Inhibition by pertussis toxin of guanyl nucleotides exchange on transducin in bovine rod cell membranes

The effect of pertussis toxin on GTP-binding protein of bovine rod cell outer segments (transducin) was studied. Pertussis toxin was shown to ADP ribosylate either alpha subunit of free transducin or transducin-GDP complex, whereas GTP and its analogue Gpp(NH)p strongly inhibit ADP ribosylation of transducin. Pertussis toxin inhibits rod outer segment membrane GTPase and GTPase of homogeneous transducin by 40% and 70-80%, respectively. Activation of rod cell cyclic nucleotide phosphodiesterase by transducin is reduced after its preincubation with pertussis toxin. In transducin modified by pertussis toxin, 83% of GDP becomes tightly bound and cannot be exchanged with Gpp(NH)p. The stabilization of complex transducin-GDP after ADP ribosylation can explain the inhibitory effect of pertussis toxin on GTP hydrolysis by transducin, and on phosphodiesterase activation by guanyl nucleotides.     

Forskolin-activated adenylate cyclase. Inhibition by guanyl-5'-yl imidodiphosphate

Forskolin activated adenylate cyclase of purified rat adipocyte membranes in the absence of exogenous guanine nucleotides. Guanyl-5'-yl imidodiphosphate (Gpp(NH)p) inhibited the forskolin-activated cyclase immediately upon addition of the nucleotide at concentrations too low to activate adenylate cyclase (10(-9) to 10(-7) M). Inhibition seen with a very high concentration of Gpp(NH)p (10(-4) M) lasted for 3-4 min and was followed by an increase in the synthetic rate which remained constant for at least 15 min. The length of the transient inhibition did not vary with forskolin concentrations above 0.05 microM but low Gpp(NH)p (10(-8) M) exhibited a lengthened (6-7 min) inhibitory phase. The transient inhibitory effects of Gpp(NH)p were eliminated by 10(-7) M isoproterenol, high (40 mM) Mg2+, or preincubation with Gpp(NH)p in the absence of forskolin. While forskolin stimulated fat cell cyclase in the presence of Mn2+, this ion blocked the inhibitory effects of Gpp(NH)p. The well documented inhibitory effects of GTP on the fat cell adenylate cyclase system were also observed in the presence of forskolin. However, the inhibition by GTP is not transitory. These findings indicate that Gpp(NH)p regulation of forskolin-stimulated cyclase has at least two components: 1) an inhibitory component which acts through an undetermined mechanism and which acts immediately to decrease cyclase activity; and 2) an activating component which modulates the inhibited cyclase activity through the guanine nucleotide regulatory protein.     

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.     

Purification and partial characterization of two forms of urinary trypsin inhibitor

The activation of bovine thyroid adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) by Gpp(NH)p has been studied using steady-state kinetic methods. This activation is complex and may be characterized by two Gpp(NH)p binding sites of different affinities with measured constants: Ka1 = 0.1 micro M and Ka2 = 2.9 micro M. GDP beta S does not completely inhibit the Gpp(NH)p activation: analysis of the data is consistent with a single GDP beta S inhibitory site which is competitive with the weaker Gpp(NH)p site. Guanine nucleotide effects upon F- activation of adenylate cyclase have been studied. When App(NH)p is the substrate, 10 micro M GTP along with 10 mM NaF gives higher activity than NaF alone, while GDP together with NaF inhibits the activity by 50% relative to NaF. These features are not observed when the complex is assayed with ATP in the presence of a nucleotide regenerating system or when analogs Gpp)NH)p or GDP beta S are used along with NaF. These effects were studied in three other membrane systems using App(NH)p as substrate: rat liver, rat ovary and turkey erythrocyte. No consistent pattern of guanine nucleotide effects upon fluoride activation could be observed in the different membrane preparations. Previous experiments showed that the size of soluble thyroid adenylate cyclase changed whether membranes were preincubated with Gpp(NH)p or NaF. This size change roughly corresponded to the molecular weight of the nucleotide regulatory protein. This finding, coupled with the present data, suggests that two guanine nucleotide binding sites may be involved in regulating thyroid cyclase and that these sites may be on different protein chains.     

Involvement of SH-groups in the regulation of adenylate cyclase signal system by isoproterenol and serotonin in cultured murine fibroblasts L (subline LSM)

The adenylyl cyclase system (ACS) plays a key role in transduction of a hormonal signal into eukaryotic cells. The functional activity of the system depends on SH-groups of proteins involved in the ACS: receptor, G-protein, and enzyme adenylyl cyclase (AC). We studied the influence of thiols and SH-blockers on the regulation of AC activity by nonhormonal (NaF and Gpp[NH]p) and hormonal (biogenic amines isoproterenol and serotonin) agents in homogenates of cultured murine fibroblasts of line L (subline LSM). In the presence of thiols 2-mercaptoethanol (5 mM) and dithiothreitol (1 mM) the basal AC activity somewhat increased, whereas the stimulating effects of NaF, Gpp[NH]p, and hormones decreased. No potentiating action of Gpp[NH]p on hormonal effect in this case was found. The SH-blockers 25 mkM p-chloromercuribenzoic acid (CMBA) and 0.2 mM N-ethylmaleimide significantly inhibited both the basal AC activity and that stimulated by different agents. Thiols partially restored CMBA inhibited AC activity (in the case of N-ethylmaleimide restoring effects of thiols were insignificant). This, the ACS of murine fibroblasts of subline LSM is SH-sensitive. The forms of SH-groups in proteins involved in the ACS determine their functional activities and a possibility of transduction of the hormonal signal on the effector systems.     

Effect of GDP on transducin interaction with cyclic nucleotide phosphodiesterase and rhodopsin from bovine retinal rods

In the presence of guanyl nucleotides and rhodopsin-containing retinal rod outer segment membranes, transducin stimulates the light-sensitive cyclic nucleotide phosphodiesterase 5.5-7 times. The activation constant (Ka) for GTP and Gpp(NH)p is 0.25 microM, that for GDP and GDP beta S is 14 and 110 microM, respectively. GDP purified from other nucleotide contaminations at concentrations up to 1 mM does not stimulate phosphodiesterase but binds to transducin and inhibits the Gpp(NH)p-dependent activation of phosphodiesterase. The mode of transducin interaction with bleached rhodopsin also depends on the nature of the bound guanyl nucleotide: in the presence of GDP rhodopsin-containing membranes bind 70-100% of transducin, whereas in the presence of Gpp(NH)p the membranes bind only 13% of the protein. The experimental results suggest that GDP and GTP convert transducin into two different functional states, i.e., the transducin X GTP complex binds to phosphodiesterase causing its stimulation, while the transducin X GDP complex is predominantly bound to rhodopsin.     

Chromium(III) beta, gamma-bidentate guanine nucleotide complexes as probes of the GTP-activated cGMP cascade of retinal rod outer segments

The exchange-inert Cr(III) beta, gamma-bidentate guanine nucleotide complexes Cr(III)GTP and Cr(III)Gpp(NH)p were used to probe the role of transducin in activating the retinal cGMP cascade. The Cr(III) nucleotide complexes were found to have lower binding affinity for transducin as compared to the Mg2+ complexes. However, the rate of hydrolysis of the transducin-bound Cr(III)GTP was similar to that of Mg(II)GTP. Cr(III)Gpp(NH)p activated the cGMP phosphodiesterase of photolyzed rod outer segment membranes up to 75% of the Mg(II)Gpp(NH)p level but lacked the ability to dissociated the transducin subunits from the rod outer segment membrane. This result implies that the activation of the phosphodiesterase by transducin-GTP complex is a membrane-associated event and the formation of a soluble complex of transducin-GTP with the inhibitory peptide of the phosphodiesterase may not be an obligatory step. Both the delta and lambda screw sense stereoisomers of Cr(III)Gpp(NH)p were capable of activating the cGMP cascade with no apparent stereoselectivity. The nature of the interaction of the metal ion and GTP at the nucleotide-binding site of transducin is discussed together with the results from previous studies using the phosphorothioate GTP analogues [Yamanaka, G., Eckstein, F., & Stryer, L. (1985) Biochemistry 24, 8094-8101] and is compared to the site found in homologous GTP-binding proteins such as elongation factor Tu [Jurnak, F. (1985) Science (Washington, D.C.) 230, 32-36; la Cour, T.F.M., Nyborg, J., Thirup, S., & Clark, B.F.C. (1985) EMBO J. 4, 2385-2388]. The implications of the observed results on the molecular mechanism of visual signal transduction are discussed.     

Mechanism of adenylate cyclase activation by the rat lung cytoplasmic factors

Epinephrine, histamine and prostaglandin E₁ stimulated adenylate cyclase activity in lung membranes and their stimulation of the enzyme activity was completely blocked by propranolol, metiamide and indomethacin, respectively. A partially-purified activator from the adult rat lung also enhanced adenylate cyclase activity in membranes. However, stimulation of adenylate cyclase by the rat lung activator was not abolished by the above receptor antagonists. Further, epinephrine, NaF and Gpp(NH)p stimulated adenylate cyclase activity rather readily, whereas stimulation of the enzyme activity by the lung activator was evident after an initial lag phase of 10 min. Also, the lung activator produced additive activation of adenylate cyclase with epinephrine, NaF and Gpp(NH)p. These results indicate that the lung activator potentiates adenylate cyclase activity in membranes by a mechanism independent from those known for epinephrine, NaF and Gpp(NH)p. Incubation of lung membranes for 30 min at 40°C resulted in a loss of adenylate cyclase activation by NaF and Gpp(NH)p. Addition of the released proteins to the heat-treated membranes did not restore the enzyme response to these agonists. However, heat treatment of lung membranes in the presence of 2-mercaptoethanol or dithiothreitol prevented the loss of adenylate cyclase response to NaF and Gpp (NH)p. N-ethylmaleimide abolished adenylate cyclase activation by epinephrine, NaF, Gpp(NH)p and the lung activator. These results indicate that the sulfhydryl groups are important for adenylate cyclase function in rat lung membranes.Abbreviations Gpp(NH)p 5-Guanylimidodiphosphate     

Properties of detergent-dispersed adenylate cyclase from cerebral cortex. Presence of an inhibitor protein

Adenylate cyclase was solubilized from washed particulate fraction of rabbit cerebral cortex with the nonionic detergent Lubrol 12A9 and subjected to either gel filtration on Ultrogel AcA 34 or chromatography on DEAE Bio-Gel A. By both procedures the enzyme was resolved into two components, one insensitive to guanyl 5'-yl imidodiphosphate [Gpp(NH)p] and NaF but stimulated by Ca2+ and calmodulin, and another that was sensitive to Gpp(NH)p and NaF but relatively insensitive to Ca2+ and calmodulin. The data support the possibility that two independent forms of adenylate cyclase exist in cerebral cortex, one regulated by guanine nucleotide regulatory protein and another by Ca2+-calmodulin. Fractions containing the guanylnucleotide-sensitive activity were found to contain a factor that inhibited basal and Ca2+-stimulated adenylate cyclase in the Ca2+-sensitive fraction. The inhibitor was inactivated by heating at 60 degrees C and by incubation with trypsin. Inhibition was not time-dependent, and it was not due to destruction of cAMP by phosphodiesterase or of ATP by ATPase. Inhibitory action was not reversed by calmodulin and therefore it does not appear to be a calmodulin binding protein. Sucrose density gradient sedimentation indicated a sedimentation coefficient of 4S for the inhibitor; by this technique it co-sedimented with the adenylate cyclase sensitive to Gpp(NH)p and NaF.     

Characterization of transducin from bovine retinal rod outer segments. The role of sulfhydryl groups

The properties and functions of the sulfhydryl groups of transducin were examined by 5,5' -dithiobis-(2-nitrobenzoic acid) titration and N-ethylmaleimide modification. The T beta gamma subunit of transducin contained a total of six free sulfhydryl groups and two were reactive under native conditions. Both reactive sulfhydryl groups were located in the beta polypeptide. The functions of transducin were not affected by the modification of these two sulfhydryl groups. The T alpha subunit of transducin contained three accessible sulfhydryl groups under both native and denaturing conditions. When 1.3 sulfhydryl groups were covalently modified by N-ethylmaleimide, the GTPase activity, the guanosine 5' -(beta, gamma-imido)triphosphate (Gpp(NH)p) uptake, and the rhodopsin-binding property of transducin were inhibited. The binding of Gpp(NH)p to T alpha blocked two of the three sulfhydryl groups from chemical modification and increased the reactivity of the remaining one. Modification of this specific sulfhydryl group of T alpha -Gpp(NH)p inhibited the exchange of the bound Gpp(NH)p for GTP. However, the modified T alpha-Gpp(NH)p was able to activate cGMP phosphodiesterase in solution and on positively charged liposomes. These findings demonstrated that a conformational change of T alpha occurs upon the binding of Gpp(NH)p and a specific sulfhydryl group of T alpha plays an important role in the activation of transducin in retinal rod outer segments.     

Action of Mn2+ and vanadium compounds on hormone and forskolin induced stimulation of juvenile rat ovarian adenylate cyclase

The effect of forskolin on the hormonal (LH, FSH) activation and on the stimulation provided by other effectors (Gpp(NH)p,NaF) of the juvenile rat ovarian adenylate cyclase was investigated. Forskolin exhibited a synergistic action with LH, FSH and Gpp(NH)p but not with NaF. Addition of Ca2+ was inhibitory over a concentration range from 10(-5) to 10(-2) M whereas EGTA enhanced the response at 5.10(-5) M and inhibited it at higher concentration. The cAMP production was increased by addition of Mn2+ at low concentration (up to 5 mM) but markedly decreased at higher concentration (30 mM). FSH induced cAMP production was completely abolished at 30 mM Mn2+. The effect of vanadyl ion was very similar to that of Mn2+ Vanadate anion on the contrary was without effect on FSH stimulation.     

Uroporphyria development in cultured chick embryo fibroblasts long-term treated with chloramphenicol and ethidium bromide

In brain cortex, low concentrations of GTP or Gpp(NH)p activated the membrane-bound low Km cyclic AMP phosphodiesterase while higher concentrations of GTP, but not of Gpp(NH)p, reversed this activation. The adenosine analog N6-phenylisopropyladenosine (N6-PIA) elicited biphasic effect on this enzyme (activation up to 10(-8) M, complete reversion at 10(-5) M), provided that GTP was present. N6-PIA activation was reduced in the presence of Gpp(NH)p and blocked by sodium (80 mM). In contrast, the soluble low Km cyclic AMP phosphodiesterase was insensitive to GTP or N6-PIA. This study suggests that guanine nucleotides and N6-PIA exert their effects on the membrane-bound enzyme through guanine nucleotide regulatory proteins.     

Inhibition of forskolin-stimulated cardiac adenylate cyclase activity by short-chain alcohols

The diterpene forskolin stimulated rat cardiac adenylate cyclase activity at least 20-fold and potentiated the effect of NaF. The stimulatory effect of forskolin was reduced in the presence of Gpp(NH)p. Ethanol markedly reduced the stimulation of adenylate cyclase by forskolin while potentiating NaF and Gpp(NH)p stimulation. The inhibitory effect of ethanol on forskolin stimulation appeared to be of a mixed type with both a competitive and a non-competitive component. Three other short-chain linear alcohols (methanol, propanol, butanol) also inhibited forskolin-stimulation, this effect being proportional to the number of carbon atoms.     

Properties of Detergent-Dispersed Adenylate Cyclase from Cerebral Cortex. Presence of an Inhibitor Protein

Abstract: Adenylate cyclase was solubilized from washed paniculate fraction of rabbit cerebral cortex with the nonionic detergent Lubrol 12A9 and subjected to either gel filtration on Ultrogel AcA 34 or chromatography on DEAE Bio-Gel A. By both procedures the enzyme was resolved into two components, one insensitive to guanyl 5'-yl imidodiphosphate [Gpp(NH)p] and NaF but stimulated by Ca²⁺ and calmodulin, and another that was sensitive to Gpp(NH)p and NaF but relatively insensitive to Ca²⁺ and calmodulin. The data support the possibility that two independent forms of adenylate cyclase exist in cerebral cortex, one regulated by guanine nucleotide regulatory protein and another by Ca²⁺-calmodulin. Fractions containing the guanylnucleotide-sensitive activity were found to contain a factor that inhibited basal and Ca²⁺-stimulated adenylate cyclase in the Ca²⁺-sensitive fraction. The inhibitor was inactivated by heating at 60°C and by incubation with trypsin. Inhibition was not time-dependent, and it was not due to destruction of cAMP by phosphodiesterase or of ATP by ATPase. Inhibitory action was not reversed by calmodulin and therefore it does not appear to be a calmodulin binding protein. Sucrose density gradient sedimentation indicated a sedimentation coefficient of 4S for the inhibitor; by this technique it co-sedimented with the adenylate cyclase sensitive to Gpp(NH)p and NaF.     

Attenuation of catecholamine-coupled adenylate cyclase following surgical isolation of rat caudate

Six weeks following complete unilateral surgical isolation of the rat caudate nucleus, activation of adenylate cyclase was reduced in response to dopamine (DA), norepinephrine (NE), 5' -guanylyl-imidodiphosphate [Gpp(NH)p], DA + Gpp(NH)p, and NaF. The low Km form of cyclic AMP phosphodiesterase was elevated in the isolated side when compared to the intact caudate. No changes in activities of guanylate cyclase or in high Km cyclic AMP phosphodiesterase (with or without the calcium-dependent regulator protein, calmodulin or CDR) were observed between the control and isolated caudate. Histologically, the neural damage to the isolated caudate was principally confined to reduced numbers of dendritic spines of the remaining intrinsic caudate neurons.     

Characterization of an ATP-Mg2+-dependent guanine nucleotide-stimulated adenylate cyclase from Neurospora crassa

A novel adenylate cyclase activity was found in crude homogenates of Neurospora crassa. The adenylate cyclase had substantial activity with ATP-Mg2+ as substrate differing significantly from the strictly ATP-Mn2+-dependent enzyme characterized previously. Additionally, the ATP-Mg2+-dependent activity was stimulated two- to fourfold by GTP or guanyl-5'-yl-imido-diphosphate (Gpp(NH)p). We propose that the ATP-Mg2+-dependent, guanine nucleotide-stimulated activity is due to a labile regulatory component (G component) of the adenylate cyclase which was present in carefully prepared extracts. The adenylate cyclase had a pH optimum of 5.8 and both the catalytic and G component were particulate. The Km for ATP-Mg2+ was 2.2 mM in the presence of 4.5 mM excess Mg2+. Low Mn2+ concentrations had no effect on adenylate cyclase activity whereas high concentrations of Mn2+ or Mg2+ stimulated the enzyme. Maximal Gpp(NH)p stimulation required preincubation of the enzyme in the presence of the guanine nucleotide and the K1/2 for Gpp(NH)p stimulation was 110 nM. Neither fluoride nor any of a variety of glycolytic intermediates or hormones, including glucagon, epinephrine, and dopamine, had an effect on ATP-Mg2+-dependent adenylate cyclase activity. However, the enzymatic activity was stimulated not only by GTP but also by 5'-AMP and was inhibited by NADH.     

A persistent active state of the adenylate cyclase system produced by the combined actions of isoproterenol and guanylyl imidodiphosphate in frog erythrocyte membranes.

Isoproterenol plus guanylyl imidodiphosphate (Gpp(NH)p) activate frog erythrocyte adenylate cyclase to a level much higher than the sum of the activities produced by the catecholamine and the synthetic nucleotide tested separately. Propranolol, the beta-receptor blocking agent, failed to inhibit activity when added after the enzyme system had been preincubated with both isoproterenol and Gpp(NH)p. However, if propranolol was added after only one of the two components had been added, it inhibited the effect of isoproterenol. Production of the propranolol-resistant state by treatment with isoproterenol and Gpp(NH)p did not require the presence of the productive substrate (MgATP). The activated propranolol-resistant state persisted following various treatments of the enzyme preparation including extensive washings of the membranes; considerable activity was retained even after sonication or treatment with the detergent Lubrol-PX, treatments which caused inactivation of the native enzyme. Extensive dilution of the membranes following pretreatment with isoproterenol and Gpp(NH)p did not significantly reduce to the activity of the enzyme. Readdition of isoproterenol after dilution caused some inhibition of adenylate cyclase activity, indicating apparently that the beta-receptor has not become inaccessible. In contrast, preincubation with isoproterenol alone failed to render the enzyme system refractive to propranolol, and dilution readily reduced the activity to negligibly low values. Preincubation with Gpp(NH)p alone also produced a persistent active state but the activity was much lower than that obtained throught the combined action of isoproterenol and Gpp(NH)p. The findings suggest that the hormone may be required only to facilitate the initial interaction of the enzyme with Gpp(NH)p. The differences, in this respect, between Gpp(NH)p and the more labile natural nucleotide, GTP, are discussed.     

Mechanism of action of forskolin on adenylate cyclase: effect on bovine sperm complemented with erythrocyte membranes

The mechanism of action of forskolin stimulation of adenylate cyclase was investigated by examining its effects on the enzyme's Mg2+ activated catalytic unit (C) from bovine sperm, both preceding and following complementation with human erythrocyte membranes as a source of guanine nucleotide regulatory protein (N). Prior to complementation, sperm C was not activated by either NaF (10 mM) or 5'-guanylyl-beta-gamma-imidodiphosphate (Gpp(NH)p, 10 microM), suggesting that functional N was not present in this preparation. Forskolin (100 microM) was also without effect on C. Following complementation of the sperm membranes with those of erythrocytes, Mg2+-dependent sensitivity to forskolin, NaF, and Gpp(NH)p was imparted to C. Our findings are incompatible with the current hypothesis that forskolin stimulates adenylate cyclase by direct activation of C. Rather, the data suggest that the activation process occurs through an effect on N or by augmentation of the interaction between the components of the adenylate cyclase complex.     

Alterations in adenylate cyclase activity due to streptozotocin-induced diabetic cardiomyopathy

The effect of chronic experimental diabetes on the adenylate cyclase system (AC) in the rat heart was investigated. Rats were made diabetic by an intravenous injection of streptozotocin (65 mg/kg), hearts were removed 8 weeks later and washed cell particles were isolated. AC activity was measured in the absence and presence of different concentrations of forskolin, NaF, GTP analogue [Gpp(NH)p] or epinephrine. A significant depression in the epinephrine stimulated AC activity was observed in diabetic hearts. Basal AC activity and stimulation of AC with forskolin, NaF and Gpp(NH)p were not significantly different between control and diabetic preparations. These results indicate no apparent alterations in the regulatory or catalytic properties of AC in hearts from chronic diabetic rats. The observed depression in epinephrine stimulated AC activity may account for the depressed inotropic action of catecholamines in the diabetic cardiomyopathy.     

The beta-adrenoceptor is precoupled to Gs in chicken erythrocyte membranes

In this study we seek to elucidate the mechanism of hormone-independent adenylate cyclase stimulation by Gpp(NH)p in chicken erythrocyte membranes, and the inhibition of this stimulation by propranolol. Membrane treatment with isoprenaline + GMP increased Gpp(NH)p stimulation to near maximal levels [obtainable with isoprenaline + Gpp(NH)p], but reduced stimulation by NaF. The stimulation by Gpp(NH)p was stereoselectively inhibited by propranolol, but not by equal concentrations of the local anaesthetic lignocaine. Propranolol's inhibitory action was abolished following membrane treatment with isoprenaline/GMP. In contrast to its inhibition of Gpp(NH)p stimulation, propranolol did not alter Gpp(NH)p-mediated 3H-GDP release from membranes. The polyene antibiotic filipin, which uncouples receptor (R) from Gs, also abolished Gpp(NH)p stimulation and this effect was partly overcome by membrane treatment. These results are consistent with a model in which free R exists in equilibrium with precoupled R.Gs complexes in the absence of hormone. These complexes are activated by Gpp(NH)p and dissociated by antagonists. The existence of such complexes is a prerequisite for Gpp(NH)p stimulatory action.