Hormone-induced guanyl nucleotide binding and activation of adenylate cyclase in the Leydig cell.

The adenylate cyclase activity of Leydig cell homogenates and membrane fractions is highly dependent on guanyl nucleotides, and enzyme responses to luteinizing hormone or human chorionic gonadotropin are small in the absence of guanyl nucleotides. However, in the presence of 10 microM guanosine 5'-[beta, gamma-imido]triphosphate Gpp[NH]p, both hormones consistently stimulated testicular adenylate cyclase activity by up to 200%. Leydig cell membranes bound [3H]Gpp[NH]p at 30 degrees C with high affinity (Ka = 1.5 X 10(7) M-1) and binding capacity of 60 pmol/mg of protein. During kinetic studies, the association rate constant was 1.7 X 10(6) M-1 min-1, and the dissociation constant was 0.038 min-1. In the presence of gonadotropin (10 pM to 10 nM), concentration-dependent increases of 40% to 100% in Gpp[NH]p binding were observed in Leydig cell membranes. Kinetic studies showed that gonadotropin decreased the association rate constant to 0.73 X 10(6) M-1 min-1 and the dissociation rate constant to 0.017 min-1, with no effect on the equilibrium binding constant. Thus, the increase in Gpp[NH]p binding was not due to a change in receptor affinity but was attributable to increased availability of nucleotide binding sites. The 50% effective dose for adenylate cyclase activation by gonadotropin in the presence of Gpp[NH]p was identical with that observed for gonadotropin-induced binding of the GTP analog (50 nM). Gonadotropin-induced binding of Gpp[NH]p in Leydig cell membranes may represent interaction with the guanyl nucleotide regulatory site during hormonal activation of adenylate cyclase.     

Mechanism of adenylate cyclase activation by cholera toxin: Inhibition of GTP hydrolysis at the regulatory site

Treatment of turkey erthrocyte membranes with cholera toxin caused an enhancement of the basal and catecholamine-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activities. Both of these activities required the presence of GTP. The toxin effect on the adenylate cyclase activity concided with an inhibition of the catecholamine-stimulated guanosinetriphosphatase activity. Inhibition of the guanosinetriphosphatase, as well as enhancement of the adenylate cyclase activity, showed the same dependence on cholera toxin concentrations, and the effect of the toxin on both activities was dependent on the presence of NAD.It is proposed that continuous GTP hydrolysis at the regulatory guanyl nucleotide site is an essential turn-off mechanism, terminating activation of the adenylate cyclase. Cholera toxin inhibits the turn-off guanosinetriphosphatase reaction and thereby causes activation of the adenylate cyclase. According to this mechanism GTP should activate the toxin-treated preparation of adenylate cyclase, as does the hydrolysis-resistant analog guanosine 5'-(beta,gamma-immino)triphosphate [Gpp(NH)p]. Indeed, the toxin-treated adenylate cyclase was maximally activated, in the presence of isoproternol, by either GTP or Gpp(NH)p, while adenylate cyclase not treated with toxin was stimulated by hormone plus GTP to only one-fifth of the activity achieved with hormone plus Gpp(NH)p. Furthermore, the toxin-treated adenylate cyclase activated by isoproterenol plus GTP remained active for and extended period (half-time of 3 min) upon subsequent addition of the beta-adrenergic blocker, propranolol. The native enzyme, however, was refractory to propranolol only if activated by Gpp(NH)p but not by GTP.     

Ca2+ potentiates corticotropin-induced, but not isoproterenol-induced, [3H]guanosine diphosphate release in rat adipocyte membranes.

EGTA abolished corticotropin (ACTH)-stimulated adenylate cyclase in rat adipocyte membranes. In contrast, the potency of guanosine triphosphate (GTP) stimulation of adenylate cyclase activated with ACTH was greater in the presence of Ca2+ (1 mmol/L). EGTA (1 mmol/L) powerfully inhibited ACTH-stimulated [3H]guanosine diphosphate (GDP) release from membranes prelabeled with [3H]GTP in the presence of isoproterenol (ISO) or ACTH, whereas Ca2+ significantly increased it. In contrast, neither EGTA nor Ca2+ affected ISO-stimulated [3H]GDP release. These data clearly show that Ca2+ is necessary for the binding of ACTH to its receptor, and that Ca2+ stimulates the interaction of the ACTH-occupied receptor with GTP-binding proteins.     

GTP is not required for calmodulin stimulation of bovine brain adenylate cyclase.

The importance of guanyl nucleotides for calmodulin stimulation of bovine cerebral cortex adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] was examined by using a partially purified calmodulin-sensitive adenylate cyclase that was resolved from calmodulin-insensitive forms of the enzyme. By using 5'-adenylyl imidodiphosphate as a substrate, in the absence of an ATP-regenerating system, it was determined that GTP is not required for calmodulin stimulation of the enzyme. Maximal activation by 5'-guanylyl imidodiphosphate (p[NH]ppG) was 5.3-fold, whereas the combination of p[NH]ppG and calmodulin stimulated the enzyme 27-fold. Although GDP inhibited p[NH]ppG stimulation of the calmodulin-sensitive adenylate cyclase, it did not affect calmodulin stimulation. In addition, calmodulin did not alter the kinetics for activation of the enzyme by p[NH]ppG. It is concluded that GTP is not required for calmodulin stimulation of brain adenylate cyclase and that calmodulin regulation of this enzyme is probably not due to effects of calmodulin on the affinity of the guanyl nucleotide regulatory complex for guanyl nucleotides.     

Modulation of follicle-stimulating hormone-sensitive rat testicular adenylate cyclase activity by guanyl nucleotides.

We have studied modulation of FSH-sensitive adenylate cyclase activity in testes of immature rats by guanyl nucleotides. Highly purified hFSH alone stimulated adenylate cyclase activity 2.2-fold over basal levels. Addition of the GTP analog, 5'-guanylyl imidodiphosphate [Gpp(NH)p], caused an additional 2.8-fold augmentation of adenylate cyclase activity to 6 times over basal levels and 3.7 times greater than that seen in the presence of Gpp(NH)p alone. GTP did not significantly stimulate basal levels of adenylate cyclase and augmented FSH stimulated activity by 1.4-fold; other nucleotides were without effect. Half-maximum activation of adenylate cyclase in each instance was produced by approximately similar concentrations of either guanyl nucleotide (about 10 microM). The Km for hormone activation of adenylate cyclase was nearly the same in the presence and absence of Gpp(NH)p. Maximum adenylate cyclase stimulation in the presence of nucleotide and/or hRSH was always less than obtained by fluoride alone. Of all nucleotides tested, only GTP and its analog, Gpp(NH)p, significantly augmented FSH stimulation of testicular adenylate cyclase activity. Gpp(NH)p also markedly inhibited binding of radiolabeled hFSH to testicular receptor, but at a concentration 15-fold greater than that required for significant stimulation of testicular adenylate cyclase activity. The results suggest a specific role for guanyl nucleoside triphosphate in regulation of FSH effects on testicular adenylate cyclase activity.     

Regulation of thyroid adenylate cyclase: guanyl nucleotide modulation of thyrotropin receptor-adenylate cyclase function

The role of guanyl nucleotides in regulating the hormonal responsiveness of adenylate cyclase was studied in thyroid plasma membranes. Guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] alone stimulated the enzyme. At a low ATP concentration (0.2 mM), TSH alone had little or no effect, but when added with Gpp(NH)p, it resulted in a dose-dependent increase of enzyme activity. Kinetic studies revealed that Gpp(NH)p alone stimulated adenylate cyclase activity only after a 10-min lag. TSH abolished the lag, resulting in an apparent increase in activity and a lowering of the activation constant for Gpp(NH)p. GTP caused an initial increase in activity at 2 min, followed by a gradual decline below basal levels. This inhibition was not prevented by TSH. Further examination revealed that GDP caused inhibition of Gpp(NH)p-stimulated activity in a competitive manner, suggesting that conversion of GTP to GDP may be responsible for the time-dependent decay seen with GTP. To study the effects of guanyl nucleotides on coupling of the TSH receptor to adenylate cyclase, plasma membranes were preactivated with saturating amounts of Gpp(NH)p and washed extensively to remove unbound Gpp(NH)p. Incubation of preactivated membranes with either Gpp(NH)p or TSH gave no further stimulation of adenylate cyclase. However, Gpp(NH)p plus TSH produced 30% more stimulation. In contrast, the addition of TSH plus GDP to preactivated membranes led to a dose-dependent decrease in activity. Furthermore, promotion of further stimulation by TSH plus Gpp(NH)p was competitively inhibited by GDP, in the same manner as untreated membranes. This suggested that the dual action of TSH, i.e, stimulation and inhibition, is governed and mediated by specific guanyl nucleotides. Analysis of guanyl nucleotides effects on the binding of [125I[iodo-TSH to membranes revealed that although some inhibition of binding exists, this effect is 1) unrelated to the concentration effect of nucleotides on adenylate cyclase, and 2) not specific for guanosine phosphates. Scatchard analysis of TSH binding in the presence of guanyl nucleotides demonstrated that even at high concentrations, GTP had no effect on the high affinity, low capacity receptor for TSH. These results suggest that GDP or Gpp(NH)p plays no role in modulating TSH-receptor interaction. Thus, guanyl nucleotide regulation of TSH action appears to be limited to adenylate cyclase.     

Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells.

The diterpene, forskolin [half-maximal effective concentration (EC50), 5-10 microM] activates adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in rat cerebral cortical membranes in a rapid and reversible manner. Activation is not dependent on exogenous guanyl nucleotides and is not inhibited by guanosine 5'-O-(2-thiodiphosphate) when assayed with adenosine 5'-[beta, gamma-imido]triphosphate as substrate. GTP and GDP potentiate responses to forskolin. The activations of adenylate cyclase by forskolin and guanosine 5'-[beta, gamma-imido]triphosphate p[NH]ppG are not additive, whereas activations by forskolin and fluoride are additive or partially additive. The responses of adenylate cyclase to forskolin or fluoride are not inhibited by manganese ions, whereas the response to p[NH]ppG is completely blocked. Activation of adenylate cyclase by forskolin is considerably greater than the activation by fluoride in membranes from rat cerebellum, striatum, heart, and liver, while being about equal or less than the activation by fluoride in other tissues. Forskolin (EC50, 25 microM) causes a rapid and readily reversible 35-fold elevation of cyclic AMP in rat cerebral cortical slices that is not blocked by a variety of neurotransmitter antagonists. Low concentrations of forskolin (1 microM) augment the response of cyclic AMP-generating systems in brain slices to norepinephrine, isoproterenol, histamine, adenosine, prostaglandin E2, and vasoactive intestinal peptide. Forskolin would appear to activate adenylate cyclase through a unique mechanism involving both direct activation of the enzyme and facilitation or potentiation of the modulation of enzyme activity by receptors or the guanyl nucleotide-binding subunit, or both.     

Photolyzed rhodopsin catalyzes the exchange of GTP for bound GDP in retinal rod outer segments.

We have studied the binding of guanyl nucleotides to retinal rod outer segment membranes to determine how light activates a cyclic GMP phosphodiesterase and a GTPase. We found that rod outer segment membranes contain tightly bound radioactive GDP after incubation in the dark with [3H]GDP or [alpha-32P]GTP. Reconstituted membranes containing only rhodopsin and phospholipid bind almost no GDP. More than 80% of the radioactive GDP bound to rod outer segment membranes could be released by subsequent illumination. At low light levels, the rate and extent of GDP release were markedly enhanced by the presence of GTP or p[NH]ppG, a nonhydrolyzable analog of GTP. The kinetics of binding of p[NH]ppG paralleled the kinetics of release of bound GDP, indicating that p[NH]ppG was exchanged for bound GDP. The maximal amount of bound p[NH]ppG was 1 per 30 rhodopsins when photolyzed membranes were incubated with 10 micro M nucleotide. Under these conditions, p[NH]ppG binding was half-maximal when only 1 in 90,000 rhodopsins was photolyzed. This corresponds to the catalyzed exchange of 500 p[NH]ppG for bound GDP per photolyzed rhodopsin. We propose a light-activated GTP-GDP amplification cycle involving a guanyl nucleotide binding protein with GTPase activity (E). The essence of this cycle is that photolyzed rhodopsin catalyzes the formation of E . GTP from E . GDP (the major species in the dark) by nucleotide exchange. The formation of several hundred E . GTP per photolyzed rhodopsin may be the first stage of amplification in visual excitation.     

Guanyl nucleotide potentiation of parathyroid hormone-stimulated adenylate cyclase in chicken renal plasma membranes: a receptor-independent effect

We investigated the interaction of guanyl nucleotides with the parathyroid hormone (PTH) receptor-adenylate cyclase system in chicken renal plasma membranes. Micromolar concentrations of guanosine triphosphate and its hydrolysis-resistant analog 5'-guanylimidodiphosphate [Gpp(NH)p] increased both basal and PTH-stimulated adenylate cyclase activity. The enzyme activation produced by the amino-terminal 1--34 peptide of bovine PTH [bPTH-(1--34)] was potentiated by both guanyl nucleotides, although quantitatively greater effects were seen with Gpp(NH)p. The apparent activation constant for bPTH-(1--34) stimulation of adenylate cyclase, 16 nM, was reduced to 3.7 nM in the presence of 1.0 microM Gpp(NH)p. The interaction of guanyl nucleotides with the PTH receptor was evaluated by measurement of specific 125I-labeled bPTH-(1--34) binding to chicken renal plasma membranes in the presence and absence of Gpp(NH)p. There was no effect of the guanyl nucleotide on the rate of binding or dissociation of 125I-labeled bPTH-(1--34) from its renal receptor. Scatchard analysis of steady state PTH binding revealed that 1.0 microM Gpp(NH)p had minimal effect on either the affinity of PTH receptors (Kd increased from 25 nM to 30 nM) or their number (total number of binding sites increased from 8.6 to 9.4 pmol/mg protein). Separate experiments demonstrated a concentration-dependent effect on Gpp(NH)p to decrease the apparent activation constant for bPTH-(1--34) stimulation of adenylate cyclase, with no detectable guanyl nucleotide effect on the affinity of PTH receptors. The results suggest that guanyl nucleotides may enhance the coupling of occupied PTH receptors to adenylate cyclase independent of direct nucleotide effects on renal PTH receptors.     

GDP activates rabbit heart adenylate cyclase, but does not support stimulation by isoproterenol: a re-appraisal of the control mechanism

The effect of GDP on rabbit heart adenylate cyclase has been determined under conditions where only 0.08% to 0.26% of an added 100 microM was converted to GTP in the course of the assay. At concentrations of 100 microM, GDP stimulated basal cyclase activity to the same extent as GTP and guanosine-5'-O-(2-thiodiphosphate) (GDP beta S). Isoproterenol increased activity in the presence of GTP or guanylyl-imidodiphosphate (Gpp(NH)p), but not in the presence of GDP or GDP beta S. It is suggested that the hydrolysis of GTP to GDP is the "turn-off" mechanism for beta-receptor stimulation of cardiac adenylate cyclase, but not for stimulation by GTP alone. The effects of GDP and GDP beta S are readily removed by washing, implying that their binding to Ns (the guanine nucleotide binding protein) is weak. GDP beta S initially competes with Gpp(NH)p, reducing Gpp(NH)p-stimulated activity. As stimulation of cyclase activity by Gpp(NH)p develops, in the course of 30 min, Gpp(NH)p becomes no longer displaceable by GDP beta S. Isoproterenol does not release 3H-Gpp(NH)p or reduce Gpp(NH)p-stimulated activity, once the nucleotide has become tightly bound. Nor does isoproterenol change the relative affinities of GDP beta S and Gpp(NH)p when these analogs are given together. There is, therefore, no evidence that isoproterenol acts by releasing tightly bound GDP from Ns, or that it 'unlocks' the guanine nucleotide binding site in the myocardial sarcolemma. In this, the cardiac adenylate cyclase system differs from the avian erythrocyte system. The action of isoproterenol is best explained by an increased dissociation of alpha(GTP) and beta,gamma-subunits of the Ns protein.     

Coupling of the glucagon receptor to adenylyl cyclase by GDP: evidence for two levels of regulation of adenylyl cyclase.

In rat liver plasma membranes preactivated with guanosine 5'-[beta,gamma-imido[triphosphate (GuoPP[NH]P), GDP promoted coupling of occupied glucagon receptor to adenylyl cyclase [adenylate cyclase; ATP, pyrophosphate-lyase (cyclizing), EC 4.6.1.1] with an apparent association constant Ka of 0.1-0.15 microM. The apparent Ka for the same effect of GTP was 0.2 microM. The effect of GDP was shown not to be due to GTP formed by putative transphosphorylation reaction(s) when ATP was present in the assay as substrate. In membranes not preactivated with GuoPP[NH]P, GDP both competitively inhibited GuoPP[NH]P stimulation of adenylyl cyclase (Ki 0.10 microM) and supported stimulation of cyclizing activity (apparent Ka 0.10 microM) by glucagon. These effects of GDP occurred in the absence of added GTP and in the absence of sufficient formation of GTP by putative transphosphorylation reaction(s) to account for them. It is concluded that two levels of regulation of liver adenylyl cyclase (cyclizing) activity must exit. One level is termed "receptor regulation"; it depends on occupancy of a receptor-related R site by nucleotide and is specific for either GDP or GTP. The second level of regulation is termed "GTPase regulation"; it is inhibited by GDP, depends on both GTP and GTPase, and accounts for activation of cyclizing activity by nonhydrolyzable analogs of GTP. The data suggest that both levels of regulation coexist and may synergize, one mediating responses to stimuli external to the cell (receptor regulation) and the other mediating stimuli of intracellular origin (GTPase regulation).     

Fatty acids as modulators of membrane functions: catecholamine-activated adenylate cyclase of the turkey erythrocyte.

Activation of the adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1[ from turkey erythrocytes by isoproterenol decreased precipitously below 26 degrees. Certain unsaturated fatty acids enhanced the activation by isoproterenol up to 25-fold at reduced temperatures. The fatty acid also enhanced the formation of a persistent active state of the enzyme which was produced by preincubation with guanosine 5'-(beta,gamma-imino)triphosphate [Gpp(NH)p]. Once the enzyme had been activated by Gpp(NH)p plus isoproterenol the reaction rate was no longer as temperature sensitive and the fatty acid had little effect. The synthetic Gpp(NH)p apparently substituted for the natural GTP, which is known to play a regulatory role in the adenylate cyclase system. The findings suggest that the function of GTP which is mediated by the hormone is the temperature-sensitive event which is enhanced by the fatty acid. The use of free fatty acid to probe membrane-associated reactions in intact cells and in isolated membrane preparations is proposed.     

Thermodependence of basal and stimulated cardiac adenylate cyclase activity in normotensive and spontaneously hypertensive rats

The cardiac adenylate cyclase activity from normotensive and spontaneously hypertensive (SHR) rats was studied as a function of temperature between 17° and 37°C. Arrhenius plots of adenylate cyclase activity displayed a break around 31°C when tested under basal conditions or in the presence of GTP but were linearized after activation with p[NH]ppG, NaF, secretin, glucagon or isoproterenol. The energy of activation of adenylate cyclase activity in the presence of GTP (9.5 ± 0.5 kcal/mol) was significantly lower than in the presence of p[NH]ppG (17.7 ± 0.8 kcal/mol). A hormone was without effect on the energy of activation observed with either GTP or p[NH]ppG but the simultaneous presence of hormone and nucleotide increased markedly the activity of the enzyme. The energies of activation were analyzed in terms of variation of enthalpy and entropy and discussed in relation with the process of activation and coupling of the guanine nucleotide regulatory protein. These thermodynamic characteristics were similar in cardiac membranes from normotensive and spontaneously hypertensive rats, suggesting that the impairment of hormone-stimulated adenylate cyclase activity observed in the heart membranes of hypertensive rats was not a consequence of a defect in the activation process of the enzyme.     

Role of guanidylic nucleotides in the adenylate cyclase activity of the rat liver]

Glucagon and adrenaline exert their action upon the liver via the cyclic AMP synthetizing system located in the plasma membrane. The enzyme adenylate cyclase is further regulated by guanyl nucleotides. It has been recently shown that the rat liver plasma membrane system could respond to GTP by simultaneous increase in the cyclase activity in response to glucagon and by the dissociation of this hormone from its binding sites (1). Unambiguous relationship between the activating effect of GTP upon the cyclase and its action upon glucagon binding has not been determined yet (2). This problem was approached using the in vitro action of epinephrine as a model. When 1 to 100 muM GTP or DGP were added to rat liver plasma membranes isolated from adrenalectomized animals, they increased markedly the response of the cyclase system to epinephrine. These effects could be observed in the absence of an ATP-regenerating system and were mimicked by 5'-guanylyl diphosphonate; GTP and GDP were the most active compounds followed by ITP, CTP and by a series of guanyl derivatives. UTP, as well as guanosine, GMP, cyclic GMP and ppGpp were inactive. Guanyl nucleotides did not increase the affinity of the cyclase system for the activating hormones, but enhanced the affinity for ATP-Mg and also the Vmax of the reaction. Finally, GTP, ATP, CTP, UTP but not GDP displaced epinephrine bound to plasma membranes by a mere chelation phenomenon. It is concluded that 1) guanyl nucleotides do not act primarily by influencing the binding of hormones to the membranes; 2) they act directly upon the catalytic subunit of the cyclase; 3) the low concentrations of GTP required for its action strongly suggest that this nucleotide plays a role in the physiological regulation of the intrahepatic cyclic AMP level.     

Guanyl nucleotide regulation of human chorionic gonadotropin binding to rat luteal tissue

Guanyl nucleotides are known to have a dual effect on most hormone-sensitive adenylate cyclase systems, regulating activation of the adenylate cyclase enzyme and binding of hormone to receptor. In the ovary, guanyl nucleotides have been shown to be required for hCG stimulation of luteal adenylate cyclase, but no effect on binding has been observed. Evidence has been obtained which suggests that guanyl nucleotide regulation of hCG binding is masked in most luteal membrane preparations by the presence of a large excess of unregulated receptor. A highly purified urea-washed membrane fraction has been prepared. Binding of hCG to this preparation was decreased (approximately 45%) in the presence of 5'-guanylylimidodiphosphate (GMPPnP). The maximal effect of GMPPnP occurred at 10 microM, with the half-maximal effect at 0.2 microM. These levels compare favorably with the GTP concentrations required for hCG stimulation of luteal adenylate cyclase. Analysis of equilibrium binding experiments showed that GMPPnP acted by reducing the number of binding sites by 45%. However, kinetic experiments suggested that this effect was due to a significant decrease in the affinity of a fraction of the hCG-binding sites. Association of hCG and its receptor was unaffected by the presence of GMPPnP (100 microM), whereas the dissociation of 40-50% of bound hormone was significantly accelerated (30-fold) by its presence. The guanyl nucleotide effect required the presence of MG+2; other divalent cations (Ca+2, Mn+2, and Co+2 could not be substituted. The ratio of beta-adrenergic to hCG-binding sites in the urea-washed heavy membrane preparation was elevated, suggesting that a sizable fraction of uncoupled hCG receptor had been removed. The results show that hCG binding to its luteal receptor is modulated by guanyl nucleotide and suggest that the modulation only occurs in those receptors that are directly coupled to the adenylate cyclase enzyme.     

The somatostatin receptor is directly coupled to adenylate cyclase in GH4C1 pituitary cell membranes

Somatostatin (SRIF) inhibits vasoactive intestinal peptide (VIP)-stimulated cAMP accumulation in the GH4C1 strain of rat pituitary tumor cells, and this effect is responsible for SRIF inhibition of VIP-stimulated hormone release. In this study we examined the interaction between the SRIF receptor and adenylate cyclase in GH4C1 cell membranes. Maximal concentrations of VIP (50 nM) increased membrane adenylate cyclase activity 4.2-fold; half-maximal stimulation was observed with 0.75 nM VIP. SRIF noncompetitively inhibited the stimulatory effect of VIP, but it did not alter basal adenylate cyclase activity. The relative potencies of SRIF and two SRIF analogs as inhibitors of VIP-stimulated adenylate cyclase activity in membranes and of VIP-stimulated cAMP accumulation in intact cells were similar. Furthermore, the concentration of SRIF that caused half-maximal inhibition of adenylate cyclase activity (ED50 = 2.3 nM) was close to the equilibrium dissociation constant for SRIF (Kd = 0.40 nM) measured in membrane preparations in the presence of GTP. Therefore, SRIF inhibition of adenylate cyclase appears to be receptor mediated. As with receptors known to regulate adenylate cyclase by interaction with a guanine nucleotide regulatory subunit, SRIF receptor binding was decreased in the presence of guanine nucleotides. Addition of GTP (150 microM) or the nonhydrolyzable GTP analog guanyl-5'-yl-imidodiphosphate (100 microM) decreased the specific binding of [125I-Tyr1]SRIF to 31% and 13% of the control value, respectively. This decrease in specific binding was due entirely to decreased receptor affinity for SRIF. GTP (150 microM) increased the equilibrium dissociation constant for SRIF from 0.11 to 0.40 nM, whereas the number of binding sites was unaffected by the nucleotide (Bmax = 0.2 pmol/mg protein). Analysis of dissociation kinetics demonstrated that in the absence of guanyl nucleotides, the rate of [125I-Tyr1]SRIF dissociation was first order (t 1/2 = 180 min). However, in the presence of a half-maximal concentration of guanyl-5'-yl-imidodiphosphate (0.3 microM), [125I-Tyr1]SRIF dissociation occurred with biphasic kinetics. Fifty percent of the specifically bound peptide dissociated at the same rate as that observed in the absence of nucleotide, whereas the remainder dissociated 15 times more rapidly (t 1/2 = 9.6 min).(ABSTRACT TRUNCATED AT 400 WORDS)     

Catecholamine stimulated myocardial adenylate cyclase: Effects of nucleotides

The effects of several nucleotides on canine myocardial adenylate cyclase have been investigated. Basal, fluoride and isoproterenol stimulated enzyme activities were studied. With adenosine 5′ triphosphate at ? 1.5 mm guanosine 5′-triphosphate increased basal and isoproterenol stimulated cyclase activity severalfold. Flouride stimulated activity was inhibited. Significant stimulation occurred at 10 nm guanosine 5′-triphosphate and effects were maximal by 1 μm. In order of decreasing potency the nucleoside 5′-triphosphates were guanosine > deoxyguanosine > uridine > thymidine > cytidine. Insosine 5′-triphosphate and xanthosine 5′-triphosphate were inactive. Although guanosine 5′-triphosphate was most potent, all other guanyl nucleotides tested, including guanosine 5′-diphosphate, guanosine 5′-monophosphate and cyclic guanosine, 3′,5′-monophosphate shared this effect. Guanosine was inert. The stimulatory action of guanosine 5′-triphosphate was exhibited after a lag period of several minutes. When substrate (adenosine 5′-triphosphate) concentrations were lowered to 0.05 mm, stimulation by isoproterenol was virtually dependent on the presence of guanosine 5′-triphosphate. Nucleotides did not affect the apparent K_m for enzyme stimulation by isoproterenol which was 1–2 × 10^?6m with or without guanosine 5′-triphosphate. Propranolol effectively blocked the augmented stimulation by isoproterenol observed in the presence of guanosine 5′-triphosphate. The data indicate that guanyl nucleotides are capable of markedly altering the sensitivity of myocardial adenylate cyclase to catecholamine stimulation.     

The effects of trimetoquinol on the intact rabbit heart and myocardial adenylate cyclase activity: evidence for spare myocardial beta receptors

We have compared and contrasted the actions of (-)isoproterenol and (+/-) trimetoquinol on rabbit heart preparations. In the presence of either GTP or Gpp[NH]p (guanosine-5'-(beta, gamma imino) triphosphate), trimetoquinol displayed partial agonist activity in stimulating adenylate cyclase activity in a particulate rabbit heart preparation. Trimetoquinol enhanced adenylate cyclase activity 20% or 65% of the maximum obtainable by isoproterenol in the presence of GTP or Gpp[NH]p respectively. In the presence of GTP, concentrations of catecholamines required to enhance cyclase activity 15% of the maximum obtainable with isoproterenol (EC15) were 2.0 X 10(-7) M and 5.5 X 10(-8) M for trimetoquinol and isoproterenol, respectively. In the presence of Gpp[NH]p EC30 values were 2.0 X 10(-7) and 3.5 X 10(-8) M for trimetoquinol and isoproterenol respectively. Trimetoquinol also displayed partial agonist activity for the ability to increase cAMP levels in the isolated perfused rabbit heart. By contrast trimetoquinol was equieffective to isoproterenol at increasing tension development and rate of contraction of the isolated perfused heart. Concentrations of catecholamines required to increase tension and rate of contraction 50% of the maximum obtainable with isoproterenol were 1.5 X 10(-7) M and 1.7 X 10(-8) M for trimetoquinol and isoproterenol, respectively. These data show that only a partial stimulation of adenylate cyclase activity and cAMP levels by trimetoquinol is sufficient to produce maximal changes in mechanical activity of the heart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of guanyl nucleotides on parathyroid hormone-responsive adenylate cyclase in chick kidney.

Both guanosine 5'-triphosphate (GTP) AND 5'-guanylylimidodiphosphate (Gpp(NH)p) activated adenylate cyclase (EC4.6.1.1) in chick kidney plasma membranes. Half-maximal stimulation occurred at 3-1 X 10(-6)M for both agents. The maximum increases in adenylate cyclase activity produced by GTP and Gpp(NH)p were respectively 130 and 720% over basal activity. At the end of a 12 min incubation period GTP concentration was 85% of that originally added in the presence of an ATP-regenerating system but less than 20% in its absence. GTP and guanosine 5'-diphosphate inhibited the activation of adenylate cyclase by Gpp(NH)p, suggesting that they all acted at a common site. Gpp(NH)p facilitated the stimulation of adenylate cyclase activity by bovine parathyroid hormone (BPTH) and by the synthetic amino terminal fragment BPTH (1-34), decreasing the concentrations required for half-maximal enzyme activation by a factor of approximately eight in both cases. This property was not shared by the native nucleotide GTP. Gpp(NH)p rendered active (at certain concentrations) a synthetic parathyroid hormone peptide fragment, BPTH (2-34), which was incapable of Activating adenylate cyclase in the absence of the nucleotide analogue. This suggested that the GTP analogue, in addition to a direct effect upon adenylate cyclase activity, was capable of influencing hormone interaction with the enzyme complex.     

Guanyl nucleotide regulation of hormonally-responsive adenylyl cyclases.

A large number of hormones and neurotransmitters activate adenylyl cyclase [ATP, pyrophosphate lyase (cyclizing; EC 4.6.1.1.)] catalyzing the formation of cAMP and PPi from ATP in the presence of Mg2+. The cAMP formed is in turn responsible for eliciting the physiological responses of these hormones and neurotransmitters. In addition to hormones and neurotransmitters, fluoride ion, cholera toxin and guanyl nucleotides (GTP and GTP analogs such as GTP gamma S and GMP-P(NH)P) also stimulate adenylyl cyclase activity (Perkins, 1974; Birnbaumer, 1977; Gill, 1977). It has become evident that hormonally-responsive adenylyl cyclase is a multi-component system consisting of at least 3 physically distinct units. The first is the hormone receptor containing a specific site for a given hormone. The second is the catalytic moiety (C component) of adenylyl cyclase bearing the site responsible for catalysis of the cyclizing reaction. The third is the guanyl nucleotide regulatory subunit (G component) which binds guanyl nucleotide. Recently, a GTPase activity has been found to be associated with the G component of adenylyl cyclase (Cassel and Selinger, 1976; Cassel et al., 1977a, b; Lambert et al., 1979). In this review we will present information on the regulation of hormonally-responsive adenylyl cyclases. This is not intended to be a comprehensive review of the literature. Rather, it represents our views on the current status of the regulation of cAMP formation.