Epinephrine-sensitive adenylate cyclase of human fat cell ghosts: Stabilization of enzyme activity by fluoride and nucleotides

The effects of fluoride and nucleotides on the stability of the human fat cell adenylate cyclase were studied. Preincubation of fat cell ghosts in 1 mM KHCO₃ at 30°C for 20 min resulted in a substantial loss of basal, epinephrine- and NaF-stimulated enzyme activities. Inactivation was almost completely prevented by nucleotides (ATP, GTP and GMP-P(NH)P). ¹ Epinephrine was without influence on the time course and degree of inactivation. NaF (20 mM), however, like the nucleotides protected from inactivation. In contrast to epinephrine NaF failed to stimulate enzyme activity in membranes pretreated with GMP-P(NH)P (0.1 mM). Our results show that NaF exerts two distinct effects on the human fat cell adenylate cyclase: stimulation of enzyme activity and protection from inactivation. Both effects appear to be related to the binding of nucleotides.     

Partial purification and characterization of a macromolecule which enhances fluoride activation of adenylate cyclase.

Fluoride activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] is significantly enhanced (2 to 5 times) by a protein factor isolated from rat brain. The fluoride-dependent adenylate cyclase stimulator (FCS) is nondialyzable, trypsin-labile, and stable at 90 degrees C for 10 min. FCS stimulates adenylate cyclase activity only in the presence of NaF (2-25 mM) and this effect is independent of added GTP, 5'-guanylylimidodiphosphate, or calcium. FCS has been purified roughly 3000-fold from a 12,000 X g supernatant fraction of rat brain homogenate. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and sucrose density gradient sedimentation suggest that FCS is a monomer with an apparent Mr of 59,000. Isoelectric focusing indicates FCS has a pI of 8.9. FCS from rat brain stimulates fluoride-activated adenylate cyclase from a variety of cell types, and FCS can also be isolated from rat liver. The effects of FCS are not reversed by washing membranes when the membranes and FCS are preincubated with NaF. The Km of adenylate cyclase for ATP and the fluoride concentration causing half-maximal activation are unchanged by FCS; however, FCS increases the Vmax by 2.5-fold. FCS may act to increase the catalytic efficiency of fluoride-activated complexes of the GTP-binding unit with adenylate cyclase or to enhance the formation of additional active complexes.     

Catecholamine-sensitive adenylate cyclase of human fat cell ghosts: a comparative study using different beta-adrenergic agents.

Some of the effects of beta-adrenergic agonists and antagonists on the adenylate cyclase system of human fat cell ghosts were studied. Isoproterenol, by causing about a fourfold increase of enzyme activity, was more potent than epinephrine and norepinephrine (about 2.5--3.0-fold stimulation). The beta2-adrenergic agonists salbutamol, terbutalin, and fenoterol were considerably less effective than the naturally occurring catecholamines. The stimulatory actions of isoproterenol and beta2-adrenergic agonists were competitively inhibited by the beta-blocking agent propranolol. Isoproterenol stimulation was also inhibited by the selective beta1-adrenergic antagonist practolol. This compound, however, was less potent than propranolol. The results are suggestive for an adenylate cyclase system in human fat cell ghosts coupled to beta1-adrenergic receptor sites. These receptors differ from the cardiac beta receptors with respect to practolol affinity.     

Cytosolic activator of adenylate cyclase: Reconstitution, characterization, and mechanism of action.

Rat liver and isolated hepatocytes contain high levels of a soluble adenylate cyclase stimulator, whereas rat erythrocytes lack this activity. Accordingly, a reconstitution system was developed with adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from erythrocyte ghosts and the soluble activator from liver cytosol. Pretreatment of erythrocyte ghosts with the cytosolic factor resulted in a 5- to 15-fold activation of adenylate cyclase in the presence or absence of NaF, 5'-guanylyl imidodiphosphate, or isoproterenol and GTP. The sequence of addition of the cytosolic component and the other activators was critical in determining the maximal activity of the enzyme. The cytosolic factor appears to be a heat-labile Mr 105,000 protein, which activates adenylate cyclase in a saturable reaction involving binding of the protein to the erythrocyte ghosts. This molecular interaction was accompanied by stabilization of a labile thiol group that was essential for catalytic activity. The cytosolic component also unmasks latent adenylate cyclase activity in human erythrocyte ghosts and in cytoskeletal preparations from rat erythrocyte ghosts. These observations suggest that the cytosolic activator may also occur as a native, peripheral membrane component of adenylate cyclase systems and may be required for the expression and stabilization of catalytic activity.     

Opiates inhibit adenylate cyclase by stimulating GTP hydrolysis.

Specific, GTP hydrolysis catalyzed by membranes prepared from neuroblastoma--glioma (NG108-15) hybrid cells can be measured in the presence of adenosine-5'-[beta, gamma-imido] triphosphate (p[NH]ppA), ATP, and a nucleotide triphosphate-regenerating system. Opiates and opioid peptides stimulate low Km GTP hydrolysis when measured in the presence of Na+ and Mg2+. Opiate stimulation is rapid, stereospecific, and reserved by the antagonist naloxone. Potencies of opiates as stimulators of GTP hydrolysis and as inhibitors of adenylate cyclase are closely correlated. Agents that stimulate adenylate cyclase, including prostaglandin E1, 2-Cl-adenosine, secretin, and NaF, have little or no effect upon the rate of GTP hydrolysis. Opiates have no effect upon either adenylate cyclase or GTPase activity in membranes prepared from C6-BU1 glioma cells, which lack opiate receptors. In view of the pivotal role of GTP in the activation of adenylate cyclase, we conclude that receptor-mediated stimulation of GTP hydrolysis is the mechanism by which opiates and other inhibitory hormones lower adenylate cyclase activity in NG108-15 cell membranes.     

Coupling of inhibitory GTP binding protein to somatostatin receptors on rat cerebrocortical membranes

We studied the interaction between somatostatin receptors and inhibitory GTP binding protein in rat cerebrocortical membranes. Guanine nucleotides reduced [125I-Tyr1] somatostatin binding to cerebrocortical membranes in a dose-dependent manner with rank order of potency being guanyl-5'-yl-imidodiphosphate (Gpp(NH)p) greater than GTP greater than GMP. Maximum reduction of the binding to 32% of control was observed in the presence of 10(-5) M Gpp(NH)p. Scatchard analysis of the labeled somatostatin binding revealed that the decrease in the binding by Gpp(NH)p was due to the decrease in the binding affinity for somatostatin. Divalent cations, such as Mg++, Mn++, and Ca++, caused an increase in labeled somatostatin binding to membranes with the maximum binding observed at a concentration of 10, 10, 1 mM, respectively. However, Na+ decreased a labeled somatostatin binding in a dose-dependent manner, and half maximum inhibition of the binding was observed at 10 mM Na+. Moreover, Gpp(NH)p and Na+ lowered labeled somatostatin binding in an additive fashion. When cerebrocortical membranes were treated at 37 degrees C for 40 min with various concentrations of Islet-Activating-Protein (IAP), which had been preactivated with dithiothreitol, subsequent labeled somatostatin binding to the membranes was decreased in a dose-dependent manner. 30 micrograms/ml IAP treatment caused a decrease in the binding to 50% of control, which was characterized by the decreased binding affinity without a significant change in the binding capacity. Furthermore, exposure of IAP plus NAD to cerebrocortical membranes caused ADP-ribosylation of a membrane protein with Mr = 41,000 on autoradiogram. Such an IAP treatment of cerebrocortical membranes abolished the inhibitory effect of somatostatin on vasoactive intestinal peptide-stimulated increase in adenylate cyclase activity. These results suggest that somatostatin receptors in the brain couple to inhibitory GTP binding protein, which mediates adenylate cyclase inhibition by somatostatin.     

Stimulation of epinephrine-sensitive fat cell adenylate cyclase by cytosol: effect of cholera toxin.

Cytosol prepared from rat epididymal fat cells by centrifugation at 100,000 X g for 1 hr was found to enhance the basal and epinephrine-sensitive adenylate cyclase [EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing)] of fat cell ghosts. Cholera toxin also stimulated adenylate cyclase and increased the response to epinephrine in fat cells. A possible relationship between the adenylate cyclase modifying activities of cytosol and the effects of cholera toxin was sought. Cytosol from freshly prepared fat cells added to ghosts prepared from cells that had been exposed to toxin for varying periods showed a progressive loss of responsiveness to cytosol epinephrine-enhancing activity. The effect appeared within 15 min after toxin exposure, a full 30 min before any direct effect of toxin on adenylate cyclase was seen. Since exposure to toxin decreased membrane response to cytosol epinephrine-enhancing activity, the possibility that epinephrine-enhancing activity in cytosol might be altered by toxin was explored. Cytosol from cells exposed to toxin for varying periods lost epinephrine-enhancing activity to an appreciable degree within 15 min. Examination of these early events after exposure to toxin should clarify the way in which this bacterial substance affects mammalian cells. The cytosol epinephrine-enhancing activity was destroyed by boiling for 3 min and was partially inactivated by trypsin. It was nondialyzable and stable at -70 degrees.     

Adenylate cyclase of catfish hepatocyte membranes: basal properties and sensitivity to catecholamines and glucagon

Some characteristics of adenylate cyclase of catfish (Ictalurus melas) liver membranes were studied, and the effects of catecholamines and of glucagon were tested. The enzyme has an optimum temperature of 40 °C, and a K_m for ATP of 0.16 mM at 30 °C, and requires Mg²⁺ for its activity. The enzyme activity is inhibited with a Ca²⁺ concentration higher than 5 × 10⁻⁵ M, and enhanced with F⁻ higher than 10⁻⁴ M. The response of adenylate cyclase to GTP is biphasic, with a maximum of activity at 10⁻⁵ M GTP. Catecholamines (epinephrine, norepinephrine, isoproterenol, phenylephrine) enhance cyclase activity. Propranolol inhibits the increase in enzyme activity induced by catecholamines, whereas phentolamine is ineffective. This indicates that catecholamines (phenylephrine included) activate adenylate cyclase through a β-adrenergic mechanism. Glucagon (mammalian) has a smaller effect than epinephrine in increasing the enzyme activity of catfish hepatocyte membranes. This fact is the opposite of that observed for the cyclase activity of rat liver membranes.     

Epinephrine stimulation of fat cell adenylate cyclase: regulation by guanosine-5'-triphosphate and magnesium ion

Guanosine-5′-triphosphate (GTP) affects the activity and responsiveness of the fat cell adenylate cyclase to epinephrine. In the absence of free magnesium ion, the presence of GTP is an absolute requirement for epinephrine stimulation. Half-maximal activation of both basal and stimulated adenylate cyclase activity occurs at a GTP concentration of 0.6 mM. In the presence of 5 mM MgCl₂, GTP is no longer required but enhances epinephrine stimulation. The half-maximal concentration for this effect occurs at approximately 10 μM GTP. At high (5 mM) magnesium ion concentrations GTP inhibits basal adenylate cyclase activity.GTP lowers the apparent affinity of adenylate cyclase for ATP while simultaneously increasing the velocity of the catalytic reaction, possibly by competing with ATP at the active site as well as by binding at a regulatory site. This effect is observed on both basal and epinephrine-stimulated activities. Epinephrine itself raises the apparent K_M for ATP and the V_max of adenylate cyclase. The interdependence of these effects suggests that transient changes in the levels of GTP, ATP, and magnesium ions in the fat cell may directly regulate the responsiveness of adenylate cyclase to epinephrine.     

Magnesium promotes both parathyroid hormone secretion and adenosine 3',5'-monophosphate production in rat parathyroid tissues and reverses the inhibitory effects of calcium on adenylate cyclase

Reduced extracellular Ca2+ is known to promote PTH secretion, while severe Mg2+ depletion has the opposite effect. We have correlated the effects of Mg2+ and Ca2+ on parathyroid hormone (PTH) secretion and cAMP accumulation by rat parathyroid tissues in vitro with the effects of these two metals on adenylate cyclase activity in broken membrane preparations. PTH secretion was maximal at 0.5 mM Ca2+, falling to low levels as the Ca2+ concentration was increased to 2.5 mM. Deletion of Mg2+ from the medium resulted in a marked decrease in PTH secretion at any given Ca2+ concentration. At a constant Ca2+ concentration of 1 mM, both PTH secretion and cAMP production rose to maximal rates as the Mg2+ concentration was increased from 0 to 2 mM. The adenylate cyclase of rat parathyroid membranes was stimulated by both GTP and guanyl-5'-yl-imidodiphosphate [Gpp(NH)p]. EDTA-treated membranes could not be stimulated by Gpp(NH)p. Repletion with Mg2+ was more effective than repletion with Ca2+ in restoring responsiveness to the guanine nucleotide. When membranes were maximally preactivated by Gpp(NH)p and then assayed in the presence of variable concentrations of metal ions, enzyme activity was directly inhibited by Ca2+ and stimulated by Mg2+. Adenylate cyclase sensitivity to Ca2+ inhibition was dependent upon the Mg2+ concentration; in the presence of 0.6 mM Mg2+ a 50% inhibition was produced by 0.05 mM Ca2+, while in the presence of 8 mM Mg2+ a 10-fold higher Ca2+ concentration was required for a similar inhibitory effect. The results suggest that Ca2+ may decrease PTH secretion at least in part by a direct inhibition of adenylate cyclase. Mg2+ may promote PTH secretion either by enhancing the activation of adenylate cyclase by endogenous guanine nucleotides or by competing with Ca2+ for binding to a distinct regulatory site on the enzyme.     

Coupling of opiate receptors to adenylate cyclase: requirement for Na+ and GTP.

Inhibition of the adenylate cyclase activity in homogenates of mouse neuroblastoma-glioma hybrid cells (NG108-15) by the opioid peptide [D-Ala2,Met5]enkephalin amide (AMEA) requires the presence of Na+ and GTP. In this process, the selectivity for monovalent cations is Na+ greater than or equal Li+ greater than K+ greater than choline+; ITP will replace GTP but ATP, UTP, or CTP will not. The apparent Km for Na+ is 20 mM and for GTP it is 1 microM. Under saturating Na+ and GTP conditions, the apparent Ki for AMEA-directed inhibition is 20 nM for basal and 100 nM for prostaglandin E1-activated adenylate cyclase activity. For both cyclase activities, maximal inhibition is only partial (i.e., approximately 55% of control in each case). In intact viable NG108-15 cells, the decrease in basal and prostaglandin E1-stimulated intracellular cyclic AMP concentrations by AMEA is also dependent upon extracellular Na+. The enkephalin-directed reductions in cyclic AMP concentrations are at least 75%. The specificity of the monovalent cation requirement for enkephalin action on intact cells is the same as for enkephalin regulation of homogenate adenylate cyclase activity. Based on these data, a model is presented in which the transfer of information from opiate receptors to adenylate cyclase requires active separate membrane components, which correspond to the sites of action of Na+ and GTP in this process.     

Stimulation of adenylate cyclase activity in rat thymocytes in vitro by 3,5,3'-triiodothyronine

In view of our previous demonstration that T3 promptly increases the cAMP concentration in freshly isolated rat thymocytes in vitro, we studied the effects of T3 on adenylate cyclase activity in a crude thymocyte plasma membrane preparation. In common with adenylate cyclase in other tissues, the enzyme in rat thymocytes was activated by NaF, GTP, 5'-guanylylimidodiphosphate, and beta-adrenergic agonists and was inhibited by high concentrations of calcium. In the presence of 1 microM Ca+2, T3 induced a time-dependent increase in adenylate cyclase activity that was statistically significant between 1 and 2 min and maximum between 2 and 5 min after hormone addition. As judged from observations made at 5 min, the effect of T3 was dose dependent over the range 1 nM to 1 microM. The stimulatory effect of T3 was calcium dependent, since it was abolished by EGTA at a concentration (0.5 mM) that did not alter basal enzyme activity, and the effect of T3 in the presence of EGTA was restored by the addition of either 0.1 or 1 mM Ca+2. As judged from the lack of hydrolysis of added cAMP, phosphodiesterase activity in the assay mixture was nil in both the presence and absence of T3. Both epinephrine and the specific beta-adrenergic agonist isoproterenol, but not the alpha-agonist phenylephrine, increased adenylate cyclase activity, and their effects appeared to be additive to that of T3. The beta-adrenergic antagonist L-alprenolol, in doses that did not influence basal adenylate cyclase activity, produced a dose-related inhibition of the stimulatory effect of T3 and of the effects of epinephrine and isoproterenol as well. Neither D-alprenolol nor the alpha-antagonist phentolamine had any effect. Various thyronine analogs displayed a rank order of potency in stimulating adenylate cyclase activity very similar to their relative potencies in increasing cAMP concentration in the intact thymocyte. These findings reveal that T3 stimulates adenylate cyclase activity in rat thymocyte plasma membrane preparations. With respect to calcium dependence, inhibition by alprenolol, and response to thyronine analogs, this effect has properties similar to those of the increase in cellular cAMP concentration induced by T3 in the intact thymocyte. It can be concluded, therefore, that the effect of T3 to increase 2-deoxyglucose uptake by the rat thymocyte in vitro, a response consequent to an increase in thymocyte cAMP concentration, derives from a stimulatory effect of the hormone on adenylate cyclase itself.     

Differential roles of high and low affinity guanosine 5'-triphosphate binding sites in the regulation of follicle-stimulating hormone binding to receptor and signal transduction in bovine calf testis membranes

We have previously shown that FSH receptors are physically and functionally associated with a guanine nucleotide regulatory protein (Gs) in membranes of calf testis. Using N-ethylmaleimide (NEM), forskolin, and cholera toxin as probes, we have investigated the role of low and high affinity GTP-binding sites of stimulatory guanine nucleotide-binding protein of adenylate cyclase (Gs) in the activation of adenylate cyclase. When calf testis membranes were exposed to NEM (1 mM), FSH binding to receptors was slightly (30%) decreased, but the receptors showed continued sensitivity to GTP, resulting in a further decrease in [125I]human FSH binding to receptors. Pretreatment of membranes with NEM (up to 20 microM) produced no effect on GTP-binding. A dose-dependent decrease in high affinity GTP-binding sites, however, was observed at higher (greater than 50 microM) NEM. Adenylate cyclase activity was reduced in response to GTP gamma S or NaF concomitant to a decrease in high affinity GTP-binding sites in membranes treated with 50-100 microM NEM, or completely abolished in membranes exposed to 300 microM NEM. Stimulation by forskolin indicated that the significant inhibition of adenylate cyclase activity occurring in membranes exposed to low NEM (50-100 microM) was not due to inactivation of catalytic unit of adenylate cyclase by NEM. Pretreatment of membranes with 100 micrograms/ml cholera toxin and NAD slightly (18%) reduced specific FSH binding but did not affect Gpp(NH)p-binding. However, adenylate cyclase stimulation by GTP plus FSH in these membranes was significantly enhanced. When membranes were treated with higher concentration of cholera toxin (250 micrograms/ml), the adenylate cyclase stimulation by GTP plus FSH was abolished due to uncoupling of FSH receptors from Gs and a significant decrease in high affinity GTP-binding sites. Our results suggest that high affinity GTP-binding sites of Gs coupled to FSH receptors are essential for FSH and guanine nucleotide activation of adenylate cyclase. The low affinity binding sites bind GTP and thereby regulate FSH binding but are not involved in the activation of adenylate cyclase.     

GTP-independent stimulation of rabbit heart adenylate cyclase by isoproterenol at physiological ATP concentrations

Summary Isoproterenol increased the activity of the adenylate cyclase of rabbit heart sarcolemmal membranes in the absence of added GTP. ATP, the ATP-regenerating system, and the sarcolemmal membrane preparation were eliminated as possible sources of contaminating GTP. Isoproterenol-stimulation increased as ATP was raised. At 0.5 mM ATP, isoproterenol increased activity by 19% whereas at 5 mM ATP isoproterenol increased activity by 121%. There was no change in basal activity between 0.5 and 5 mM ATP. Stimulation by Gpp(NH)p and NaF increased slightly between 0.5 and 5 mM ATP; stimulation by KCl was unaffected. GTP does not activate cyclase d. GTP does not activate cyclase to the same extent as Gpp(NH)p even though the two act at the same site on Ns (the stimulatory guanine nucleotide-binding protein). GTP decreased cyclase activation by Gpp(NH)p in a concentration-dependent fashion when the two were added to the assay simultaneously. Increasing ATP from 0.5 to 5 mM did not reduce activation by Gpp(NH)p when both were added simultaneously to the assay. This suggests that ATP does not interact with the same site as Gpp(NH)p. ATPS, an analogue of ATP which irreversibly thiophosphorylates proteins, did not irreversibly support activation by isoproterenol. The effect of ATP in supporting isoproterenol stimulation is not, therefore, thought to be due to phosphorylation of a protein.     

Evidence that testosterone modulates in vivo the adenylate cyclase activity in fat cells

In male hamster fat cell membranes, the alpha 2-adrenoreceptor-mediated inhibitory response of adenylate cyclase was almost completely suppressed by castration and was restored to control values after testosterone treatment, whereas the cyclase inhibitory response to nicotinic acid was insensitive to androgenic status. Basal and forskolin-, guanylylimidodiphosphate- and isoproterenol-stimulated cyclase activities were decreased by 30-40% after castration and restored to control values after testosterone treatment. In addition, Mn2+ + forskolin-stimulated activity in the presence or absence of GDP beta S was lower (-30%) after castration and normalized after testosterone treatment. Finally, the effects of testosterone described above were completely abolished when the potent androgen receptor antagonist RU 23908 was administered together with testosterone. These results indicate that both the inhibitory and stimulatory responses of adenylate cyclase are promoted by testosterone through an androgen receptor-dependent mechanism; promotion of the inhibitory response concerns specifically the alpha 2-receptor-mediated pathway, whereas promotion of the stimulatory response appears unspecific and mainly due to increased activity of the cyclase catalytic subunit.     

5′-Guanylylimidodiphosphate, A Potent Activator of Adenylate Cyclase Systems in Eukaryotic Cells

5'-Guanylylimidodiphosphate (Gpp(NH)-p) stimulates adenylate cyclase [ATP-pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in plasma membranes isolated from frog and salmon erythrocytes, from rat adrenal, hepatic, and fat cells, and from bovine thyroid cells. The nucleotide acts cooperatively with the various hormones (glucagon, secretin, ACTH, thyrotropin, and catecholamines) that stimulate these adenylate cyclase systems with resultant activities that equal or exceed those obtained with hormone plus GTP or with fluoride ion. In the absence of hormones, Gpp(NH)p is a considerably more effective activator than GTP, and, under certain conditions of incubation, stimulates rat fat cell adenylate cyclase to levels of activity (about 20 nmoles of 3',5'-adenosine monophosphate mg protein per min) far higher than reported hitherto for any adenylate cyclase system examined. The nucleotide activates frog erythrocyte adenylate cyclase when the catecholamine receptor is blocked by the competitive antagonist, propranolol, and activates the enzyme from an adrenal tumor cell line which lacks functional ACTH receptors. In contrast, Gpp(NH)p does not stimulate adenylate cyclase in extracts from Escherichia coli B. Gpp(NH)p appears to be a useful probe for investigating the mechanism of hormone and nucleotide action on adenylate cyclase systems in eukaryotic cells.     

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)     

Regulation by calmodulin of adenylate cyclase activity in anterior pituitary

The control of adenylate cyclase activity is exerted through separate components: receptors, guanyl nucleotide-binding protein, catalytic subunit, calmodulin (CaM), and divalent cations. We examined the roles of CaM, Mg+2, and Ca+2 in the regulation of adenylate cyclase activity in plasma membranes from anterior pituitary. Adenylate cyclase activity was reduced with 2.5 mM EGTA, 125 micrograms/ml compound 48/80, and 200 microM trifluoperazine, which are known inhibitors of CaM in a variety of tissues. Mg+2, in excess of ATP, stimulated adenylate cyclase activity. Ca+2 produced a biphasic effect on adenylate cyclase activity over the concentration range of 0.1-10 mM, exhibiting inhibition up to 2.0 mM and stimulation above that. GTP, 5'-guanylylimidodiphosphate, and F- each enhanced adenylate cyclase activity, but activity stimulated after each of these agents was reduced or returned toward control values by administration of compound 48/80 or trifluoperazine. In the absence of free Mg+2 (i.e. Mg+2 in excess of ATP concentrations), 10 mM Ca+2 produced marked stimulation of adenylate activity which was not reduced by trifluoperazine. We concluded that the plasma membranes from anterior pituitary possess a CaM-dependent adenylate cyclase and that activation of adenylate cyclase by guanyl nucleotide-binding protein requires CaM. Ca+2 may have allosteric binding sites on the catalytic subunit, and Ca+2 and Mg+2 appear to have antagonistic effects at different binding sites.     

beta-Adrenergic receptor agonists increase phospholipid methylation, membrane fluidity, and beta-adrenergic receptor-adenylate cyclase coupling.

The beta-adrenergic agonist L-isoproterenol stimulated the enzymic synthesis of phosphatidyl-N-monomethylethanolamine and phosphatidylcholine in rat reticulocyte ghosts containing the methyl donor S-adenosyl-L-methionine. The stimulation was stereospecific, dose-dependent, and inhibited by the beta-adrenergic agonist propranolol. The addition of GTP inside the resealed ghosts shifted the dose-response of phospholipid methylation by L-isoproterenol to the left by 2 orders of magnitude. Direct stimulation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] with sodium fluoride or cholera toxin did not increase the methylation of phospholipids. At a concentration of S-adenosyl-L-methionine that stimulates synthesis of phosphatidyl-N-monomethylethanolamine, the activity of isoproterenol-sensitive adenylate cyclase was increased 2-fold without changes in the basal activity of adenylate cyclase and the number of beta-adrenergic receptors. The increase of phospholipid methylation by L-isoproterenol decreased membrane viscosity and increased translocation of methylated lipids. These findings indicate that enhancement of phospholipid methylation by L-isoproterenol decreases membrane microviscosity and thus increases lateral movement of the beta-adrenergic receptors and coupling with adenylate cyclase.     

Involvement of the regulatory protein (Ns) in the maturation of ACTH-sensitive adenylate cyclase of ovine fetal adrenal during late gestation

The responsiveness to several stimuli of the adenylate cyclase of crude adrenal membranes from fetal and neonate lambs has been measured under different conditions. The response to Gpp(NH)p, ACTH1-24 + Gpp(NH)p and NaF was significantly lower in membranes from fetuses than from neonate lambs, whereas the response to forskolin was similar. Addition of human erythrocyte ghosts to fetal adrenal membranes enhanced the stimulation of cyclase by NaF and Gpp(NH)p, which became similar to that observed in membranes from neonates. However, such a "complementation' did not enhance the response of the enzyme to ACTH1-24. The adenylate cyclase activity displayed a biphasic response to GTP under basal conditions or when stimulated by forskolin. The magnitude of the inhibition achieved with 10(-3) M GTP was similar in fetal and neonatal adrenal membranes. These data, together with previous results (Endocrinology, 1981, 108, 2114-2119), show that the maturation of the ACTH-sensitive adenylate cyclase system of the ovine fetal adrenal gland involves an enhancement of the number of ACTH receptors and a development of the Ns subunit, whereas the catalytic site is hardly (if at all) involved in this process.