Site of pertussis toxin-induced ADP-ribosylation on Gi is critical for receptor modulation of GDP interaction with Gi

In this study, the influence of the inhibitory mu-opioid receptor on the potencies of 5'-guanosine alpha-thiotriphosphate (GTP gamma S) and GDP at the inhibitory GTP-binding protein (Gi) were investigated in an adenylyl cyclase system. It was hoped that a receptor-mediated change in the potency of either GTP gamma S or GDP in affecting adenylyl cyclase activity may elucidate how a receptor alters cyclase activity via its G-protein. In an adenylyl cyclase system employing 5'-adenylyl imidodiphosphate as substrate, GTP gamma S, a nonhydrolyzable analog of GTP, inhibited forskolin-stimulated adenylyl cyclase activity in the absence of morphine; morphine failed to significantly affect the apparent potency of GTP gamma S. GDP blocked the GTP gamma S-induced inhibition of adenylyl cyclase; morphine profoundly diminished the ability of GDP to block the inhibitory effect of GTP gamma S. The IC50 values of GTP gamma S were 0.02 +/- 0.01, 0.18 +/- 0.04, and 2.2 +/- 0.5 microM in the absence of other drugs, in the presence of a combination of 100 microM GDP and morphine, and in the presence of 100 microM GDP, respectively. GDP blocked the inhibitory effect of GTP gamma S (0.3 microM) in a concentration-dependent manner; the EC50 for GDP was 16 +/- 2.6 microM in the absence of morphine and 170 +/- 32 microM in the presence of morphine. Exposure of 7315c cells to pertussis toxin for 3 h resulted in a small decrease in the potency of GTP gamma S in inhibiting cyclase. However, the relative potency of GDP in blocking the GTP gamma S-mediated inhibition of cyclase was increased: the EC50 values of GDP were 11 +/- 4 and 0.81 +/- 0.2 microM in untreated and pertussis toxin-treated membranes, respectively. In untreated membranes, there was a brief lag in the GTP gamma S-induced inhibition of adenylyl cyclase; morphine diminished this lag. In membranes treated with pertussis toxin, there was an exaggerated lag in the onset of GTP gamma S inhibition of adenylyl cyclase activity; morphine could no longer affect this lag. Thus, uncoupling the mu-opioid receptor from Gi appeared to increase the affinity of Gi for GDP. These data suggest that the effect of an inhibitory receptor is to decrease the affinity of Gi for GDP by virtue of its interaction with the carboxy-terminal region of Gi alpha.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of adenylate cyclase in human keratinocytes by protein kinase C

Adenylate cyclase (ATP-pyrophosphate lyase (cyclizing); EC 4.6.1.1) in the human keratinocyte cell line SCC 12F was potentiated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), phorbol-12,13-diacetate, and 1,2-dioctanoylglycerol. Keratinocytes exposed to TPA showed a 2-fold enhancement of adenylate cyclase activity when assayed in the presence of isoproterenol or GTP. The half-maximal effective concentration (EC50) for both isoproterenol and GTP were unaltered by TPA treatment of the cells. Basal adenylate cyclase activity in membranes from TPA-treated cultures was also increased 2-fold relative to activity in control membranes. Potentiation of adenylate cyclase activity was dependent on the concentration of TPA to which the keratinocytes were exposed (EC50 for TPA = 3 nM). TPA actions on adenylate cyclase were maximal after 15 min of incubation of the cells with the compound, correlating well with the time course of translocation of protein kinase C (Ca2+/phospholipid-dependent enzyme) from cytosol to membrane. The action of cholera toxin on adenylate cyclase was additive with TPA. In contrast, pertussis toxin actions on adenylate cyclase were not additive with TPA. Treatment of control cells with pertussis toxin activated adenylate cyclase 1.5-fold, whereas cells exposed to pertussis toxin for 6 h followed by TPA for 15 min showed the same 2-fold increase in adenylate cyclase activity as observed in membranes from cells exposed to TPA without prior exposure to pertussis toxin. Pertussis toxin catalyzed ADP-ribosylation was increased 2-fold in membranes from SCC 12F cells exposed to TPA, indicating an increase in the alpha beta gamma form of Gi. These data suggest that exposure of human keratinocytes to phorbol esters increases adenylate cyclase activity by a protein kinase C-mediated increase in the heterotrimeric alpha beta gamma form of Gi resulting in decreased inhibition of basal adenylate cyclase activity.     

Alpha-adrenergic inhibition of renal cortical adenylate cyclase

Adenylate cyclase in homogenates of rat renal cortex was inhibited by alpha-adrenergic agonists. Inhibition required sodium ion and GTP. A maximum inhibition of 17.8 +/- 1.4% (S.E.M.) was produced by l-epinephrine in the presence of 0.2 M NaCl, 10 microM GTP and 10 microM propranolol. Similar inhibition was produced by l-norepinephrine and alpha-methylnorepinephrine. The EC50 values for l-epinephrine, l-norepinephrine and alpha-methylnorepinephrine were respectively 1.9 +/- 0.7 microM, 2.3 +/- 1.6 microM and 5.1 +/- 1.8 microM. Clonidine was a partial agonist causing 50% as much inhibition as epinephrine. Phenylephrine and methoxamine did not inhibit at concentrations up to 100 microM. Micromolar concentrations of phentolamine and yohimbine prevented the inhibition of adenylate cyclase by epinephrine. However, prazosin was ineffective. Thus the adenylate cyclase coupled alpha-receptors have alpha-2 specificity. Inhibition of adenylate cyclase by alpha-adrenergic agonists was not observed in homogenates of renal medulla.     

Ca2+-Independent Stimulation of Adenylate Cyclase Activity by Muscarinic Receptors in Rat Olfactory Bulb

In rat olfactory bulb homogenate, carbachol stimulated adenylate cyclase activity in a concentration-dependent manner (EC50 = 1.1 microM). The carbachol stimulation occurred fully in membranes that had been prepared in the presence of 1 mM EGTA and incubated in a Ca2(+)-free enzyme reaction medium. Under these conditions, exogenous calmodulin (1 microM) failed to stimulate adenylate cyclase activity. In miniprisms of olfactory bulb, carbachol (1 mM) increased accumulation of inositol phosphates, but this response was markedly reduced in a Ca2(+)-free medium. Moreover, the carbachol stimulation of adenylate cyclase activity was not affected by staurosporine at a concentration (1 microM) that completely blocked the stimulatory effect of phorbol 12-myristate 13-acetate, an activator of Ca2+/phospholipid-dependent protein kinase. Quinacrine, a nonselective phospholipase A2 inhibitor, reduced the carbachol stimulation of adenylate cyclase activity, but this inhibition appeared to be competitive with a Ki of 0.2 microM. Nordihydroguaiaretic acid and indomethacin, two inhibitors of arachidonic acid metabolism, failed to affect the carbachol response. These results indicate that in rat olfactory bulb, muscarinic receptors stimulate adenylate cyclase activity through a mechanism that is independent of Ca2+ and phospholipid hydrolysis.     

Biphasic Ca2+ response of adenylate cyclase. The role of calmodulin in its activation by Ca2+ ions

The Ca2+-dependent regulation of human platelet membrane adenylate cyclase has been studied. This enzyme exhibited a biphasic response to Ca2+ within a narrow range of Ca2+ concentrations (0.1-1.0 microM). At low Ca2+ (0.08-0.3 microM) adenylate cyclase was stimulated (Ka = 0.10 microM), whereas at higher Ca2+ (greater than 0.3 microM) the enzyme was inhibited to 70-80% control (Ki = 0.8 microM). Membrane fractions, prepared by washing in the presence of LaCl3 to remove endogenous calmodulin (approximately equal to 70-80% depletion), exhibited no stimulation of adenylate cyclase by Ca2+ but did show the inhibitory phase (Ki = 0.4 microM). The activation phase could be restored to La3+-washed membranes by addition of calmodulin (Ka = 3.0 nM). Under these conditions it was apparent that calmodulin reduced the sensitivity of adenylate cyclase to Ca2+ (Ki = 0.8 microM). Prostaglandin E1 (PGE1) did not alter Ki or Ka values for Ca2+. Calmodulin did not alter the EC50 for PGE1 stimulation of adenylate cyclase but increased the Vmax (1.5-fold). The calmodulin antagonist trifluoperazine potently inhibited adenylate cyclase in native membranes (80%) and to a much lesser extent in La3+-washed membranes (15%). This inhibition was due to interaction of trifluoperazine with endogenous calmodulin since trifluoperazine competitively antagonized the stimulatory effect of calmodulin on adenylate cyclase in La3+-washed membranes. We propose that biphasic Ca2+ regulation of platelet adenylate cyclase functions to both dampen (low Ca2+) and facilitate (high Ca2+) the haemostatic function of platelets.     

Insulin exerts actions through a distinct species of guanine nucleotide regulatory protein: inhibition of adenylate cyclase.

Insulin failed to exert an effect on the basal and glucagon- and guanosine 5'-[beta, gamma-imido]-triphosphate-stimulated adenylate cyclase activities of hepatocyte membranes. In the presence of high GTP (0.1 mM) concentrations, however, insulin was shown to inhibit adenylate cyclase activity. This effect was dose-dependent, exhibiting an EC50 (median effective concentration) of 3 microM for GTP. Elevated glucagon concentrations blocked the inhibitory effect of insulin in a dose-dependent fashion, with an EC50 of 1 nM. The insulin inhibition was dose-dependent (EC50 = 90 pM). The inhibitory effects of insulin were abolished using membranes from either glucagon-desensitized hepatocytes or cholera-toxin-treated hepatocytes. If either Mn2+ replaced Mg2+ in adenylate cyclase assays or Na+ was removed from the assay mixtures then insulin failed to exert any inhibitory effect. It is suggested that insulin exerts its action on adenylate cyclase through an inhibitory guanine nucleotide protein. This is integrated with the proposal [Heyworth, Rawal & Houslay (1983) FEBS Lett. 154, 87-91; Heyworth, Wallace & Houslay (1983) Biochem. J. in the press] that insulin mediates a variety of cellular effects through a specific guanine nucleotide regulatory protein and associated protein kinase(s).     

Modulation of guanine nucleotide affinity does not affect the first order rate constant of activation of adenylate cyclase in native membranes

A new method was developed to follow the rate of activation of adenylate cyclase in rat brain membranes by rapid freezing and N-ethylmaleimide treatment at 0 degrees C. This method was used to investigate the relationship between the rate of activation of adenylate cyclase by p(NH)ppG and GTP gamma S and their apparent affinities. These studies established the following. 1) The kinetics of activation by p(NH)ppG and GTP gamma S were indistinguishable although the apparent affinity of p(NH)ppG was 20-fold lower than the affinity of GTP gamma S. Activation was first order, kobs varying approximately 1.5-fold (average t 1/2 = 3.5 min, 30 degrees C) between 20-90% occupancy by either guanine nucleotide. 2) Final levels of activity were strictly dependent on the concentration of the nucleotides in a saturable manner. 3) Mg2+ increased the apparent affinity of either guanine nucleotide by 10-20-fold between 0.1 microM and 3 mM free Mg2+ in the presence of 2 mM EDTA but did not enhance the rate or maximal extent of activation. 4) The effects of Mg2+ were expressed through two independent classes of sites with affinities in the nanomolar and micromolar range. 5) A Mg2+ X guanine nucleotide complex was not the substrate for activation. The affinity of Mg2+ for nucleotides was determined as 6.25 mM GTP gamma S, 0.930 mM GTP, 0.156 mM p(NH)ppG. 6) Full activation by p(NH)ppG was completely reversible but activation by GTP gamma S was only partially reversible. These results suggest that: activation of adenylate cyclase in native membranes does not require Mg2+ or irreversible binding of the guanine nucleotide and there are two independent pathways for formation of active adenylate cyclase. A minimal mechanism for activation is discussed in light of current models.     

A novel site of action of a high affinity A1 adenosine receptor antagonist

XAC, a high affinity antagonist of the A1 adenosine receptor, enhances adenylate cyclase activity by 1.3-2 fold with an EC50 of approximately 47 nM in adipocyte membranes pretreated with adenosine deaminase to eliminate adenosine and in the presence of total phosphodiesterase inhibition by 100 microM papaverine. This effect of XAC is observed only at concentrations of GTP sufficient to activate Gi (approximately 5 x 10(-6) M GTP) and is not evident in the absence or presence of lower GTP concentrations. ADP ribosylation of Gi by pertussis toxin treatment also abolishes this stimulatory action of XAC. Furthermore, in the presence of GTP activation of inhibitory prostaglandin E1 receptors diminishes the stimulatory effect of XAC on adenylate cyclase. In addition, XAC interferes with GTP-mediated inhibition of forskolin-stimulated adenylate cyclase activity in a noncompetitive manner. Finally, XAC is only a weak inhibitor of the low Km cyclic AMP phosphodiesterase, producing approximately 40% inhibition of phosphodiesterase activity at a concentration of 100 microM. These data suggest that XAC increases adenylate cyclase activity in absence of endogenous adenosine by inhibiting tonic Gi activity in a reversible manner.     

Inhibition of adenylate cyclase of catfish and rat hepatocyte membranes by 9-(tetrahydro-2-furyl)adenine (SQ 22536).

The adenosine analogue 9-(Tetrahydro-2-furyl)adenine, SQ 22536, inhibited adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity of crude membrane preparations from catfish (Ictalurus melas) and rat isolated hepatocytes in a non-competitive manner. The IC50s were reduced in the presence of NaF. SQ 22536 reduced the activity of adenylate cyclase also in the presence of increasing concentrations of GTP, as well as Mg++ and Mn++. In the presence of catecholamines (epinephrine, norepinephrine, isoproterenol, phenylephrine) SQ 22536 reduced their activating effect on adenylate cyclase in both catfish and rat membranes. SQ 22536 also inhibited the effect of glucagon (0.1 microM) on rat membrane cyclase activity.     

Alteration in the protein components of catecholamine-sensitive adenylate cyclase during maturation of rat reticulocytes

The maturing rat reticulocyte was used as a model system in which to study developmental changes in the protein components of hormone-sensitive adenylate cyclase. Plasma membranes from rat erythrocytes display 10 to 20% of the adenylate cyclase activity and 30 to 50% of the beta-adrenergic receptors which are measured in membranes from rat reticulocytes, as noted by others. Reticulocyte membranes also display equal activities in response to (-)-isoproterenol in the presence of either GTP or GTP gamma S, whereas erythrocyte membrane adenylate cyclase is twice as active in the presence of isoproterenol plus GTP gamma S as in the presence of isoproterenol plus GTP. We have studied this system in greater detail by developing or applying independent assays for the catalytic protein (C) and the guanine nucleotide-binding regulatory protein (G/F) of adenylate cyclase. C was assayed in membranes by its intrinsic Mn2+-stimulated activity. It was also measured by reconstituting membranes with saturating amounts of GTP gamma S-activated G/F, yielding an operationally defined Vmax for the catalyst. By either assay, reticulocytes display about 3-fold greater C activity than do erythrocytes. G/F was assayed by its ability to confer GTP gamma S-stimulated activity upon C (which was supplied by membranes of cyc- S49 lymphoma cells). This assay indicates that reticulocyte membranes contain about 3 times as much G/F as do erythrocyte membranes. Cholera toxin and [32P]NAD were used to [32P]ADP-ribosylate the 45,000- and 52,000-dalton subunits of G/F. Total incorporation of 32P into these subunits decreased 3- to 4-fold with reticulocyte maturation. The ratio of label in the 52,000-dalton peptide to that in the 45,000-dalton peptide decreased from 0.29 in reticulocyte membranes to 0.14 in erythrocyte membranes. The apparently coordinate decrease in the amounts of C, G/F, and beta-adrenergic receptors suggest that the stoichiometry between these components is maintained during maturation, and may account for the decrease in adenylate cyclase in the membranes. However, the qualitative changes in responsiveness to hormones in the presence of GTP or GTP gamma S may be related to loss or proteolysis of the 52,000-dalton subunit of G/F.     

Mechanism of glucagon activation of adenylate cyclase in the presence of Mn2+

For a variety of ligand states, adenylate cyclase activity in the presence of Mn2+ was greater than with Mg2+. Trypsin treatment of intact hepatocytes, under conditions which destroy cell surface glucagon receptors, led to a first order loss of glucagon-stimulated adenylate cyclase activity in isolated membranes assayed in the presence of Mn2+ whether or not GTP (100 microM) was present in the assays. Arrhenius plots of basal activity exhibited a break at around 22 degrees C, those with NaF were linear and those with glucagon +/- GTP (100 microM) were biphasic with a break at around 28 degrees C. It is suggested that Mn2+ perturbs the coupling interaction between the glucagon receptor and catalytic unit of adenylate cyclase at the level of the guanine nucleotide regulatory protein. This appears to take the form of Mn2+ preventing GTP from initiating glucagon's activation of adenylate cyclase through a collision coupling mechanism.     

Developmental alterations in guanine nucleotide regulation of the beta-adrenergic receptor-adenylate cyclase system of skeletal muscle

The postnatal development of skeletal muscle is accompanied by an increased capacity for glycogenolysis and anaerobic glycolysis. In the present study, regulatory features of cAMP synthesis were examined in neonatal and adult rabbit sarcolemmal membranes. Adult sarcolemma exhibited a 3-, 6-, and 10-fold greater adenylate cyclase activity than neonate for basal, NaF, and isoproterenol plus GTP, respectively. The Km for activation by isoproterenol was 1.4 X 10(-8) M and 6 X 10(-8) M for GTP. The number of beta-receptors was similar (0.9-1.2 pmol/mg). 10 microM GTP shifted isoproterenol EC50 from 1 X 10(-8) M to 1 X 10(-7) M in adult; neonatal agonist affinity was unaffected by GTP. Cholera toxin stimulated adenylate cyclase activity 2-fold and catalyzed 32P ribosylation of a Mr = 42,000 peptide in adult sarcolemma; both activities were low or absent in neonate. Isoproterenol-stimulated GTPase activity was elevated 4-fold in adult compared to neonatal sarcolemma. Mn2+ ion-stimulated basal activity, an indicator of catalytic function of adenylate cyclase, was also elevated in adult. Together, these findings suggest that the development of catecholamine-sensitive cAMP synthesis in muscle is governed by the coordinate expression of the regulatory and catalytic proteins of adenylate cyclase, but not the beta-receptor.     

The odorant-sensitive adenylate cyclase of olfactory receptor cells. Differential stimulation by distinct classes of odorants

We have characterized odorant-stimulated adenylate cyclase activity in isolated chemosensory cilia prepared from frog and rat olfactory epithelium. Cilia from both species exhibit high levels of adenylate cyclase activity. Basal activity is stimulated approximately 2-fold by GTP and approximately 5-fold by guanosine 5'-(3-O-thio)triphosphate and forskolin. Odorants augment enzyme activity 30-65% above the basal level in a tissue-specific and GTP-dependent manner. Calcium reduces GTP-stimulated activity with a 50% effective concentration at 10 microM. Odorants vary in their influence upon olfactory adenylate cyclase activity. Most fruity, floral, minty, and herbaceous odorants stimulate the enzyme. 3,7-Dimethyl-2,6-octadienenitrile (citralva), menthone, D-carvone, L-carvone, and 2-isobutyl-3-methoxypyrazine display similar potencies in activating the adenylate cyclase upto concentrations of 100 microM. Putrid odorants, such as isovaleric acid, triethylamine, pyridine, thiazole, and methoxypyrazine, and odorous chemical solvents, do not stimulate enzyme activity. In homologous series of pyrazine, thiazole, and pyridine odorants, compounds with the longest hydrocarbon side chains are best able to enhance enzyme activity. The failure of certain odorants to affect adenylate cyclase activity suggests that additional transduction mechanisms besides the formation of cAMP are involved in olfaction.     

Stimulation of adenylate cyclase in the heart of Aplysia californica by biogenic amines

The effects of serotonin (5-HT), dopamine (DA), several peptides including FMRFamide and arginine vasotocin, the diterpene forskolin and Ca2+ were examined on adenylate cyclase in a particulate fraction from hearts of Aplysia californica. Enzyme activity was stimulated 6-7-fold by 5-HT (EC50, 1 microM) in the presence of GTP. Several 5-HT analogs particularly 5-methoxytryptamine and 5-methoxy-N-N-dimethyltryptamine were also active. The stimulatory action of 5-HT was antagonized by the 5-HT receptor blockers methergoline and metitepine and by the DA receptor blocker chlorpromazine. Dopamine had weak stimulatory action (EC50, 10 microM) and an efficacy relative to that of 5-HT of 0.3. The action of DA was antagonized by chloropromazine and metitepine. Several peptides including FMRFamide and arginine vasotocin had no effect on adenylate cyclase when tested over the concentration range 0.1-100 microM. The enzyme was stimulated 6-fold by the diterpene forskolin (EC50, 2 microM). 5-HT-stimulated activity was strongly inhibited by Ca2+. Calmodulin had no action on the enzyme in the presence of Ca2+.     

Differential Regulation by Calmodulin of Basal, GTP-, and Dopamine-Stimulated Adenylate Cyclase Activities in Bovine Striatum

The concentration requirements of calmodulin in altering basal, GTP-, and dopamine-stimulated adenylate cyclase activities in an EGTA-washed particulate fraction from bovine striatum were examined. In the bovine striatal particulate fraction, calmodulin activated basal adenylate cyclase activity 3.5-fold, with an EC50 of 110 nM. Calmodulin also potentiated the activation of adenylate cyclase by GTP by decreasing the EC50 for GTP from 303 +/- 56 nM to 60 +/- 10 nM. Calmodulin did not alter the maximal response to GTP. The EC50 for calmodulin in potentiating the GTP response was only 11 nM as compared to 110 nM for activation of basal activity. Similarly, calmodulin increased the maximal stimulation of adenylate cyclase by dopamine by 50-60%. The EC50 for calmodulin in eliciting this response was 35 nM. These data demonstrate that calmodulin can both activate basal adenylate cyclase and potentiate adenylate cyclase activities that involve the activating GTP-binding protein, Ns. Mechanisms that involve potentiation of Ns-mediated effects are much more sensitive to calmodulin than is the activation of basal adenylate cyclase activity. Potentiation of GTP-stimulated adenylate cyclase activity by calmodulin was apparent at 3 and 5 mM MgCl2, but not at 1 or 10 mM MgCl2. These data further support a role for calmodulin in hormonal signalling and suggest that calmodulin can regulate cyclic AMP formation by more than one mechanism.     

Cyclic AMP-Dependent Protein Phosphorylation in Chemosensory Neurons: Identification of Cyclic Nucleotide-Regulated Phosphoproteins in Olfactory Cilia

Chemosensory dendritic membranes (olfactory cilia) contain protein kinase activity that is stimulated by cyclic AMP and more efficiently by the nonhydrolyzable GTP analog guanosine-5'-O-(3-thio)triphosphate (GTP gamma S). In control nonsensory (respiratory) cilia, the cyclic AMP-dependent protein kinase is practically GTP gamma S-insensitive. GTP gamma S activation of the olfactory enzyme appears to be mediated by a stimulatory GTP-binding protein (G-protein) and adenylate cyclase previously shown to be enriched in the sensory membranes. Protein kinase C activity cannot be detected in the chemosensory cilia preparation under the conditions tested. Incubation of olfactory cilia with [gamma-32P]ATP leads to the incorporation of [32P]phosphate into many polypeptides, four of which undergo covalent modification in a cyclic nucleotide-dependent manner. The phosphorylation of one polypeptide, pp24, is strongly and specifically enhanced by cyclic AMP at concentrations lower than 1 microM. This phosphoprotein is not present in respiratory cilia, but is seen also in membranes prepared from olfactory neuroepithelium after cilia removal. Cyclic AMP-dependent protein kinase and phosphoprotein pp24 may be candidate components of the molecular machinery that transduces odor signals.     

Inhibitory and stimulatory effects of neuropeptide Y(17-36) on rat cardiac adenylate cyclase activity. Structure-function studies.

Neuropeptide Y (NPY) inhibits cardiac adenylate cyclase activity by interacting with specific receptors coupled to a pertussis toxin-sensitive G protein. Structure-activity studies revealed that only C-terminal fragments can exhibit an NPY-like inhibitory effect on 125I-NPY binding and adenylate cyclase activity of rat cardiac ventricular membranes. Although NPY(17-36) inhibited 125I-NPY binding with high potency, it produced a biphasic effect on basal (GTP, 10 and 100 microM or guanosine 5'-gamma-O-(thio)triphosphate (GTP gamma S, 10 microM) adenylate cyclase activity. Low concentrations (less than 1 nM) of NPY(17-36) inhibited the adenylate cyclase activity whereas high concentrations (greater than 1 nM) reversed this action. GTP gamma S (100 microM) reversed the biphasic effect of NPY(17-36). NPY(17-36) exhibited only a stimulatory effect in the membranes from pertussis toxin-treated rats and an inhibitory effect with membranes from cholera toxin-treated rats. Low concentrations (less than 1 nM) of NPY(17-36) inhibited isoproterenol-stimulated adenylate cyclase activity whereas high doses (greater than 1 nM) reversed this activity. The cardiac NPY receptor antagonist, NPY(18-36) (1 microM), completely blocked the biphasic effect of NPY(17-36) on isoproterenol-stimulated activity. The inhibitory dose-response curve of NPY on isoproterenol-stimulated adenylate cyclase activity was shifted parallel to the right by NPY(17-36) (1 microM), suggesting that it is an antagonist of NPY at high concentrations. N-alpha-acetylated and C-terminally deamidated analogs of NPY(17-36) had no effect on the adenylate cyclase activity. [im-DNP-His26] NPY exhibited a more pronounced biphasic effect whereas N-alpha-myristoyl-NPY(17-36) elicited only a stimulatory effect. These investigations suggest that: 1) the inhibitory and stimulatory effects of NPY(17-36) are mediated by high affinity NPY receptors coupled to a pertussis toxin-sensitive G protein and a distinct population of low affinity receptors coupled to a cholera toxin-sensitive G protein, respectively; and 2) the stimulatory effect of NPY(17-36) is dissociable.     

Immunohistochemical Localization of 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase and Myelin Basic Protein in the Chick Retina

A GTP-dependent regulatory component of adenylate cyclase was found in myelin from rat brain. The fraction solubilized from myelin contained a component that reconstituted guanine nucleotide-responsive adenylate cyclase activity when combined with the catalytic unit of adenylate cyclase prepared from rat brain. Purified myelin demonstrated little adenylate cyclase activity, even in the presence of F- or Mn2+. The reconstituted activity was dependent on the amount of the solubilized myelin fraction and required the presence of 5'-guanylylimidodiphosphate, a hydrolysis-resistant analog of GTP. The elution pattern of the component solubilized from myelin in gel filtration was very similar to that of a GTP-dependent regulatory component from synaptic plasma membranes. The content of the regulatory component-like activity in myelin was estimated to be 50-60% of that in synaptic plasma membranes. Cholera toxin ADP-ribosylated proteins having molecular weights of 48,000, 38,000, 23,000, 20,000, and 15,000 and other minor peptides in myelin, some of which were also present in synaptic plasma membranes. We conclude that myelin contained a GTP-dependent regulatory component of adenylate cyclase despite the apparent lack of adenylate cyclase activity in myelin.     

Rapid kinetics of alpha 2-adrenergic inhibition of adenylate cyclase. Evidence for a distal rate-limiting step

Activation and inhibition of adenylate cyclase in the presence of GTP, the natural guanine nucleotide regulator, are too fast to study by standard biochemical methods. In order to identify the rate-limiting steps in adenylate cyclase regulation, we measured the kinetics of stimulation and inhibition of the enzyme on a subsecond to second time scale using a novel rapid-mix quench technique. Even using our rapid-mix quench method, activation by PGE1 and forskolin was instantaneous (cAMP accumulation was linear between 0.5 and 30 s). In contrast, we found a lag period of 1.2-10 s for epinephrine-mediated inhibition. The length of the lag depended on the concentration of GTP and monovalent cations present. In the absence of NaCl, the rate constant for the onset of inhibition (kinh) increased only slightly with GTP concentration saturating at a value of 0.16 s-1 (t1/2 4.3 s) at 1 microM GTP. In the presence of 100 mM NaCl, kinh was strongly dependent on GTP concentration, reaching a maximum value of 0.57 s-1 (t1/2 1.2 s) at 100 microM GTP. Thus, activation of both Gi and Gs in intact platelet membranes is much faster (t1/2 less than 5 s) than previously reported for reconstituted systems. Also, the strong dependence of the rate of adenylate cyclase inhibition on GTP concentration implies that the rate-limiting step in inhibition is distal to GTP binding. The effect of NaCl to increase the maximal rate of inhibition is specific for sodium since KCl has no effect on kinh.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of a Low Km GTPase Activity in Rat Striatum by Calmodulin

In rat striatum, the activation of adenylate cyclase by the endogenous Ca2+-binding protein, calmodulin, is additive with that of GTP but is not additive with that of the nonhydrolyzable GTP analog, guanosine-5'-(beta, gamma-imido)triphosphate (GppNHp). One possible mechanism for this difference could be an effect of calmodulin on GTPase activity which has been demonstrated to "turn-off" adenylate cyclase activity. We examined the effects of Ca2+ and calmodulin on GTPase activity in EGTA-washed rat striatal particulate fractions depleted of Ca2+ and calmodulin. Calmodulin inhibited GTP hydrolysis at concentrations of 10(-9)-10(-6) M but had no effect on the hydrolysis of 10(-5) and 10(-6) M GTP, suggesting that calmodulin inhibited a low Km GTPase activity. The inhibition of GTPase activity by calmodulin was Ca2+-dependent and was maximal at 0.12 microM free Ca2+. Maximal inhibition by calmodulin was 40% in the presence of 10(-7) M GTP. The IC50 for calmodulin was 100 nM. In five tissues tested, calmodulin inhibited GTP hydrolysis only in those tissues where it could also activate adenylate cyclase. Calmodulin could affect the activation of adenylate cyclase by GTP in the presence of 3,4-dihydroxyphenylethylamine (DA, dopamine). Calmodulin decreased by nearly 10-fold the concentration of GTP required to provide maximal stimulation of adenylate cyclase activity by DA in the striatal membranes. The characteristics of the effect of calmodulin on GTPase activity with respect to Ca2+ and calmodulin dependence and tissue specificity parallel those of the activation of adenylate cyclase by calmodulin, suggesting that the two activities are closely related.(ABSTRACT TRUNCATED AT 250 WORDS)