Effects of prostaglandins and catecholamines on rat spleen adenylate cyclase in vitro

The results reported here show some characteristics of adenylate cyclase (EC 4.6.1.1) derived from homogenates of rat spleen, and describe the in vitro stimulation of this enzyme by prostaglandins, nucleotides, and F⁻ under conditions where cyclic nucleotide degradative pathways are effectively inhibited.Particulate fractions from rat spleen homogenates contain high adenylate cyclase activities, and the highest specific activity is recovered in a particulate fraction prepared by low speed (1200 × g) centrifugation. Activity found in all particulate fractions is stimulated by fluoride, prostaglandins E₁ and E₂, catecholamines, and purine nucleotides. No stimulation is caused by prostaglandins F_1α and F_2α. Stimulation by prostaglandin E₁ or E₂ is augmented by GTP and other purine nucleotides, and stimulation by the combination of GTP and prostaglandin E₁ is equal to that caused by optimal fluoride concentrations. Stimulation c caused by L-isoproterenol is additive to that caused by GTP but is not increased by GTP.     

Changes in adenylate cyclase and phosphodiesterase activities during the growth cycle of adult rat hepatocytes in primary culture

Long-term primary adult rat hepatocyte cultures show growth-state-dependent changes in adenylate cyclase and cAMP phosphodiesterase activities. Cellular adenylate cyclase activity decreases to undetectable levels within 1 day postplating, reappears on Days 4-5, and becomes maximal on Day 9. Membrane adenylate cyclase and cellular cAMP formation are insensitive to glucagon during log phase (Days 4-8) but not during lag (Day 1) or stationary phase (Day 12). Cyclic AMP phosphodiesterase activities (soluble and particulate) fall approximately equal to 70% by Day 2 but recover as proliferation begins. By contrast, the particulate phosphodiesterase assayed at 100 microM cAMP, decreased during Days 0-2. These observations simulate changes seen during liver proliferative transitions in vivo and, therefore, further support the use of these cultures as a developmental model.     

Inhibition of pituitary adenylate cyclase by atrial natriuretic factor

The effect of synthetic rat atrial natriuretic factor (ANF) on adenylate cyclase activity was studied in rat anterior and posterior pituitary homogenates. ANF (Arg 101-Tyr 126) inhibited adenylate cyclase activity in anterior and posterior pituitary homogenates in a concentration dependent manner. The maximum inhibitions observed were 42% in anterior pituitary with an apparent Ki of 10(-10) M, and 25% with an apparent Ki of 10(-11) M in posterior pituitary. Corticotropin-releasing factor (CRF), vasoactive intestinal peptide (VIP) and prostaglandins (PGE1) stimulated adenylate cyclase to various degrees in anterior pituitary homogenates and ANF inhibited the stimulatory effect of all these hormones. In addition ANF was also able to inhibit the stimulation exerted by NaF and forskolin which activate adenylate cyclase by receptor independent mechanism. Similarly, the stimulatory effects of N-Ethylcarboxamide adenosine (NECA), NaF and forskolin on adenylate cyclase in posterior pituitary homogenates were also inhibited by ANF. This is the first study demonstrating the inhibitory effect of ANF on pituitary adenylate cyclase.     

A simple method for routine determination of adenylate cyclase activity in intact fat cells

The role of metal ions in the biological activity of natural and synthetic metalloproteins is an area of active scientific interest. It would be advantageous in such work to be able to analyze for many elements simultaneously. Methods for this purpose should be rapid, require little or no elaborate pretreatment, and consume only small amounts of material. Of particular interest is the resulting metal-ion distribution in synthetically prepared preparations where the metal-free form of the enzyme can act as a scavenger for unwanted elements during the synthesis.Proton-induced X-ray emission analysis (PIXEA) has been applied, in our laboratory and in others, to the analysis of trace amounts of metallic and nonmetallic elements in a variety of matrices (1–3).In general, the sensitivity in terms of μg/g of this method is comparable to conventional tube-excited X-ray fluorescence, but PIXEA offers the advantage of a higher absolute sensitivity. The technique is nondestructive in the sense that the sample is not consumed in the process and, in addition, a broad range of elements can be simultaneously detected at the submicrogram level. Of particular significance is the small total-sample requirement in that, for the studies reported here, only milligram amounts of protein were required for a total analysis.     

Stimulation of adenylate cyclase by adenosine and other agonists in mesenteric artery smooth muscle cells in culture

An adenosine-sensitive adenylate cyclase has been characterized in cultured mesenteric artery smooth muscle cells. N-Ethylcarboxamide-adenosine (NECA), N-Methylcarboxamide-adenosine (MECA), L-N6-phenylisopropyladenosine (PIA) and 2-chloroadenosine (2-cl-Ado) all stimulated adenylate cyclase in a concentration dependent manner. NECA was the most potent analog (EC50, 1 microM), whereas PIA (EC50, 15 microM), 2-Cl-Ado (EC50, 15 microM) and MECA (EC50, 24 microM), were less potent and had efficacies relative to NECA of 0.61, 0.61 and 0.65, respectively. Adenosine showed a biphasic effect: stimulation at lower concentrations and inhibition at higher concentrations, whereas 2' deoxyadenosine only inhibited adenylate cyclase activity. The stimulatory effect of NECA on adenylate cyclase was dependent on metal ion concentration and was blocked by 3-isobutyl-l-methylxanthine (IBMX) and 8-phenyltheophylline (8-PT). Adenylate cyclase from these cultured cells was also stimulated by other agonists such as epinephrine, norepinephrine, prostaglandins, dopamine, NaF and forskolin. The stimulation of adenylate cyclase by isoproterenol, epinephrine and norepinephrine was blocked by propranolol but not by phentolamine. On the other hand, phentolamine, propranolol and flupentixol all inhibited dopamine-stimulated adenylate cyclase activity. In addition, the stimulation by an optimal concentration of PIA was additive or almost additive with maximal stimulation caused by catecholamines and prostaglandins. These data indicate the presence of adenosine (Stimulatory "Ra"), catecholamine and prostaglandin receptors in mesenteric artery smooth muscle cells and suggest that these agents may exert their physiological actions through their interaction with their respective receptors coupled to adenylate cyclase.     

Studies on the mechanism of inhibition of amphibian oocyte adenylate cyclase by progesterone

Progesterone treatment induces the meiotic maturation of Xenopus laevis oocytes. Previous evidence indicates that this hormonal effect may be due to inhibition of oocyte adenylate cyclase. The present work studies several aspects of the mechanism of adenylate cyclase inhibition by this hormone. Forskolin greatly stimulates oocyte adenylate cyclase in the absence of guanine nucleotides and this activity is not sensitive to progesterone inhibition. In addition the forskolin-activated enzyme is not inhibited by a wide range of guanine nucleotide, in the presence or absence of hormone. The time course of cAMP synthesis catalyzed by oocyte adenylate cyclase in the presence of guanyl-5′_l-imidodiphosphate (Gpp(NH)p) shows an initial lag period that does not depend on the concentration of Gpp(NH)p. Progesterone causes a very significant increase in the hysteresis of the reaction, at least doubling the half-time of enzyme activation. The hormonal effect on the lag cannot be reversed by saturating concentrations of Gpp(NH)p. Progesterone also decreases the steady-state rates of the reaction. This effect, however, depends on the concentration of Gpp(NH)p. High concentrations of Gpp(NH)p almost completely reverse the inhibition of the steady-state rates. Progesterone does not inhibit if it is added to the reaction after the initial lag period. Guanosine-5′-O-(2-thiodiphosphate) (GDP-β-S) is an efficient competitive inhibitor of Gpp(NH)p activation of adenylate cyclase. Progesterone inhibition is observed at all concentrations of GDP-β-S and is potentiated at high ratios of GDP-β-S to Gpp(NH)p. These data indicate that progesterone inhibits by interfering with the activation of the N_s subunit of the enzyme by guanine nucleotides, rather than through a mechanism involving a separate N_i subunit.     

Solubilization and chromatographic separation of gonadotropin receptor from adenylate cyclase in ovarian preparations

The distribution of gonadotropin receptor and adenylate cyclase activities has been monitored in Sepharose 6B chromatographic eluates of a Lubrol solubilized fraction of 25 day old rat ovarian tissue. The receptor was resolved from the adenylate cyclase activity. Chromatography of a gonadotropin desensitized rat ovarian preparation showed that the desensitized preparation contained normal amounts of the adenylate cyclase activity when compared with the control ovarian preparation, but there was a marked reduction in the receptor activity in the desensitized ovaries. This study demonstrates that in ovarian tissue adenylate cyclase is a separate entity distinct from the receptor molecule and that the desensitization causes a reduction in the receptor activity with no detectable change in the catalytic and chromatographic properties of the adenylate cyclase.     

The subcellular localization of calmodulin, cyclic AMP phosphodiesterase, and adenylate cyclase in bovine adrenal medulla

The subcellular localization of calmodulin, cyclic nucleotide phosphodiesterase, and adenylate cyclase was studied in bovine adrenal medulla. Approximately 70% of the calmodulin and 90% of the cAMP phosphodiesterase activities were found colocalized in the cytoplasm. The subcellular distribution of adenylate cyclase closely paralleled the distribution of acetylcholinesterase, a marker for plasma membranes. The fraction of calmodulin which is particulate in nature has a distribution profile very similar to that of adenylate cyclase. The chromaffin granule fraction contained only 0.86% of the total cAMP phosphodiesterase, 0.41% of the total adenylate cyclase, and 1.4% of the total calmodulin.     

cAMP-stimulated adenylate cyclase activation in Dictyostelium discoideum is inhibited by agents acting at the cell surface

The ability of Dictyostelium discoideum amoebae to synthesize and secrete cAMP in response to exogenous cAMP is called cAMP signaling. Concanavalin A is a potent, rapid, noncompetitive inhibitor of this response, with the rate of inhibition consistent with its rate of binding. The concanavalin A does not deplete cellular ATP, alter cAMP binding to its surface receptors, or affect basal adenylate cyclase activity, but blocks the cAMP-stimulated activation of adenylate cyclase. Therefore, concanavalin A appears to inhibit a step between the receptor and the adenylate cyclase which is necessary for the transduction of the cAMP signal. Wheat germ agglutinin, a polyclonal antibody against an 80-kDa glycoprotein, four monoclonal antibodies against the amoebal surface, and a chemical cross-linking agent which reacts with cell surface primary amines also inhibit signaling. To determine the importance of cross-linking in the inhibition, succinylated concanavalin A and the unlinked, reactive portion of the chemical cross-linker were tested and found to be relatively ineffective inhibitors. Thus it appears that ligands capable of cross-linking molecules on the external surface of D. discoideum amoebae inhibit cAMP signaling. It is proposed that these cross-linking agents prevent membrane or cytoskeletal rearrangement and that this rearrangement must occur before the adenylate cyclase is activated.     

Differences in the cation sensitivity of adenylate cyclase from heart and skeletal muscle: modification by guanyl nucleotides and isoproterenol

Low concentrations of Mn²⁺ supported the basal adenylate cyclase activity in crude and purified sarcolemmal membranes from cardiac muscle more effectively than did relatively high concentrations of Mg²⁺; at saturating concentrations the cyclase activities obtained with Mg²⁺ or Mn²⁺ were similar. In contrast, Mg²⁺ supported the basal cyclase activities of crude membrane fractions and purified sarcolemmal membranes from skeletal muscle far more effectively than did Mn²⁺; at saturating concentrations of either metal ion the Mg²⁺-supported cyclase activities were 5- to 10-fold greater than Mn²⁺-supported activities. Further, compared to Mg²⁺, Mn²⁺ supported the cyclase activities very poorly in all the primary subcellular fractions of skeletal muscle, whereas this cation was at least as effective as Mg²⁺ in all fractions of cardiac muscle. The apparent affinities of the cyclase for Mn²⁺ in heart as well as skeletal muscle appeared to be greater compared to those for Mg²⁺. The skeletal muscle cyclase displayed greater apparent affinity for MnATP^2? (app. K_m 0.10 mm) compared to MgATP^2? (app. K_m 0.32 mm) whereas the heart enzyme displayed greater apparent affinity for MgATP^2? (app. K_m 0.07 mm) compared to MnATP^2? (app. K_m 0.19 mm). Following preactivation with guanyl-5′-yl imidodiphosphate and isoproterenol, Mn²⁺ (0.15 to 2 mm) supported the cyclase activity of skeletal muscle even more effectively than did optimally effective concentrations of Mg²⁺. With the heart enzyme the relatively greater potency of Mn²⁺ persisted following preactivation. Significant enhancement in the Mn²⁺-sensitivity of skeletal muscle cyclase was also observed when assayed in the presence of GTP and isoproterenol or in the presence of NaF. Preactivation of both heart and skeletal muscle cyclases caused selective enhancement in the enzyme's apparent affinity for free Me²⁺ (Mg²⁺ or Mn²⁺) without influencing the apparent K_m for MeATP^2? (MgATP^2? or MnATP^2?). Evidences were obtained to show that the poor effectiveness of Mn²⁺ in supporting the basal activity of skeletal muscle cyclase is not related to (a) potentiation by Mn²⁺ of adenosine-mediated inhibition of the cyclase, (b) Mn²⁺-induced lability of the cyclase, (c) indirect effects of Mn²⁺ on ATP-regenerating system, or (d) the presence of different cation-specific molecular forms of the cyclase. It is also shown that the onset of enhanced Mn²⁺ sensitivity of the skeletal muscle enzyme following preactivation is not accompanied by a general loss of cation specificity of the cyclase. These results suggest that cations support the catalytic activity of adenylate cyclase by interacting with an enzymeregulatory free metal binding site and that the differential cation sensitivity of nonactivated (basal) cyclases from heart and skeletal muscle is likely due to differences in the properties of such an allosteric metal site. Furthermore, the metal site appears to undergo a conformational change following interaction of the cyclase system with the guanyl nucleotide and isoproterenol since the cation sensitivity of the cyclase and the relative potency of cations depend on the conformational status of the enzyme.     

Effect of specific trinitrophenylation of the lysine epsilon amino group of glucagon on receptor binding and adenylate cyclase activation

The trinitrophenyl group was specifically introduced into the ?-amino group of glucagon by reaction of N^α-citraconyl glucagon with trinitrobenzenesulfonic acid. The N^α-citraconyl blocking group was subsequently removed by acid treatment yielding N^?-trinitrophenyl glucagon which was purified by anion-exchange chromatography. The derivative showed less secondary structure as measured by circular dichroism than the native hormone at pH 8.0 and at pH 2.0 in the presence of sodium dodecyl sulfate. The analog possessed 4–5% the potency of glucagon in stimulating adenylate cyclase with 90% maximal stimulation and possessed 30% the potency of glucagon in competing for glucagon-specific receptor sites in hepatic plasma membranes. Although the structure of N^?-trinitrophenyl glucagon is very similar to N^?-4-azido-2-nitrophenyl glucagon, the photoaffinity antagonist synthesized by M. D. Bregman and D. Levy [(1977) Biochem. Biophys. Res. Commun., 78, 584–590.], the biological activities of the two are different. Possible explanations for these differences are discussed.     

Activating effect of destomycin A on adenylate cyclase from several animal tissues

Highly purified sarcolemmal membranes were prepared from pig heart homogenates by differential and density gradient centrifugations. The membrane fragments exhibit ATP-dependent Ca²⁺-transport and Na⁺/Ca²⁺-exchange activities. ATP-dependent Ca²⁺-transport (K^Ca2+_0.5 = 0.3 μM; V_max = 4.6 nmol Ca²⁺?mg protein^?1 ?min^?1)_is not stimulated by oxalate. Ca²⁺-uptake is also not supported by p-nitrophenylphosphate. Preincubation of sarcolemma with MgATP, calmodulin and catalytic subunit of cyclic AMP-dependent protein kinase stimulates active Ca²⁺-transport 1.8-fold. The effects of calmodulin and catalytic subunit are potentiating rather than additive. A large portion of the Ca²⁺ additionally accumulated after prephosphorylation of membranes is exchangable for Na⁺ via the Na⁺/Ca²⁺-exchange system.     

The effect of temperature on the activity of the adenylate cyclase system of liver plasma membranes

The rat liver adenylate cyclase system shows a discontinuity in the Arrhenius plots at 20°C in the nonstimulated activity (basal) with activation energies of 16 and 28 Kcal/mole. The discontinuity disappears when the enzyme is stimulated either by glucagon, sodium fluoride, 5′ guanylyl-imidodiphosphate or glucagon plus 5′ guanylyl-imidodiphosphate and the energy of activation was the same with all the compounds tested. If the activator was initially in contact with the membranes at 0°C the energy of activation was similar to that observed below the break (26 Kcal/mole) but it changed to that above the break if the compound contacted the membranes at temperatures above the break (22–24°C). We discuss the possibility of two different conformations of the enzyme; both conformations can be “frozen” by any of the compounds tested, “isolating” the enzyme from any subsequent physical change of the membrane due to temperature.     

Structure of membrane-associated and solubilized uterine adenylate cyclase. Evidence against activation through dissociable subunit interactions in the lipid bilayer

Adenylate cyclase was extracted from the rat uterus with Lubrol PX in a form which remained soluble following centrifugation for 60 min at 100,000g. The soluble enzyme was stimulated by both Mn+2 and by guanyl-5'-yl-imidodiphosphate (Gpp(NH)p), indicating that both the catalytic subunit (C) and the guanyl nucleotide-binding coupling factor (N) had been extracted. Catalytic activity was bound by a GTP-affinity resin only under conditions which resulted in irreversible activation of the native (particulate) form of the enzyme and could be eluted under acidic conditions shown to reverse the activated state. The S020,w of the soluble enzyme in both its activated and unactivated state was determined by linear sucrose gradient centrifugation. Activation by prolonged treatment with Gpp(NH)p did not alter the S020,w of the enzyme whether treatment was carried out before or after solubilization. The chaotrope LiBr (0.4 M) reduced the S020,w of the soluble enzyme but its smaller size was still not altered by activation with Gpp(NH)p. These results indicate that most adenylate cyclase activity in uterine membranes exists as a preformed complex between the catalytic subunit and the coupling factor: NC. The existence of this complex explains some of the temperature-dependent properties previously described for this form of the enzyme and suggests that dissociable interactions between the subunits do not play a role in the activation of C by guanyl nucleotides.     

The immunologic control mechanism against cholera toxin: II. Stimulation of adenylate cyclase without fluid secretion

Cholera toxin-stimulated fluid secretion by the ileum is believed to be mediated by an adenylate cyclase-cyclic AMP mechanism. Immunization against cholera toxin (CT) reduces CT binding to microvillus membranes and suppresses fluid secretion following CT challenge. The present study disclosed no alteration in the fluid absorption rates. However, suppression of fluid secretion occurred despite maximal stimulation of mucosal adenylate cyclase in immunized animals.     

Guanine nucleotide activation and inhibition of adenylate cyclase as modified by islet-activating protein, pertussis toxin, in mouse 3T3 fibroblasts

Guanine nucleotide regulation of membrane adenylate cyclase activity was uniquely modified after exposure of 3T3 mouse fibroblasts to low concentrations of islet-activating protein (IAP), pertussis toxin. The action of IAP, which occurred after a lag time, was durable and irreversible, and was associated with ADP-ribosylation of a membrane Mr = 41,000 protein. GTP, but not Gpp(NH)p, was more efficient and persistent in activating adenylate cyclase in membranes from IAP-treated cells than membranes from control cells. GTP and Gpp(NH)p caused marked inhibition of adenylate cyclase when the enzyme system was converted to its highly activated state by cholera toxin treatment or fluoride addition, presumably as a result of their interaction with the specific binding protein which is responsible for inhibition of adenylate cyclase. This inhibition was totally abolished by IAP treatment of cells, making it very likely that IAP preferentially modulates GTP inhibitory responses, thereby increasing GTP-dependent activation and negating GTP-mediated inhibition of adenylate cyclase.     

Stimulatory and inhibitory effects of guanyl-5'-yl imidodiphosphate on adenylate cyclase activity of cardiac sarcolemma.

A nucleotide phosphohydrolase-resistant analog of GTP, guanyl-5′-yl imidodiphosphate [GMP-P(NH)P], caused stimulation of basal adenylate cyclase activity of cardiac sarcolemma when ethylene glycol bis(β-aminoethyl ether)- N,N′-tetraacetic acid (EGTA) was absent in the assay mixture, whereas the nucleotide, in the presence of EGTA, inhibited basal cyclase activity. GTP, GDP, GMP, and guanosine failed to show such an inhibition of basal enzyme activity. The degree of both stimulatory and inhibitory effects of GMP-P(NH)P depended on the concentration of magnesium ions. The apparent affinities toward magnesium ions of the metal binding site and toward MgATP^2? of the catalytic site of control and ?GMP-P(NH)P-inhibited” enzyme were similar. Isoproterenol reversed the inhibitory effect, whereas calcium ions failed to revert it. Both in the presence and absence of EGTA, GMP-P(NH)P plus isoproterenol caused a synergistic stimulation of the enzyme activity, the degree of stimulation being lower with EGTA present. Exposure of sarcolemma to GMP-P(NH)P (with and without isoproterenol and in the absence and presence of EGTA) caused an activation of adenylate cyclase, the degree of activation being higher with isoproterenol present. The activated enzyme displayed increased affinity toward Mg²⁺ at the metal binding site. When activated enzyme preparations were assayed in the presence of EGTA, reversal of the activated state was observed in the case of the GMP-P(NH)P-activated enzyme but not in the case of the GMP-P(NH)P + isoproterenol-activated enzyme.     

Insulin action on Escherichia coli Regulation of the adenylate cyclase and phosphotransferase enzymes

Insulin action on Escherichia coli was studied using wild type E. coli B/r and K12 strains and a number of phosphoenolpyruvate phosphotranferase mutants. In vivo, the effects of insulin on the differential rate of tryptophanase synthesis, The rate of α-methyglucoside uptake and the rate of growth on glucose were determined in E. coli B/r. in vitro, the effect of insulin on the adenylate cyclase and the phosphotransferase activities was determined using toluenized cell preparations of E. coli B/r, E. coli K12 and phosphotransferase mutant strains. The specificity of insulin action on E. coli was determined using glucagon, vasopressin and somatropin as well as insulin antisera. Results show the specific action of insulin n E. coli, inhibiting tryptophanase induction and adenylate cyclase activity, while stimulating growth on glucose and uptake and phosphorylation of α-methylglucosode     

Gossypol inhibition of adenylate cyclase

Gossypol, a polyphenolic binaphthalene -dialdehyde reputed to exert contraceptive action in males, reversibly inhibits adenylate cyclase [ATP pyrophosphate lyase (cyclizing), EC 4.6.1.1] in a concentration-dependent manner. In membranes prepared from a variety of organs, the half-maximal inhibitory concentration (IC50) ranges from 75 microM (rat Leydig tumor cells) to 250 microM (rat liver membranes). Kinetic studies using partially purified catalytic subunit isolated from bovine testis show that gossypol is competitive with ATP with an apparent Ki of 110 microM. These data suggest that gossypol inhibition of adenylate cyclase is due to direct interaction at the nucleotide-binding domain of the catalytic subunit of the enzyme.