Guanine Nucleotide-binding Regulatory Component Research Articles

A cytosolic peptide potentiates the GTP effect on beta-adrenergic response of adenylate cyclase.

A cytosolic peptide-GTP complex that stimulates l-isoproterenol-responsive adenylate cyclase activity was identified in the rat liver. The peptide component was purified and characterized with regard to its interaction with GTP. The peptide was isolated as a complex form with GTP on a Sephadex G-25 column in 1 mM NaHCO3, and was purified as a dissociated form, with relative molecular weight (M(r)) approximately 3,000 and GTP-binding ability, in 200 mM ammonium acetate. The purified peptide alone displayed little stimulatory effect on adenylate cyclase activity, but its reassociated form with GTP clearly enhanced the effect of GTP on the enzyme activity. The isoproterenol competition curve using l-[3H]dihydroalprenolol as an antagonist ligand shifted to lower affinity by the addition of the peptide reassociated with GTP (16.5-fold shift), whereas the same concentration of GTP (1 microM) or the peptide alone had little or no effect (1.5- or 0.9-fold shift, respectively). Furthermore, the peptide enhanced the GTP effect in response to l-isoproterenol but not to glucagon, prostaglandin E1, or fluoride. These results suggest that the cytosolic peptide potentiates the effect of GTP on the agonist-beta-adrenergic receptor-stimulatory guanine nucleotide-binding regulatory component of the adenylate cyclase ternary complex.

The 5-HT receptor--G-protein--effector system complex in depression. I. Effect of glucocorticoids.

Hormonal modulation of neurotransmission emerged as a concept from the recognition that adrenocortical steroids exert profound effects at the level of receptors, G-proteins and effector units. G-proteins, a family of guanine nucleotide binding regulatory components that couple neurotransmitter receptors to various types of intracellular effector systems, appear to be a key target of glucocorticoid (GC) action in the CNS. It is thought that Gs/Gi mediates stimulation/inhibition of adenylate cyclase (AC system), which forms cyclic AMP as second messenger, while receptors stimulating phospholipase C do so through Go to produce two second messengers, inositol 1,4,5-triphosphate and diacylglycerol (PI system). Recent evidence suggests that GC increase Gs alpha-and decrease Gi alpha-protein subunit expression without affecting Go alpha. Activation of central pre- and postsynaptic 5-HT1A receptors which are linked to the Gi-AC complex, induces hypothermia and ACTH/cortisol release in rodents and humans. Compared with controls, patients with a major depressive disorder exhibit increased basal cortisol secretion associated with decreased hypothermic and ACTH/cortisol responses. The attenuated neuroendocrine and thermoregulatory response to 5-HT1A receptor activation may reflect a GC-dependent feedback inhibition of the hypothalamic-pituitary-adrenal (HPA) system and subsensitivity of the presynaptic 5-HT1A-Gi-AC complex function. Differential regulation of 5-HT1A and 5-HT2 function leading to a relative 5-HT2-Go-PI complex supersensitivity may maintain HPA hyperactivity during the course of depression. These findings corroborate recent reports that GC, via GC-GC receptor (GR) complex activated promotion of gene transcription, modify the expression 5-HT1A-coupled Gi (but not 5-HT2-coupled Go) resulting in altered sensitivity of 5-HT1A-mediated signal transduction and further support the hypothesis of a differential regulation of 5-HT1A and 5-HT2 receptor function and a GC-GR/5-HT1A-G-protein--effector system-related abnormality in depression.

Evidence for a rabbit luteal ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase

1.1. An ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase was identified in a plasma membrane fraction from rabbit corpora lutea and partially characterized by comparing the properties of the luteal transferase with those of cholera toxin.2.2. Incubation of luteal membranes in the presence of GTP and varying concentrations of NAD resulted in concentration-dependent increases in adenylyl cyclase activity.3.3. Stimulation of adenylyl cyclase by NAD and cholera toxin plus NAD was observed in the presence of GTP but not in the presence of guanosine-5′-0-(2-thiodiphosphate) or guanyl-5′-yl imidodiphosphate.4.4. NAD or cholera toxin plus NAD reduced the Kact values for luteinizing hormone to activate adenylyl cyclase 3- to 3.5-fold.5.5. NAD or cholera toxin plus NAD increased the extent to which cholate extracts from luteal membranes were able to reconstitute adenylyl cyclase activity in S49 cyc mouse lymphoma membranes.6.6. It was necessary to add ADP-ribose and arginine to the incubation mixture in order to demonstrate cholera toxin-specific ADP-ribosylation of a protein corresponding to the a subunit of the stimulatory guanine nucleotide-binding regulatory component (αGs).7.7. Treatment of luteal membranes with NAD prior to incubation in the presence of [32P]NAD plus cholera toxin resulted in reduced labeling of αGs.8.8. Endogenous ADP-ribosylation of gaGs was enhanced by Mg but was not altered by guanine nucleotide, NaF or luteinizing hormone and was inhibited by cAMP.9.9. Incubation of luteal membranes in the presence of [32P]ADP-ribose in the absence and presence of cholera toxin did not result in the labeling of any membrane proteins.

Transforming growth factor β enhances parathyroid hormone stimulation of adenylate cyclase in clonal osteoblast‐like cells

The effects of transforming growth factor beta (TGF beta) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Purified TGF beta incubated with UMR-106 cells for 48 hr produced a concentration-dependent increase in PTH stimulation of adenylate cyclase, with maximal increase in PTH response (37%) occurring at 1 ng/ml TGF beta. TGF beta also enhanced receptor-mediated activation of adenylate cyclase by isoproterenol and prostaglandin E2 (PGE2) and nonreceptor-mediated enzyme activation by cholera toxin and forskolin. In cells in which PTH-stimulated adenylate cyclase activity was augmented by treatment with pertussis toxin, the incremental increase in PTH response produced by TGF beta was reduced by 33%. However, TGF beta neither mimicked nor altered the ability of pertussis toxin to catalyze the ADP-ribosylation of a 41,000-Da protein, presumably the alpha subunit of the inhibitory guanine nucleotide-binding regulatory component (Gi) of adenylate cyclase, in cholate-extracted UMR-106 cell membranes. TGF beta also had no effect on the levels of alpha or beta subunits of Gi, as assessed by immunotransfer blotting. In time course studies, brief (less than or equal to 30 min) exposure of cells to TGF beta during early culture was sufficient to increase PTH response but only after exposed cells were subsequently allowed to grow for prolonged periods. TGF beta enhancement of PTH and isoproterenol responses was blocked by prior treatment of cells with cycloheximide but not indomethacin. The results suggest that TGF beta enhances PTH response in osteoblast-like cells by action(s) exerted at nonreceptor components of adenylate cyclase. The effect of TGF beta may involve Gi, although in a manner unrelated to either pertussis toxin-catalyzed ADP-ribosylation of the alpha subunit of Gi or changes in levels of Gi subunits. The regulatory action of TGF beta on adenylate cyclase is likely to be mediated by the rapid generation of cellular signals excluding prostaglandins, followed by a prolonged sequence of events involving protein synthesis. These observations suggest a mechanism by which TGF beta may regulate osteoblast responses to systemic hormones.

Inhibition of adenylate cyclase from luteinized rat ovary by monovalent cations: Roles of the stimulatory guanine nucleotide-binding regulatory component and stimulatory hormone receptor

Sodium and other monovalent cations (added as chloride salts) inhibited adenylate cyclase of luteinized rat ovary. Sodium chloride (150 m m) inhibited basal enzyme activity by 20%. Sodium chloride inhibition was enhanced to 34–54% under conditions of enzyme stimulation by guanine nucleotides (GTP and its nonhydrolyzable analog 5′-guanylyl imidodiphosphate), fluoride anion, and agonists (ovine luteinizing hormone (oLH) and the β-adrenergic catecholamine isoproterenol) acting at stimulatory receptors linked to adenylate cyclase. Sodium chloride inhibition was dependent on salt concentration over a wide range (25–800 m m) as well as the concentrations of GTP and oLH. Inhibition by NaCl was of rapid onset and appeared to be reversible. The order of inhibitory potency of monovalent cations was Li + > Na + > K +. The role of individual components of adenylate cyclase in the inhibitory action of monovalent cations was examined. Exotoxins of Vibrio cholerae and Bordetella pertussis were used to determine respectively the involvement of the stimulatory and inhibitory guanine nucleotide-binding regulatory components (N s and N i) in NaCl inhibition. Sodium chloride inhibited cholera toxin-activated adenylate cyclase activity by 29%, N i did not appear to mediate cation inhibition of adenylate cyclase because pertussis toxin did not attenuate inhibition by NaCl. Enzyme stimulation by agents (forskolin and Mn 2+) thought to activate the catalytic component directly was not inhibited by NaCl but was instead significantly enhanced. Sodium chloride (150 m m) increased both the K d for high-affinity binding of oLH to 125I-human chorionic gonadotropin binding sites and the K act for oLH stimulation of adenylate cyclase by sevenfold. In contrast, NaCl had no appreciable effect on either isoproterenol binding to (−)-[ 125I]iodopindolol binding sites or the K act for isoproterenol stimulation of adenylate cyclase. The results suggest that in luteinized rat ovary monovalent cations uncouple, or dissociate, N s from the catalytic component and, in a distinct action, reduce gonadotropin receptor affinity for hormone. Dissociation of the inhibitory influence of N i from direct catalytic activation could account for NaCl enhancement of forskolin- and Mn 2+-associated activities. On the basis of these results, the spectrum of divergent stimulatory and inhibitory effects of monovalent cations on adenylate cyclase activities in a variety of tissues may be interpreted in terms of differential enzyme susceptibilities to cation-induced uncoupling of N and catalytic component functions.

Inhibition of parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by transforming growth factor alpha and epidermal growth factor.

The effects of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TFG-alpha and EGF incubated with UMR-106 cells for 48 h each produced concentration-dependent inhibition of PTH-responsive adenylate cyclase, with maximal inhibition of 38-44% at 1-3 ng/ml of either growth factor. TGF-alpha and EGF also inhibited beta-adrenergic agonist (isoproterenol)-stimulated adenylate cyclase by 32%, but neither growth factor affected enzyme response to prostaglandin or basal (unstimulated) activity. Nonreceptor-mediated activation of adenylate cyclase by forskolin and cholera toxin was inhibited 18-20% by TGF-alpha and EGF. Pertussis toxin augmented PTH-stimulated adenylate cyclase, suggesting modulation of PTH response by a functional inhibitory guanine nucleotide-binding regulatory component of the enzyme. However, pertussis toxin had no effect on TGF-alpha inhibition of PTH response. Growth factor inhibition of PTH response was time-dependent, with maximal inhibition by 4-12 h of TGF-alpha exposure, and was reduced by prior treatment of UMR-106 cells with cycloheximide. TGF-alpha was not mitogenic for UMR-106 cells. The results indicate that TGF-alpha and EGF selectively impair PTH- and beta-adrenergic agonist-responsive adenylate cyclase of osteoblast-like cells. Growth factor inhibition of adenylate cyclase may be exerted at the receptor for stimulatory agonist and at nonreceptor components excluding pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins. The inhibitory action of growth factors may also require protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Intestinal brush border membranes contain regulatory subunits of adenylyl cyclase.

Cholera toxin alters intestinal function by stimulation of adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing) or adenylate cyclase, EC 4.6.1.1]. The mechanism of this activation is unknown and particularly puzzling because adenylyl cyclase is confined to the basal lateral membrane of enterocytes, whereas it is the brush border membrane that binds the toxin and contains proteins that undergo cholera toxin-catalyzed ADP ribosylation. It is shown that cholate extracts from cholera toxin-treated brush border membranes can efficiently reconstitute adenylyl cyclase activity in the guanine nucleotide-binding regulatory component (Gs)-deficient cyc- variant of the S49 mouse lymphoma cell line (cyc- cells lack the alpha subunit of Gs needed to activate the catalytic subunit of adenylyl cyclase). Moreover, NaF (in the presence of Al3+) and guanyl-5'-yl imidodiphosphate mediate strong activation of cyc- adenylyl cyclase provided the cholate extracts of brush border membranes are also present. Therefore, it appears that brush border membranes contain high levels of regulatory subunits of adenylyl cyclase in the absence of catalytic subunits. This represents a previously unrecognized feature of this transduction system that presumably plays an important role in the derangement of intestinal cell function by cholera toxin.

A1 and A2 adenosine receptors in rabbit cortical collecting tubule cells. Modulation of hormone-stimulated cAMP.

Adenosine analogs were used to investigate the cellular mechanisms by which adenosine may alter renal tubular function. Cultured rabbit cortical collecting tubule (RCCT) cells, isolated by immunodissection, were treated with 5'-N-ethylcarboxamideadenosine (NECA), N6-cyclohexyladenosine (CHA), and R-N6-phenylisopropyladenosine (PIA). All three analogs produced both dose-dependent inhibition and stimulation of RCCT cell cyclic AMP (cAMP) production. Stimulation of cAMP accumulation occurred at analog concentrations of 0.1 microM to 100 microM with the rank order of potency NECA greater than PIA greater than CHA. Inhibition occurred at concentrations of 1 nM to 1 microM with the rank order of potency CHA greater than PIA greater than NECA. These effects on cAMP production were inhibited by 1,3-diethyl-8-phenylxanthine and isobutylmethylxanthine. CHA (50 nM) blunted AVP- and isoproterenol-stimulated cAMP accumulation. This modulation of hormone-induced cAMP production was abolished by pretreatment of RCCT cells with pertussis toxin. Prostaglandin E2 production was unaffected by 0.1 mM CHA. These findings indicate the presence of both inhibitory (A1) and stimulatory (A2) receptors for adenosine in RCCT cells. Moreover, occupancy of the A1 receptor causes inhibition of both basal and hormone-stimulated cAMP formation through an action on the inhibitory guanine nucleotide-binding regulatory component, Ni, of the adenylate cyclase system.

Mechanism of action of prostaglandin F 2α-induced luteolysis: evidence for a rapid effect on the guanine nucleotide binding regulatory component of adenylate cyclase in rat luteal tissue

The initial events in prostaglandin F 2α-(PGF 2α)-induced luteolysis were studied in pregnant mare serum gonadotropin/human chorionic gonadotropin-(PMSG/hCG)-treated rats with luteinized ovaries. Injection with a potent PGF 2α analog (cloprostenol, 5 μg/ml) induced functional luteolysis, as assessed by plasma levels of progesterone and 20α-dihydroprogesterone. At 0.5 and 3 h after cloprostenol administration the luteolytic effect was also evident as a reduced response of luteal adenylate cyclase to all stimulatory agents tested, LH, isoproterenol, fluoride, guanylylimidodiphosphate and forskolin. 24 h after cloprostenol the response to all agents, except to LH, had returned to normal. This general and transient block of the luteal adenylate cyclase system indicates that a common factor, possibly the stimulatory guanine nucleotide binding protein (N s), is involved in the mechanism of action of PGF 2α. To test this hypothesis, we measured the functional coupling of the N s protein to the β-adrenergic receptor in luteal membranes. Binding competition curves showed a marked shift to the right in membranes prepared from rats injected with cloprostenol 0.5 and 3 h before membrane preparation, while at 24 h after cloprostenol the shift had disappeared. The total number of β-adrenergic receptors was, however, not affected by the cloprostenol treatment. Computer analysis of the data indicates that, at 0.5 and 3 h after cloprostenol treatment, there was a reduced number of high affinity binding sites, 38 and 41%, respectively, compared to 53% for control membranes. The cellular mechanism for this action of PGF 2α on the N s protein remains to be elucidated.

Actions of 1,1,1-trichloroethane on the cAMP metabolism in mouse brain.

Inhalation or intraperitoneal administration of 1,1,1-trichloroethane (TCE) increased the cAMP content in the brain stem in a dose-related and time-dependent fashion. TCE had no effect on the cAMP level in the cerebellum and hippocampus, but slightly reduced that in the cerebral cortex. Following intraperitoneal administration of TCE the activity of norepinephrine- and F- -activated adenylate cyclase (AC) in a brain stem homogenate was enhanced, while the serotonin-stimulated AC activity was decreased. Neither the basal AC activity nor the guanine nucleotide- and forskolin-activated enzyme was affected by TCE. TCE had no effect on the soluble cAMP-dependent phosphodiesterase activity. It is suggested that the increased cAMP content in the brain stem induced by TCE may be mediated via adrenoreceptor interaction with the guanine nucleotide-binding regulatory component, resulting in activation of the catalytic unit of the adenylate cyclase and thereby an increase in the cAMP level.

Inhibition by islet-activating protein of a chemotactic peptide-induced early breakdown of inositol phospholipids and Ca2+ mobilization in guinea pig neutrophils.

Receptors for a chemotactic peptide (fMet-Leu-Phe) in guinea pig neutrophils were primarily coupled to phospholipase C catalyzing breakdown of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate, which was in turn responsible for intracellular Ca2+ mobilization. These early responses of neutrophils to fMet-Leu-Phe, eventually leading to O2- generation, were abolished by prior exposure of cells to islet-activating protein (IAP), pertussis toxin, which had been reported to bring about ADP-ribosylation of a membrane Mr = 41,000 protein (Okajima, F., and Ui, M. (1984) J. Biol. Chem. 259, 13863-13871). The IAP substrate, probably the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase (Ni) or an analogous protein, is hence proposed to mediate fMet-Leu-Phe receptor-linked activation of the phospholipase C. In support of this proposal, A23187 and phorbol myristate acetate which stimulate arachidonate release or O2- generation by-passing these early processes of signaling were effective in IAP-treated cells as well. Release of arachidonic acid and accumulation of inositol 1-monophosphate in delayed response to fMet-Leu-Phe were also abolished by the IAP treatment of cells, despite the fact that slowly-onset inflow of Ca2+ which must be responsible for these delayed responses was observed in these IAP-treated cells. Thus, the IAP substrate may play an additional role in Ca2+-dependent activation of somehow compartmentalized phospholipases.

Protein kinase C interferes with N i-mediated inhibition of human platelet adenylate cyclase

Addition of phorbol ester-activated, partially purified protein kinase C to membranes of human platelets had no effect on forskolin stimulation of the adenylate cyclase and increased stimulation by prostaglandin E 1 only at high GTP concentrations by preventing inhibition by GTP. Hormonal inhibition of the platelet adenylate cyclase by epinephrine was eliminated or largely impaired. At low GTP concentrations, epinephrine even caused a small increase in cyclase activity. The data suggest that activated protein kinase C interferes with GTP- and hormone-induced adenylate cyclase inhibition probably by phosphorylating the inhibitory guanine nucleotide-binding regulatory component N i.

Stimulation and inhibition of rat basophilic leukemia cell adenylate cyclase by forskolin

The influence of the diterpene, forskolin, was studied on adenylate cyclase activity in membranes of rat basophilic leukemia cells. Forskolin increased basal adenylate cyclase activity maximally 2-fold at 100 μM. However, adenylate cyclase activity stimulated via the stimulatory guanine nucleotide-binding protein, N s, by fluoride and the stable GTP analog, guanosine 5′- O-(3-thiotriphosphate), was inhibited by forskolin. Half-maximal and maximal inhibition occurred at about 1 and 10 μM forskolin, respectively. The inhibition occurred without an apparent lag phase, whereas the enzyme stimulation by forskolin was preceded by a considerable lag period. The inhibition was not affected by treating intact cells or membranes with pertussis toxin and proteolytic enzymes, respectively, which have been shown in other cell types to prevent adenylate cyclase inhibition mediated by the guanine nucleotide-binding regulatory component, N i. The forskolin inhibition of the stable GTP analog-activated adenylate cyclase was impaired by increasing the Mg 2+ concentration and was reversed into a stimulation by Mn 2+. Under optimal inhibitory conditions, forskolin even decreased basal adenylate cyclase activity. Finally, forskolin largely reduced the apparent affinity of the rat basophilic leukemia cell adenylate cyclase for its substrate, MgATP, which reduction resulted in an apparent inhibition at low MgATP concentrations and a loss of the inhibition at higher MgATP concentrations. The data indicate that forskolin can cause both stimulation and inhibition of adenylate cyclase and, furthermore, they suggest that the inhibition may not be mediated by the N i protein, but may be caused by a direct action of forskolin at the adenylate cyclase catalytic moiety.

Solubilization and characterization of rat brain alpha 2-adrenergic receptor.

alpha 2-Adrenergic receptors labelled by [3H]-clonidine (alpha 2-agonist) can be solubilized from the rat brain in a form sensitive to guanine nucleotides with a zwitterionic detergent, 3-[3-(cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). About 40% of the original [3H]CLO binding sites in the membranes were solubilized with 6 mM CHAPS. Separation of the soluble [3H]CLO-bound complex was performed by the vacuum filtration method using polyethylenimine-treated GF/B filters. Solubilized [3H]CLO binding sites retained the same pharmacological characteristics of membrane-bound alpha 2-adrenergic receptors. Scatchard plots of [3H]CLO binding to solubilized alpha 2-receptors were curvilinear, indicating the existence of the two distinct binding components. Solubilized receptors were eluted as a single peak from Bio-Gel A-1.5 m column with a Stokes radius of 6.6 nm. The isoelectric point was 5.6-5.8. Regulations of the receptor binding by guanine nucleotides, monovalent cations, and sulfhydryl-reactive agents were maintained intact in the soluble state, whereas those by divalent cations were lost. The apparent retention of receptors and guanine nucleotide binding regulatory component(s) in the soluble state may allow a investigation of the regulation mechanisms of the brain alpha 2-adrenergic receptor system at the molecular level.

Agonist/N-ethylmaleimide mediated inactivation of β-adrenergic receptors: Molecular aspects

Incubation of turkey erythrocyte and rat lung membranes with the β-adrenergic agonist isoproterenol in the presence of the group specific reagent N-ethylmaleimide (NEM) causes the apparent inactivation of a well defined fraction of the β-adrenergic receptors. This inactivation process requires the coupling between the receptors and the guanine nucleotide binding regulatory component of the adenylate cyclase system. Accordingly, only the isoproterenol/NEM sensitive receptor population is susceptible to undergo this functional coupling. Incubation of the treated membranes in alkaline buffer and/or in the presence of GTP reactivates the receptors. The reactivated receptors show the initial binding properties for both isoproterenol and the radiolabelled antagonist [ 3H]-dihydroalprenolol and are still dithiothreitol sensitive. Moreover the reactivated receptors can again couple to the regulator in the presence of agonists. The present study makes it possible to discriminate between the different models advanced for the N-ethylmaleimide action and is valid for β 1- as well as β 2-adrenergic receptors. The reagent appears indeed to freeze the hormone-receptor-regulator complex by alkylating the latter component, as previously proposed by Korner et al. (1982) J. biol. Chem. 257, 3389–3396. It is also suggested that the existence of a limited fraction of coupling-prone receptors results from differences in the membrane microenvironment or from a structural heterogeneity of the receptors rather than from a stoichiometric limitation of the regulator concentration.

The Membrane-Bound Spermatozoal Adenylyl Cyclase System Does not Share Coupling Characteristics with Somatic Cell Adenylyl Cyclases*

Membrane-bound adenylyl cyclases from ram, dog, and human sperm are unresponsive to fluoride and guanylylimidodiphosphate [GMP-P(NH)P], two agents that stimulate the adenylyl cyclases of somatic cells by an action on the stimulatory guanine nucleotide-binding regulatory (Ns) component of adenylyl cyclase. We have investigated whether this is because the sperm cell catalytic unit is functionally uncoupled from Ns but, nevertheless, capable of interacting with it, or because the sperm cell adenylyl cyclase system is unique and regulated differently from that of somatic cells. Sperm cells were found to be deficient in Ns, as evidenced by the inability of detergent extracts from sperm cell membranes and fractions to reconstitute Ns-mediated regulation of the adenylyl cyclase of cyc- S49 cells. In addition, attempts to label Ns in sperm cell membranes by [32P]ADP ribosylation with cholera toxin revealed that, if present, Ns is less than 1% of that found in human erythrocyte membranes. This, however, was not the only reason for the unresponsiveness of sperm cell adenylyl cyclase, since fluoride stimulation of the sperm cell enzyme could not be induced by reconstituting it with Ns purified from human erythrocytes (hRBC). When intact hRBC membranes were added to sperm cell fractions in the presence of fluoride, the activities that resulted were greater than the sum of the individual activities. This apparent reconstitution of fluoride regulation of sperm cell adenylyl cyclase could be blocked by lima bean trypsin inhibitor and appears to have resulted from proteolytic activation of the hRBC adenylyl cyclase by sperm proteases. Sperm cell membranes also appear to lack a functional inhibitory regulatory protein of the adenylyl cyclase system (Ni), since they did not contain an ADP-ribosylatable substrate for pertussis toxin action. These results suggest that the sperm cell adenylyl cyclase system is unique and different from that of somatic cells. Sperm cells appear to neither contain Ns or Ni nor possess the ability of their adenylyl cyclase system to interact with Ns from an exogenous source.

Cholera toxin action on rabbit corpus luteum membranes: effects on adenylyl cyclase activity and adenosine diphospho-ribosylation of the stimulatory guanine nucleotide-binding regulatory component.

Cholera toxin elicited 5- to 7-fold stimulation of adenylyl cyclase activity. Half-maximal activation was at 4.42 micrograms/ml cholera toxin. Cholera toxin-mediated activation was time dependent. At 0.1 mM ATP, both guanosine triphosphate (GTP) and nicotinamide adenine dinucleotide (NAD+) were required for cholera toxin activation of luteal adenylyl cyclase. The concentrations of GTP and NAD+ required for half-maximal activation were 1 and 200 microM, respectively. The GTP requirement could be eliminated by increasing the ATP concentration to 1.0 mM. Guanosine-5'-O-(2-thiodiphosphate) [GDP beta S] did not support cholera toxin activation of the luteal enzyme. Cholera toxin treatment increased GTP-stimulated activity, did not significantly alter guanyl-5'-yl imidodiphosphate [GMP-P(NH)P]-stimulated activity, and depressed NaF-stimulated activity. Furthermore, toxin treatment resulted in a 3.4-fold reduction in the Kact values for ovine luteinizing hormone (oLH) to activate adenylyl cyclase. A similar reduction in Kact values for oLH was obtained when concentration-effect curves performed in the presence of GMP-P(NH)P were compared to those performed in the presence of GTP. In addition, luteal membranes treated with cholera toxin and [32P]NAD+ were subjected to autoradiographic analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This treatment resulted in the [32P] adenosine diphospho (ADP)-ribosylation of a 45,000-dalton protein doublet, corresponding to the alpha subunit of the stimulatory guanine nucleotide-binding regulatory component (Ns). As with activation of adenylyl cyclase activity, cholera toxin-specific [32P] ADP-ribosylation was time dependent and increased with increasing concentrations of cholera toxin. GTP, GMP-P(NH)P, and NaF, but not GDP beta S, were capable of supporting [32P] ADP-ribosylation of the protein doublet. oLH did not alter the ability of cholera toxin to ADP-ribosylate the protein activation of luteal adenylyl cyclase activity is due to the ADP-ribosylation of the alpha subunit of Ns and the concomitant inhibition of a GTPase associated with adenylyl cyclase.

Forskolin stimulation of adenylate cyclase in human thyroid membranes.

Forskolin stimulates adenylate cyclase in human thyroid membranes approximately 7-fold with half-maximal stimulation occurring at 5-10 microM. Guanine nucleotides are not required for stimulation of the enzyme by forskolin. Forskolin-stimulation is additive or greater than additive with that of TSH or Gpp(NH)p- (above 1 microM). Different from TSH- or Gpp(NH)p-stimulation of adenylate cyclase, uncoupling of the guanine nucleotide-binding regulatory component by increasing concentrations of MnCl2 did not result in uncoupling of forskolin stimulation. The finding indicates that forskolin may mainly act on the catalytic component of adenylate cyclase. From the present study, it is suggested that the diterpene forskolin stimulates adenylate cyclase in human thyroid membranes by a novel mechanism that differs from TSH- or Gpp(NH)p-stimulation, and that the diterpene may be a useful tool to investigate the metabolism of thyroid and its regulation in normal and pathological situations.

Ni-mediated inhibition of human platelet adenylate cyclase by thrombin.

The influence of the proteolytic enzyme, thrombin, was studied on adenylate cyclase activity in human platelet membranes. Thrombin had a biphasic inhibitory effect on the enzyme. Up to a concentration of about 1 U/ml, thrombin inhibited the enzyme in a strictly GTP-dependent manner by maximal 60-70%, with half-maximal inhibition occurring at about 0.005 U/ml thrombin. At higher concentrations, thrombin-induced inhibition of platelet adenylate cyclase was independent of GTP. The inhibitory effect of thrombin observed at low concentrations was further evaluated. The inhibition was half-maximal and maximal at about 0.1 and 3 microM GTP, respectively, occurred without major lag phase and was observed with the unstimulated and the forskolin or prostaglandin E1-stimulated platelet adenylate cyclase. Additionally, thrombin accelerated and potentiated the enzyme inhibition caused by the stable GTP analog, guanosine 5'-(gamma-thio)triphosphate. Furthermore, at identical concentrations causing adenylate cyclase inhibition, thrombin effectively stimulated GTP hydrolysis in platelet membranes. Finally, the thrombin inhibition of human platelet adenylate cyclase was impaired or abolished by Mn2+ and by treatment of the platelet membranes with N-ethylmaleimide or pertussis toxin. All of these data indicate that thrombin at low concentrations inhibits platelet adenylate cyclase by a process involving the inhibitory guanine nucleotide-binding regulatory component, Ni, in a manner similar to hormonal factors. However, in contrast to typical hormonal inhibition, thrombin inhibition of the platelet enzyme was not reversed by washing of the platelet membranes or by subsequent addition of the thrombin inactivator, hirudin, which prevented the inhibition when added together with thrombin. These data suggest that thrombin by its proteolytic capacity causes a persistent activation of its receptor site which leads to a persistent activation of Ni with subsequent persistent adenylate cyclase inhibition.

Inhibition of receptor-mediated release of arachidonic acid by pertussis toxin.

Treatment of guinea pig neutrophils with pertussis toxin (islet-activating protein; IAP) results in inhibition of N-formyl peptide receptor-mediated release of arachidonic acid and granular enzymes. Inhibition by the toxin is specific, in that responses to the calcium ionophore A23187 are not affected. The action of the toxin is not associated with alterations in cellular concentrations of cyclic AMP but is correlated with the ability of the toxin to catalyze the ADP-ribosylation of a 41,000 dalton membrane protein. This protein comigrates on SDS-polyacrylamide gels with the alpha subunit of Gi, the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. It is likely that this G protein is involved in receptor-mediated signal transduction in neutrophils by mechanisms that do not involve cyclic AMP.