Showing papers on "Cyclase published in 1978"
TL;DR: The results indicate that cholera toxin affects the adenylate cyclase system by catalyzing an ADP-ribosylation of the 42,000-M(r) component bearing the guanyl nucleotide regulatory site.
Abstract: Treatment of pigeon erythrocyte membranes with cholera toxin and NAD+ enhanced the GTP stimulation and suppressed the F- activation of the adenylate cylase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. In the presence of NAD+ labeled with 32P in the AMP moiety the toxin catalyzed the covalent incorporation of radioactivity into membrane proteins with molecular weights (Mrs) of 200,000, 86,000, and 42,000. Extraction of toxin-treated membranes with Lubrol PX followed by affinity chromatography on a GTP-Sepharose column resulted in a 200-fold purification of the 42,000-Mr labeled protein and in its complete separation from the other labeled proteins. The fraction containing the purified GTP-binding component from toxin-treated membranes conferred an enhanced GTP-stimulated activity on adenylate cyclase solubilized from nontreated membranes. Likewise, the addition of GTP-binding fraction from nontreated membranes to an enzyme solubilized from toxin-treated membranes restored F- stimulation of the adenylate cyclase. The toxin-induced modification of adenylate cyclase and the incorporation of radioactivity into the 42,000-Mr protein were partially reversed upon incubation with toxin and nicotinamide at pH 6.1. The results indicate that cholera toxin affects the adenylate cyclase system by catalyzing an ADP-ribosylation of the 42,000-Mr component bearing the guanyl nucleotide regulatory site.
815 citations
TL;DR: Evidence is presented that suggests that the most readily modified membrane protein is the adenylate cyclase-associated GTP-binding protein, which is stimulated by guanine nucleotides in cholera toxin fragment A1.
Abstract: In the presence of ATP and a cytosolic factor, cholera toxin fragment A1 catalyzes the transfer of ADP-ribose from NAD to a number of soluble and membrane-bound proteins of the pigeon erythrocyte. Evidence is presented that suggests that the most readily modified membrane protein (Mr 42,000) is the adenylate cyclase-associated GTP-binding protein. Its modification by toxin is stimulated by guanine nucleotides. Adenylate cyclase activity increases in parallel with the addition of ADP-ribose to this protein and decreases in parallel with the subsequent reversal of ADP-ribosylation by toxin and nicotinamide. The protein is only accessible to toxin A subunits if the erythrocytes are lysed. When adenylate cyclase activity reaches a maximum, the number of ADP-ribose residues bound to this protein (about 1500 per cell) is similar to the reported number of beta-adrenergic receptors.
671 citations
TL;DR: The thermostable moiety of the enzyme appears to consist of two functional components, based upon differential thermal lability of its ability to reconstitute hormone-, NaF-, or Gpp(NH)p-stimulated adenylate cyclase activity.
445 citations
TL;DR: The findings indicate that the inactive adenylate cyclase possesses tightly bound GDP, produced by the hydrolysis of GTP at the regulatory site, which the hormone stimulates by inducing an "opening" of the guanyl nucleotide site.
Abstract: The fate of the guanyl nucleotide bound to the regulatory site of adenylate cyclase was studied on a preparation of turkey erythrocyte membranes that was incubated with [3H]GTP plus isoproterenol and subsequently washed to remove hormone and free guanyl nucleotide. Further incubation of this preparation in the presence of beta-adrenergic agonists resulted in the release from the membrane of tritiated nucleotide, identified as [3H]GDP. The catecholamine-induced release of [3H]GDP was increased 2 to 3 times in the presence of the unlabeled guanyl nucleotides GTP, guanosine 5'-(beta,gamma-imino)triphosphate [gpp(NH)p], GDP, and GMP, whereas adenine nucleotides had little effect. In the presence of Gpp(NH)p, isoproterenol induced the release of [3H]GDP and the activation of adenylate cyclase, both effects following similar time courses. The findings indicate that the inactive adenylate cyclase possesses tightly bound (GDP, produced by the hydrolysis of GTP at the regulatory site. The hormone stimulates adenylate cyclase activity by inducing an "opening" of the guanyl nucleotide site, resulting in dissociation of the bound GDP and binding of the activating guanosine triphosphate.
346 citations
TL;DR: The data strongly suggest that the collision coupling is the mode of coupling between the beta receptor and cyclase coupling in turkey erythrocyte membranes.
Abstract: The mode of coupling of the beta-adrenergic receptor to the enzyme adenylate cyclase in turkey erythrocyte membranes was analyzed in detail. A number of experimental techniques have been used: (1) measurement of the kinetics of cyclase activation to its permanetly active state in the presence of guanylyl imidodiphosphate, as a function of hormone concentrations; (2) measurement of antagonist and agoinst binding to the beta-adrenergic receptor prior and subsequent to the enzyme activation by hormone and guanylyl imidodiphosphate. On the bases of these two approaches, all the models of receptor to enzyme coupling which involve an equilibrium between the enzyme and the receptor can be rejected. The binding and the kinetic data, however, can be fitted by two diametrically opposed models of receptor to enzyme coupling: (a) the precouped enzyme-receptor model where activation of the enzyme occurs, according to the following scheme: formula (see text) where H is the hormone, RE is the precoupled respetor-enzyme complex, k1 and k2 are the rate constants describing hormone binding, and k is the rate constant characterizing the formation of HRE' from the intermediate HRE. According to this model, the activated complex is composed of all of the interacting species. (b) The other model is the collision coupling mechanism: formula (see test) wheere KH is the horome-receptor dissociation constant, k1 is the bimolecular rate constant governing the formation of HRE, and k3 the rate constant governing the activation of the enzyme. In this case the intermediate never accumulates and constitutes only a small fraction of the total receptor and adenylate cyclase concentrations. In order to establish which of the two mechanisms governs the mode of adenylate cyclase activation by its receptor, a diagnostic experiment was performed: Progressive inactivation of the beta receptor by a specific affinity label was found to cause a decrease in the maximal binding capacity of the receptor and a proportional decrease in the rate of activation, but no change in the maximum level of activity was attained. Progressive inactivation of the enzyme by p-hydroxymercuribenzoate was found not to change the rate of activation nor the capacity of the receptor to bind hormone. Only the maximal level of activation was found to be decreased. These results are not compatible with the precoupled model of receptor and cyclase nor with floating receptor models in which an intermediate of hormone, receptor, and cyclase is in equilibrium with its reactants. The data strongly suggest that the collision coupling is the mode of coupling between the beta receptor and cyclase coupling in turkey erythrocyte membranes.
338 citations
TL;DR: regulation by calcium of calcium-dependent protein kinase activity may be mediated physiologically by the calcium-binding protein postulated to regulate cyclic nucleotide phosphodiesterase and adenylate cyclase.
Abstract: THE important role of Ca2+ in the physiology of the nervous system is well documented1,2. However, the biochemical mechanisms underlying certain of its physiological effects, such as stimulus–secretion coupling3,4 and synthesis of cate-cholamines5,6, have not yet been elucidated. Calcium has been implicated in several biochemical reactions of potential importance to synaptic function. Thus, calcium and a heat-stable calcium-binding protein activate the cyclic nucleotide phosphodiesterase from mammalian brain7,8. The calcium-dependent regulator (CDR) from porcine brain9, bovine heart10 and bovine brain11 has been purified and characterised. CDR seems to be a calcium receptor as it binds calcium strongly and specifically. There is evidence that a CDR·Ca2+ complex is the true activator of cyclic nucleotide phosphodiesterase12–14. A detergent-solubilised preparation of brain adenylate cyclase can also be activated by this calcium-binding protein15. Calcium stimulates protein phosphorylation in both intact16,17 and lysed18,19 synaptosomes and this cyclic nucleotide-independent mechanism may mediate or modulate some of the intracellular effects of Ca2+ on the function of presynaptic nerve terminals. We report here that calcium-dependent phosphorylation of synaptosomal membrane fractions from rat cerebral cortex requires an endogenous protein factor present in the synaptosomal cytoplasm. Calcium stimulated phosphorylation is lost on purification of synaptic membranes and can be effectively recovered by reconstitution with either the synaptosomal cytoplasm or with a purified preparation of CDR. Thus, regulation by calcium of calcium-dependent protein kinase activity may be mediated physiologically by the calcium-binding protein postulated to regulate cyclic nucleotide phosphodiesterase and adenylate cyclase.
314 citations
TL;DR: Self-regulation of target-cell responsiveness, variously termed refractoriness, desensitization and down-regulation, has been observed with catecholamines, as well as with a number of other hormonal agonists.
Abstract: β-ADRENERGIC catecholamines, which modulate function of a variety of tissues, are assumed to act by initially binding to receptors on target-cell plasma membranes, thereby activating adenylate cyclase and stimulating generation of adenosine 3′:5′-monophosphate (cyclic AMP). In addition to activating adenylate cyclase, β-adrenergic agonists commonly have a second action — regulation of responsiveness of the target cells. Thus, continued treatment with such agonists often results in decreased cellular response. This self-regulation of target-cell responsiveness, variously termed refractoriness, desensitization and down-regulation, has been observed with catecholamines, as well as with a number of other hormonal agonists.1 2 3 For catecholamines, studies in several experimental . . .
265 citations
TL;DR: One or both of the toxin substrates thus appears to be involved in regulation of adenylate cyclase by guanyl nucleotides and fluoride ion.
254 citations
TL;DR: It is reported here that the inhibition by adenosine of the increase in the intracellular level of cyclic AMP evoked by isoprenaline in cultured glioblasts is mediated by extracellular receptors.
Abstract: ADENOSINE stimulates the accumulation of cyclic AMP in some tissues and cell types, but has inhibitory or biphasic effects on the accumulation of cyclic AMP in other cells. Londos and Wolff1 have attempted to explain these diverse effects by the existence of two adenosine reactive sites on adenylate cyclase. The first is the R-site, occupancy of which usually leads to activation on adenylate cyclase and which for activity requires integrity of the ribose ring. Much evidence suggests that this site is identical to an extracellular receptor for adenosine2–6. The second type is the P-site, which mediates inhibition of adenylate cyclase and requires integrity of the purine ring for activity. Preliminary evidence suggests that this site is only accessible from the interior of the cell1,3. However, we report here that the inhibition by adenosine of the increase in the intracellular level of cyclic AMP evoked by isoprenaline in cultured glioblasts is mediated by extracellular receptors.
241 citations
TL;DR: The results show that cyclic AMP plays no significant role in the responses of platelets to aggregating agents in the absence of compounds that increase the platelet cyclicAMP concentration above the resting value.
Abstract: Whereas adenosine itself exerted independent stimulatory and inhibitory effects on the adenylate cyclase activity of a platelet particulate fraction at low and high concentrations respectively, 2-substituted and N6-monosubstituted adenosines had stimulatory but greatly decreased inhibitory effects. Deoxyadenosines, on the other hand, had enhanced inhibitory but no stimulatory effects. The most potent inhibitors found were, in order of increasing activity, 9-(tetrahydro-2-furyl)adenine (SQ 22536), 2',5'-dideoxyadenosine and 2'-deoxyadenosine 3'-monophosphate. Kinetic studies on prostaglandin E1-activated adenylate cyclase showed that the inhibition caused by either 2',5'-dideoxyadenosine or compound SQ 22536 was non-competitive with MgATP and that the former compound, at least, showed negative co-operativity; 50% inhibition was observed with 4 micron-2',5'-dideoxyadenosine or 13 micron-SQ 22536. These two compounds also inhibited both the basal and prostaglandin E1-activated adenylate cyclase activities of intact platelets, when these were measured as the increases in cyclic [3H]AMP in platelets that had been labelled with [3H]adenine and were then incubated briefly with papaverine or papaverine and prostaglandin E1. Both compounds, but particularly 2',5'-dideoxyadenosine, markedly decreased the inhibition by prostaglandin E1 of platelet aggregation induced by ADP or [arginine]vasopressin as well as the associated increases in platelet cyclic AMP, so providing further evidence that the effects of prostaglandin E1 on platelet aggregation are mediated by cyclic AMP. 2'-Deoxyadenosine 3'-monophosphate did not affect the inhibition of aggregation by prostaglandin E1, suggesting that the site of action of deoxyadenosine derivatives on adenylate cyclase is intracellular. Neither 2',5'-dideoxyadenosine nor compound SQ 22536 alone induced platelet aggregation. Moreover, neither compound potentiated platelet aggregation or the platelet release reaction when suboptimal concentrations of ADP, [arginine]vasopressin, collagen or arachidonate were added to heparinized or citrated platelet-rich plasma in the absence of prostaglandin E1. These results show that cyclic AMP plays no significant role in the responses of platelets to aggregating agents in the absence of compounds that increase the platelet cyclic AMP concentration above the resting value.
230 citations
TL;DR: Ribose-modified adenosine analogs inhibit adenylate cyclase through a process that is neither dependent upon the GTP concentration nor antagonized by methylxanthines, demonstrating the importance of GTP in mediating inhibition by agents that act at cell surface receptors.
Abstract: Adenylate cyclase in purified membranes from rat adipocytes is inhibited by low concentrations of purine-modified adenosine analogs, particularly those modified in the N6 position. Such inhibition is antagonized competitively by methylxanthines, but not by other cyclic nucleotide phosphodiesterase inhibitors, and it is dependent on "inhibitory" concentrations of GTP in the assay medium. Ribose-modified adenosine analogs inhibit adenylate cyclase through a process that is neither dependent upon the GTP concentration nor antagonized by methylxanthines. These results explain the potent effects of adenosine and methylxanthines on fat cell metabolism and demonstrate the importance of GTP in mediating inhibition by agents that act at cell surface receptors.
TL;DR: The results of the present study suggest that muscarinic cholinergic agonists can regulate both beta-adrenergic receptors and adenylate cyclase by modulating the effects of GTP.
TL;DR: The localization of multiple binding sites in the crude synaptosomal fraction correlates well with the localization of dopamine-sensitive adenylate cyclase in this fraction.
TL;DR: It is demonstrated here that the effects of modulating membrane fluidity on the rate of adenlyate cyclase activation can be used to distinguish between the two modes of coupling.
Abstract: THE hormone- and neurotransmitter-dependent adenylate cyclases represent transmembrane regulatory systems in which the receptor unit faces the outside of the cell and the cyclase catalytic unit faces the inside. When the hormone or neurotransmitter binds to the receptor, the catalytic unit of the cyclase is activated and produces cyclic AMP at the inner surface of the membrane1. It has recently become clear that some hormone receptors are permanently coupled to adenylate cyclase whereas others are not. We demonstrate here that the effects of modulating membrane fluidity on the rate of adenlyate cyclase activation can be used to distinguish between the two modes of coupling.
Journal Article•
TL;DR: It is shown that highly purified Schwann cells in dissociated cell cultures of newborn rat nerve, which divide infrequently in 10% serum, are stimulated to divide when exposed to cholera toxin, 3-isobutyl-L-methyl-xanthine (IBMX, a 3′,5′-cyclic nucleotide phosphodiesterase inhibitor5), or dibutyryl cyclic AMP.
Abstract: THE possible role of intracellular cyclic AMP in the control of cell division is the subject of much controversial literature (reviewed in refs 1, 2). In most such studies fibroblastic cell lines have been used, and various manipulations which raise intracellular concentration of cyclic AMP have been reported to inhibit cell proliferation1. Here we show that highly purified Schwann cells in dissociated cell cultures of newborn rat nerve, which divide infrequently in 10% serum, are stimulated to divide when exposed to cholera toxin (an irreversible activator of adenyl cyclase activity3,4), 3-isobutyl-L-methyl-xanthine (IBMX, a 3′,5′-cyclic nucleotide phosphodiesterase inhibitor5), or dibutyryl cyclic AMP, all of which would be expected to increase intracellular cyclic AMP levels.
TL;DR: A role for VIP as a neuromodulatory or neuro-transmitter compound in the central nervous system, (CNS), mediating its action through the adenylate cyclase/cAMP system is supported.
TL;DR: The presence of transferase and glycohydrolase activities in structurally distinct toxins that activate adenylate cyclase strengthens the hypothesis that the ADP-ribosylation of arginine is a model for the NAD-dependent activation of adenyate cyclases.
Abstract: Highly purified, polymyxin-released, low molecular weight Escherichia coli heat-labile enterotoxin (LT) catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide. This NAD glycohydrolase activity was stimulated by dithiothreitol and was independent of cellular components. Nicotinamide formation was enhanced by arginine methyl ester > d-arginine congruent with l-arginine congruent with guanidine. A 20-fold increase in activity was noted with arginine methyl ester, and maximal activity again required dithiothreitol. When the reaction was initiated with toxin, a delay was observed before a constant rate was established. The reaction products found after incubation of [adenine-U-(14)C]NAD and l-[(3)H]arginine or unlabeled arginine methyl ester with the enterotoxin had mobilities on thin-layer chromatograms similar to the reaction products obtained after incubation of choleragen with these substrates and are consistent with the formation of ADP-ribose-l-arginine and ADP-ribose-l-arginine methyl ester, respectively. Both toxins, which catalyze the NAD-dependent activation of adenylate cyclase, thus appear to possess NAD glycohydrolase and ADP-ribosyltransferase activities. Although the activities of both toxins are dependent on dithiothreitol, Escherichia coli enterotoxin exhibited optimal activity in Tris (Cl(-)) (pH 7.5) and was inhibited by high concentrations of potassium phosphate (pH 7.0) or low pH (sodium acetate, pH 6.2). It appears that the optimal assay conditions as well as the kinetic constants for the reactants differ from those previously noted with choleragen. It is probable therefore that although the two toxins catalyze similar reactions, they differ in primary structure. The presence of transferase and glycohydrolase activities in structurally distinct toxins that activate adenylate cyclase strengthens our hypothesis that the ADP-ribosylation of arginine is a model for the NAD-dependent activation of adenylate cyclase; activation may result from ADP-ribosylation of the cyclase itself or of a protein that regulates its activity.
TL;DR: The presence in the avian erythrocyte of a protein that, like choleragen and Escherichia coli heat-labile enterotoxin, apparently activates adenylate cyclase and possesses ADP-ribosyl transferase activity is consistent with the view that the mechanisms through which the bacterial toxins produce pathology are not entirely foreign to vertebrate cells.
Abstract: An ADP-ribosyltransferase was purified ∼500-fold from the supernatant fraction of turkey erythrocytes. The enzyme hydrolyzed [carbonyl-14C]NAD to ADP-ribose and [carbonyl-14C]nicotinamide at a low rate. Nicotinamide formation from NAD was enhanced by arginine methyl ester > D-arginine ∼ L-arginine > guanidine; lysine, histidine, and citrulline were ineffective. Incubation of [adenine-U-14C]NAD and arginine methyl ester or arginine with the purified enzyme resulted in the formation of new compounds that contained 14C, reacted with ninhydrin, and quenched background fluorescence of thin-layer plates viewed in ultraviolet light. Their mobilities on thin-layer chromatograms were indistinguishable from those of ADP-ribosylarginine methyl ester and ADP-ribosylarginine formed during incubation of choleragen with NAD and arginine methyl ester or arginine, respectively [Moss, J. & Vaughan, M. (1977) J. Biol. Chem. 252, 2455-2457]. The purified transferase also catalyzed the incorporation of label from [adenine-14C]-NAD into lysozyme, histones and polyarginine. When the 14C-labeled lysozyme was incubated with snake venom phosphodiesterase, the radioactivity was released and, on thin-layer chromatograms, exhibited a mobility indistinguishable from that of 5′-AMP, as would be expected of an ADP-ribosylated protein, but not of a poly(ADP-ribosylated) product. The purified transferase activated rat brain adenylate cyclase and, as is the case with choleragen, activation was absolutely dependent on NAD. The presence in the avian erythrocyte of a protein that, like choleragen and Escherichia coli heat-labile enterotoxin, apparently activates adenylate cyclase and possesses ADP-ribosyl transferase activity is consistent with the view that the mechanisms through which the bacterial toxins produce pathology are not entirely foreign to vertebrate cells, at least some of which may possess and employ an analogous mechanism for activation of adenylate cyclase.
TL;DR: The results suggest that activation of soluble guanylate cyclase from splenic cells can be achieved by the oxidation of sulfhydryl groups that may be associated with specific hydrophobic sites of the enzyme or a related regulatory component.
TL;DR: It is suggested that benzyl alcohol effects a fluidization of the bilayer, which is clearly demonstrated by its ability to lower the temperature of a lipid phase separation occurring at 28 degrees C in the outer half of the Bilayer to around 22 degrees C.
Abstract: 1. The local anaesthetic benzyl alcohol progressively activated glucagon-stimulated adenylate cyclase activity up to a maximum at 50 mM-benzyl alcohol. Further increases in benzyl alcohol concentration inhibited the activity. The fluoride-stimulated adenylate cyclase activity was similarly affected except for an inhibition of activity occurring at low benzyl alcohol concentrations (approx. 10 mM. 2. The fluoride-stimulated adenylate cyclase activity of a solubilized enzyme preparation was unaffected by any of the benzyl alcohol concentrations tested. 3. Increases in 3-phenylpropan-1-ol and 5-phenylpentan-1-ol concentrations progressively activated both the fluoride- and glucagon-stimulated adenylate cyclase activities up to a maximum, above which further increases in alcohol concentration inhibited the activities. 4. The ‘break’ points in Arrhenius plots of glucagon-stimulated adenylate cyclase activity in native plasma membranes, and in plasma membranes fused with synthetic dimyristoyl phosphatidylcholine so as to constitute 60% of the total lipid pool, were decreased by approx. 6 degrees C by addition of 40 mM-benzyl alcohol. This was accompanied by a fall in the associated activation energies. 6. Arrhenius plots of fluoride-stimulated adenylate cyclase activity in the presence and absence of 40 mM-benzyl alcohol were linear, although addition of benzyl alcohol caused a dramatic decrease in the associated activation energy of the reaction. 7. 5′-Nucleotidase activity was stimulated by benzyl alcohol, and the ‘break’ point in the Arrhenius plot of its activity was decreased by about 6 degrees C by addition of 40 mM-benzyl alcohol to the assay. 8. It is suggested that benzyl alcohol effects a fluidization of the bilayer, which is clearly demonstrated by its ability to lower the temperature of a lipid phase separation occurring at 28 degrees C in the outer half of the bilayer to around 22 degrees C. The increase in bilayer fluidity relieves a physical constraint on the membrane-bound adenylate cyclase, activating the enzyme. 9. The various inhibition phenomena are discussed in detail, together with the suggestion that the interaction between the uncoupled catalytic unit of adenylate cyclase and the lipids of the bilayer is altered on its physical coupling to the glucagon receptor.
TL;DR: The quantitative topographical distribution of the dopaminergic innervation and the DA-sensitive adenylate cyclase were estimated in the anterior cerebral cortex of the rat and found the dorsal part of the frontal cortex presented the highest number of [3H]DA uptake sites.
TL;DR: The specificity of binding and the close correlation with alpha-adrenergic receptor-mediated biochemical and physiological responses suggest that the [(3)H]dihydroergocryptine binding site represents, or is closely related to, the human platelet alpha- adrenergic receptors.
Abstract: Human platelets aggregate and undergo a release reaction when incubated with catecholamines. Indirect evidence indicates that these events are mediated through alpha-adrenergic receptors. We used [(3)H]dihydroergocryptine, an alpha-adrenergic antagonist, to identify binding sites on platelets that have the characteristics of alpha-adrenergic receptors. Catecholamines compete for the binding sites in a stereo-specific manner with the potency series of (-) epinephrine > (-) norepinephrine > (+/-) phenylephrine > (-) isoproterenol. The dissociation constant (K(d)) of (-) epinephrine is 0.34 muM. Binding is saturable using a platelet particulate fraction but not with intact platelets. There are 0.130 pmol binding sites per milligram protein in fresh platelet membranes. This number represents approximately 100 binding sites per platelet. The K(d) for [(3)H]-dihydroergocryptine was 0.003-0.01 muM. The alpha-adrenergic antagonist phentolamine (K(d) = 0.0069 muM) was much more potent than the beta-adrenergic antagonist (+/-) propranolol (K(d) = 27 muM) in competing for the binding sites. The binding data were correlated with catecholamine-induced platelet aggregation and inhibition of basal and prostaglandin E(1)-stimulated adenylate cyclase. (-) Epinephrine was more potent than (-) norepinephrine in producing aggregation whereas (-) isoproterenol was ineffective. The threshold dose for inducing aggregation by (-) epinephrine was 0.46 muM. Phentolamine and dihydroergocyrptine blocked this response, whereas (+/-) propranolol had no effect. (-) Epinephrine and (-) norepinephrine inhibited basal and prostaglandin E(1)-stimulated adenylate cyclase in a dose-related manner. The concentration of (-) epinephrine inhibiting adenylate cyclase 50% was 0.7 muM. This inhibition was also blocked by phentolamine and dihydroergocryptine but not by (+/-) propranolol. The specificity of binding and the close correlation with alpha-adrenergic receptor-mediated biochemical and physiological responses suggest that the [(3)H]dihydroergocryptine binding site represents, or is closely related to, the human platelet alpha-adrenergic receptor. The ability to assay this receptor directly and to correlate these data with independently measured sequelae of receptor activation should facilitate increased understanding of the physiology and pathophysiology of the human platelet alpha-adrenergic receptor.
Journal Article•
TL;DR: There was a satisfactory correlation between the antidiuretic activities of the peptides tested in the rat and their abilities to activate the adenylate cyclase present on renal membranes.
Abstract: Membranes prepared from the medullopapillary portion of pig and rat kidneys were used to test the relative abilities of 11 arginine-vasopressin structural analogues to activate adenylate cyclase and to inhibit [3H]lysine-vasopressin binding to these membrane preparations. The analogues tested were: [1-deaminopenicillamine, 2-O-methyltyrosine]-arginine-vasopressin; [1-deaminopenicillamine]arginine-vasopressin; [1-deaminopenicillamine, 4 valine, 8-D-arginine]-vasopressin (dP-V-DAVP); [8-D-arginine]-vasopressin; [2-O-methyltyrosine]arginine-vasopressin; [4-valine, 8-D-arginine]-vasopressin; [4-valine]arginine-vasopressin; deamino [4-threonine, 8-D-arginine]-vasopressin; deamino [8-D-arginine]-vasopressin; deamino [4-valine, 8-D-arginine]-vasopressin; [1(β-mercapto-β,β-cyclopentamethylene propionic acid), 4-valine, 8-D-arginine]-vasopressin (cyclo-dV-DAVP). Cyclo-dV-DAVP behaved like a competitive inhibitor of vasopressin-induced adenylate cyclase activation. The apparent Ki values were 10 and 310 nM for the rat and pig systems, respectively. This peptide is the most active antagonist described so far. dP-V-DAVP behaved like a competitive inhibitor on the pig system (Ki = 2.9 µM) and was found to produce a maximal enzyme activation that was 75% of that induced by arginine-vasopressin in the rat system (Kact = 3.7 nM). In both the rat and pig systems there was a good correlation between the Kbind values for binding to renal membranes and the corresponding Kact or Ki values for the adenylate cyclase response. The structural requirements for binding to the pig renal receptor and to the rat renal receptor were found to be different. When one takes into account for metabolic stability of the ADH analogues tested (as estimated by the relative duration of the antidiuretic response), there was a satisfactory correlation between the antidiuretic activities of the peptides tested in the rat and their abilities to activate the adenylate cyclase present on renal membranes.
TL;DR: GTP is repored here tha t GTP as a cons t i tuent o f the cy tosol is required for epinephrineinduced inhibi t ion o f membrane -bou nd adenyla te cyclase.
TL;DR: It is postulated that choleragen exerts its effects on cells through the NAD-dependent ADP-ribosylation of an arginine or similar amino acid in either the cycl enzyme itself or a regulatory protein of the cyclase system.
Abstract: Choleragen exerts its effect on cells through activation of adenylate cyclase. Choleragen initially interacts with cells through binding of the B subunit of the toxin to the ganglioside GM1 on the cell surface. Subsequent events are less clear. Patching or capping of toxin on the cell surface may be an obligatory step in choleragen action. Studies in cell-free systems have demonstrated that activation of adenylate cyclase by choleragen requires NAD. In addition to NAD, requirements have been observed for ATP, GTP, and calcium-dependent regulatory protein. GTP also is required for the expression of choleragen-activated adenylate cyclase. In preparations from turkey erythrocytes, choleragen appears to inhibit an isoproterenol-stimulated GTPase. It has been postulated that by decreasing the activity of a specific GTPase, choleragen would stabilize a GTP-adenylate cyclase complex and maintain the cyclase in an activated state. Although the holotoxin is most effective in intact cells, with the A subunit having 1/20th of its activity and the B subunit (choleragenoid) being inactive, in cell-free systems the A subunit, specifically the A1 fragment, is required for adenylate cyclase activation. The B protomer is inactive. Choleragen, the A subunit, or A1 fragment under suitable conditions hydrolyzes NAD to ADP-ribose and nicotinamide (NAD glycohydrolase activity) and catalyzes the transfer of the ADP-ribose moiety of NAD to the guandino group of arginine (ADP-ribosyltransferase activity). The NAD glycohydrolase activity is similar to that exhibited by other NAD-dependent bacterial toxins (diphtheria toxin, Pseudomonas exotoxin A), which act by catalyzing the ADP-ribosylation of a specific acceptor protein. If the ADP-ribosylation of arginine is a model for the reaction catalyzed by choleragen in vivo, then arginine is presumably an analog of the amino acid which is ADP-ribosylated in the acceptor protein. It is postulated that choleragen exerts its effects on cells through the NAD-dependent ADP-ribosylation of an arginine or similar amino acid in either the cyclase itself or a regulatory protein of the cyclase system.
TL;DR: This study demostrates the existence of an adenylate cyclase sensitive to vasopressin in the medullary portion of the rat thick ascending limb.
Abstract: This study demostrates the existence of an adenylate cyclase sensitive to vasopressin in the medullary portion of the rat thick ascending limb. Maximal adenylate cyclase stimulations achieved in that segment (31-fold) were higher than those obtained in collecting tubules from the same rats (22-fold). From comparisons of absolute maximal responses it can be calculated that thick ascending limbs account for about 80% of the response to vasopressin of a kidney medulla homogenate. The apparent Km value of adenylate cyclase activation (from 10(-9)-2 x 10(-8) M) in thick ascending limbs was higher in each experiment than that simultaneously measured in the collecting tubules from the same rats (2 x 10(-10)-3 x 10(-9) M). Such a lower sensitivity is probably not due to a greater hormone degradation by the thick ascending limb samples. Experiments using structural analogues of the oxytocin series ([deamino-6-carba]oxytocin and vasotocin) did not give evidence for different vasopressin receptors in the thick ascending limb and the collecting tubule. A step beyond the hormone-receptor interaction, thus, must account for the different patterns of adenylate cyclase response to vasopressin of these two segments.