Catecholamine and guanine nucleotide activation of skeletal muscle adenylate cyclase

Activation of adenylate cyclase by guanine nucleotide and catecholamines was examined in plasma membranes prepared from rabbit skeletal muscle. The GTP analog, 5′-guanylyl imidodiphosphate caused a time and temperature-dependent activation of the enzyme which was persistent, the K_a was 0.05 μM. 5′-Guanylyl imidodiphosphate binding to the membranes was time and temperature dependent, K_D 0.07 μM. Beta adrenergic amines accelerated the rate of 5′-guanylyl imidodiphosphate activation of the enzyme with an order of potency isoproterenol ≈ soterenol ≈ salbutamol > epinephrine ? norepinephrine. Catecholamine activation was antagonized by propranolol and the β₂ antagonist butoxamine; the β₁ antagonist practolol was inactive. [³H]Dihydroalprenolol bound to the membranes and binding was antagonized by β adrenergic agonists with an order of potency similar to the activation of adenylate cyclase and was antagonized by butoxamine but not by practolol. The data are consistent with the idea that adenylate cyclase in skeletal muscle plasma membranes is coupled to adrenergic receptors of the β₂ type.     

Calcium regulation of guanine nucleotide activation of hepatic adenylate cyclase

Pretreatment of isolated rat liver plasma membranes by washing with NaHCO₃ buffer or by exposure to the chelator ethyleneglycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) with or without the ionophore A23187, produced a decrease in the sensitivity of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) to subsequent stimulation by NaF or guanosine 5′-(β-γ-imino)triphosphate (GPP(NH)P). Sensitivity to activation by the nucleotide could be restored by addition of the lyophilized and ashed wash or by addition of Ca²⁺, Mg²⁺ or Mn²⁺. The factor extracted from the membranes by these various treatments which was responsible for loss of stimulation was identified as Ca²⁺. Determination of the metal ion content of isolated membranes by atomic absorption spectrometry indicated that Ca²⁺ was the only divalent cation present in sufficient concentration to support persistent activation by either NaF or GPP(NH)P.Pretreatment of liver plasma membranes with trifluoperazine, which inhibits the action of Ca²⁺-dependent regulator protein in other enzyme systems, reduced GPP(NH)P activation of adenylate cyclase and caused marked depletion of membrane Ca²⁺. The effects of low concentrations (less than 100 μM) of the phenothiazine could be reversed totally by Ca²⁺ and partly by regulator protein. At higher concentrations of trifluoperazine, slight restoration of enzyme activation was seen with either agent. The hypothesis is presented that Ca⁺ interacts with the nucleotide (GTP or GDP) regulatory site(s) of the adenylate cyclase. This interaction may be regulator-protein-dependent and may be important in determining the sensitivity of the enzyme to nucleotide activation in vivo.     

Specificity for guanine nucleotide activation and stabilization of rabbit cardiac adenylate cyclase

These studies examined the structural specificity for guanine nucleotide-facilitated hormonal activation and guanine nucleotide stabilization of cardiac adenylate cyclase. 1. The phosphonate analogues of GTP, p[CH(2)]ppG (guanosine 5'-[betagamma-methylene]-triphosphate) and pp[CH(2)]pG (guanosine 5'-[alphabeta-methylene]triphosphate), were the most effective activators of adenylate cyclase. Other nucleotides producing significant activation (P<0.01) were, in decreasing order of activation: ITP, GDP, GMP, GTP, XTP, CTP, p[NH]ppG (guanosine 5'-[betagamma-imido]triphosphate), dGTP and 2'-O-methyl-GTP. Guanosine, cyclic GMP, UTP and ppppG (guanosine tetraphosphate) had no effect, and 7-methyl-GTP caused a decrease in the activity. 2. Preincubation of membranes at 37 degrees C for 15min before assay at 24 degrees C produced an 80% decrease in adenylate cyclase activity, and preincubation with p[CH(2)]ppG and pp[CH(2)]pG protected and resulted in a net increase in activity. Other nucleotides that completely or partially preserved activity in decreasing order of effectiveness were p[NH]ppG, GDP, GTP, dGTP, ITP, ppppG, 2'-O-methyl-GTP, GMP, CTP and XTP. Several compounds had no effect, including guanosine, cyclic GMP and UTP, whereas preincubation with 7-methyl-GTP produced a further decrease (P<0.05) in activity. 3. The concentration-dependence for activation and stabilization by the naturally occurring guanine nucleotides was examined in the absence of a regenerating system and revealed GMP to have no stabilizing effect and to be less potent than either GDP or GTP in activating adenylate cyclase. 4. A significant correlation (r=0.90) was found between the properties of activation and stabilization for the compounds examined. These findings are consistent with there being a single nucleotide site through which both the activation and stabilization of adenylate cyclase are mediated.     

Catecholamine secretion and adenylate cyclase activation in sea urchin eggs

The role of neurotransmitters in sea-urchin eggs was investigated by studying their effect on adenylate cyclase of the egg membrane. Maximal stimulation of enzyme activity occurs in the presence of dopamine and GTP. 5-hydroxytriptamine, 5-methoxytriptamine and acetylcholine have no effect on activity, despite a decrease in intracellular cAMP level in eggs treated with 5-hydroxytriptamine antagonists as previously reported (Renaud et al., 1983). High-performance liquid chromatography (HPLC) revealed that dopamine is released from the sea-urchin egg into the external medium following fertilization.     

Developmental alterations in guanine nucleotide regulation of the beta-adrenergic receptor-adenylate cyclase system of skeletal muscle

The postnatal development of skeletal muscle is accompanied by an increased capacity for glycogenolysis and anaerobic glycolysis. In the present study, regulatory features of cAMP synthesis were examined in neonatal and adult rabbit sarcolemmal membranes. Adult sarcolemma exhibited a 3-, 6-, and 10-fold greater adenylate cyclase activity than neonate for basal, NaF, and isoproterenol plus GTP, respectively. The Km for activation by isoproterenol was 1.4 X 10(-8) M and 6 X 10(-8) M for GTP. The number of beta-receptors was similar (0.9-1.2 pmol/mg). 10 microM GTP shifted isoproterenol EC50 from 1 X 10(-8) M to 1 X 10(-7) M in adult; neonatal agonist affinity was unaffected by GTP. Cholera toxin stimulated adenylate cyclase activity 2-fold and catalyzed 32P ribosylation of a Mr = 42,000 peptide in adult sarcolemma; both activities were low or absent in neonate. Isoproterenol-stimulated GTPase activity was elevated 4-fold in adult compared to neonatal sarcolemma. Mn2+ ion-stimulated basal activity, an indicator of catalytic function of adenylate cyclase, was also elevated in adult. Together, these findings suggest that the development of catecholamine-sensitive cAMP synthesis in muscle is governed by the coordinate expression of the regulatory and catalytic proteins of adenylate cyclase, but not the beta-receptor.     

Genetically acquired diabetes: adipocyte guanine nucleotide regulatory protein expression and adenylate cyclase regulation.

Adipocyte membranes from diabetic (db/db) animals showed marked elevations in the levels of alpha-subunits for Gi-1 which were almost twice those found in membranes from their normal, lean littermates. In contrast, no apparent differences were noted for levels of the alpha-subunits of Gi-2 and Gi-3, the 42 and 45 kDa forms of Gs and for G-protein beta-subunits. Adenylate cyclase specific activity was similar in membranes from both normal and diabetic animals under basal conditions and also when stimulated by optimal concentrations of either NaF or forskolin. In contrast, the ability of isoprenaline, glucagon and secretin to stimulate adenylate cyclase activity was greater in membranes from normal animals compared with membranes from diabetic animals. Receptor-mediated inhibition of adenylate cyclase, as assessed using PGE1 and nicotinate, was similar using membranes from both sources, but PIA (phenylisopropyladenosine) was a slightly more effective inhibitor in membranes from diabetic animals. A doubling in the expression of Gi-1 thus appears to have little discernible effect upon the inhibitory regulation of adenylate cyclase.     

beta-adrenergic receptor and adenylate cyclase in transverse tubules of skeletal muscle.

Experiments were carried out to clarify the sites of action of beta-adrenergic agonists in skeletal muscle microsomes. Microsomes were fractionated into longitudinal reticulum, terminal cisternae, and isolated transverse tubules. Transverse tubules were selectively labeled and tracked with [3H]ouabain. beta-adrenergic receptor was identified by [3H]dihydroalprenolol binding. Assays of beta-adrenergic receptor, adenylate cyclase, and protein kinase-stimulated phosphorylation showed: 1) beta-adrenergic receptor was detected in transverse tubules with a receptor density of 0.61 pmol/mg of protein. No significant binding was detected in longitudinal reticulum or in terminal cisternae. 2) Isoproterenol-stimulated adenylate cyclase was present in microsomes but was similarly confined to the transverse tubular fraction. The activity of F- stimulated cyclase in transverse tubules was 2.3 nmol/mg of protein/min. 3) No phosphorylation of microsomes by cyclic AMP and protein kinase could be detected. We conclude that the action of epinephrine on skeletal muscle is mediated through receptors and adenylate cyclase in the external membrane.     

Purification and properties of the inhibitory guanine nucleotide regulatory unit of brain adenylate cyclase

Hormonal inhibition of adenylate cyclase is mediated by a guanine nucleotide regulatory protein (Ni) which is different from the one which mediates hormonal stimulation. There is substantial evidence that the active component of Ni (termed alpha i can be ADP-ribosylated by a toxin from Bordetella pertussis. We have found that in bovine cerebral cortex there are three proteins of similar molecular weight (39,000-41,000) which are modified by pertussis toxin. We have purified these proteins and have resolved the 41,000-dalton protein from the 40,000/39,000-dalton doublet. All three forms of pertussis toxin substrate can be isolated in free form or together with a 36,000 beta component. We have also purified this beta component. ADP-ribosylation of the three pertussis toxin substrates is greatly enhanced by the addition of the purified beta component. This makes possible an assay of beta subunit activity based on its interaction with alpha i. The three forms of pertussis toxin substrate which we have purified differ in two functions: susceptibility to ADP-ribosylation and GTPase activity. The 41,000-dalton protein is more readily ADP-ribosylated by pertussis toxin than the smaller forms. The 39,000-dalton protein has GTPase activity with a low Km (0.3 microM) for GTP. The GTPase activity can be doubled by phospholipids. The GTPase activity of the 41,000-dalton protein is almost undetectable. It is not yet known what the relationship of the forms is to each other. The smaller forms may be derived from the larger by proteolysis or it may be intrinsically different. It remains to be shown whether one of the forms represents a different type of regulatory protein which transmits a hormonal signal to effectors other than adenylate cyclase.     

Genetically acquired diabetes: adipocyte guanine nucleotide regulatory protein expression and adenylate cyclase regulation

Adipocyte membranes from diabetic (db/db) animals showed marked elevations in the levels of α-subunits for G_i-1 which were almost twice those in membranes from their normal, lean littermates. In contrast, no apparent differences were noted for levels of the α-subunits of G_i-2 and G_i-3, and 42 and 45 kDa forms of G_s and for G-protein β-subunits. Adenylate cyclase specific activity was similar in membranes from both normal and diabetic animals under basal conditions and also when stimulated by optimal concentrations of either NaF or forsckolin. In contrast, the ability of isoprenaline, glucagon and secretin to stimulate adenylate cyclase activity was greater in membranes from normal animals compared with membranes from diabetic animals. Receptor-mediated inhibition of adenylate cyclase, as assessed using PGE₁ and nicotinate, was similar using membranes from both sources, but PIA (phenylisopropyladenosine) was a slightly more effective inhibitor in membranes from diabetic animals. A doubling in the expression of G₁-1 thus appears to have little discernible effect upon the inhibitory regulation of adenylate cyclase.     

Characteristics of the guanine nucleotide regulatory component of adenylate cyclase in human erythrocyte membranes

This study probes the structure and mutual interactions of the components of adenylate cyclase. We use a complementation assay which involves the addition of an adenylate cyclase-related guanine nucleotide-binding protein component to a membrane lacking this component to measure guanine nucleotide-stimulated-adenylate cyclase. Instead of using detergent extracts we were able to achieve full complementation by mixing intact membrane preparations in the presence of the nucleotide component. Of particular interest was the human erythrocyte membrane which contains very low amounts of catalytic activity and no measurable beta-adrenergic receptor but has normal amounts of the nucleotide component. This component appears to be the same, by several criteria, as components found in pigeon and turkey erythrocytes and in rat liver plasma membrane. The component confers Gpp(NH)p, fluoride, and GTP stimulation of adenylate cyclase along a single reconstitution curve. It is labeled with NAD by cholera toxin, and has an apparent molecular weight of 39 000 upon sodium dodecyl sulfate gel electrophoresis. The presence of the nucleotide unit in the virtual absence of the active catalytic unit allowed us to determine those properties intrinsic to each unit and those conferred by the association of the units. The nucleotide component binds guanine nucleotides weakly in the human erythrocyte membrane, yet produces persistent activation of adenylate cyclase and tight binding (of Gpp(NH)p) upon combination with the catalytic unit. Treatment of the human erythrocyte membrane with N-ethylmaleimide causes a simultaneous diminution in both Gpp(NH)p and fluoride stimulation in reconstituted activities, suggesting that both activities are conferred by the same component.     

Amiloride interacts with guanine nucleotide regulatory proteins and attenuates the hormonal inhibition of adenylate cyclase

The effect of amiloride on the hormonal regulation of adenylate cyclase was studied in the rat anterior pituitary. The diuretic did not alter basal adenylate cyclase but augmented the enzyme activity in an irreversible manner in the presence of guanosine 5'-O-(thiotriphosphate) (GTP gamma S) stimulated adenylate cyclase at lower concentrations and inhibited at higher concentrations. Amiloride treatment enhanced the stimulatory and abolished the inhibitory phase of GTP gamma S action. In addition, amiloride also attenuated the inhibitory effects of atrial natriuretic factor (ANF 99-126) and angiotensin II on cAMP levels and adenylate cyclase activity. On the other hand, amiloride showed an additive effect on the stimulation exerted by corticotropin-releasing factor and vasoactive intestinal peptide on adenylate cyclase in anterior pituitary and on isoproterenol-stimulated cAMP levels in cultured vascular smooth muscle cells. Pertussis toxin, in the presence of [alpha-32 P]NAD, catalyzed the ADP-ribosylation of two protein bands of Mr 41,000 and 39,000, referred to as Gi and Go, respectively, in the anterior pituitary, and 40,000-Da protein in the aorta, referred to as Gi. Amiloride treatment inhibited the labeling of all these bands in a concentration- and time-dependent manner. Similarly, the pertussis toxin-catalyzed ADP-ribosylation of purified Gi from bovine brain was also inhibited by amiloride treatment. However, amiloride had no significant effect on the cholera toxin-catalyzed ADP-ribosylation of Gs. These data suggest that amiloride interacts with the guanine nucleotide regulatory proteins Gi and Go. Modification of Gi results in the attenuation of hormone-induced adenylate cyclase and cAMP inhibition. However, the interaction between amiloride and Go and the consequent Ca2+ mobilization and phosphatidylinositol turnover have to be investigated.     

Guanine nucleotide activation of adenylate cyclase in saponin permeabilized glioma cells

We have compared the regulation of adenylate cyclase activity in membrane fractions from C6 glioma cells and in monolayer cultures of C6 cells that had been permeabilized with saponin. Guanine nucleotides (GTP and GTP gamma S) and isoproterenol increase adenylate cyclase activity in C6 membranes and in permeabilized C6 cells. In C6 membranes, guanine nucleotides activate adenylate cyclase in the presence or absence of isoproterenol; in permeabilized cells, however, guanine nucleotides increase adenylate cyclase activity only in the presence of isoproterenol. We suggest that the properties of the permeabilized cells more closely resemble those of intact cells, and that some component which is present in permeabilized cells but is lost following cell disruption may be important for the normal regulation of adenylate cyclase activity.     

In vitro characterization of skeletal muscle beta-adrenergic receptors coupled to adenylate cyclase.

[3H]Dihydroalprenolol, a potent beta-adrenergic antagonist, was used to identify the adenylate cyclase-coupled beta-adrenoceptors in isolated membranes of rat skeletal muscle. The receptor sites, as revealed by [3H]dihydroalprenolol binding, were predominantly localized in plasmalemmal fraction. That skeletal muscle fraction may also contain the plasmalemma of other intramuscular cells, especially that of blood vessels. Hence, the [3H]dihydroalprenolol binding observed in that fraction may be due partly to its binding to the plasmalemma of blood vessels. Small but consistent binding was also observed in sarcoplasmic reticulum and mitochondria. The level of [3H]dihydroalprenolol binding in different subcellular fractions closely correlated with the level of adenylate cyclase present in those fractions. The binding of [3H]dihydroalprenolol to plasmalemma exhibited saturation kinetics. The binding was rapid, reaching equilibrium within 5 min, and it was readily dissociable. From the kinetics of binding, association (K1) and dissociation (K2) rate constants of 2.21 . 10(7) M-1 . min-1 and 3.21 . 10(-1) min-1, respectively, were obtained. The dissociation constant (Kd) of 15 mM for [3H]dihydroalprenolol obtained from saturation binding data closely agreed with the Kd derived from the ratio of dissociation and association rate constants (K2/K1). Several beta-adrenergic agents known to be active on intact skeletal muscle also competed for [3H]dihydroalprenolol binding sites in isolated plasmalemma with essentially similar selectivity and stereospecificity. Catecholamines competed for [3H]dihydroalprenolol binding sites with a potency of isoproterenol greater than epinephrine greater than norepinephrine. A similar order of potency was noted for catecholamines in the activation of adenylate cyclase. Effects of catecholamines were stereospecific, (-)-isomers being more potent than (+)-isomers. Phenylephrine, an alpha-adrenergic agonist, showed no effect either on [3H]dihydroalprenolol binding or on adenylate cyclase. Known beta-adrenergic antagonists, propranolol and alprenolol, stereospecifically inhibited the [3H]dihydroalprenolol binding and the isoproterenol-stimulated adenylate cyclase. The Ki values for the antagonists determined from inhibition of [3H]dihydroalprenolol binding agreed closely with the Ki values obtained from the inhibition of adenylate cyclase. The data suggest that the binding of [3H]dihydroalprenolol in skeletal muscle membranes possess the characteristics of a substance binding to the beta-adrenergic receptor.     

Differential effects of fluoride on adenylate cyclase activity and guanine nucleotide regulation of agonist high-affinity receptor binding.

Fluoride ion, presumably an Al3+-F- complex, has been proposed to activate the guanine nucleotide regulatory protein (G-protein) of the visual system, transducin, by associating with GDP at the nucleotide-binding site and thus mimicking the effects of non-hydrolysable GTP analogues [Bigay, Deterre, Pfister & Chabre (1985) FEBS Lett. 191, 181-85]. We have examined this proposed model by using the adenylate cyclase complexes of frog erythrocytes, S49 lymphoma cells and human platelets. Preincubation of plasma membranes from frog erythrocytes and S49 cells with 20 mM-fluoride for 20 min at 30 degrees C strongly stimulated adenylate cyclase activity. In contrast, the preactivated membranes were still able to bind beta-adrenergic agonist with high affinity, as determined by radioligand-binding techniques. Moreover, high-affinity agonist binding in fluoride-treated membranes was fully sensitive to guanine nucleotide, which decreased beta-adrenergic-receptor affinity for agonist. Very similar results were obtained for [3H]prostaglandin E1 binding to S49 membranes pretreated with fluoride. Incubation of human platelet membranes with increasing concentrations of fluoride (1-50 mM) resulted in biphasic regulation of adenylate cyclase activity, with inhibition observed at concentrations greater than 10 mM. Preincubation of platelet membranes with 20 mM-fluoride did not affect agonist high-affinity binding to alpha 2-adrenergic receptors, nor receptor regulation by guanine nucleotide. These results suggest that the model developed from the study of transducin may not be generally applicable to the G-proteins of the adenylate cyclase system.     

Hormonal activation of adenylate cyclase in macrophage membranes is regulated by guanine nucleotides

Many macrophage functions such as chemotaxis, phagocytosis, enzyme secretion, and cytotoxicity are influenced by intracellular cyclic nucleotide levels, but the regulatory mechanisms involved are poorly defined. We have developed methods that allowed us to study the activation of AC in isolated guinea pig (g.p.) macrophage membranes. AC in these membrane preparations could be stimulated approximately twofold by guanine nucleotides. We could not obtain any hormonal activation of membrane-bound AC in the absence of guanine nucleotides. In the presence of GTP, however, the hormones isoproterenol and PGE1 elicited an additional threefold rise in AC activity, which subsided after approximately 15 min. As little as 10(-8) M concentrations of these two hormones induced significant elevations of AC activity. Replacement of GTP by its nonhydrolyzable analogue Gpp(NH)p resulted in a persistent hormone-independent activation of AC, and addition of hormones enhanced this level of activation. Thus, GTP-ase activity is present in macrophage membrane preparations and serves to regulate AC activation. Hormonal stimulation of AC was receptor mediated, because the effect of the beta-adrenergic agonist isoproterenol, but not PGE1, was inhibited by the beta-adrenergic blocker propranolol. In addition, the potency series of PG corresponded to that observed for stimulation of cAMP production in intact g.p. macrophages, i.e., PGE1 = PGE2 greater than PGA1 greater than PGF2 alpha. AC activation by PG in the membrane preparation was inhibited by an alpha-adrenergic agonist, thus demonstrating one means for down regulating cAMP production in g.p. macrophages. Our studies also showed that certain hormones (e.g., beta-adrenergic agonists, PG) can exert their effect on cAMP production by stimulation of membrane-bound AC, whereas other agents such as lectins or arachidonic acid require additional intracellular components to elevate cAMP levels in macrophages. The mechanism of activation of AC by hormones in g.p. macrophage membranes appears to fit the model of a ternary complex, the components of which include the hormone receptor, AC, and guanine nucleotide regulatory protein, which transmits the signal from the receptor to AC.