Regulation of hormone-sensitive adenylate cyclase

Adenylate cyclases in the membranes of eukaryotes are regulated by stimulatory receptors (R_s) and inhibitory receptors (R_i) which activate the catalyst adenylate cyclase (C) through the heterotrimeric GTP-binding proteins, G_s and G_i. The actions of G_s and G_i are not symmetric, since G_s associates tightly with C, whereas G_i does not. Alexander Levitzki analyses the current knowledge and thinking on the mode of interaction between each of these regulatory units and describes one working model which accounts for all the experimental observations and the kinetics involved.     

Activation of hormone-sensitive adenylate cyclase by forskolin

An overview of the organization of adenylte cyclase systems and various sites for regulation is presented. These components are discussed with respect to the activation of adenylate cyclase by forskolin. Evidence for direct activation of adenylate cyclase as well as potentiation of hormone-activated adenylate cyclase by forskolin is reviewed, as these data relate to the possible site(s) of action of forskolin. Studies of the action of forskolin in different systems and interaction studies with different regulators of adenylate cyclase have yielded valuable information about the regulation of adenylate cyclase and the relationship of its various components. A better understanding of these relationships may reveal new therapeutic potential for forskolin.     

Beta-adrenergic receptors and adenylate cyclase activity in rat reticulocytes and mature erythrocytes.

The adenylate cyclase activity in membranes from rat reticulocytes exhibits a markedly greater stimulation (relative to basal activity) by catecholamines than does the activity in membranes from mature erythrocytes; this increased responsiveness of reticulocytes to catecholamines is not observed for other hormonal adenylate cyclase activators such as prostaglandins E₁and E₂ and human growth hormone. From the order of potency ofcatecholamine-stimulated adenylate cyclase activity (isoproterenol > epinephrine > norepinephrine), and from the selective action of butoxamine compared with practolol, both in inhibiting isoproterenol-stimulated adenylate cyclase activity and in blocking the binding to membranes of ¹²⁵I-labeled hydroxybenzylpindolol, it is concluded that both reticulocytes and mature rat erythrocytes possess adrenergic receptors of the beta-2 type. Practolol acts as a partial agonist in both cell types, stimulating adenylate cyclase activity. The guanine nucleotides, GTP and 5′-guanylylimidodiphosphate (GMP-PNP), affect the adenylate cyclase activity in membranes from both cell types. GTP stimulates basal activity 2-fold, but does not affect stimulation by fluoride. In reticulocyte membranes, GTP enhances isoproterenol stimulation of adenylate cyclase to a level approximately 2-fold greater than that achieved by fluoride; in contrast, in mature erythrocyte membranes, isoproterenol stimulation of adenylate cyclase in the presence of GTP is equivalent to that of fluoride. In reticulocyte membranes, the stimulation of adenylate cyclase by GMP-PNP alone is equivalent to the stimulation in the presence of either isoproterenol or fluoride. In contrast, in membranes from mature erythrocytes the stimulation by GMP-PNP alone is equivalent to stimulation by fluoride, but is markedly lower than that obtained in the simultaneous presence of isoproterenol. It is concluded that whereas the beta-2 nature of the catecholamine receptor on rat erythrocytes remains unaltered as the reticulocyte matures, there is a pronounced change during maturation in the coupling of adrenergic-receptor occupation to adenylate cyclase activation, as indicated by the effects of guanine nucleotides.     

Involvement of the inhibitory GTP-binding regulatory protein and a low-affinity benzodiazepine receptor in the inhibitory effect of diazepam on rat brain adenylate cyclase system

The effect of diazepam on the adenylate cyclase system was studied in rat synaptosomal membranes. Micromolar concentrations of diazepam inhibited the cyclase activities in the presence or absence of guanylyl-5'-imidodiphosphate (GppNHp). The inhibitory effect of diazepam was greater on the cyclase activity in the presence of GppNHp than on that in the basal state. This effect of diazepam was not antagonized by Ro15-1788, an antagonist of a high affinity benzodiazepine receptor in the central nervous system. Furthermore, micromolar concentrations of Ro15-1788 had no inhibitory effect on cyclase activities in the presence or absence of GppNHp. In addition, the bromide ion enhanced the inhibition by diazepam of the cyclase activity in the presence of GppNHp, but not the basal activity, although the bromide ion had no effect on both activities in the absence of diazepam. On the other hand, the pretreatment of synaptosomal membranes with GppNHp increased the KD value for [3H]diazepam binding from 98 microM to 198 microM. These data led us to conclude that diazepam inhibits rat brain adenylate cyclase through the effects on both a low affinity benzodiazepine receptor coupled with the inhibitory GTP-binding regulatory protein (Gi) and the catalytic protein.     

Regulation of the adenylate cyclase system by catecholamines in the rat prostate

Summary Tolerance to catecholamines in the beta-adrenergic and dopaminergic receptor-adenylate cyclase system from the rat prostatic membrane has been studied. Preincubation of the tissue with isoproterenol produced not only a decrease in beta-adrenergic receptors and its sensitive adenylate cyclase activity, but also a reduction in dopaminergic response. Similarly, exposure to dopamine resulted in a decline both of isoproterenol- and dopamine-stimulated adenylate cyclase activity. A beta adrenergic antagonist, propranolol, exclusively blocked desensitization in the beta-adrenergic and dopaminergic adenylate cyclase system caused by isoproterenol, while dopamine-induced refractoriness was prevented by a dopaminergic antagonist, haloperidol. These results suggest that desensitization of the beta-adrenergic and dopaminergic adenylate cyclase system begins on receptor-occupation by its specific agonist.This research was supported by Grant No. 377096 from the Ministry of Education, Science and Culture, JapanThe postgraduate fellow from Department of Neuropsychiatry, St. Marianna University School of Medicine     

Age-dependent changes in the adenylate cyclase system of rat ventral prostate]

Changes in beta-adrenoceptors, GTP binding (G) proteins and adenylate cyclase (AC) activity of ventral prostates of rats during aging were studied in these experiments. The density of beta-adrenoceptors increased markedly after birth, reaching a maximum in the tissues at 16 weeks of age and remained at this level for about 90 weeks. When stimulated by isoproterenol, the AC activity increased 4- and 22-fold in 2- and 8-week-old rats, respectively, but only 7-fold in both 16- and 104-week-old rats. Activation of AC by forskolin was the greatest during the 2 weeks after birth and then showed a sharp decrease, reaching a plateau in the tissues at 8 weeks. Bmax value of [35S]GTP gamma S binding to the tissues was the largest at 8 weeks. Pretreatment of the tissues with pertussis or cholera toxin caused age-dependent changes in both the binding abilities of Gi and Gs proteins to GTP that coincided with changes in the binding of G proteins to GTP. There was no difference between the abilities of Gi and Gs proteins to bind to GTP in the tissues of rats of the respective age. These results show that changes in the binding ability of G proteins to GTP influence the function of ventral prostates of rats during the aging process, and this is mediated through the regulation of AC activity.     

Diuretics and the renal adenylate cyclase system

1 The relationship between the diuretic effectiveness and the effect on the renal adenylate cyclase of three diuretics, acetazolamide, frusemide and ethacrynic acid, was examined. The hypothesis that acetazolamide and parathyroid hormone (PTH), inhibit renal carbonic anhydrase by a cyclic adenosine 3′,5′-monophosphate (cyclic AMP)-dependent mechanism was also tested.

2 In vitro, acetazolamide, frusemide and ethacrynic acid at high concentrations (10^-3M) all produced some inhibition of basal and stimulated rat kidney plasma membrane adenylate cyclase. The effect of acetazolamide was much less than that of frusemide and ethacrynic acid. These plasma membrane effects were reproduced in studies of cyclic AMP formation in isolated kidney tubules of rats.

3 Intravenous injections of acetazolamide did not change the total cyclic AMP content of the kidneys of rats killed by microwave irradiation.

4 Acetazolamide produced a diuresis in the rat and a slight inhibition of the antidiuretic effect of Pitressin. Frusemide produced a diuresis and greatly reduced the antidiuretic response to Pitressin. Ethacrynic acid was ineffective as a diuretic in the rat and actually enhanced the antidiuretic response to Pitressin.

5 In investigating the possible influence of diuretics and PTH on the activity and state of phosphorylation of carbonic anhydrase it was found that: there was no correlation between the ability of diuretics to inhibit carbonic anhydrase activity and to inhibit carbonic anhydrase phosphorylation; neither PTH nor cyclic AMP (in the presence of adenosine triphosphate, Mg²⁺, K⁺ and incubation at 37°C) inhibited rat cortex homogenate carbonic anhydrase activity.

6 It seems unlikely that any of the tested diuretics exerts its pharmacological effect by means of changes in kidney cyclic AMP metabolism.

     

Hormonal changes and adenylate cyclase system in rat bearing 7800 Morris hepatoma

Adenylate cyclase activity was measured in plasma membranes isolated from Morris Hepatoma 7800 and from control and host livers. The only difference found in tumor enzyme activity was the lack of response to glucagon. The membrane-binding capacities for the pancreatic hormones insulin and glucagon were measured. Hepatoma membranes did not bind glucagon. Insulin-binding parameters could not be determined because of high non-specific binding. The plasma levels of insulin in the tumor-bearing animals were approximately half of those found in controls, whereas the glucagon levels in plasma were 50% higher in tumor-bearing animals. Thyroxine and triiodothyronine plasma levels were reduced in tumor-bearing rats, while the thyroid-stimulating hormone level was within normal limits. The amount of cAMP (275 pmol g-1) and cGMP (3.6 pmol g-1) in the tumor were lower than in the host and control livers, but the ratio of cGMP to cAMP in the tumor was increased by a factor of 2. These results are discussed with respect to control mechanisms of cell proliferation in comparison with other hepato-proliferative states.     

Effects of chronic propranolol treatment on hepatic adenylate cyclase system in the rat

The biochemical aspects of hepatic beta-adrenergic receptors and adenylate cyclase activity in male adult rats were examined during chronic treatment of a beta-adrenergic antagonist, propranolol. The blockade of beta-adrenergic nervous systems for 7 to 10 days produced a considerable elevation of basal, glucagon, sodium fluoride, and 5'-guanylylimidodiphosphate, Gpp (NH)p-stimulated enzyme activity with a negligible response to a beta-adrenergic agonist, isoproterenol. There was no alteration in the density or the affinity of beta-adrenergic receptors for the agonist during the treatment. Guanine nucleotides have failed to induce a transformation of the higher affinity to the lower affinity state of beta-adrenergic receptors of the hepatic membrane derived either from control or the propranolol-treated animals. The activity of stimulatory guanine nucleotide regulatory proteins (Ns) in the enzyme, assessed by ADP-ribosylation was also not altered by the antagonist. These results suggest that the mechanism of the observed sensitization of adenylate cyclase induced by chronic beta-adrenergic blockade involves facilitation of Ns interaction with the catalytic subunit of the enzyme with no change in the beta-adrenergic receptor functions.     

Stimulatory and inhibitory regulation of myocardial adenylate cyclase by 5'-guanylyl-imidodiphosphate

Particulate and soluble rat myocardial adenylate cyclase enzymes were characterized with respect to their stimulatory and inhibitory regulation by Gpp(NH)p. Gpp(NH)p (60 microM) stimulated Mg2+- and Mn2+-dependent adenylate cyclase. High concentrations of Gpp(NH)p (600 microM) attenuated the maximal stimulatory response to Gpp(NH)p but only at low cation concentrations. The attenuating effects of 600 microM Gpp(NH)p resulted predominantly from the introduction of a prolonged lag in the kinetics of activation of adenylate cyclase. Steady-state rates of adenylate cyclase activities were similar with either 60 or 600 microM Gpp(NH)p. At any concentration of Gpp(NH)p, the lag was eliminated by Mg ions or isoproterenol. No antihysteretic property for free Mn ions was evident. Forskolin-sensitive particulate adenylate cyclase was not stimulated further by Gpp(NH)p. A 600 microM concentration of Gpp(NH)p inhibited particulate forskolin-sensitive adenylate cyclase at low Mg ion concentrations. In contrast, Gpp(NH)p at 60 microM consistently activated forskolin-sensitive adenylate cyclase after solubilization. The early transient inhibitory properties of 600 microM Gpp(NH)p which resulted in attenuation of adenylate cyclase by 600 microM Gpp(NH)p were diminished by detergent extraction, resulting in only a minor effect of 600 microM Gpp(NH)p to inhibit solubilized adenylate cyclase. These findings indicate that guanine nucleotides exert both stimulatory and inhibitory control upon the myocardial adenylate cyclase enzyme; that solubilization shifts the balance between the stimulatory and inhibitory properties of Gpp(NH)p to allow more dominant expression of the stimulatory response; and that Mg ions critically modify the nature of the myocardial adenylate cyclase response to Gpp(NH)p.     

Effects of the desensitization by morphine of the opiate-dependent adenylate cyclase system in the rat striatum on the activity of the inhibitory regulatory G protein

Opiates act through a specific receptor to inhibit the striatal adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4,6.1.1] and stimulate a high-affinity GTPase (EC 3.6.1). The present study analyzes the functions of the striatal adenylate cyclase complex following chronic morphine treatment in the rat. The inhibitory effects of GTP on basal adenylate cyclase activity, between 10(-6) and 10(-4) M, were reduced. Moreover, the half-maximal inhibitory concentration of the opiate receptor agonist (D-Ala2-Met5)-enkephalinamide (DAME) on striatal adenylate cyclase activity was increased by about four times, whereas the maximal effect was reduced in membranes from treated rats. In parallel, the half-maximal stimulatory concentration of DAME on GTPase was increased by two times, and the maximal stimulation was reduced from 60 to 25%. Binding studies performed with [3,5-3H]DAME (saturation curves) and with [3H]naloxone (competition curves) did not show any change in opiate receptor numbers and affinity. Moreover, the kinetics of the activation of the inhibitory GTP binding protein (Gi) which transduces the opiate receptor effect on adenylate cyclase showed a small but significant delay. Therefore, hypofunction of Gi can be, at least in part, responsible for the observed desensitization by morphine of the opiate-dependent GTPase and adenylate cyclase.     

Coupling of the stimulatory GTP-binding protein Gs to rat synaptic membrane adenylate cyclase is enhanced subsequent to chronic antidepressant treatment

In an attempt to resolve a unified postreceptor mechanism of action for antidepressant therapy, rats were treated with amitriptyline, desipramine or iprindole. Chronic treatment with these antidepressant drugs increased guanylylimidodiphosphate-[Gpp(NH)p-], NaF-, or forskolin-activated adenylate cyclase in synaptic membranes prepared from cerebral cortexes of treated rats. Gpp(NH)p-dependent inhibition of adenylate cyclase was unaffected. Maximal binding of the photoaffinity GTP analog azidoanilido-GTP (AAGTP) to the adenylate cyclase stimulatory (Gs alpha) and inhibitory (Gi alpha) G proteins was unaffected by antidepressant treatment. The chemical elimination of Gs (low pH treatment) eliminated all differences between control and antidepressant-treated groups. Further, nonneural tissues from rats receiving chronic antidepressants showed no changes in adenylate cyclase activity or AAGTP binding. The results of these studies suggest that chronic antidepressant administration promoted increased coupling between Gs and catalytic unit of adenylate cyclase. Thus, the molecular locus of antidepressant action may reside at the stimulatory GTP-binding protein, Gs.     

Novel mechanism of heterologous desensitization of adenylate cyclase: prostaglandins bind with different affinities to both stimulatory and inhibitory receptors on platelets.

Prostaglandins E1, I2, and D2 (PGE1, PGI2, and PGD2) all stimulate and desensitize platelet adenylate cyclase, giving rise to peak and plateau effects in the time course of cyclic AMP metabolism in the intact cell. The peak and plateau effects vary with prostaglandin concentration to a different extent for each prostaglandin. However, at high concentrations, all prostaglandins give rise to the same time course of cyclic AMP formation. Differences in the extent of activation and desensitization can be modeled in terms of distinct stimulatory and slow-acting inhibitory receptors that differ in affinity for each prostaglandin but lead to the same maximum extent of activation and desensitization for all prostaglandins. The affinity for the stimulatory receptor is in the order PGI2 greater than PGE1 much greater than PGD2; the affinity for the inhibitory receptor is in the order of PGE1 greater than PGI2 much greater than PGD2. In addition, the inhibitory receptor binds PGE1 more tightly than the stimulatory receptor, whereas in the case of PGI2 or PGD2, the stimulatory receptor binds agonist more tightly than the inhibitory receptor. It is shown that the model gives rise to heterologous desensitization such that PGE1 readily inhibits PGI2- and PGD2-stimulated cyclic AMP formation, because it has high affinity for the inhibitory receptor. At the same time, because the final steady state concentration of cyclic AMP depends on the fractional occupancy of both the stimulatory and inhibitory receptors, PGE1 can cause either a rise or fall in cyclic AMP level, depending on the concentration of PGI2 or PGD2 used to challenge the platelets before PGE1 addition. The presence of a distinct inhibitory receptor may represent a general mechanism of autocoid desensitization, buffering cellular response against transient localized increases in agonist concentration that may occur when agonists are produced close to their sites of action.     

Dopamine-sensitive adenylate cyclase in the rat kidney particulate preparation.

Dopamine, apomorphine, isoproterenol and norepinephrine all increased the concentration of adenosine 3',5'-monophosphate in the rat kidney particulate preparation, which was composed of tubules, glomeruli and blood vessels. The concentrations of dopamine, apomorphine, isoproterenol and norepinephrine causing a half-maximal increase were 50, 83, 0.1 and 10 muM, respectively. The alpha-blocker, phentolamine, at a concentration as high as 1 mM, did not significantly reduce the effect of these drugs on the adenosine 3',5'-monophosphate concentration. The beta-blocker, propranolol (50 muM), blocked the effect of isoproterenol and norepinephrine, but not that of dopamine. The effect of dopamine was selectively blocked by spiroperidol (50 muM), a dopamine receptor antagonist, whereas the effects of isoproterenol and norepinephrine was not blocked by spiroperidol. These results suggest that in the rat kidney particulate preparation there is a specific dopamine receptor which can lead to the increase in the concentration of adenosine 3',5'-monophosphate.     

Effects of prostaglandins on rat cardiac adenylate cyclase

A significant stimulatory action of PGE₁ on adenylate cyclase of the particulate membrane preparation of rat heart was observed at 1 × 10^?4 M. PGE₁ caused a positive inotropic effect on isolated spontaneously beating rat atria at 1 × 10^?5 M, and an increase in atrial cyclic AMP level at 1 × 10^?6 M. PGE_2α had no effect on these enzyme preparations. Our results suggest that: (i) The inotropic response of isolated spontaneously beating rat atria to PGE₁ may be connected with an increase in cyclic AMP level; (ii) the effect of prostaglandins on heart in vivo must be indirect, because the concentrations needed to stimulate isolated atria and particulate adenylate cyclase were high, compared with the doses effective in vivo.     

Isobutylmethylxanthine stimulates adenylate cyclase by blocking the inhibitory regulatory protein, Gi

The methylxanthines, such as caffeine and theophylline, are an important and widely used class of drugs, which are believed to mediate many of their physiological effects by increasing intracellular concentrations of cAMP. These agents are known to inhibit phosphodiesterases and to block inhibitory A1 adenosine receptors in a competitive manner. Thus, the methylxanthines may increase cAMP accumulation by slowing its inactivation or by enhancing its production. Using a rat adipocyte membrane model we demonstrate that isobutylmethylxanthine (IBMX) induces a dose-dependent 34% increase in cAMP production above that produced by complete phosphodiesterase inhibition with papaverine. This stimulatory effect is dependent upon the inhibitory guanine nucleotide regulatory protein G1, in that inactivation of Gi by pertussis intoxication ablates IBMX-mediated stimulation of adenylate cyclase activity. Because the Gi-dependent effect of IBMX results in increased cAMP production, the mode of action is likely blockade of Gi activity. Accordingly, the capacity of GTP itself to inhibit adenylate cyclase activity is attenuated by IBMX. In contrast to Gi blockade induced by pertussis toxin, this heretofore unappreciated stimulatory mechanism is completely reversed by inhibitory receptor agonists. This mechanism of action may be responsible for certain physiological effects of methylxanthines, which are not easily explained by phosphodiesterase inhibition or antagonism of A1 adenosine receptors.