Calmodulin activation of rat lung adenylate cyclase is independent of the cytoplasmic factors modulating the enzyme.

Adenylate cyclase activity in the rat lung membranes washed with 150 microM-EGTA was stimulated by calmodulin in the presence of 100 microM-Ca2+. The calmodulin activation of the enzyme was concentration-dependent; however, at high concentrations the activation was diminished. Activation of adenylate cyclase by calmodulin was immediate, reversible and due to an increase in the Vmax. without apparent effect on the affinity of the enzyme for ATP. The rat lung supernatant produced additive activation of the adenylate cyclase that was already maximally stimulated by calmodulin, indicating that either calmodulin and cytoplasmic factors act at different sites on adenylate cyclase or different adenylate cyclases may be involved. The data further support our previous conclusion that calmodulin is not involved in the activation of adenylate cyclase by cytoplasmic factors in rat lungs.     

Endocrine control of cytosolic factors stimulating adenylate cyclase in rat lung

Endocrine control of cytoplasmic factors modulating adenylate cyclase activity in rat lung membranes was investigated. Hypophysectomy, adrenalectomy and thyroidectomy showed an adverse effect on the body and organ weights. Lung protein, glycogen and DNA contents were decreased in the endocrine ablated animals which were restored to the normal values on hormone treatment. Phosphodiesterase and phosphorylase activities were increased and decreased in adrenalectomized and thyroidectomized animals, respectively. The activities of these enzymes were restored to normal values on hormone treatment. Adrenalectomy and thyroidectomy affected ATPases differently. Basal adenylate cyclase activity in rat lung membranes was not affected by adrenalectomy and hormone treatment. However, the total enzyme activity was increased by both dexamethasone (DEX) and thyroxine (T4) treatments. The activation of the particulate adenylate cyclase by the cytoplasmic factors was markedly decreased in the lung from hypophysectomized, adrenalectomized and thyroidectomized rats. This decrease in the cytoplasmic activation of adenylate cyclase was restored to or above the control values on hormone treatment. Alteration in the activation of enzyme by cytoplasmic factors did not appear to be due to the change in the responsiveness of the enzyme. Glucocorticoids appeared to have a specific effect on the cytoplasmic factors modulating the enzyme.     

Saccharin induces changes in adenylate cyclase activity in liver and muscle membranes in rats

The non-nutritive sweetener, saccharin, was found to stimulate significantly the activity of adenylate cyclase in membranes derived from skeletal muscle of rat. Sodium saccharin enhanced adenylate cyclase activity in a dose-related manner, and this activation appeared to be dependent on the presence of guanine nucleotides, suggesting the involvement of GTP-binding proteins. In membranes derived from the liver, however, sodium saccharin had an effect which was dependent on the concentration of membranes used in the adenylate cyclase assay. In high concentrations of membranes, sodium saccharin had a stimulatory effect, while in low concentration an inhibition was observed.     

Mechanism of adenylate cyclase activation by the rat lung cytoplasmic factors

Epinephrine, histamine and prostaglandin E₁ stimulated adenylate cyclase activity in lung membranes and their stimulation of the enzyme activity was completely blocked by propranolol, metiamide and indomethacin, respectively. A partially-purified activator from the adult rat lung also enhanced adenylate cyclase activity in membranes. However, stimulation of adenylate cyclase by the rat lung activator was not abolished by the above receptor antagonists. Further, epinephrine, NaF and Gpp(NH)p stimulated adenylate cyclase activity rather readily, whereas stimulation of the enzyme activity by the lung activator was evident after an initial lag phase of 10 min. Also, the lung activator produced additive activation of adenylate cyclase with epinephrine, NaF and Gpp(NH)p. These results indicate that the lung activator potentiates adenylate cyclase activity in membranes by a mechanism independent from those known for epinephrine, NaF and Gpp(NH)p. Incubation of lung membranes for 30 min at 40°C resulted in a loss of adenylate cyclase activation by NaF and Gpp(NH)p. Addition of the released proteins to the heat-treated membranes did not restore the enzyme response to these agonists. However, heat treatment of lung membranes in the presence of 2-mercaptoethanol or dithiothreitol prevented the loss of adenylate cyclase response to NaF and Gpp (NH)p. N-ethylmaleimide abolished adenylate cyclase activation by epinephrine, NaF, Gpp(NH)p and the lung activator. These results indicate that the sulfhydryl groups are important for adenylate cyclase function in rat lung membranes.Abbreviations Gpp(NH)p 5-Guanylimidodiphosphate     

The sweet taste inhibitor methyl 4,6-dichloro-4,6-dideoxy-{alpha}-D-galactopyranoside inhibits sucrose stimulation of the chorda tympani nerve and of the adenylate cyclase in anterior lingual membranes of rats

The effects of the sweet taste inhibitor methyl 4,6-dichloro-4,6-dideoxy--D-galactopyranoside(MAD-diCl-Gal) and a few disaccharides, on the electrophysiologicalresponses of the chorda tympani nerve and on adenylate cyclasein membranes prepared from the anterior tongue epithelium, werestudied in rats. MAD-diCl-Gal inhibited the sucrose stimulationof whole chords tympani responses, and this inhibition was reversible.In addition, MAD-diCl-Gal inhibited the sucrose stimulationof adenylate cyclase activity in lingual (gustatory) membranesin a dose-dependent manner. High concentrations of MAD-diCl-Galabolished the sucrose induced adenylate cyclase activity. Thedisaccharides sucrose, maltose, trehalose and melibiose stimulatedboth chords tympani nerve responses and adenylate cyclase activity.These stimulations were dose dependent. Sucrose was the mostpotent stimulator of the chorda tympani nerve. Other disaccharidesresulted in lower responses than sucrose. Sucrose was also amore effective stimulus than maltose for adenylate cyclase activity.In contrast to electrophysiological data, trehalose and melibiosestimulated the adenylate cyclase activity to the same extentas sucrose. The results of this study support the suggestionof cAMP involvement in the cellular transduction of sweet tastein the rat.     

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.     

Antibodies directed against transducin beta subunits interfere with the regulation of adenylate cyclase activity in brain membranes

In an attempt to study the mechanisms of action of membrane-bound adenylate cyclase, we have applied to rat brain synaptosomal membranes antibodies raised against purified bovine transducin (T) beta gamma subunits. The antibodies recognized one 36-kDa protein in Western blots of the membranes. Adenylate cyclase activation by GTP non-hydrolyzable analogues was greatly decreased in immune, as compared to preimmune, antibody-treated membranes, whereas the enzyme basal activity was unaffected by both types of antibodies. The inhibition of forskolin-stimulated adenylate cyclase by guanine 5'-(beta, gamma-imino)triphosphate (Gpp-(NH)p) was decreased in membranes preincubated with immune, but not preimmune, antibodies. Anti-T beta antibodies moderately decreased the extent of subsequent adenylate cyclase activation by forskolin, while not affecting activation by Al3+/F-. The enzyme activation by Gpp(NH)p in untreated membranes remained the same upon further incubation in the presence of either type of antibodies. Such results were consistent with the decreased exchange of guanine nucleotides which occurred in membrane treated with immune, but not preimmune antibodies, upon addition of GTP. The blockade of the regulation of adenylate cyclase by Gpp(NH)p observed in membranes pretreated by anti-T beta antibodies thus appears to be caused by the impairment of the guanine nucleotide exchange occurring on Gs alpha subunits. The G beta subunits in the adenylate cyclase complex seem to be instrumental in the guanine nucleotide exchange on G alpha subunits, just as T beta subunits are in the transducin complex.     

Protein kinase C stimulates adenylate cyclase activity in prolactin-secreting rat adenoma (GH4C1) pituicytes by inactivating the inhibitory GTP-binding protein Gi

The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and thyroliberin exerted additive stimulatory effects on prolactin release and synthesis in rat adenoma GH4C1 pituicytes in culture. Both TPA and thyroliberin activated the adenylate cyclase in broken cell membranes. When combined, the secretagogues displayed additive effects. TPA did not alter the time course (time lag) of adenylate cyclase activation by hormones, guanosine 5'-[beta,gamma-imino]triphosphate or forskolin, nor did it affect the enzyme's apparent affinity (basal, 7.2 mM; thyroliberin-enhanced, 2.2 mM) for free Mg2+. The TPA-mediated adenylate cyclase activation was entirely dependent on exogenously added guanosine triphosphate. ED50 (dose yielding half-maximal activation) was 60 microM. Access to free Ca2+ was necessary to express TPA activation of the enzyme, however, the presence of calmodulin was not mandatory. TPA-stimulated adenylate cyclase activity was abolished by the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, by the protein kinase C inhibitor polymyxin B and by pertussis toxin, while thyroliberin-sensitive adenylate cyclase remained unaffected. Experimental conditions known to translocate protein kinase C to the plasma membrane and without inducing adenylate cyclase desensitization, increased both basal and thyroliberin-stimulated enzyme activities, while absolute TPA-enhanced adenylate cyclase was maintained. Association of extracted GTP-binding inhibitory protein, Gi, from S49 cyc- murine lymphoma cells with GH4C1 cell membranes yielded a reduction of basal and hormone-stimulated adenylate cyclase activities, while net inhibition of the cyclase of somatostatin was dramatically enhanced. However, TPA restored completely basal and hormone-elicited adenylate cyclase activities in the Gi-enriched membranes. Finally, TPA completely abolished the somatostatin-induced inhibition of adenylate cyclase in both hybrid and non-hybrid membranes. These data suggest that, in GH4C1 cells, protein kinase C stimulation by phorbol esters completely inactivates the n alpha i subunit of the inhibitory GTP-binding protein, leaving the n beta subunit functionally intact. It can also be inferred that thyroliberin conveys its main effect on the adenylate cyclase through activation of the stimulatory GTP-binding protein, Gs.     

Relationship Among Calmodulin-, Forskolin-, and Guanine Nucleotide-Dependent Adenylate Cyclase Activities in Cerebellar Membranes: Studies by Limited Proteolysis

Adenylate cyclase activity in bovine cerebellar membranes is regulated by calmodulin, forskolin, and both stimulatory (Ns) and inhibitory (Ni) guanine nucleotide-binding components. The susceptibility of the enzyme to chymotrypsin proteolysis was used as a probe of structure-function relationships for these different regulatory pathways. Pretreatment of membranes with low concentrations of chymotrypsin (1-2 micrograms/ml) caused a three- to fourfold increase in basal adenylate cyclase activity and abolished the Ca2+-dependent activation of the enzyme by calmodulin. In contrast, the stimulation of the enzyme by GTP plus isoproterenol was strongly potentiated after protease treatment, an effect that mimics the synergistic activation of adenylate cyclase by Ns and calmodulin in unproteolyzed membranes. Limited proteolysis revealed low- and high-affinity components in the activation of adenylate cyclase by forskolin. The low-affinity component was readily lost on proteolysis, together with calmodulin stimulation of the enzyme. The activation via the high-affinity component was resistant to proteolysis and nonadditive with the Ns-mediated activation of the enzyme, suggesting that both effectors utilize a common pathway. The inhibitory effect of low concentrations (10(-7) M) of guanyl-5'-yl imidodiphosphate [Gpp(NH)p] on forskolin-activated adenylate cyclase was retained after limited proteolysis of the membranes, indicating that the proteolytic activation does not result from an impairment of the Ni subunit. Moreover, in the rat cerebellum, proteolysis as well as calmodulin was found to enhance strongly the inhibitory effect of Gpp(NH)p on basal adenylate cyclase activity. Our results suggest that calmodulin and Ns/Ni interact with two structurally distinct but allosterically linked domains of the enzyme. Both domains appear to be involved in the mode of action of forskolin.     

Inhibition of rat brain adenylate cyclase activity by benzodiazepine through the effects on Gi and catalytic proteins

The effect of benzodiazepines on adenylate cyclase system was examined in rat brain. Micromolar concentrations of diazepam inhibited the enzyme activity in synaptic membranes in dose- and time-dependent manners. The inhibitory effect of diazepam was more evident on the enzyme activity in the presence of guanylyl-5'-imidodiphosphate (GppNHp) or NaF-AlCl3 than on that in the basal state. In the pertussis toxin-treated membranes, the effect of diazepam in the presence of GppNHp or NaF-AlCl3 was markedly suppressed. In addition, other benzodiazepines, such as medazepam, flurazepam, flunitrazepam, and clonazepam, had similar effects to those of diazepam, whereas Ro15-1788, an antagonist of a high affinity receptor in the central nervous system, had no effect on adenylate cyclase activity and did not antagonize the effect of diazepam. These findings indicate that benzodiazepines inhibit rat brain adenylate cyclase activity through the effects on both a low affinity benzodiazepine receptor coupled with the inhibitory GTP-binding regulatory protein (Gi) and catalytic protein.     

Calmodulin stimulation of adenylate cyclase in rat brain membranes does not require GTP

Calcium stimulates adenylate cyclase activity in rat cerebral cortical membranes with either ATP or AppNHp as substrate. In contrast, isoproterenol stimulates the cerebral cortical enzyme with ATP as substrate but not with AppNHp as substrate unless exogenous GTP is added. In rat striatal membranes, calcium or dopamine stimulate adenylate cyclase activity with ATP as substrate, but not with AppNHp as substrate. GTP restores the dopamine but not the calcium response. The inhibitory guanine nucleotide GDP-βS antagonizes dopamine and GppNHp stimulation of the brain adenylate cyclases, but not stimulation by calcium of either rat cerebral cortical or striatal enzymes. Results indicate that GTP is not requisite to calcium-calmodulin activation of adenylate cyclases in brain membranes. In addition, calcium-calmodulin cannot activate striatal adenylate cyclases with a nonphosphorylating nucleotide, AppNHp, as substrate.     

Mechanism of molybdate activation of adenylate cyclase

Molybdate activation of rat liver plasma membrane adenylate cyclase has been examined and compared with the effect of glucagon, Gpp(NG)p and fluoride. Glucagon does not stimulate the detergent solubilized enzyme, though molybdate, fluoride, and Gpp(NH)p are effective in this regard. The stimulatory effects of either fluoride or molybdate are additive with those of GTP and do not require guanyl nucleotide to evoke their activation. Neither fluoride nor molybdate can substitute for GTP when glucagon is the activator of rat liver adenylate cyclase. The stimulatory effects of either ion on adenylate cyclase are additive with that produced by glucagon. Activation of adenylate cyclase by either molybdate or fluoride occurs by a mechanism distinct from that of glucagon or guanyl nucleotide. The data presented here suggest that fluoride and molybdate may act via a similar mechanism of action. Neither ion displays a lag in activation of adenylate cyclase. The pH profiles of fluoride and molybdate-stimulated adenylate cyclase activity are similar, and distinct from guanyl nucleotide-stimulated activity. Cholera toxin treatment of adenylate cyclase blocks fluoride and molybdate stimulation of the enzyme to the same extent, while enhancing the activation obtained with GTP and hormones.     

Information from e.x.a.f.s. spectroscopy on the structures of different forms of molybdenum in xanthine oxidase and the catalytic mechanism of the enzyme.

In isolated perfused rat hearts, epidermal growth factor (EGF; 15 nM) increased cellular cyclic AMP (cAMP) content by 9.5-fold. In rat cardiac membranes, EGF also stimulated adenylate cyclase activity in a dose-dependent manner, with maximal stimulation (35% above control) being observed at 10 nM-EGF. Half-maximal stimulation of adenylate cyclase was observed at 40 pM-EGF. Although the beta-adrenergic-receptor antagonist propranolol markedly attenuated the isoprenaline-mediated increase in cAMP content of perfused hearts and stimulation of adenylate cyclase activity, it did not alter the ability of EGF to elevate tissue cAMP content and stimulate adenylate cyclase. The involvement of a guanine-nucleotide-binding protein (G-protein) in the activation of adenylate cyclase by EGF was indicated by the following evidence. First, the EGF-mediated stimulation of adenylate cyclase required the presence of the non-hydrolysable GTP analogue, guanyl-5'-yl-imidodiphosphate (p[NH]ppG). Maximal stimulation was observed in the presence of 10 microM-p[NH]ppG. Secondly, in the presence of 10 microM-p[NH]ppG, the stable GDP analogue guanosine 5'-[beta-thio]diphosphate at a concentration of 10 microM blocked the stimulation of the adenylate cyclase by 1 nM- and 10 nM-EGF. Third, NaF + AlCl3-stimulated adenylate cyclase activity was not altered by EGF. The ability of EGF to stimulate adenylate cyclase was not affected by pertussis-toxin treatment of cardiac membranes. However, in cholera-toxin-treated cardiac membranes, when the adenylate cyclase activity was stimulated by 2-fold, EGF was ineffective. Finally, PMA by itself did not alter the activity of cardiac adenylate cyclase, but abolished the EGF-mediated stimulation of this enzyme activity. The experimental evidence in the present paper demonstrates, for the first time, that EGF stimulates adenylate cyclase in rat cardiac membranes through a stimulatory GTP-binding regulatory protein, and this effect is manifested in elevated cellular cAMP levels in perfused hearts exposed to EGF.     

Possible Involvement of Inhibitory GTP Binding Regulatory Protein in α2-Adrenoceptor-Mediated Inhibition of Adenylate Cyclase Activity in Cerebral Cortical Membranes of Rats

Influences of alpha 2-adrenoceptor stimulation on adenylate cyclase activity were investigated in cerebral cortical membranes of rats. Pretreatment of the membranes with islet-activating protein and NAD resulted in a significant increase in basal activity as well as in GTP- or forskolin/GTP-induced elevation of adenylate cyclase activity. Strong activation of adenylate cyclase was also caused in membranes pretreated with cholera toxin together with NAD in comparison to that in control membranes, suggesting that adenylate cyclase activity is perhaps regulated by stimulatory and inhibitory GTP binding regulatory protein existing in synaptic membranes. In addition, adrenaline (with propranolol) or clonidine significantly reduced adenylate cyclase activity stimulated by pretreatment with forskolin and GTP. The inhibitory effects of adrenaline were also observed in membranes pretreated with cholera toxin and NAD. Moreover, the inhibition by adrenaline or clonidine was completely abolished by treatment with (a) yohimbine or (b) islet-activating protein and NAD. It is suggested that alpha 2-receptor stimulation causes inhibitory influences on adenylate cyclase activity mediated by the inhibitory GTP binding regulatory protein in synaptic membranes of rat cerebral cortex.     

Phosphorylated adenosine derivatives as low-affinity adenosine-receptor agonists. Methodological implications for the adenylate cyclase assay.

In cellular systems provided with activatory (Ra-site) receptors for adenosine, such as rat cerebral microvessels and rat liver plasma membranes, the adenosine-receptor antagonist 8-phenyltheophylline (10 microM) significantly decreased adenylate cyclase activity if ATP was the substrate and only if GTP was present. With dATP as substrate, adenylate cyclase activities in both preparations remained unaffected by 8-phenyltheophylline. In rat cerebral-cortical membranes, with inhibitory (Ri-site) receptors for adenosine, 8-phenyltheophylline significantly enhanced adenylate cyclase activity only in the presence of GTP and if ATP was the substrate. In rat cardiac ventricular membranes, which are devoid of any adenylate cyclase-coupled adenosine receptor, the methylxanthine had no GTP-dependent effect, irrespective of the substrate used. All assay systems contained sufficiently high amounts of adenosine deaminase (2.5 units/ml), since no endogenous adenosine, formed from ATP, was found chromatographically. In order to demonstrate a direct influence of phosphorylated adenosine derivatives on adenylate cyclase activity, we investigated AMP in a dATP assay system. AMP was verified chromatographically to remain reasonably stable under the adenylate cyclase assay conditions. In the microvessels, AMP increased enzyme activity in the range 0.03-1.0 mM, an effect competitively antagonized by 8-phenyltheophylline. In the cortical membranes, 0.1 mM-AMP inhibited adenylate cyclase, which was partially reversed by the methylxanthine. The presence of GTP was again necessary for all observations. In the ventricular membranes, AMP had no effect. Since the efficacy of adenosine-receptor agonists and, probably, that of other hormones on adenylate cyclase activity can be more efficiently measured with dATP as the enzyme substrate, this nucleotide seems preferable for adenylate cyclase measurements in systems susceptible to modulation by adenosine.     

Activation of solubilized liver membrane adenylate cyclase by guanyl nucleotides.

Liver plasma membranes of hypophysectomized rats were purified, treated with 0.1 m Lubrol-PX and centrifuged at 165,000g for 1 h. The detergent solubilized 50% of the membrane protein; adenylate cyclase activity was present in the supernatant fraction. Optimal substrate concentration of the soluble enzyme was 0.32 mm ATP. Basal activity of 25 preparations of the solubilized enzyme ranged from 124 to 39 pmol cyclic AMP/mg protein/10 min. The solubilized enzyme retained the same sensitivity to activation by guanyl nucleotides as was present in the membrane preparation from which it was derived. Relative sensitivity of the solubilized enzyme with 0.1 mm nucleotides or -side was GDP > GTP > GMP > guanosine; GMP-PNP = GMP-PCP > ITP > GTP. GTP, GMP-PCP, GMP-PNP and other nucleotides were hydrolyzed by phosphohydrolases present in liver membranes that were solubilized with Lubrol-PX along with adenylate cyclase. The presence of the ATP regenerating system in the adenylate cyclase assay also aided in maintaining guanyl nucleotide concentrations. The degree of adenylate cyclase activation by guanyl nucleotides was not related to the sparing effects of nucleotides on substrate ATP hydrolysis. These findings demonstrate that activation of adenylate cyclase by nucleotides is a consequence of a nucleotide-enzyme interaction that is independent of membrane integrity.     

Effect of proteases and protease inhibitors on adenylate cyclase activity in rat cerebral cortical membranes

Mild proteolysis of membrane preparations from rat cerebral cortex with low concentrations of endopeptidases such as trypsin or chymotrypsin caused a 50–400% increase in the basal adenylate cyclase activity. Maximal activation of adenylate cyclase was obtained by including the protease in the adenylate cyclase assay, although an activated preparation could be obtained by pretreatment of the membranes with proteolytic enzymes. The proteolytically activated enzyme showed an increased V, with very little change in the K_m for the substrate, ATP. The proteolytically activated enzyme retained responsiveness to activation by sodium fluoride and 5′-guanylylimidodiphosphate (GppNHp), but was no longer activated by gangliosides or calcium-dependent activator protein. Activation by alcohols and detergent was lost or reduced in magnitude. The activity of adenylate cyclase after protease treatment showed a very marked temperature dependence, with maximal activity expressed in the 30–40 °C range and no activation due to the prior protease treatment expressed at either 10 or 50 °C. Basal adenylate cyclase activity was usually slightly inhibited in the presence of various protease inhibitors. Activation by fluoride, gangliosides, or GppNHp was little affected by protease inhibitors although one inhibitor, N-α-tosyl-l-lysine chloromethyl ketone, caused an inhibition of the ganglioside and GppNHp responses, slightly inhibited the fluoride response, and blocked the norepinephrine response normally seen in the presence of gangliosides or GppNHp. This inhibitor caused a loss of β-adrenergic binding sites for dihydroalprenolol in rat cortical membranes which paralleled the loss of the responsiveness of adenylate cyclase to a GppNHp-norepinephrine combination.     

Activation of adenylate cyclase in cultured fibroblasts by trypsin

Adenylate cyclase activity measured in membranes of cultured normal rat kidney (NRK) fibroblasts was markedly increased by prior treatment of the intact cells with trypsin. Cell population density influenced the extent of activation observed. Trypsin treatment of sparse cells significantly enhanced adenylate cyclase activity, whereas similar treatment of confluent cells caused only a slight increase in adenylate cyclase activity. The degree of activation noted after trypsin treatment also varied depending on the adenylate cyclase function measured. Activity determined in the presence of GTP alone showed the greatest increase after trypsin treatment. Similar enhancement of adenylate cyclase activity of a washed cell membrane preparation was achieved by the addition of low concentrations of trypsin directly to the adenylate cyclase reaction mixture. The membranes of confluent NRK fibroblasts initially exhibited higher adenylate cyclase activity than did membranes of sparse cells. The present results suggest that this change in adenylate cyclase activity at cell confluence is not due to an increase in the amount of adenylate cyclase in the cell membrane but rather to a change in membrane components that regulate its activity. Proteolytic activation of adenylate cyclase appears to result from degradation of cell membrane proteins that modulate the activity of this enzyme.     

Isolation, purification and characterization of regulatory properties of adenylate cyclase from rabbit heart]

Rabbit heart membranes possessing the adenylate cyclase activity were isolated and purified by extraction with high ionic strength solutions and centrifugation in the sucrose density gradient. It was shown that the membranes are characterized by a high percentage of cholesterol (molar ratio cholesterol/phospholipids is 0.24) and an increased activity of Na, K-ATPase, which suggests the localization of adenylate cyclase in the sarcolemma. During centrifugation in the sucrose density gradient the activities of andenylate cyclase and Na,K-ATPase are not separated. Treatment of heart sarcolemma with a 0.3% solution of lubrol WX results in 10--20% solubilization of adenylate cyclase. Purification of the enzyme in the membrane fraction is accompanied by a decrease in the activity of phosphodiesterase; however, about 2% of the heart diesterase total activity cannot be removed from the sarcolemma even after its treatment with 0.3% lubrol WX. Epinephrine and NaF activate adenylate cyclase without changing the pH dependence of the enzyme. The alpha-adrenergic antagonist phentolamine has no effect on the adenylate cyclase activation by catecholamines, glucagon and histamine; the beta-adrenergic antagonist alprenolol competitively inhibits the effects of isoproterenol, epinephrine and norepinephrine, having no effect on the enzyme activation by glucagon and histamine. There is no competition between epinephrine, glucagon and histamine for the binding site of the hormone; however, there may occur a competition between the hormone receptors for the binding to the enzyme. A combined action of several hormones on the membranes results in the averaging of their individual activating effects. When the hormones were added one after another, the extent of adenylate cyclase activation corresponded to that induced by the first hormone; the activation was insensitive to the effect of the second hormone added. It is assumed that the outer membrane of myocardium cells contains a adenylate cyclase and three types of receptors, each being capable to interact with the same form of enzyme. The activity of adenylate cyclase is determined by the type of the receptor, to which it is bound and by the amount of the enzyme-receptor complex.     

Regulation of rat lung adenylate cyclase by cytoplasmic factor(s) during development.

Basal adenylate cyclase activity in rat lung homogenate was low prenatally but increased several-fold after birth and remained elevated to maturity. The results also demonstrate the appearance of some factor(s) in the lung cytoplasm at a certain age which markedly activated adenylate cyclase. During late gestation and early neonatal life, when the cytoplasmic factor(s) was low or absent, basal adenylate cyclase activity was low and norepinephrine and NaF produced maximum activation of the enzyme. However, when the cytoplasmic factor(s) appeared in the adult lungs, basal adenylate cyclase activity was elevated and both norepinephrine and NaF produced little or no activation of the enzyme. These data suggest a role for the cytoplasmic factor(s) in regulating rat lung adenylate cyclase. The cytoplasmic factor(s) appeared to be a protein since it was inactivated by trypsin digestion and by heating to 75 degrees C. Activation of adenylate cyclase was not due to small ions or other low molecular weight components of the cytoplasm as dialysis of the supernatant did not alter its activation of adenylate cyclase. The cytoplasmic factor(s) did not appear to be either GTP or calcium-dependent regulator of cyclic AMP phosphodiesterase as these did not activate the rat lung adenylate cyclase.