Binding of [3H]forskolin to solubilized preparations of adenylate cyclase

The binding of [3H]forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating [3H]forskolin bound to protein from free [3H]forskolin by rapid filtration. The Kd for [3H]forskolin binding to solubilized proteins was 14 nM which was similar to that for [3H]forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for [3H]forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. [3H]forskolin bound to proteins solubilized from membranes with a Bmax of 38 fmol/mg protein which increased to 94 fmol/mg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on [3H]forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmol/mg/min which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmol/mg/min which was not stimulated by GppNHp or forskolin. Thus, the number of high affinity binding sites for [3H]forskolin in solubilized preparations correlated with the activation of adenylate cyclase by GppNHp via the guanine nucleotide binding protein (GS).     

Binding of [3H]forskolin to human platelet membranes. Regulation by guanyl-5'-yl imidodiphosphate, NaF, and prostaglandins E1 and D2

[3H]Forskolin binds to human platelet membranes in the presence of 5 mM MgCl2 with a Bmax of 125 fmol/mg of protein and a Kd of 20 nM. The Bmax for [3H]forskolin binding is increased to 455 and 425 fmol/mg of protein in the presence of 100 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and 10 mM NaF, respectively. The increase in the Bmax for [3H]forskolin in the presence of Gpp(NH)p or NaF is not observed in the absence of MgCl2. The EC50 values for the increase in the number of binding sites for [3H]forskolin by Gpp(NH)p and NaF are 600 nM and 4 mM, respectively. The EC50 value for Gpp(NH)p to increase the number of [3H]forskolin binding sites is reduced to 35 mM and 150 nM in the presence of 50 microM PGE1 or PGD2, respectively. The increase in the number of [3H]forskolin binding sites observed in the presence of NaF is unaffected by prostaglandins. The binding of [3H]forskolin to membranes that are preincubated with Gpp(NH)p for 120 min or assayed in the presence of PGE1 reaches equilibrium within 15 min. In contrast, a slow linear increase in [3H]forskolin binding is observed over a period of 60 min when Gpp(NH)p and [3H]forskolin are added simultaneously to membranes. A slow linear increase in adenylate cyclase activity is also observed as a result of preincubating membranes with Gpp(NH)p. In human platelet membranes, agents that activate adenylate cyclase via the guanine nucleotide stimulatory protein (Ns) increase the number of binding sites for [3H]forskolin in a magnesium-dependent manner. This is consistent with the high affinity binding sites for [3H]forskolin being associated with the formation of an activated complex of the Ns protein and adenylate cyclase. This state of the adenylate cyclase may be representative of that formed by a synergistic combination of hormones and forskolin.     

Prostaglandin E-induced heterologous desensitization of hepatic adenylate cyclase. Consequences on the guanyl nucleotide regulatory complex

Prostaglandin E (PGE) receptor density in hepatic plasma membranes can be down-regulated by in vivo exposure to the 16,16-dimethyl analog of PGE2, and this is associated with desensitization of PGE-sensitive adenylate cyclase. These studies examined adenylate cyclase response to other agonists in membranes whose PGE receptor density was 51% decreased and whose maximal PGE-stimulated adenylate cyclase activity was 31% decreased. Down-regulated membranes had a 37% decrease in their maximal response to glucagon, indicating that treatment with the PGE analog had induced both homologous and heterologous desensitization. To determine whether adenylate cyclase had been affected, stimulation with NaF, guanyl 5'-yl imidodiphosphate (GppNHp), and forskolin was examined in both intact and solubilized membranes. Intact membranes had decreased adenylate cyclase responses to all three stimulators (NaF, -41%; GppNHp, -25%; forskolin, -41%) as did solubilized membranes (NaF, -51%; GppNHp, -50%; forskolin, -50%), suggesting alterations in adenylate cyclase rather than indirect membrane effects. Cholera toxin activation and labeling were examined to more directly assess whether the guanine nucleotide (G/F) regulatory component of adenylate cyclase had been affected. Cholera toxin activation was 42% less in down-regulated membranes, and these membranes incorporated less label when the incubation was performed in the presence of [32]NAD. Solubilized G/F subunit activity from down-regulated membranes was less effective in reconstitution of adenylate cyclase activity from cyc- cell membranes than G/F activity from control membranes. These data indicate that in vivo exposure to the PGE analog causes both homologous and heterologous desensitization of adenylate cyclase as well as an apparent quantitative decrease in G/F.     

Adenylate cyclase of platelets from spontaneously hypertensive rats: reduction of the inhibition by GTP

We have compared the effects of Gpp[NH]p on adenylate cyclase activity of platelet membranes in SHR and WKY rats. In the presence of 50 microM forskolin, low concentrations of Gpp[NH]p (0.01 to 0.3 microM) inhibited the enzyme activity in both strains, but the maximal level of inhibition was significantly lower in SHR (- 20%). In the absence of forskolin, 0.1 microM Gpp[NH]p was inhibitory only in WKY and the adenylate cyclase activity was greater in hypertensive rats at this nucleotide concentration. Increasing Gpp[NH]p from 0.1 to 3 microM induced the same increase of enzyme activity in both strains. In SHR, GTP itself induced a lower inhibition of the enzyme stimulated by 50 microM forskolin or 0.1 microM prostaglandin E1. These results suggest that the modulatory effect of the guanine nucleotide inhibitory protein on adenylate cyclase may be reduced in platelets from SHR.     

Binding of [14,15-3H]14,15-dihydroforskolin to rat liver membranes: comparison with the stimulatory effect of forskolin on adenylate cyclase

The binding of [14,15-3H]14,15-dihydroforskolin ([3H]DHF) to rat liver membranes has been further characterized and was compared with the stimulatory effect of forskolin on adenylate cyclase. The binding equilibrium dissociation constant (KD) for 14,15-dihydroforskolin obtained in inhibition experiments was 0.6 microM, with a maximal binding capacity (Bmax) of 114 pmol/mg protein. A similar KD value (0.5 microM) was derived from kinetics studies that revealed very rapid association and dissociation reactions. For structure-activity relationship studies several forskolin derivatives were synthesized and tested for their ability to inhibit [3H]DHF binding and increase adenylate cyclase activity. Among the tested compounds, forskolin itself was the most potent agonist (K1 = 0.2 microM). Further modification of the molecule in position 7 and (or) 1 decreased or abolished its agonist properties in both adenylate cyclase and binding studies. [3H]DHF binding was not affected by several nucleotides, carbohydrates, lectins, and hormone receptor agonists including isoproterenol, glucagon, and adenosine, but the steroids 17-beta-estradiol, progesterone, and testosterone showed slight inhibitory effects at unphysiologically high concentrations. [3H]DHF binding and forskolin-stimulated adenylate cyclase were sensitive to heat and N-ethylmaleimide treatment. Forskolin protected adenylate cyclase against inactivation by heat but not by N-ethylmaleimide. Preincubation of the membrane with trypsin decreased [3H]DHF binding. The results presented in this study demonstrate that the binding sites identified with [3H]DHF have a high specificity for forskolin and provide evidence that these binding sites are involved in the stimulation of adenylate cyclase by forskolin.     

Inhibition of prostaglandin E1-induced activation of adenylate cyclase in human blood platelet membrane

Activation of human blood platelet adenylate cyclase is initiated through the binding of prostaglandin E1 to the membrane receptors. Incubation of platelet membrane with [3H]prostaglandin E1 at pH 7.5 in the presence of 5 mM MgCl2 showed that the binding of the autacoid was rapid, reversible and highly specific. The binding was linearly proportional to the activation of adenylate cyclase. Although the membrane-bound radioligand could not be removed either by GTP or its stable analogue 5'-guanylylimido diphosphate, 150 nM cyclic AMP displaced about 40% of the bound agonist from the membrane. Scatchard analyses of the binding of the prostanoid to the membrane in the presence or absence of cyclic AMP showed that the nucleotide specifically inhibited the high-affinity binding sites without affecting the low-affinity binding sites. Incubation of the membrane with 150 mM cyclic AMP and varying amounts of prostaglandin E1 (25 nM to 1.0 microM) showed that the percent removal of the membrane-bound autacoid was similar to the percent inhibition of adenylate cyclase at each concentration of the agonist. At a concentration of 25 nM prostaglandin E1, both the binding of the agonist and the activity of adenylate cyclase were maximally inhibited by 40%. With the increase of the agonist concentration in the assay mixture, the inhibitory effects of the nucleotide gradually decreased and at a concentration of 1.0 microM prostaglandin E1 the effect of the nucleotide became negligible. These results show that cyclic AMP inhibits the activation of adenylate cyclase by low concentrations of prostaglandin E1 through the inhibition of the binding of the agonist to high-affinity binding sites.     

Prostaglandin receptor-adenylate cyclase system in plasma membranes of rat liver and ascites hepatomas, and the effect of GTP upon it.

1. Adenylate cyclase in plasma membranes from rat liver was stimulated by prostaglandin E1, and to a lesser extent by prostaglandin E2. Prostaglandin F1alpha and A1 did not stimulate the cyclase. The prostaglandin E1-mediated activation was found to require GTP when the substrate ATP concentration was reduced from 3 mM to 0.3 mM in the reaction mixture. Adenylate cyclase of the plasma membranes from rat ascites hepatomas AH-130 and AH-7974 was not stimulated by prostaglandin E1 in the presence or the absence of GTP, although the basal activity of adenylate cyclase as well as its stimulation by GTP alone were similar to normal liver plasma membranes. 2. Liver plasma membranes were found to have two specific binders for [3H] prostaglandin E1 with dissociation constants of 17.6-10(-9) M and 13.6-10(8) M (37 degrees C) and one specific binder for [3H]prostaglandin F2alpha with a dissociation constant of 2.31-10(8) M (37 degrees C). The specific binders for prostaglandin E1 could not be detected in the hepatoma plasma membranes. 3. Binding of [3H] prostaglandin E1 to the liver plasma membranes was exchange by, GTP dGPT, GDP, ATP and GMP-P(N)P, but not by GMP, CGMP, DTTP, UTP or CTP. The increase in the binding of [3H] prostaglandin E1 was found to be due to the increased affinity of the specific binders to prostaglandin F2alpha was not affected by GTP. 4. GTP alone was found to increase V of adenylate cyclase of liver plasma membranes, while GTP plus prostaglandin E1 was found to decrease Km of adenylate cyclase in addition to the increase of V to a further extent.     

Synthesis of 2',3'-O-(2,4,6-trinitrocyclohexadienylidine)guanosine 5'-triphosphate and a study of its inhibitory properties with adenylate cyclase

A fluorescent GTP analog 2',3'-O-(2,4,6-trinitrocyclohexadienylidine) guanosine 5'-triphosphate (TNP-GTP) has been prepared and some of its physical properties characterized. TNP-GTP was found to be a potent inhibitor of chick embryo heart adenylate cyclase as activated by guanyl 5'-(beta,gamma-imido)triphosphate (GppNHp), F-, and forskolin with Ki values in the 8-15 microM range. It also appeared to inhibit substantially basal adenylate cyclase in this system. TNP-GTP demonstrated an effective competition with [3H]GppNHp, binding to membranes equivalently to GppNHp and about three times better than GTP. 8-Azidoguanosine 5'-triphosphate (8N3GTP) mimics GTP activation of chick embryo heart adenylate cyclase and [gamma-32P]8N3GTP is effectively photoincorporated into a 42,000- to 44,000-Mr doublet when proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. TNP-GTP effectively prevents this photoincorporation, as does GTP, at concentrations that agree with their respective apparent inhibition and activation binding constants. The data suggest that TNP-GTP could prove to be a valuable tool for studying the mechanisms of GTP regulation of adenylate cyclase and other GTP-regulated systems.     

Characterization of [3H]forskolin binding sites in the iris-ciliary body of the albino rabbit

[3H]Forskolin binding sites were identified using membranes prepared from the iris-ciliary body of adult, albino rabbits. Scatchard analysis of saturation binding experiments demonstrated that [3H]forskolin bound to a single population of high affinity sites. The Kd and Bmax values were 8.7 +/- 0.9 nM and 119.0 +/- 30.9 fmol/mg prot. using membranes prepared from frozen tissue and 17.0 +/- 6.2 nM and 184.4 +/- 47.2 fmol/mg prot. using fresh tissue. The binding of [3H]forskolin was magnesium-dependent. The Bmax was enhanced by sodium fluoride and Gpp(NH)p, a nonhydrolyzable guanine nucleotide analog. Forskolin was the most potent inhibitor of [3H]forskolin binding; two commercially-available analogs were weaker inhibitors. In an adenylate cyclase assay, there was the same rank order of potency to enhance enzyme activity. Based upon binding affinities, magnesium-dependence, sensitivity to sodium fluoride and Gpp(NH)p, rank order of potencies of analogs and correlation of binding with adenylate cyclase activity, these studies suggest that the [3H]forskolin binding site in the iris-ciliary body is similar to the binding site in other tissues.     

Differential effect of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) on [3H]SCH23390 and [3H]forskolin binding in rat striatum

The binding of [3H]forskolin to a homogeneous population of binding sites in rat striatum was enhanced by NaF, guanine nucleotides and MgCl2. These effects of NaF and guanylylimidodiphosphate (Gpp(NH)p) were synergistic with MgCl2, but NaF and Gpp(NH)p together elicited no greater enhancement of [3H]forskolin binding. These data suggest that [3H]forskolin may label a site which is modulated by the guanine nucleotide regulatory subunit which mediates the stimulation of adenylate cyclase (NS). The D1 dopamine receptor is known to stimulate adenylate cyclase via NS. In rat striatum, the Bmax of [3H]forskolin binding sites in the presence of MgCl2 and NaF was approximately two fold greater than the Bmax of [3H]SCH23390-labeled D1 dopamine receptors. Incubation of striatal homogenates with the protein modifying reagent EEDQ elicited a concentration-dependent decrease in the binding of both [3H]SCH23390 and [3H]forskolin, although EEDQ was approximately 14 fold more potent at inactivating the D1 dopamine receptor. Following in vivo administration of EEDQ there was no significant effect on [3H]forskolin binding sites using a dose of EEDQ that irreversibly inactivated greater than 90% of D1 dopamine receptors. These data suggest that EEDQ is a suitable tool for investigating changes in the stoichiometry of receptors and their second messenger systems.     

Differential effects of fluoride on adenylate cyclase activity and guanine nucleotide regulation of agonist high-affinity receptor binding.

Fluoride ion, presumably an Al3+-F- complex, has been proposed to activate the guanine nucleotide regulatory protein (G-protein) of the visual system, transducin, by associating with GDP at the nucleotide-binding site and thus mimicking the effects of non-hydrolysable GTP analogues [Bigay, Deterre, Pfister & Chabre (1985) FEBS Lett. 191, 181-85]. We have examined this proposed model by using the adenylate cyclase complexes of frog erythrocytes, S49 lymphoma cells and human platelets. Preincubation of plasma membranes from frog erythrocytes and S49 cells with 20 mM-fluoride for 20 min at 30 degrees C strongly stimulated adenylate cyclase activity. In contrast, the preactivated membranes were still able to bind beta-adrenergic agonist with high affinity, as determined by radioligand-binding techniques. Moreover, high-affinity agonist binding in fluoride-treated membranes was fully sensitive to guanine nucleotide, which decreased beta-adrenergic-receptor affinity for agonist. Very similar results were obtained for [3H]prostaglandin E1 binding to S49 membranes pretreated with fluoride. Incubation of human platelet membranes with increasing concentrations of fluoride (1-50 mM) resulted in biphasic regulation of adenylate cyclase activity, with inhibition observed at concentrations greater than 10 mM. Preincubation of platelet membranes with 20 mM-fluoride did not affect agonist high-affinity binding to alpha 2-adrenergic receptors, nor receptor regulation by guanine nucleotide. These results suggest that the model developed from the study of transducin may not be generally applicable to the G-proteins of the adenylate cyclase system.     

Electroconvulsive Shock (ECS) and the Adenosine Neuromodulatory System: Effect of Single and Repeated ECS on the Adenosine A1 and A2 Receptors, Adenylate Cyclase, and the Adenosine Uptake Site

The effect of a single electroconvulsive shock (ECS) (30 min and 24 h after treatment) and repeated ECS (10 once-daily) on the adenosine neuromodulatory system was investigated in rat cerebral cortex, cerebellum, hippocampus, and striatum. The present study examined the adenosine A1 receptor using N6-[3H]cyclohexyladenosine ([3H]CHA), the A2 receptor using 5'-N-[3H]ethylcarboxyamidoadenosine ([ 3H]NECA), adenylate cyclase using [3H]forskolin, and the adenosine uptake site using [3H]nitrobenzylthioinosine ([3H]NBI). At 30 min after a single ECS, the Bmax of the [3H]NBI binding in striatum was increased by 20%, which is in good agreement with the well-known postictal adenosine release. The Bmax of [3H]forskolin binding in striatum and cerebellum was increased by 60 and 20%, respectively. In contrast to earlier reported changes following chemically induced seizures, [3H]CHA binding was not altered postictally. At 24 h after a single ECS, there were no changes for any ligand in any brain region. Following repeated ECS, there was a 20% increase of [3H]CHA binding sites in cerebral cortex, which lasted for at least 14 days after the last ECS. [3H]Forskolin binding in hippocampus and striatum was 20% lowered 24 h after 10 once-daily ECS but had already returned to control levels 48 h after the last treatment. Evidence is provided that the upregulated adenosine A1 receptors are coupled to guanine nucleotide binding proteins and, furthermore, that this upregulation is not paralleled by an increase in adenylate cyclase activity as labeled by [3H]forskolin.     

Purification and properties of prostaglandin E1/prostacyclin receptor of human blood platelets

Activation of platelet adenylate cyclase by prostaglandin E1 or prostacyclin is initiated through the interaction of the agonists with the same receptors on membrane. Prostaglandin E1/prostacyclin receptors of human platelets were solubilized in buffer, containing 0.05% Triton X-100 and protease inhibitors. The soluble membrane protein was chromatographed on a DEAE-cellulose column and assayed by a microfiber filter by equilibrium binding technique. The active fractions eluted at 0.7 M KCl were pooled, and the receptors were purified to homogeneity by Sephadex G-200 gel filtration with an overall recovery of 30%. The isolated receptor was 2,200-fold purified over the starting platelets. As evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the receptor showed a molecular mass of 190,000 daltons and is composed of two nonidentical subunits with molecular masses of 85,000 and 95,000 daltons. The interaction of prostaglandin E1 with the purified receptor was rapid, saturable, reversible, and highly specific. Among all prostaglandins tested, only prostacyclin was capable of displacing [3H]prostaglandin E1 bound to the receptor. Scatchard analysis of [3H]prostaglandin E1 binding to the purified receptor suggested the presence of a single class of high affinity binding sites (Kd = 9.8 nM) and a second population of low affinity binding sites (Kd = 0.7 microM) in the same protein molecule. Incubation of the purified receptor with platelets stripped of the receptor by washing with low concentrations of Triton X-100 efficiently restored the ability of prostaglandin E1 and prostacyclin to activate adenylate cyclase in these cells.     

Binding of (3H)prostaglandin E1 to putative receptors linked to adenylate cyclase of cultured cell clones.

A method for assessing the binding of 3H-labeled prostaglandin E1 ([3H]PGE1) to cell membranes has been developed and used to study the interaction of [3H]PGE1 with membranes from cultured mammalian cells. Receptor sites were identified by correlation of the potency of a series of compounds to compete for [3H]PGE1 binding sites and to stimulate adenylate cyclase activity, by correlation of rates of binding and change in enzyme activity, and by the correspondence of [3H]PGE1-binding activity with the presence or absence of PGE1-sensitive adenylate cyclase in several clones. In clone B82, a murine L-cell, [3H]PGE1 binds with an activation energy of 14 kcal/mol to a class of sites with an affinity of 0.5 X 10(8) M-1 and a capacity of 150 fmol/mg of protein. Concentration dependence of adenylate cyclase activation by PGE1 (KD =30 nM) and kinetic analysis of [3H]PGE1 binding (k1 = 4 X 10(6) liters/mol/min, k-1 0.15/min) verify this affinity. Concentration dependence and specificity of binding and activation of adenylate cyclases in neuroblastoma clone N4TG1 and N18TG2 substantiate the method. In several clones that lack PGE1-responsive adenylate cyclase, no specific [3H]PGE1 binding is detectable.     

Activation of adenylate cyclase in bovine corpus-luteum membranes by human choriogonadotropin, guanine nucleotides and NaF

1. Preincubation of luteal membranes with human choriogonadotropin results in the formation of an activated state of adenylate cyclase which is not reversed by washing and which is limited only by the absence of guanine nucleotides, whereas preincubation with GTP yields only a partially activated adenylate cyclase which requires the presence of both GTP and human choriogonadotropin during assay to demonstrate maximal activity. 2. Preincubation of luteal membranes with GTP and human choriogonadotropin does not lead to a synergistic increase in wash-resistant activity. 3. Luteal membranes that had been preincubated with GTP and hormone exhibited a decreasing rate of cyclic AMP synthesis during the adenylate cyclase assay incubation; addition of GTP during the assay incubation reversed the decrease. 4. Membranes that had been preincubated in the absence of guanine nucleotide and hormone showed a `burst' phase of cyclic AMP synthesis when GTP was present in the assay incubation and a `lag' phase with p[NH]ppG (guanosine 5′-[β,γ-imido]triphosphate) present in the assay. The presence of human choriogonadotropin with either nucleotide in the assay incubation eliminated the curvatures in plots observed with guanine nucleotides alone. 5. Luteal adenylate cyclase was persistently activated by preincubation with p[NH]ppG alone or in combination with human choriogonadotropin; the activation caused by p[NH]ppG alone was still increasing after 70min of preincubation, whereas that caused by p[NH]ppG in the presence of hormone was essentially complete within 10min of preincubation. 6. Luteal adenylate cyclase that had been partially preactivated by preincubation with p[NH]ppG was slightly increased in activity by the inclusion of further p[NH]ppG in the adenylate cyclase assay incubation, but more so with p[NH]ppG and hormone. Human choriogonadotropin alone caused no further increase in the activity of the partially stimulated preparation unless p[NH]ppG was also added to the assay incubation. 7. GTP decreased the activity of adenylate cyclase in membranes that had been partially preactivated in the presence of p[NH]ppG; the decrease in activity was greater when GTP and hormone were present simultaneously in the assay. 8. The results indicate that stable activation states of adenylate cyclase can be induced by preincubation of luteal membranes in vitro with human choriogonadotropin or p[NH]ppG, and that in the presence of p[NH]ppG the hormone may accelerate events subsequent to guanine nucleotide binding. Stable activation of luteal adenylate cyclase by prior exposure to GTP is not achieved. The involvement of GTPase activity and of hormone-promoted guanine nucleotide exchange in the modulation of luteal adenylate cyclase activity is discussed.     

The phorbol ester TPA stimulates the expression of functional beta-adrenoceptors in human T lymphoblasts Molt 3.

In crude membranes from human T lymphoblasts Molt 3 cultured under standard conditions, the adenylate cyclase system was stimulated by GTP, its beta gamma-imido analogue (p[NH]ppG,) NaF and forskolin, but not by isoprenaline, prostaglandin E1 and vasoactive intestinal peptide. TPA (tumour-promoting agent phorbol ester) added at low concentration (3.2 nM) to the culture medium induced a marked increase in functional beta 2-adrenoceptors. Competition curves of [125I]cyanopindolol with the antagonist ICI 118.551 and four beta-adrenergic agonists indicated that the emergence of functional beta 2-adrenoceptors corresponded to one class of binding sites, shifting from a high-affinity state for agonists to a low-affinity state in the presence of p[NH]ppG. This expression of beta 2-adrenoceptors after a 4 h lag period depended on newly formed mRNA and protein synthesis as judged by the inhibitory effects of actinomycin D and cycloheximide. Further effects of TPA included alterations of the stimulatory G-protein Gs and/or the catalytic unit of adenylate cyclase.     

Involvement of Ni protein in the functional coupling of the atrial natriuretic factor (ANF) receptor to adenylate cyclase in rat lung plasma membranes

In the presence of 1 microM atrial natriuretic factor (ANF) and low (0.1 mM) Mg2+ concentrations, the initial rate of binding of [3H]guanosine 5'-[beta, gamma-imido)triphosphate [( 3H]p[NH]ppG) to rat lung plasma membranes was increased twofold to threefold. ANF-dependent stimulation of the initial rate of [3H]p[NH]ppG binding was reduced at high (5 mM) Mg2+ concentrations. Preincubation of membranes with p[NH]ppG (5 min at 37 degrees C) eliminated the ANF-dependent effect on [3H]p[NH]ppG binding whereas ANF-dependent [3H]p[NH]ppG binding was unaffected by similar pretreatment with guanosine 5'-[beta-thio]diphosphate (GDP[beta S]). An increase in ANF concentration from 10 pM to 1 microM caused a 40% decrease in forskolin-stimulated or isoproterenol-stimulated adenylate cyclase activities (IC50 5 nM) in rat lung plasma membranes. GTP (100 microM) was obligatory for the ANF-dependent inhibition of adenylate cyclase, which could be completely overcome by the presence of 100 microM GDP[beta S] or the addition of 10 mM Mn2+. Reduction of Na2+ concentration from 120 mM to 20 mM had the same effect. Pertussis toxin eliminated ANF-dependent inhibition of adenylate cyclase by catalyzing ADP-ribosylation of membrane-bound Ni protein (41-kDa alpha subunit of the inhibitory guanyl-nucleotide-binding protein of adenylate cyclase). The data support the notion that one of the ANF receptors in rat lung plasma membranes is negatively coupled to a hormone-sensitive adenylate cyclase complex via the GTP-binding Ni protein.     

Isolation and characterization of 9-hydroxy-10-trans,12-cis-octadecadienoic acid, a novel regulator of platelet adenylate cyclase from Glechoma hederacea L. Labiatae

We have identified and characterized a fatty acid, (9S,10E,12Z)-9-hydroxy-10,12-octadecadienoic acid (9-HODE) as a regulator of adenylate cyclase activity of human platelet membranes. This fatty acid was isolated from a methanolic extract of the plant Glechoma hederacea L. Labiatae (commonly known as 'lierre terrestre', 'ground ivy' or 'creeping Charlie'; it was identified by nuclear magnetic resonance and mass spectroscopy. This compound increased basal adenylate cyclase activity in platelet membranes about threefold and had an EC50 of 10-20 microM. This increase in adenylate cyclase activity occurred without a temporal lag, was reversible, and represented an increase in Vmax without a substantial change in Km for ATP, Mg2+ or Mn2+. In addition, 9-HODE additively or synergistically increased platelet adenylate cyclase activity in response to guanosine 5'-[beta,gamma-imido]triphosphate and forskolin, but the fatty acid failed to alter inhibition of adenylate cyclase activity mediated by epinephrine (alpha 2-adrenergic receptor). Studies of the interaction of 9-HODE with activation of platelet adenylate cyclase activity mediated by prostaglandin E1 (PGE1) and prostaglandin D2 (PGD2) indicated that this fatty acid produced a parallel shift in the concentration/response curve of PGE1 and PGD2 without altering maximal response, which was substantially greater than that observed with 9-HODE alone. From these results, we conclude that 9-HODE appears to be a partial agonist at PGE1 and PGD2 receptors on human platelets. We believe that this is a novel example of a plant-derived fatty acid which acts on cells to regulate adenylate cyclase via prostaglandin receptors.     

Human platelet adenylate cyclase: persistent binding of guanine nucleotide is central to the regulation of both hormone-stimulated and basal activities

Prostaglandin E1 stimulation of human platelet adenylate cyclase, in purified plasma membranes, occurs without the addition of exogenous GTP. Possible contamination of the adenylate cyclase assay mixture by GTP either from nonspecifically bound nucleotide in the plasma membrane or from the substrate ATP was ruled out as follows: (a) variation of the membrane concentration, repeated washing, inclusion of EDTA, GDP beta S, or GMP in the wash step, or UDP in the assay, are all without effect, and (b) analysis of the substrate by high-performance liquid chromatography revealed no contaminating GTP. Other prostaglandins (I2, E2, D2) also activate cyclase without the addition of GTP. In sharp contrast, stimulation of adenylate cyclase in the human neutrophil plasma membrane by prostaglandin E1 shows an obligatory requirement for GTP, under identical assay conditions. GDP beta S pretreatment amplifies the fold cyclase stimulation by GTP in the presence and absence of prostaglandin E1, by lowering the basal activity. This alteration occurs without lowering the GTP-independent prostaglandin E1 activation, and is specific for inhibitory guanine nucleotides (GDP beta S, GMP, GDP) in the pretreatment. Extensive washing with buffer or incubation with other nucleotides, epinephrine, or prostaglandin E1 prior to the assay, is without effect. GTP gamma S treatment of the membrane induces a high-activity state and abolishes the GDP beta S effect on basal activity as well as prostaglandin E1 activation of cyclase. The results suggest distinct patterns of prostaglandin stimulation in platelet and neutrophil cyclase systems, and further imply that guanine nucleotide, prebound to specific sites within the GTP-regulatory proteins, may modify the kinetic characteristics of platelet adenylate cyclase.     

Ca2+-Guanine Nucleotide Interactions in Brain Membranes. I. Modulation of Central 5-Hydroxytryptamine Receptors in the Rat

Abstract: The present study indicates that central 5-hydroxytryptamine (5-HT; serotonin) receptors can be modulated in opposite directions by Ca²⁺ and guanine nucleotides [guanosine triphosphate (GTP), β, γ-imidoguanosine 5′-triphosphate (GppNHp)]. Thus CaCl₂ (≥0.5 mm ) inhibited whereas GTP and GppNHp (10 μm ) stimulated the 5-HT-sensitive adenylate cyclase in the hippocampus of newborn rats. Both the affinity (K_d ^?1) and the number (B_max) of [³H]5-HT binding sites in hippocampal membranes from adult rats were increased in the presence of Ca²⁺ (≥0.25 mm ); GTP (≥0.1 mm ) and GppNHp (≥0.3; μm ) produced reverse effects. The efficacy of guanine nucleotides in inhibiting specific [³H]5-HT binding was counteracted by Ca²⁺: the addition of this cation (5mm -CaCl₂) to the assay mixture resulted in a 40-fold increase in the IC₅₀ for GTP; the IC₅₀ for GppNHp increased five-fold under the same condition. The examination of the respective effects of Ca²⁺ and of GTP on the specific binding of [³H]5-HT to various hippocampal membrane preparations (from developing rats, from subcellular fractions of adult tissues, and from adult rats after the selective degeneration of serotoninergic innervation in the forebrain) indicated that the amplitudes of the Ca²⁺-induced increase and of the GTP-induced decrease were generally correlated. This conclusion did not apply to striatal membranes of kainic acid-treated rats because [³H]5-HT binding sites persisting after the intrastriatal injection of kainic acid (i.e., half of the total number in striatal membranes from control rats) were markedly less affected by GTP but at least as responsive as control membranes to the Ca²⁺-induced increase. These data are compatible with the hypothesis of a possible coupling of some–but not all–[³H]5-HT binding sites to adenylate cyclase in the rat brain.