced dose-dependent increases in heart rate whereas the hypotensive response produced by the nitrovasodilator, sodium nitroprusside (10 microg/kg, i.v.), was associated with a minimal tachycardia. The PACAP-27-induced tachycardia was unaffected by chlorisondamine, but was virtually abolished by propranolol. These results suggest that the vasodilator effects of PACAP-27 are due to actions in the microcirculation rather than to the release of adrenal catecholamines and that this vasodilation may not involve the release of endothelium-derived NO. These results also suggest that PACAP-27 produces tachycardia by directly releasing norepinephrine from cardiac sympathetic nerve terminals rather than by direct or baroreceptor reflex-mediated increases in sympathetic nerve activity.     

Pituitary adenylate cyclase activating polypeptide induces multiple signaling pathways in rat peritoneal mast cells

Pituitary adenylate cyclase activating polypeptide (PACAP) is a high-affinity ligand for at least two types of G-protein coupled receptors, the PACAP type 1 and type 2 receptor. In this study it is demonstrated that the C-terminal PACAP-fragment PACAP(6-27) stimulates serotonin release from rat peritoneal mast cells with higher potency (EC50: 0.2 vs. 2.0 microM) than the PACAP receptor ligand PACAP(1-27). PACAP-induced degranulation of rat peritoneal mast cells was abolished by pertussis toxin and by benzalkonium chloride (IC50: 9.1 microg/ml) indicating the involvement of heterotrimeric G-proteins of the Gi-type. The PACAP effect was also reduced by inhibitors of the phosphatidylinositol specific phospholipase C ((U73122), IC50: 4 microM; (ET-18-O-CH3), IC50: 18 microM), by D609, a specific inhibitor of the phosphatidylcholine specific phospholipase C (IC50: 41 microM), by the protein kinase C-inhibitor staurosporine (IC50: 0.6 microM) and by the lipoxygenase inhibitor nordihydroguaiaretic acid (NGDA) but not by indomethacin. It is concluded that PACAP peptides stimulate secretion in rat peritoneal mast cells in a PACAP receptor-independent manner, probably via direct activation of heterotrimeric G-proteins of the Gi-type; these G-proteins may lead to a sequential activation of different signaling cascades (see above), which may converge at the level of one or more staurosporine-sensitive protein kinase.     

Gene expression of pituitary adenylate cyclase activating polypeptide (PACAP) in the rat hypothalamus

Pituitary adenylate cyclase activating polypeptide (PACAP) isolated from ovine hypothalamus is considered to be a member of the vasoactive intestinal peptide/glucagon/secretin/growth hormone-releasing hormone family of peptides. Two forms of PACAP, PACAP38 and PACAP27, have been demonstrated in the rat hypothalamus. The PACAP precursor contains another peptide called PACAP-related peptide (PRP), but so far no information on this peptide in tissue exists. We have developed three radioimmunoassays specific for PACAP38, PACAP27 and PRP and demonstrate that all three preproPACAP peptides are expressed in the rat hypothalamus, the PACAP38/PACAP27 ratio being around 60 and the PACAP38/PRP ratio being around 10. HPLC analysis of hypothalamic extract showed that PACAP38 and PACAP27 are found in only one form corresponding to the respective synthetic peptides, whereas PRP eluted in two peaks, the predominant form corresponding to synthetic PRP1-29. The cellular distribution of PACAP38, PACAP27, and PRP and corresponding mRNA in the hypothalamus was determined with immunohistochemistry and in situ hybridization histochemistry. PACAP- and PRP-immunoreactive neuronal perikarya were observed in the medial parvocellular part of the paraventricular nucleus (PVN) in colchicine pretreated rats. Some cell bodies of magnocellular variety were found in the PVN. PACAP mRNA containing cells were observed in moderate numbers in the same parts of the paraventricular nucleus. PACAP- and PRP immunoreactive fibres and varicosities were distributed in the PVN and in the periventricular nucleus. These data show that PACAP38, PACAP27 and PRP are expressed in the parvocellular part of the PVN, implying roles as hypothalamic regulatory peptides.