Serotonin sensitive adenylate cyclase in horse brain synaptosomal membranes.

In different membranal preparations isolated from horse brain stritum we have shown the existence of an adenylate cyclase system sensitive to serotonin (5-HT). Activation of the adenylate cyclase was determined by measuring cAMP using a radioimmunoassay. This serotoninergic sensitive enzyme is characterized by a high apparent affinity constant (in the nanomolar range), located on synaptosomal membranes. It is inhibited by antiserotoninergic drugs (cyproheptadine, cinanserin, methysergide, LSD), and synergistically activated by GTP. This serotoninergic activation is clearly additive to the activation induced by dopamine. It appears different from the adenylate cyclase system previously described in the literature which is also activated by 5-HT, but which has a low apparent affinity constant (in the micromolar range); the latter is apparently located in non-synaptosomal membranes, and its activation by 5-HT is non-additive to the activation induced by dopamine.The serotoninergic sensitive adenylate cyclase reported in this study, might be related to the serotoninergic binding system which we have previously described which has similar affinity constant, a similar subcellular distribution and which is inhibited in the same concentration ranges by antiserotoninergic drugs. These two systems might represent a synaptosomal serotoninergic receptor complex.     

Inhibition of gonadotropin sensitive adenylate cyclase by ovarian follicular fluid.

Follicular fluid obtained from medium or large bovine ovarian follicles inhibited ovarian luteinizing hormone/human chorionic gonadotropin sensitive adenylate cyclase in a dose-dependent manner (I₅₀ = 3 mg follicular fluid protein/ml). The inhibitory activity was excluded by Sephadex G-10 and was fully retained following treatment with charcoal. Fluoride-stimulated enzyme activity was not inhibited. Binding of ¹²⁵I human chorionic gonadotropin to ovarian plasma membranes was only slightly reduced by the follicular fluid. The post-microsomal supernatant of homogenates from ovaries of immature (27-day-old) rats collected 24–36 h after treatment with 15 i.u. of pregnant mare serum gonadotropin also inhibited luteinizing hormone-sensitive adenylate cyclase. The extent of this inhibition seemed to decline with follicular maturation. The possibility is raised that ovarian sulfated glycosaminoglycans are responsible for the observed inhibition of adenylate cyclase.     

Corticotropin-releasing factor stimulates adenylate cyclase activity in the anterior pituitary gland

Ovine corticotropin-releasing factor (CRF) stimulates adenylate cyclase activity in rat anterior pituitary homogenate at an ED₅₀ value of 70 nM. GTP increases the stimulatory effect of CRF on [³²P] cyclic AMP formation in a rat adenohypophysial particulate fraction and in bovine anterior pituitary plasma membranes. The present data show that CRF stimulates adenylate cyclase activity in the anterior pituitary gland at least partly through a guanyl nucleotide-dependent mechanism.     

Cytochemical localization of adenylate cyclase activity in the rat anterior pituitary

Summary The cytochemical localization of adenylate cyclase was studied in relation to the secretory function of the anterior pituitary glands of male rats. The reaction product of adenylate cyclase was localized on the outside of plasma membranes, but was not detected intracellularly. High activity of adenylate cyclase was detected on somatotrophs and microvilli of follicular cells, whereas no activity was found on thyrotrophs or corticotrophs. Although most of the gonadotrophs showed little or no adenylate-cyclase activity, some was detected in a small number of gonadotrophs in the central portion of the gland. In somatotrophs, activity was not detected on the plasma membranes facing perivascular spaces where exocytotic extrusion of secretory granules was frequently observed, although the remaining areas of plasma membranes of the same somatotrophs were associated with high levels of adenylate-cyclase activity. These findings indicate that the association of a high level of adenylate-cyclase activity is not directly related to the ability of the plasma membranes to fuse with secretory granule membranes.     

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans. I. Inhibition by heparin of gonadotrophin-stimulated ovarian adenylate cyclase.

Heparin inhibits (I50 = 2 microgram/ml) the activity of luteinizing hormone and human chorionic gonadotropin-stimulated adenylate cyclase in purified rat ovarian plasma membranes. Unstimulated enzyme activity and activity stimulated by NaF, GTP or guanosine 5'-(beta,gamma-imido)triphosphate were inhibited to a lesser extent. Human chorionic gonadotropin binding to this membrane preparation was inhibited by heparin (I50 = 6 microgram/ml). The inhibition with respect to hormone concentration was of a mixed type for hormone binding and adenylate cyclase stimulation. Inhibition by heparin was not eliminated at saturating hormone concentration. The degree of inhibition was unaffected by the order in which enzyme, hormone and heparin were introduced into the assay system. Heparin (3 microgram/ml) did not affect the pH activity relationship of basal and hormone-stimulated adenylate cyclase activity and did not change the dependence of enzyme activity on magnesium ion concentration. The inhibitory action of heparin cannot be solely attributed to interference with either catalysis or hormone binding. The possibility is considered that the highly charged heparin molecule interferes with enzyme receptor coupling, by restricting the mobility of these components or by effecting their conformation.     

Growth hormone-releasing factor stimulates adenylate cyclase activity in the anterior pituitary gland

Synthetic human pancreatic growth hormone-releasing factor (hpGRF) (1–40)-NH₂ stimulates adenylate cyclase activity in rat anterior pituitary particulate fraction at an ED₅₀ value of approximately 150 nM. GTP more than doubles the stimulatory effect of hpGRF aand PGE₂ on [³²p] cyclic AMP formation. The present data show that hpGRF as well as PGE₂, another potent stimulus of GH secretion, act at least partly, through GTP-dependent mechanisms in their coupling with adenylate cyclase.     

Soluble adenylate cyclase activity in Neurospora crassa.

A soluble form of adenylate cyclase was extracted from mycelia of Neurospora crassa wild-type strains. This enzyme activity was purified by chromatography on hexyl-amino-Sepharose, agarose and Blue Sepharose and preparative polyacrylamide-gel electrophoresis. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, peak fractions from the later purification steps showed a main polypeptide band with an apparent molecular weight of about 66 000. The following hydrodynamic and molecular parameters were established for the Neurospora soluble adenylate cyclase activity: sedimentation coefficient, 6.25 S; Stokes radius, 7.3 nm; partial specific volume, 0.74 ml/g; molecular weight, 202 000; frictional ratio, 1.65. The isoelectric point of this enzyme activity was 4.65. The enzyme was not activated by GTP, [beta gamma-imido]GTP, fluoride or cholera toxin.     

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans I. Inhibition by heparin of gonadotropin- stimulated ovarian adenylate cyclase

Heparin inhibits (I₅₀ = 2 μg/ml) the activity of luteinizing hormone and human chorionic gonadotropin-stimulated adenylate cyclase in purified rat ovarian plasma membranes. Unstimulated enzyme activity and activity stimulated by NaF, GTP or guanosine 5′-(β,γ-imido)triphosphate were inhibited to a lesser extent. Human chorionic gonadotropin binding to this membrane preparation was inhibited by hepatin (I₅₀ = 6 μg/ml). The inhibition with respect to hormone concentration was of a mixed type for hormone binding and adenylate cyclase stimulation. Inhibition by heparin was not eliminated at saturating hormone concentration. The degree of inhibition was unaffected by the order in which enzyme, hormone and heparin were introduced into the assay system. Herapin (3 μg/ml) did not affect the pH activity relationship of basal and hormone-stimulated adenylate cyclase activity and did not change the dependence of enzyme activity on magnesium ion concentration. The inhibitory action of heparin cannot be solely attributed to interference with either catalysis or hormone binding. The possibility is considered that the highly charged herapin molecule interferes with enzyme receptor coupling, by restricting the mobility of these components or by effecting their conformation.     

Desensitization of vasopressin sensitive adenylate cyclase by vasopressin and phorbol esters

Desensitization of vasopressin V2 receptor-mediated adenylate cyclase was studied in canine kidney cell line, MDCK cells. Overnight treatment of MDCK cells with arginine vasopressin (AVP) resulted in a loss of vasopressin receptors and an inhibition of cAMP accumulation in response to AVP. Both the loss of receptor and reduction in cAMP accumulation were time- and AVP concentration-dependent. Desensitization was selective for AVP because cAMP formation in response to isoproterenol, prostaglandin E1 (PGE1) and forskolin was not affected by AVP pre-treatment. Pre-treatment of MDCK cells with phorbol dibutyrate (PDBu) also caused a dose-dependent inhibition of AVP mediated cAMP accumulation, but not of isoproterenol-, PGE1- and forskolin-induced cAMP accumulation. PDBu pre-treatment did not cause loss of vasopressin receptors. Instead, the affinity for vasopressin was changed by PDBu treatment. Pre-treatment of the cells with pertussis toxin (PT) had no effect on the desensitization and downregulation of vasopressin (V2) receptors, suggesting that the desensitization may not be mediated by pertussis toxin sensitive G-protein. Our data suggest that pre-treatment of MDCK cells with AVP or PDBu caused desensitization of AVP-mediated cAMP accumulation and that downregulation of V2 receptors required agonist occupancy of the receptors, whereas the affinity of the receptors was changed by phorbol ester treatment.     

Inhibition of forskolin-activated adenylate cyclase by ethanol and other solvents

Ethanol and a variety of solvents are known to activate basal and Gpp(NH)p- and hormone-stimulated adenylate cyclase. We report here that ethanol and other solvents inhibit the activation of adenylate cyclase by forskolin. In the presence of 10 microM forskolin, 2% ethanol gives about 20% inhibition and 5% ethanol gives 40% inhibition of enzyme activity. Analysis of ethanol inhibition at several forskolin concentrations suggests that inhibition is competitive versus forskolin. Thus the effect of ethanol is greater at low forskolin concentrations and minimal at high concentrations. In addition to ethanol, inhibition of forskolin activation was observed with acetone, n-butanol, t-butanol, dimethyl formamide, dioxane, methanol and n-propanol. Dimethyl sulfoxide was inhibitory only at high concentrations (10%). Since some solvent is needed to prepare forskolin solutions and to maintain solubility at higher concentrations, the inhibitory effects reported here are an important consideration in studies employing forskolin activation. To minimize solvent inhibition we recommend that dimethyl sulfoxide be used to prepare forskolin solutions. At concentrations of 5% and less, dimethyl sulfoxide gives little if any inhibition of forskolin activation and causes only small increases in basal activity.     

Mouse neuroblastoma adenylate cyclase. Adenosine and adenosine analogues as potent effectors of adenylate cyclase activity.

1. Intact mouse neuroblastoma NS20 cells, in the presence of cyclic adenosine 3':5'-monophosphate (cAMP) phosphodiesterase inhibitor, responded to adenosine (200 muM) and 2-chloroadenosine (200 muM) with a 20-fold increase in intracellular cAMP levels. AMP (200 muM) additions caused only a 3.5-fold cAMP level elevation. ATP, ADP, guanosine, cytidine, uridine, and guanine, all at 200 muM, had no effect on the cAMP level of these cells. 2. Homogenate NS20 adenylate cyclase activity was increased 2.5- to 4-fold by addition of 200 muM adenosine, 2-chloroadenosine, 2-hydroxyadenosine, or 8-methylaminoadenosine. Prostaglandin E1 additions (1.4 muM) produced about an 8-fold stimulation of homogenate cyclase activity. The Km of homogenate cyclase activation by adenosine and 2-chloroadenosine was 67.6 and 6.7 muM, respectively. Addition of 7-deazaadenosine, tolazoline, yohimbine, guanosine, cytosine, guanine, 2-deoxy-AMP, and adenine 9-beta-D-xylopyranoside, all at 200 muM were found to be without effect on homogenate NS20 adenylate cyclase. Two classes of inhibitors of homogenate NS20 adenylate cyclase activity were observed. One class, which included AMP, adenine, and theophylline, blocked 2-chloroadenosine but not prostaglandin E1 stimulation of cyclase. Theophylline was shown to be a competitive inhibitor of 2-chloroadenosine, with a Ki of 35 muM. The second class of inhibitors, which included 2'- and 5'-deoxyadenosine, inhibited unstimulated, 2-chloroadenosine and prostaglandin E1-stimulated homogenate cyclase activity to about the same degree. 3. Activation of NS20 homogenate adenylate cyclase by adenosine appears to be noncooperative. 4. The inhibitory action of putative "purinergic" neurotransmitters is postulated to be due to their effects on adenylate cyclase activity.     

Guanylate cyclase activity in Escherichia coli mutants defective in adenylate cyclase.

Guanylate cyclase, which catalyzes the synthesis of guanosine 3',5'-monophosphate, has been assayed in several strains of Escherichia coli. They include wild-type cells and mutants defective in adenylate cyclase, which is responsible for the synthesis of adenosine 3',5'-phosphate. Our results demonstrate that adenylate cyclase and guanylate cyclase are two different enzymes in E. coli and suggest that the gene that encodes adenylate cyclase also plays a regulatory role in the synthesis of guanylate cyclase.     

Calcium-dependent adenylate cyclase from rat cerebral cortex: Activation by guanine nucleotides

The adenylate cyclase activity of a participate preparation of rat cerebral cortex is composed of at least two contributing components, one of which requires a Ca²⁺-dependent regulator protein (CDR) for activity (Brostrom, C. O., Brostrom, M. A., and Wolff, D. J. (1977) J. Biol. Chem.252, 5677–5685). Each of these components of the activity was activated by GTP and its synthetic analog, 5-guanylylimidodiphosphate (Gpp(NH)p). The component of the adenylate cyclase activity which did not respond to CDR (CDR-independent activity) was stimulated approximately 60% by 100 μm GTP and 3.5-fold by 100 μm Gpp(NH)p. Concentrations of GTP required for maximal activation of the CDR-dependent adenylate cyclase component decreased as CDR concentrations in the assay were increased. Similarly, GTP pr Gpp(NH)p lowered the concentration of CDR required to produce half-maximal activation of this enzyme form. At saturating CDR concentrations, however, increases in activity were not observed with the addition of these nucleotides. The CDR-dependent component responded biphasically (activation followed by inhibition) to increasing free Ca²⁺ concentrations; both phases of this response occurred at lower free Ca²⁺ concentrations with GTP present in the assay. The concentration of chlorpromazine which inhibited activation of adenylate cyclase by CDR was elevated when GTP was present. The CDR-dependent form of activity, which is stabilized by CDR to thermal inactivation, was also stabilized by Gpp(NH)p. The increase in stability produced by Gpp(NH)p did not require the presence of CDR, and stabilization with both Gpp(NH)p and CDR was greater than that obtained with either Gpp(NH)p or CDR alone.     

Trypanosoma cruzi adenylate cyclase activity. Purification and characterization.

Adenylate cyclase activity associated with Trypanosoma cruzi sedimentable fractions was solubilized by treatment with the non-ionic detergent Lubrol PX and 0.5 M-(NH4)2SO4. The following hydrodynamic and molecular parameters were established for a partially purified enzyme-detergent complex: sedimentation coefficient 6.2 S; Stokes radius 5.65 nm; partial specific volume 0.83 ml/g; Mr 244 000; frictional ratio 1.33. A Mr of about 124 000 was calculated for the detergent-free protein from these parameters. The pI of this enzyme activity was 6.2. A monoclonal antibody to T. cruzi adenylate cyclase was obtained, which inhibited cyclase activities from several lower eukaryotic organisms. The T. cruzi adenylate cyclase was further purified by using this antibody in immunoaffinity chromatographic columns. Fractions obtained after this chromatography showed, on SDS/polyacrylamide-gel electrophoresis, a main polypeptide band with an apparent Mr of about 56 000, which specifically reacted with the monoclonal antibody.     

Presence of adenylate cyclase activity in Golgi and other fractions from rat liver. I. Biochemical determination

The distribution of adenylate cyclase (AC) in Golgi and other cell fractions from rat liver was studied using the Golgi isolation procedure of Ehrenreich et al. In liver homogenate the AC activity was found to decay with time, but addition of 1 mM EGTA reduced the rate of enzyme loss. The incorporation of 1 mM EGTA into the sucrose medium used in the initial two centrifugal steps of the Golgi isolation method stabilized the enzyme activity throughout the entire procedure and resulted in good enzyme recovery. In such preparations, AC activity was demonstrated to be associated not only with plasma membranes but also with Golgi membranes and smooth microsomal membranes as well. Furthermore, under the conditions used, enzyme activity was also associated with the 105,000 g x 90 min supernatant fraction. The specific activity of the liver homogenate was found to be 2.9 pmol-mg protein-1-min-1, the nonsedimentabel and microsomal activity was of the same order of magnitude, but the Golgi and plasma membrane activities were much higher. The specific activity of plasma membrane AC was 29 pmol-mg proten-1-min-1. The Golgi activity varied in the three fractions, with the highest activity (14 pmol) in GF1 lowest activity (1.8) in GF2, and intermediate activity (5.5) in GF3, when the Golgi activity was corrected for the presence of content protein, the activity in GF1 became much higher (9 x) than that of the plasma membrane while the activities in GF2 and GF3 were comparable to that of plasma membrane. In all locations studied, the AC was sensitive to NaF stimulation, especially the enzyme associated with Golgi membranes. The activities in plasma and microsomal membranes were stimulated by glucagon, whereas the Golgi and nonsedimentable AC were not.     

Effects of Mn2+ and other divalent cations on adenylate cyclase activity in rat brain.

Abstract— Mn²⁺ caused an 8-to 16-fold stimulation of adenylate cyclase activity in homogenates as well as synaptosomcs. isolated synaptic membranes, and slices prepared from rat brain. The stimulation occurred at low concentrations of Mn²⁺. with a doubling of activity at 50-60μM. and was unaffected by a 60-fold excess of Mg²⁺. Whether or not Mg²⁺ was added, inclusion of a low concentration of Mn²⁺ reduced, but did not prevent the stimulation of adenylate cyclase caused by dopaminc in homogenates of corpus striatum. In contrast, Ca²⁺. at a concentration that had little effect on basal cyclase activity, completely prevented the stimulation by dopamine. The increase of cyclase activity produced by Mn²⁺ in brain homogenates was potentiated by F^?. Other ions, notably Hg²⁺. Pb²⁺. Cu²⁺ and Zn²⁺. in order of decreasing potency, inhibited both basal and Mn^2--stimulated cyclase activity. It is proposed that the effect of Mn²⁺ on adenylate cyclase activity may involve only the catalytic subunit of the enzyme, and that the mechanism is different from that by which either dopamine or F^? stimulates the enzyme. These results suggest that the effects of low concentrations of Mn²⁺ and certain other divalent metal ions on adenylate cyclase activity may be involved in their neuropsychiatrie or other toxic effects, and that such ions may also participate in normal physiological mechanisms involving cyclic nucleotides.     

Calmodulin-dependent adenylate cyclase activity in rat cerebral cortex: effects of divalent cations, forskolin and isoprenaline

The role of calcium-calmodulin (Ca2+-CaM) in the modulation of beta-adrenergic adenylate cyclase activity in rat cerebral cortex has been studied. In addition, the effects of manganese (Mn2+) and forskolin on CaM-dependent enzyme activity were investigated. At 2 mM magnesium (Mg2+) low concentrations of Ca2+ stimulated the enzyme activity (Ka 0.25 +/- 0.08 microM), whereas higher Ca2+ levels (greater than 2 microM) inhibited the activity. No activating effect of Ca2+ was observed in CaM-depleted membranes, but the inhibitory effect persisted and the stimulatory action of Ca2+ could be restored by addition of exogenous CaM. The ability of Ca2+ to activate the enzyme was reduced by increasing concentrations of Mg2+. At 10 mM Mg2+ the apparent Ka of Ca2+ was 0.55 +/- 0.16 microM and half-maximal inhibition was observed at 80-120 microM Ca2+. A synergistic effect was observed between Ca2+ and isoprenaline on the adenylate cyclase activity. Calcium did not alter the apparent Ka of isoprenaline (0.9 +/- 0.27 microM) and isoprenaline did not change the apparent Ka of Ca2+. However, isoprenaline decreased the apparent Ka of CaM; 0.11 +/- 0.07 micrograms vs. 0.32 +/- 0.1 micrograms (0.5 ml assay mixture)-1, with and without isoprenaline, respectively. A synergistic effect was also observed between Ca2+ and forskolin, but no change in their apparent Ka values was found. Furthermore, Mn2+ was found to activate the enzyme through CaM. These data demonstrate that Ca2+ -CaM potentiates beta-adrenergic adenylate cyclase activity and thus is able to modulate neurotransmitter stimulation in cortex. Furthermore, both forskolin and Mn2+ affect CaM-dependent enzyme activity. Forskolin potentiates Ca2+-CaM stimulation, while Mn2+ increases the activity by activating the enzyme through CaM.     

Solubilization and other studies on adenylate cyclase of baker''s yeast.

1. Adenylate cyclase of Saccharomyces cerevisiae was sedimented from mechanically disintegrated preparations of yeast over an unusually wide range of centrifugal forces. 2. The enzyme was readily solubilized by Ficoll and by Lubrol PX. Lubrol caused a 2-fold activation. 3. Both particle-bound and Lubrol-solubilized enzyme had an apparent Km for ATP of 1.6 mM in the presence of 0.4 mM-cyclic AMP and 5 mM-MnCl2 at pH 6.2 and 30 degrees C. 4. The Lubrol-solubilized enzyme behaved on gel filtration as a monodisperse protein with an apparent mol.wt. of about 450000.