Muscarinic cholinergic receptor modulation of beta-adrenergic receptor affinity for catecholamines.

The effects of the muscarinic cholinergic agonist methacholine on affinity of beta-adrenergic receptors for isoproterenol and on isoproterenol-induced stimulation of adenylate cyclase activity were assessed in canine myocardium. GTP and guanyl-5'-yl imidoiphosphate both decreased the affinity of beta-adrenergic receptors for isoproterenol without altering the affinity of these receptors for propranolol. Methacholine (10 nM to 10 micronM) antagonized the guanine nucleotide-induced reduction in beta-adrenergic receptor affinity for isoproterenol. This effect of methacholine was reversed by atropine. The choline ester had no effect on the affinity of beta-adrenergic receptors for isoproterenol in the absence of guanine nucleotides. Likewise, methacholine had no effect on the affinity of beta-adrenergic receptors for propranolol, either in the presence or absence of guanine nucleotides. Methacholine also attenuated GTP-induced activation of adenylate cyclase or isoproterenol-induced activation of the enzyme in the presence of GTP. The effects of methacholine on myocardial adenylate cyclase activity were apparent only in the presence of GTP. These effects were also reversed by atropine. The choline ester had no effect on adenylate cyclase activity in the presence of guanyl-5'-yl imidodiphosphate or NaF. 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.     

Interaction of guanine nucleotides with adenylate cyclase in normal and spontaneously transformed RL-PR-C cloned rat hepatocytes

Spontaneous transformation of RL-PR-C hepatocytes leads to alterations in the adenylate cyclase complex which include a lower than normal basal level of activity, a loss of sensitivity to exogenous GTP, and a decreased sensitivity to isoproterenol. Both normal and transformed membranes posses substantial TGPase activity. Treatment of transformed hepatocyte membranes with either isoproterenol plus GMP or with cholera toxin, under conditions that displace tightly bound GDP, restored the GTP effect on adenylate cyclase, and eliminated the lag in the activation by guanyl-5′-yl-imidodiphosphate. Such pretreatment also enhanced guanine nucleotide effects on the adenylate cyclase of normal hepatocytes. These results are explainable on the basis that transformation increases adenylate cyclase-associated GTPase activity, and increase occupancy of nuceotide regulatory sites by inactive or inhibitory guanine nucleotides, e.g., GDP. Seemingly, both catecholamines and cholera toxin promote an exchange reaction at the regulatory sites, resulting in clearance of these sites of inhibitory nucleotides.     

Guanine nucleotide regulation of adenylate cyclase in permeabilized cells of Saccharomyces cerevisiae

Adenylate cyclase in permeabilized cells of Saccharomyces cerevisiae was examined. Among various permeabilization procedures, including organic solvents, detergents and other reagents, dimethylsulfoxide (DMSO) and digitonin treatments resulted in the highest recovery of adenylate cyclase activity. Incubation of cells at 30 degrees C with digitonin at 0.01% to 0.1%, or DMSO at 20% to 40% for 15 to 30 min gave optimal adenylate cyclase activity. The enzyme activity in digitonin-permeabilized cells could be supported only by Mn2+, whereas Mg2+ with or without guanine nucleotides did not support cyclase activity. DMSO-permeabilized cells exhibit efficient Mn2+- and Mg2+/Gpp[NH]p-dependent stimulation. Furthermore, digitonin added to yeast membranes at a 1:50 detergent to protein ratio (w/w) abolishes guanyl nucleotide regulation without significantly affecting the Mn2+-supported cyclase activity. The superiority of DMSO is further supported by the fact that recovery of adenylate cyclase activity is better in the DMSO-treated cells than in the digitonin-treated cells. DMSO most probably causes less disturbance of the fabric of the native cell. We conclude that digitonin, but not DMSO, uncouples the catalytic unit of adenylate cyclase from the regulatory GTP binding (ras) proteins.     

Interaction of guanine nucleotides with adenylate cyclase in normal and spontaneously transformed RL-RP-C cloned rat hepatocytes

Spontaneous transformation of RL-PR-C hepatocytes leads to alterations in the adenylate cyclase complex which include a lower than normal basal level of activity, a loss of sensitivity to exogenous GTP, and a decreased sensitivity to isoproterenol. Both normal and transformed membranes possess substantial GTPase activity. Treatment of transformed hepatocyte membranes with either isoproterenol plus GMP or with cholera toxin, under conditions that displace tightly bound GDP, restored the GTP effect on adenylate cyclase, and eliminated the lag in the activation by guanyl-5'-yl-imidodiphosphate. Such pretreatment also enhanced guanine nucleotide effects on the adenylate cyclase of normal hepatocytes. These results are explainable on the basis that transformation increases adenylate cyclase-associated GTPase activity, and increases occupancy of nucleotide regulatory sites by inactive or inhibitory guanine nucleotides, e.g., GDP. Seemingly, both catecholamines and cholera toxin promote an exchange reaction at the regulatory sites, resulting in clearance of these sites of inhibitory nucleotides.     

Multiple effects of guanine nucleotides on human platelet adenylated cyclase.

We report that the adenylate cyclase system in human platelets is subject to multiple regulation by guanine nucleotides. Previously it has been reported that GTP is either required for or has little effect on the response of the enzyme to prostaglandin E1. We have found that when platelet lysates were prepared in the presence of 5 mM EDTA, GTP lowered the basal and prostaglandin E1-stimulated adenylate cyclase activity, but at a higher concentration of Mn2+, it caused an increase in enzyme activity exceeding that occurring in the presence of prostaglandin E1. In the presence of Mn2+, dGTP mimics the effect of GTP and is 50% as effective as GTP. Our data suggest that the inhibitory effect of GTP on prostaglandin E1-stimulated adenylate cyclase is mainly due to its direct effect on the enzyme itself, whereas the stimulatory effect of GTP on prostaglandin E1-stimulated adenylate cyclase is due to enhancement of the coupling between the prostaglandin E1 receptor and adenylate cyclase. These studies also indicate that the method of preparation of platelet lysates can profoundly alter the nature of guanine nucleotide regulation of adenylate cyclase.     

Activation of adenylate cyclase in bovine corpus-luteum membranes by human choriogonadotropin, guanine nucleotides and NaF

1. Preincubation of luteal membranes with human choriogonadotropin results in the formation of an activated state of adenylate cyclase which is not reversed by washing and which is limited only by the absence of guanine nucleotides, whereas preincubation with GTP yields only a partially activated adenylate cyclase which requires the presence of both GTP and human choriogonadotropin during assay to demonstrate maximal activity. 2. Preincubation of luteal membranes with GTP and human choriogonadotropin does not lead to a synergistic increase in wash-resistant activity. 3. Luteal membranes that had been preincubated with GTP and hormone exhibited a decreasing rate of cyclic AMP synthesis during the adenylate cyclase assay incubation; addition of GTP during the assay incubation reversed the decrease. 4. Membranes that had been preincubated in the absence of guanine nucleotide and hormone showed a `burst' phase of cyclic AMP synthesis when GTP was present in the assay incubation and a `lag' phase with p[NH]ppG (guanosine 5′-[β,γ-imido]triphosphate) present in the assay. The presence of human choriogonadotropin with either nucleotide in the assay incubation eliminated the curvatures in plots observed with guanine nucleotides alone. 5. Luteal adenylate cyclase was persistently activated by preincubation with p[NH]ppG alone or in combination with human choriogonadotropin; the activation caused by p[NH]ppG alone was still increasing after 70min of preincubation, whereas that caused by p[NH]ppG in the presence of hormone was essentially complete within 10min of preincubation. 6. Luteal adenylate cyclase that had been partially preactivated by preincubation with p[NH]ppG was slightly increased in activity by the inclusion of further p[NH]ppG in the adenylate cyclase assay incubation, but more so with p[NH]ppG and hormone. Human choriogonadotropin alone caused no further increase in the activity of the partially stimulated preparation unless p[NH]ppG was also added to the assay incubation. 7. GTP decreased the activity of adenylate cyclase in membranes that had been partially preactivated in the presence of p[NH]ppG; the decrease in activity was greater when GTP and hormone were present simultaneously in the assay. 8. The results indicate that stable activation states of adenylate cyclase can be induced by preincubation of luteal membranes in vitro with human choriogonadotropin or p[NH]ppG, and that in the presence of p[NH]ppG the hormone may accelerate events subsequent to guanine nucleotide binding. Stable activation of luteal adenylate cyclase by prior exposure to GTP is not achieved. The involvement of GTPase activity and of hormone-promoted guanine nucleotide exchange in the modulation of luteal adenylate cyclase activity is discussed.     

The regulation of adenylate cyclase by guanine nucleotides in Dictyostelium discoideum membranes

Extracellular cAMP induces the activation of adenylate cyclase in Dictyostelium discoideum cells. Conditions for both stimulation and inhibition of adenylate cyclase by guanine nucleotides in membranes are reported. Stimulation and inhibition were induced by GTP and non-hydrolysable guanosine triphosphates. GDP and non-hydrolysable guanosine diphosphates were antagonists. Stimulation was maximally twofold, required a cytosolic factor and was observed only at temperatures below 10 degrees C. An agonist of the cAMP-receptor-activated basal and GTP-stimulated adenylate cyclase 1.3-fold. Adenylate cyclase in mutant N7 could not be activated by cAMP in vivo; in vitro adenylate cyclase was activated by guanine nucleotides in the presence of the cytosolic factor of wild-type but of not mutant cells. Preincubation of membranes under phosphorylation conditions has been shown to alter the interaction between cAMP receptor and G protein [Van Haastert (1986) J. Biol. Chem. in the press]. These phosphorylation conditions converted stimulation to inhibition of adenylate cyclase by guanine nucleotides. Inhibition was maximally 30% and was not affected by the cytosolic factor involved in stimulation. In membranes obtained from cells that were treated with pertussis toxin, adenylate cyclase stimulation by guanine nucleotides was as in control cells, whereas inhibition by guanine nucleotides was lost. When cells were desensitized by exposure to cAMP agonists for 15 min, and adenylate cyclase was measured in isolated membranes, stimulation by guanine nucleotides was lost while inhibition was retained. These results suggest that Dictyostelium discoideum adenylate cyclase may be regulated by Gs-like and Gi-like activities, and that the action of Gs but not Gi is lost during desensitization in vivo and by phosphorylation conditions in vitro.     

Alteration in the protein components of catecholamine-sensitive adenylate cyclase during maturation of rat reticulocytes

The maturing rat reticulocyte was used as a model system in which to study developmental changes in the protein components of hormone-sensitive adenylate cyclase. Plasma membranes from rat erythrocytes display 10 to 20% of the adenylate cyclase activity and 30 to 50% of the beta-adrenergic receptors which are measured in membranes from rat reticulocytes, as noted by others. Reticulocyte membranes also display equal activities in response to (-)-isoproterenol in the presence of either GTP or GTP gamma S, whereas erythrocyte membrane adenylate cyclase is twice as active in the presence of isoproterenol plus GTP gamma S as in the presence of isoproterenol plus GTP. We have studied this system in greater detail by developing or applying independent assays for the catalytic protein (C) and the guanine nucleotide-binding regulatory protein (G/F) of adenylate cyclase. C was assayed in membranes by its intrinsic Mn2+-stimulated activity. It was also measured by reconstituting membranes with saturating amounts of GTP gamma S-activated G/F, yielding an operationally defined Vmax for the catalyst. By either assay, reticulocytes display about 3-fold greater C activity than do erythrocytes. G/F was assayed by its ability to confer GTP gamma S-stimulated activity upon C (which was supplied by membranes of cyc- S49 lymphoma cells). This assay indicates that reticulocyte membranes contain about 3 times as much G/F as do erythrocyte membranes. Cholera toxin and [32P]NAD were used to [32P]ADP-ribosylate the 45,000- and 52,000-dalton subunits of G/F. Total incorporation of 32P into these subunits decreased 3- to 4-fold with reticulocyte maturation. The ratio of label in the 52,000-dalton peptide to that in the 45,000-dalton peptide decreased from 0.29 in reticulocyte membranes to 0.14 in erythrocyte membranes. The apparently coordinate decrease in the amounts of C, G/F, and beta-adrenergic receptors suggest that the stoichiometry between these components is maintained during maturation, and may account for the decrease in adenylate cyclase in the membranes. However, the qualitative changes in responsiveness to hormones in the presence of GTP or GTP gamma S may be related to loss or proteolysis of the 52,000-dalton subunit of G/F.     

Guanosine 5', alpha-beta-methylene, triphosphate, a novel GTP analog, causes persistent activation of adenylate cyclase: evidence against pyrophosphorylation mechanism.

To test the hypothesis that guanine nucleotides activate adenylate cyclase by a covalent mechanism involving pyrophosphorylation of the enzyme, we studied the effect of a novel GTP analog, guanosine 5′, α-β-methylene triphosphate (Gp(CH₂)pp), with a methylene bond in the α-β-position that is stable to enzymatic hydrolysis. Gp(CH₂)pp was as effective as GTP in stimulating rat reticulocyte adenylate cyclase in the presence of isoproterenol. Previously only guanine nucleotides with modified terminal phosphates such as guanylyl 5′-imidodiphosphate (Gpp(NH)p) were thought capable of causing persistent activation of adenylate cyclase. Gp(CH₂)pp, however, caused persistent activation of rat reticulocyte and turkey erythrocyte adenylate cyclase. We conclude that guanine nucleotides do not activate adenylate cyclase by a pyrophosphorylation mechanism and that a modified γ-phosphate is not essential in guanine nucleotides for generation of the irreversibly-activated enzyme state.     

Forskolin-activated adenylate cyclase. Inhibition by guanyl-5'-yl imidodiphosphate

Forskolin activated adenylate cyclase of purified rat adipocyte membranes in the absence of exogenous guanine nucleotides. Guanyl-5'-yl imidodiphosphate (Gpp(NH)p) inhibited the forskolin-activated cyclase immediately upon addition of the nucleotide at concentrations too low to activate adenylate cyclase (10(-9) to 10(-7) M). Inhibition seen with a very high concentration of Gpp(NH)p (10(-4) M) lasted for 3-4 min and was followed by an increase in the synthetic rate which remained constant for at least 15 min. The length of the transient inhibition did not vary with forskolin concentrations above 0.05 microM but low Gpp(NH)p (10(-8) M) exhibited a lengthened (6-7 min) inhibitory phase. The transient inhibitory effects of Gpp(NH)p were eliminated by 10(-7) M isoproterenol, high (40 mM) Mg2+, or preincubation with Gpp(NH)p in the absence of forskolin. While forskolin stimulated fat cell cyclase in the presence of Mn2+, this ion blocked the inhibitory effects of Gpp(NH)p. The well documented inhibitory effects of GTP on the fat cell adenylate cyclase system were also observed in the presence of forskolin. However, the inhibition by GTP is not transitory. These findings indicate that Gpp(NH)p regulation of forskolin-stimulated cyclase has at least two components: 1) an inhibitory component which acts through an undetermined mechanism and which acts immediately to decrease cyclase activity; and 2) an activating component which modulates the inhibited cyclase activity through the guanine nucleotide regulatory protein.     

"Spare" beta-adrenergic receptors of rat white adipocyte membranes

The apparent equilibrium dissociation constants for the interaction of isoproterenol with beta-receptors and adenylate cyclase were determined under the same conditions in rat adipocyte membranes and were compared with the apparent dissociation constant for the interaction of isoproterenol with cyclic AMP accumulation in the adipocyte. From these determinations, it was calculated that the occupancy of less than 4% of the receptor population is required for half-maximal stimulation of adenylate cyclase in membranes and cyclic AMP accumulation in intact cells, provided that receptor-binding and adenylate cyclase assays are performed in the presence of guanine nucleotides. Since guanine nucleotides are also required for adenylate cyclase activation in intact cells, it is concluded that the beta-receptors of rat adipocytes are "spare" receptors.     

Essential role of GTP in the expression of adenylate cyclase activity after cholera toxin treatment.

Expression of activation of rat liver adenylate cyclase by the A1 peptide of cholera toxin and NAD is dependent on GTP. The nucleotide is effective either when added to the assay medium or during toxin (and NAD) treatment. Toxin treatment increases the Vmax for activation by GTP and the effect of GTP persists in toxin-treated membranes, a property seen in control membranes only with non-hydrolyzable analogs of GTP such as Gpp(NH)p. These observations could be explained by a recent report that cholera toxin acts to inhibit a GTPase associated with denylate cyclase. However, we have observed that one of the major effects of the toxin is to decrease the affinity of guanine nucleotides for the processes involved in the activation of adenylate cyclase and in the regulation of the binding of glucagon to its receptor. Moreover, the absence of lag time in the activation of adenylate cyclase by GTP, in contrast to by Gpp(NH)p, and the markedly reduced fluoride action after toxin treatment suggest that GTPase inhibition may not be the only action of cholera toxin on the adenylate cyclase system. We believe that the multiple effects of toxin action is a reflection of the recently revealed complexity of the regulation of adenylate cyclase by guanine nucleotides.     

Increased membrane-associated nucleoside diphosphate kinase activity as a possible basis for enhanced guanine nucleotide-dependent adenylate cyclase activity induced by picolinic acid treatment of simian virus 40-transformed normal rat kidney cells

A biochemical analysis of an increase in guanine nucleotide-dependent adenylate cyclase activity induced by treatment of cultured SV40-transformed normal rat kidney cells with picolinic acid is described. In purified membranes from drug-treated cells with an ATP regenerating system in assay, GTP- and GTP plus hormone-stimulated adenylate cyclase activities were increased, whereas basal and NaF-stimulated cyclase activities, and steady state rate with guanosine 5'-(beta, gamma-imino)triphosphate were essentially unaltered by drug treatment. In assay systems devoid of ATP regenerating system, the drug-induced increase in cyclase activity was seen with GDP as well as with GTP, it being larger with GDP than with GTP in terms of activity ratio, whereas such an increase was not observed with their analogs, guanosine 5'-O-(2-thiodiphosphate) or guanosine 5'-(beta, gamma-imino)triphosphate. Guanosine 5'-(beta, gamma-imino)triphosphate-stimulated from drug-treated membranes became less sensitive to the inhibition by GDP as shown by a rightward shift in inhibition curve, but this shift could not be reproduced with guanosine 5'-O-(2-thiodiphosphate). From these results, it was concluded that altered guanine nucleotide metabolism in membranes was involved. Neither the amount of guanine nucleotide-binding protein nor its related functions including GTPase activity were changed by drug treatment. However, we observed in the drug-treated cell membranes, an increase in activity of nucleoside diphosphate kinase, an additional factor which has been proposed to play a role in regulating adenylate cyclase by replenishing GTP near the guanine nucleotide binding site (Kimura, N., and Shimada, N. (1983) J. Biol. Chem. 258, 2278-2283). The altered features of adenylate cyclase with the natural guanine nucleotides induced by drug treatment were explained as a result of this enhanced nucleoside diphosphate kinase activity associated with the membranes.     

Modulation of adenylate cyclase in human keratinocytes by protein kinase C

Adenylate cyclase (ATP-pyrophosphate lyase (cyclizing); EC 4.6.1.1) in the human keratinocyte cell line SCC 12F was potentiated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), phorbol-12,13-diacetate, and 1,2-dioctanoylglycerol. Keratinocytes exposed to TPA showed a 2-fold enhancement of adenylate cyclase activity when assayed in the presence of isoproterenol or GTP. The half-maximal effective concentration (EC50) for both isoproterenol and GTP were unaltered by TPA treatment of the cells. Basal adenylate cyclase activity in membranes from TPA-treated cultures was also increased 2-fold relative to activity in control membranes. Potentiation of adenylate cyclase activity was dependent on the concentration of TPA to which the keratinocytes were exposed (EC50 for TPA = 3 nM). TPA actions on adenylate cyclase were maximal after 15 min of incubation of the cells with the compound, correlating well with the time course of translocation of protein kinase C (Ca2+/phospholipid-dependent enzyme) from cytosol to membrane. The action of cholera toxin on adenylate cyclase was additive with TPA. In contrast, pertussis toxin actions on adenylate cyclase were not additive with TPA. Treatment of control cells with pertussis toxin activated adenylate cyclase 1.5-fold, whereas cells exposed to pertussis toxin for 6 h followed by TPA for 15 min showed the same 2-fold increase in adenylate cyclase activity as observed in membranes from cells exposed to TPA without prior exposure to pertussis toxin. Pertussis toxin catalyzed ADP-ribosylation was increased 2-fold in membranes from SCC 12F cells exposed to TPA, indicating an increase in the alpha beta gamma form of Gi. These data suggest that exposure of human keratinocytes to phorbol esters increases adenylate cyclase activity by a protein kinase C-mediated increase in the heterotrimeric alpha beta gamma form of Gi resulting in decreased inhibition of basal adenylate cyclase activity.     

Transient complexes. A structural model for the activation of adenylate cyclase by hormone receptors (guanine nucleotides/irradiation inactivation)

1. The irradiation-inactivation procedure was used to study changes in the state of association of the protein components of adenylate cyclase in intact rat liver plasma membranes by measurement of alterations in the target size determined from the catalytic activity of the enzyme. 2. A decrease in target size at 30 degrees C in response to p[NH]ppG (guanosine 5'-[betagamma-imido]triphosphate) or GTP was demonstrated, which we take to reflect the dissociation of a regulatory subunit. The effect of GTP is potentiated by glucagon. This effect is not observed at 0 degrees C. 3. An increase in target size was observed in response to glucagon in the absence of guanine nucleotides, which we take to reflect the association of glucagon receptor with adenylate cyclase. 4. We propose a model for the activation of adenylate cyclase by glucagon in which the binding of the hormone to its receptor causes an initial association of the receptor with the catalytic unit of the enzyme and a regulatory subunit to form a ternary complex. The subsequent activation of the adenylate cyclase results from the dissociation of the ternary complex to leave a free catalytic unit in the activated state. This dissociation requires the binding of a guanine nucleotide to the regulatory subunit. 5. The effects of variation of temperature on the activation of adenylate cyclase by glucagon and guanine nucleotides were examined and are discussed in relation to the irradiation-activation data. 6. The effectiveness of hormones, guanine nucleotides and combinations of hormone and guanine nucleotides as activators of adenylate cyclase in both rat liver and rat fat-cell plasma membranes was studied and the results are discussed in relation to the model proposed, which is also considered in relation to the observations published by other workers.     

Dicyclohexylcarbodiimide influences the regulatory properties of the adenylate cyclase system in rat reticulocyte membranes

Treatment of rat reticulocyte plasma membranes with dicyclohexylcarbodiimide (DCCD) decreased the GTP-stimulated adenylate cyclase activity and reduced the number of receptors that bind the agonist with high affinity in the absence of GTP. Besides, the agonist's competition curve was shifted to the right, irrespective of the presence of guanyl nucleotides. The dissociation constant for the antagonist and the number of binding sites did not change. Preincubation of the DCCD-treated membranes with GMP in the presence of isoproterenol restored the regulation of the agonist's affinity by guanyl nucleotides; however, in this case the GTP-independent change in the agonist's affinity was retained. This suggests that one of the DCCD-modified components of the adenylate cyclase system is a regulatory protein.     

ADP-Ribosylation by Cholera Toxin of Membranes Derived from Brain Modifies the Interaction of Adenylate Cyclase with Guanine Nucleotides and NaF

We have developed a method to ADP-ribosylate the stimulatory guanine nucleotide-binding protein of adenylate cyclase (GS) in brain membranes by using cholera toxin. In particular, we used isonicotinic acid hydrazide and 3-acetylpyridine adenine dinucleotide to inhibit the potent NAD-glycohydrolase activity of brain membranes, and we used the detergent Triton X-100 (at 0.1%) to improve the accessibility of the toxin and guanine nucleotides used for supporting the ADP-ribosylation. This method reveals that GS is a very abundant protein in membranes derived from calf brain (approximately 30 pmol/mg of protein). In brain, GS exists in large excess over the previously reported amount of the adenylate cyclase catalytic subunit. The modification of GS with an ADP-ribosyl residue (a) elicits a four- to fivefold activation of adenylate cyclase by GTP, (b) increases the stabilization of adenylate cyclase by GTP, and (c) reduces adenylate cyclase activation by fluoride but does not change basal activity, activation by guanosine 5'-(beta, gamma-imido)triphosphate, or the sensitivity of adenylate cyclase to heat-induced denaturation. A correlation between ADP-ribosylation and the alterations in the activation of adenylate cyclase by guanine nucleotides and by fluoride is presented.     

In situ analysis of adenylate cyclase activity in permeabilized rat adipocytes: sensitivity to GTP, isoproterenol, and N6-(phenylisopropyl)adenosine

Adenylate cyclase activity in rat adipocyte suspensions was assayed in situ using a digitonin permeabilization technique. Recovery of activity was dependent on digitonin concentration, reaching a maximum at 20 micrograms/ml digitonin and paralleling the effect on cell permeability. Maximum adenylate cyclase activity recovered in permeabilized cells was 75% of that in comparable homogenates. Isoproterenol, a beta-adrenergic agonist, activated adenylate cyclase by 1.4, 2.2 and 4.5 fold at 10(-6), 10(-5) and 10(-3) M, respectively, despite perturbation of the plasma membrane. Exogenous GTP was not required for expression of beta-adrenergic activation, but 10(-5) M GTP maximally increased both basal and isoproterenol-dependent activity. The response to 10(-6) M isoproterenol was increased 2.1 fold by 10(-5) M GTP. N6-(Phenylisopropyl)adenosine at 10(-6) M inhibited both basal and isoproterenol-dependent adenylate cyclase activity by approximately 30%, demonstrating that the adenosine-dependent inhibitory pathway (Ni) remained functional in the digitonin-permeabilized cells. In situ analysis of adenylate cyclase is not only simple and rapid, but provides a unique approach to studying regulation of this key enzyme.     

Chymotrypsin selectively decreases forskolin stimulation of adenylate cyclase

We studied the effects of chymotrypsin on turkey erythrocyte membrane adenylate cyclase activity. Proteolysis with chymotrypsin led to a concentration- and time-dependent increase in activation of adenylate cyclase by isoproterenol + guanine nucleotides, and fluoride, and to a decrease in activation by forskolin. Maximal effects (up to 10-fold increases in fluoride- and isoproterenol + guanine nucleotide-stimulated activity, and up to 100% inhibition of forskolin-stimulated activity) occurred under similar conditions (10-20 micrograms/ml chymotrypsin for 10-15 min at 30 degrees C). Augmentation of isoproterenol + guanosine-3'-O-thiotriphosphate (GTP-gamma-S)-stimulated activity by chymotrypsin occurred only if proteolysis preceded stimulation with isoproterenol + GTP-gamma-S. Addition of isoproterenol + GTP-gamma-S to membranes before proteolysis, however, did not prevent chymotrypsin from augmenting subsequent stimulation by these agents. In contrast, addition of forskolin during proteolysis with chymotrypsin prevented the time- and concentration-dependent decline in forskolin stimulation observed with chymotrypsin. Proteolysis decreased the magnitude of stimulation at any concentration of forskolin, but did not alter the concentration dependence of forskolin stimulation (apparent half-maximum = 3 microM). The data are consistent with the existence of a chymotrypsin-sensitive site essential for forskolin stimulation of adenylate cyclase. In view of the simultaneous effect of chymotrypsin to augment fluoride- and isoproterenol + guanine nucleotide-stimulated activities, it is highly unlikely that the site is on the stimulatory guanine nucleotide binding protein. Since forskolin is thought to act directly on the catalytic unit of adenylate cyclase, and since forskolin can protect against the effect of proteolysis with chymotrypsin, the site involved may be on the catalytic unit itself.     

Multiple effects of guanine nucleotides on human platelet adenylate cyclase

We report that the adenylate cyclase system in human platelets is subject to multiple regulation by guanine nucleotides. Previously it has been reported that GTP is either required for or has little effect on the response of the enzyme to prostaglandin E₁. We have found that when platelet lysates were prepared in the presence of 5 mM EDTA, GTP lowered the basal and prostaglandin E₁-stimulated adenylate cyclase activity when the enzyme was assayed in the presence of Mg²⁺. The basal and prostaglandin E₁-stimulated adenylate cyclase activities were also increased by washing, which presumably removes endogenous GTP. The analog, guanyl-5′-yl-imidodiphosphate mimics the inhibitory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase activity but it stimulates basal enzyme activity. The onset of the inhibitory effect of GTP on the adenylate cyclase system is rapid (1 min) and is maintained at a constant rate during incubation for 10 min. GTP and guanyl-5′-yl-imidodiphosphate were noncompetitive inhibitors of prostaglandin E₁. An increase in the concentration of Mg²⁺ gradually reduces the effect of GTP while having little influence on the effect of guanyl-5′-yl-imidodiphosphate. Neither the substrate concentration nor the pH (7.2–8.5) is related to the inhibitory effect of guanine nucleotides. The inhibition by nucleotides was found to show a specificity for purine nucleotides with the order of potency being guanyl-5′-yl-imidodiphosphate > dGTP > GTP > ITP > XTP > CTP > TTP. The inhibitory effect of GTP is reversible while the effect of guanyl-5′-yl-imidodiphosphate is irreversible. The GTP inhibitory effect was abolished by preparing the lysates in the presence of Ca²⁺. However, the inhibitory effect of guanyl-5′-yl-imidodiphosphate persisted. Substitution of Mn²⁺ for Mg²⁺ in the assay medium resulted in a diminution of the inhibitory effect of GTP on basal activity and converted the inhibitory effect of GTP on prostaglandin E₁-stimulated activity to a stimulatory effect. At a lower concentration of Mn²⁺ (less than 2 mM) guanyl-5′-yl-imidodiphosphate inhibited prostaglandin E₁-stimulated adenylate cyclase activity, but at a higher concentration of Mn²⁺, it caused an increase in enzyme activity exceeding that occuring in the presence of prostaglandin E₁. In the presence of Mn²⁺, dGTP mimics the effect of GTP and is 50% as effective as GTP. Our data suggest that the inhibitory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase is mainly due to its direct effect on the enzyme itself, whereas the stimulatory effect of GTP on prostaglandin E₁-stimulated adenylate cyclase is due to enhancement of the coupling between the prostaglandin E₁ receptor and adenylate cyclase. These studies also indicate that the method of preparation of platelet lysates can profoundly alter the nature of guanine nucleotide regulation of adenylate cyclase.