Forskolin-stimulated Enzyme Activity Research Articles

Adenylyl cyclase activity and G-protein subunit levels in postmortem frontal cortex of suicide victims

Basal and stimulated adenylyl cyclase activities and Gs and Gi protein alpha-subunit levels (Gs alpha and Gi alpha) were compared in postmortem frontal cortex from 18 suicide cases and 22 matched controls. Basal, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) stimulated and forskolin stimulated enzyme activities were significantly lower in the suicide cases, compared to controls. These effects were most apparent in those suicides that had died from violent means or that had had a history of depression and appeared to reflect the lowered basal activity rather than a reduced ability of either GTP gamma S or forskolin to activate the enzyme. No significant correlations were found between adenylyl cyclase activity and either subject age or postmortem delay. Western blotting revealed no significant differences in Gs alpha and Gi alpha levels between control and suicide cases. However, levels of the smaller Gs alpha isoform (Gs alpha-S) showed a tendency to be increased in the violent death suicide and depressed suicide subgroups, compared to controls. Levels of the larger Gs alpha isoform (Gs alpha-L) showed a significant positive correlation with subject age. Gi alpha levels showed a significant negative correlation with subject age and a positive correlation with postmortem delay. These results support the hypothesis that suicidal behaviour and depressive illness may be associated with an altered regulation of adenylyl cyclase.

Significance of the carbohydrate moiety of the rat ovarian luteinizing-hormone/chorionic-gonadotropin receptor for ligand-binding specificity and signal transduction.

The contribution of the carbohydrate moiety of the rat ovarian luteinizing-hormone (LH)/chorionic-gonadotropin (CG) receptor to ligand-binding specificity and signal transduction was investigated by using glycosidases. Purified membranes from pseudo-pregnant rat ovaries were treated with neuraminidase or peptide N-glycosidase F, to remove terminal sialic acids and N-linked oligosaccharides of the receptor, respectively. Ligand blotting and densitometric scanning of the autoradiograms showed that 90-95% of the receptors were deglycosylated, and that desialylation was virtually complete. Neither the desialylated nor the deglycosylated receptors were able to bind human follicle-stimulating hormone or bovine thyroid-stimulating hormone, as revealed by competition binding experiments. The 50% effective dose of hCG for adenylate cyclase activation, as determined by measuring the formation of cyclic [32P]AMP from [alpha-32P]ATP for 15 min at 30 degrees C, was similar in the control and deglycosylated membranes: 10.2 +/- 3.3 nM and 12.2 +/- 3.8 nM respectively. The same was true for the time course of the basal, hCG- and forskolin-stimulated enzyme activity. In addition, removal of oligosaccharides from the receptor did not restore the ability of desialylated hCG, nor of the deglycosylated hormone, to stimulate adenylate cyclase. In conclusion, the carbohydrate moiety of the native membrane-inserted rat ovarian LH/CG receptor does not contribute to the ligand-binding specificity, and it is not required for the functional coupling of the occupied receptor and the adenylate cyclase system. These functions are associated with the polypeptide portion of the receptor.

Neurotransmitter-mediated inhibition of post-mortem human brain adenylyl cyclase.

The effects of a range of neurotransmitter agonists showing selectivity for receptor types inhibitorily coupled to adenylyl cyclase were compared in membrane preparations of hippocampus, frontal cortex and caudate nucleus/striatum from previously frozen post-mortem human and rat brain. Agonists were tested against basal and forskolin stimulated activities, forskolin being a potent activator of the catalytic sub-unit of the enzyme. Of those agonists tested, only somatostatin (100 microM) and neuropeptide Y (10 microM) gave consistent inhibitions of basal and forskolin stimulated enzyme activities in all three regions of both human and rat brain. Somatostatin-mediated inhibition of human brain adenylyl cyclase was reduced in the absence of GTP and in the presence of the guanine nucleotide partial agonist, guanosine 5'-O-thiodiposphate, consistent with a G-protein-linked receptor. No such GTP-dependence was found for the neuropeptide Y-mediated adenylyl cyclase inhibition. GTP-dependent somatostatin mediated inhibitions of human brain adenylyl cyclase activity were of highest magnitude in the thalamus, intermediate magnitude in the hippocampus and caudate nucleus and lowest magnitude in the frontal cortex. It is concluded that of a range of neurotransmitter receptor agonists tested, only somatostatin gives robust, GTP-dependent responses that are reproducible enough to be used with post-mortem tissue for the comparison of receptor function in human brain disorders.

Adenylyl cyclase activity in postmortem human brain: evidence of altered G protein mediation in Alzheimer's disease.

The effects of agonal status, postmortem delay, and age on human brain adenylyl cyclase activity were determined in membrane preparations of frontal cortex from a series of 18 nondemented subjects who had died with no history of neurological or psychiatric disease. Basal and guanosine 5'-O-(3-thiotriphosphate)-, aluminum fluoride-, and forskolin-stimulated enzyme activities were not significantly reduced over an interval from death to postmortem of between 3 and 37 h and were also not significantly different between individuals dying with a long terminal phase of an illness and those dying suddenly. Basal and aluminum fluoride-stimulated enzyme activities showed a negative correlation with increasing age of the individual. In subsequent experiments, basal and guanosine 5'-O-(3-thiotriphosphate)-, aluminum fluoride-, and forskolin-stimulated enzyme activities were compared in five brain regions from a series of eight Alzheimer's disease and seven matched nondemented control subjects. No significant differences were observed between the groups for either basal activity or activities in response to forskolin stimulation of the catalytic subunit of the enzyme. In contrast, enzyme activities in response to stimulation with guanosine 5'-O-(3-thiotriphosphate) and aluminum fluoride were significantly reduced in preparations of neocortex and cerebellum from the Alzheimer's disease cases compared with the nondemented controls. Lower guanosine 5'-O-(3-thiotriphosphate)-, but not aluminum fluoride-, stimulated activity was also observed in preparations of frontal cortex from a group of four disease controls compared with nondemented control values. The disease control group, which contained Parkinson's disease and progressive supranuclear palsy patients, showed increased forskolin-stimulated activity compared with both the nondemented control and the Alzheimer's disease groups. These findings indicate a widespread impairment of G protein-stimulated adenylyl cyclase activity in Alzheimer's disease brain, which occurs in the absence of altered enzyme catalytic activity and which is unlikely to be the result of non-disease-related factors associated with the nature of terminal illness of individuals.

Enhanced adenylate cyclase activity in neonatally dopamine lesioned rats is related to increased G s-protein coupling

It has previously been shown that neonatal selective lesions of the central dopamine system with 6-hydroxydopamine lead to increased basal and dopamine-stimulated adenylate cyclase activity in striatum without any alterations of dopamine receptor binding characteristics. In the present study, it was shown that adenylate cyclase activity following G-protein stimulation by the GTP analogue 5-guanylimidodiphosphate (Gpp(NH)p) was increased in striatal preparations from neonatally 6-hydroxy-dopamine-lesioned rats, compared with control animals. No difference was seen in forskolin-stimulated enzyme activity between the two groups. These results indicate that neonatal dopamine lesions induce a selective functional supersensitivity at the D1 receptor complex by enhancing the coupling efficiency of the Gs protein to adenylate cyclase, without alterating the catalytic activity of the enzyme.

Coupling of a mutated form of the human beta 2-adrenergic receptor to Gi and Gs. Requirement for multiple cytoplasmic domains in the coupling process.

We constructed five genes encoding mutant human beta 2-adrenergic receptor sequence (beta 2AR) which contained 12-22 amino acid substitutions with corresponding sequence from the human alpha 2AAR in order to assess the receptor domains involved in Gs versus Gi recognition and coupling. Mutant beta 2AR with substitutions in the N (S1)- and C-terminal (S2) portions of the third intracellular loop, the proximal cytoplasmic tail (S3), and two combinations thereof (S2,3 and S1,2,3), were stably expressed in Chinese hamster fibrobasts (CHW-1102), as were the human beta 2AR and alpha 2AAR at comparable receptor levels. All mutant receptors with S2 substitutions (i.e. S2, S2,3, S1,2,3) were significantly (approximately 85%) uncoupled from Gs. Upon exposure to pertussis toxin, which uncouples receptors from Gi, S1,2,3 exhibited a 526 +/- 99% increase in agonist-stimulated adenylylcyclase activity compared with a 59 +/- 13% increase with the wild type receptor. This enhanced ability of S1,2,3 to interact with Gs following pertussis toxin treatment indicates that, in the absence of toxin exposure, substantial coupling occurs between the mutant receptor and Gi. Mutant beta 2AR bearing only one or two alpha 2AAR-substituted sequences showed no such enhancement. Forskolin-stimulated enzyme activities were increased by pertussis toxin treatment to similar degrees in all clones examined, indicating that the observed effects are confined to the receptor-mediated pathway. In the absence of GTP, competition binding experiments with S1,2,3, beta 2AR and alpha 2AAR revealed that approximately 40-50% of the receptors formed a high affinity binding state for agonist. Pertussis toxin treatment markedly reduced this to approximately 19% with S1,2,3, while having no effect on beta 2AR and completely eliminating high affinity agonist binding to alpha 2AAR. These results suggest that S1,2,3 interacts with Gi as well as Gs, and that receptor:G protein coupling requires the concerted participation of multiple cytoplasmic receptor domains.

Effects of lithium ion on the inhibitory GTP-binding protein and its coupling response

Addition of lithium ion to the inhibitory GTP-binding (Gi) protein resulted in a decrease of its ADP-ribosylation by islet-activating protein (pertussis toxin, IAP). The possibility that this decrease was due to dissociation of the Gi protein trimer was examined. Results showed that lithium ions had no appreciable effect on either the Gi protein trimer or its dissociation into its three subunits induced by Mg2+ and GTP gamma S. Next, the effect of lithium ions on Gi protein-mediated adenylate cyclase inhibition and alpha 2-adrenoceptor in human platelet membranes was examined. Lithium ion was found to impair adenylate cyclase inhibition of alpha 2-adrenoceptor stimulation of forskolin-stimulated enzyme activities. The monovalent ion also abolished guanine nucleotide modulation (GTP shift) of agonist binding, while it had no remarkable effects on antagonist binding in alpha 2-adrenoceptor of human platelet membranes. These results suggested that lithium ion caused functional change of the Gi protein without remarkable change of its dissociation, causing modulation in a coupling between alpha 2-adrenoceptor and Gi protein.

α-Bag cell peptide inhibits bag cell adenylate cyclase via a GTP-dependent mechanism

alpha-Bag cell peptide (alpha-BCP), one of several secreted peptides encoded in the precursor to the egg-laying hormone (proELH) of the neurosecretory bag cells of Aplysia, has been variously reported to have autoexcitatory or autoinhibitory effects on the cells which secrete it. Since we had found previously that alpha-BCP reduces stimulated cAMP levels in intact bag cells, an effect that would be consistent with electrophysiological inhibition, we investigated the direct effect of the peptide on adenylate cyclase in bag cell membrane preparations. alpha-Bag cell peptide did not affect basal adenylate cyclase activity, but reduced forskolin-stimulated activity by about 30%. The potency of the peptide in this assay was within the range reported for observable physiological effects: half-maximal inhibition was seen at approximately 100 nM peptide. Both basal and forskolin-stimulated enzyme activity were dependent on GTP, and the inhibitory effect of alpha-BCP was inversely dependent on the nucleotide. The non-hydrolyzable analogue, GTP-gamma-S, stimulated both basal and forskolin-stimulated enzyme activity and enhanced alpha-BCP's effect to the extent that the peptide completely inhibited forskolin's stimulation of the enzyme. The peptide's effect could be blocked by pretreatment with pertussis toxin. We conclude that alpha-BCP inhibits bag cell adenylate cyclase, an effect which is consistent with an autoinhibitory role in bag cell function. Moreover, this inhibition appears to be mediated by a GTP-binding protein.

Calmodulin Involvement in Stress‐ and Corticosterone‐Induced Down‐Regulation of Cyclic AMP‐Generating Systems in Brain

Manipulation of the hypothalamic-pituitary-adrenal axis selectively alters alpha-adrenergic potentiation of the cyclic AMP response to beta-adrenergic receptor stimulation in rat cerebral cortex. Calcium has been implicated in this alpha-receptor-mediated response, which may involve activation of phospholipases A2 and C and/or calmodulin-dependent adenylate cyclase. We therefore investigated the effects of stress and corticosterone (CORT) on membrane calmodulin-dependent adenylate cyclase and noradrenaline-stimulated cyclic AMP accumulation in brain slices. Repeated stress for 21 days selectively attenuated the adenylate cyclase response to calcium/calmodulin in cerebral cortex membranes, without affecting basal or forskolin-stimulated enzyme activity. There was no such effect in hippocampal membranes. The same pattern of response was elicited by daily CORT injection (50 mg/kg s.c.) for 21 days, while vehicle injection had no effect. CORT in the drinking water (400 micrograms/ml) elicited the same reduction of body weight as CORT injections, but had no effect on calmodulin adenylate cyclase. In parallel with calmodulin adenylate cyclase, cyclic AMP accumulation elicited by noradrenaline in slices of cerebral cortex was suppressed by both stress and daily CORT injections, with smaller effects observed with CORT in the drinking water. Unlike calmodulin adenylate cyclase, noradrenaline-stimulated cyclic AMP accumulation in hippocampus showed the same suppression as that in cerebral cortex. These results are discussed in relation to the differential mode of coupling of alpha-adrenergic receptors to cyclic AMP-generating systems between brain regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of thyroid hormone status on expression of genes encoding G protein subunits in the rat heart.

We have studied the influence of thyroid hormone status in vivo on expression of the genes encoding guanine nucleotide-binding regulatory protein (G protein) alpha-subunits Gs alpha, Gi alpha(2), Gi alpha(3), and both the 36-kDa form (beta 1) and the 35-kDa form (beta 2) of the beta-subunit in rat ventricle. The relative amounts of immunoactive Gi alpha(2) and Gi alpha(3) were greater in ventricular membranes from hypothyroid animals than from euthyroid animals (1.9- and 2.6-fold, respectively). A corresponding 2.3-fold increase in Gi alpha(2) mRNA was observed as well as a 1.5-fold increase in Gi alpha(3) mRNA. The relative amounts of immunoactive beta 1 and beta 2 polypeptides were also increased (2.8- and 1.8-fold, respectively) in the hypothyroid state and corresponded with comparable increases in the relative levels of beta 1 and beta 2 mRNAs. No difference was seen between the amounts of Gi alpha(2), Gi alpha(3), beta 1, and beta 2 in the euthyroid state and the hyperthyroid state. In contrast to these effects of thyroid hormone status on Gi alpha and beta, the steady-state amounts of Gs alpha protein and mRNA were not altered by thyroid hormone status. Thyroid hormone status did not alter sensitivity of adenylyl cyclase to stimulation by sodium fluoride or guanyl-5'-yl imidodiphosphate (GppNHp), nor did it influence GppNHp-induced inhibition of forskolin-stimulated enzyme activity. These results demonstrate that thyroid hormone status in vivo can regulate expression of specific G protein subunits in rat myocardium. However, the physiological consequences of these changes remain unclear.

Somatostatin inhibition of adenylate cyclase activity in different brain areas

Somatostatin receptors have been identified in different brain areas but the characterization of their postreceptor effect is still lacking. In this study we analyze the somatostatin effect on adenylate cyclase activity in different brain regions, namely frontal cortex, striatum, hypothalamus and hippocampus. Somatostatin inhibited basal adenylate cyclase activity in all brain areas, being maximally effective in the frontal cortex (-42%). Moreover, somatostatin inhibited both dopamine- and norepinephrine-stimulated adenylate cyclase activity in the examined cerebral regions showing a higher effectiveness than in basal conditions. VIP stimulation of adenylate cyclase was also reduced by somatostatin. The peptide inhibited by 50% forskolin-stimulated (10 nM to 10 microM) enzyme activity in frontal cortex and hypothalamus (in hippocampus the inhibition reached only -25%) showing a non-competitive pattern of inhibition. Finally, pertussis toxin pretreatment abolished the somatostatin inhibition of forskolin-stimulated frontal cortex adenylate cyclase activity. These results show that brain somatostatin receptors are coupled in an inhibitory way with adenylate cyclase enzyme that may represent one of the postreceptor mechanisms mediating the somatostatin modulation of brain functions.

Calmodulin modulates thymocyte adenylate cyclase activity through the guanine nucleotide regulatory unit

We have previously demonstrated in rat thymocyte plasma membranes that adenylate cyclase activity and its stimulation by 3,5,3'-triiodothyronine (T3) are influenced by calmodulin, and that these effects of calmodulin require calcium. In the present study, the mechanism by which calmodulin exerts its action was examined, in situ, in fresh plasma membranes isolated from rat thymocytes. Adenylate cyclase activity was potentiated by guanyl nucleotides, NaF and forskolin. Calmodulin did not affect basal adenylate cyclase activity. However, calmodulin influenced the guanyl nucleotide- and forskolin-stimulated adenylate cyclase activity, but had no effect on the fluoride-stimulated enzyme activity. This was evident from experiments with inhibitors of calmodulin: trifluoperazine, calmidazolium, and antibodies against calmodulin. The three inhibitors did not change basal adenylate cyclase activity, but all produced a marked decrease in the guanyl nucleotide- and forskolin-stimulated adenylate cyclase activity. The inhibitory effect of all three agents was reversed completely by the addition of calmodulin. The three inhibitors, however, failed to affect the fluoride-stimulated adenylate cyclase activity. In addition, T3, like the calmodulin inhibitors, did not change basal adenylate cyclase activity, increased the guanyl nucleotide- and forskolin-stimulated enzyme activity, but had no effect on the fluoride-stimulated enzyme activity. From these results I suggest that in the rat thymocyte calmodulin activation, and thereby T3 stimulation of the calcium-sensitive adenylate cyclase system is mediated through the guanine nucleotide regulatory unit.

Effective inhibition by pentobarbital of forskolin-stimulated adenylate cyclase activity in rat brain.

The effect of pentobarbital on the adenylate cyclase system was examined in synaptosomal membranes from rat brain. Pentobarbital inhibited forskolin-stimulated enzyme activity more effectively than the basal and Mn2(+)-stimulated enzyme activities. The degree of inhibition of the enzyme activity by pentobarbital was increased by the presence of forskolin in a concentration-dependent manner. No significant difference is observed in the degree of the inhibition by pentobarbital between the basal and forskolin-stimulated activities in the membranes prepared from the peripheral tissues.

Mechanism of galanin-inhibited insulin release. Occurrence of a pertussis-toxin-sensitive inhibition of adenylate cyclase

In the insulin-secreting beta cell line Rin m 5F, galanin, a newly discovered ubiquitous neuropeptide, inhibited, by 50%, the stimulation of insulin release induced by gastric inhibitory polypeptide (GIP) or forskolin, i.e. two cAMP-generating effectors. In contrast, it failed to decrease the stimulation of insulin release elicited by either the Ca2+-mobilizing agent, carbamoylcholine, or by dibutyryl-cAMP. Concomitantly, galanin inhibited the GIP- and forskolin-stimulated cAMP production. Furthermore, adenylate cyclase in membranes from Rin m 5F cells was highly sensitive to galanin, which exerted a marked inhibitory effect on the forskolin-stimulated enzyme activity. All these galanin effects were observed at low physiological doses, in the nanomolar range. Overnight treatment of the Rin m 5F cells with pertussis toxin completely abolished the inhibitory effect of galanin on insulin release, cAMP production and adenylate cyclase activity. Moreover, pertussis toxin specifically ADP-ribosylated a 39-kDa protein present in membranes from those cells. Taken together, these data show that the galanin inhibition of insulin release most likely occurs through the inhibition of adenylate cyclase, involving a petussis-toxin-sensitive inhibitory GTP-binding regulatory protein.

Adenylate cyclase in membrane fractions of RIN-A2-cells: studies with forskolin, NaF, GppNHp and NEM.

In crude membrane fractions of rat pancreatic islets and of RIN-A2-cells, forskolin and NaF stimulated adenylate cyclase activity. Basal and stimulated enzyme activity was approximately 3 to 6 fold higher in membranes of RIN-A2-cells than in membranes of islet cells. In RIN-A2-cells GppNHp and NEM inhibited forskolin-stimulated enzyme activity. The inhibitory effect of GppNHp could be reduced by NEM. It is suggested that the adenylate cyclase system of RIN-A2-cells contains inhibitory and stimulatory N-proteins and that there are critical thiols related to Ni, Ns and/or the catalytic unit. Thus, membrane fractions of RIN-A2-cells may be an appropriate model for studies on the adenylate cyclase system of insulin-producing cells.

Alpha 2-adrenoceptor-mediated inhibition of platelet adenylate cyclase activity in heroin addicts in abstinence.

The inhibition of basal and forskolin-stimulated platelet adenylate cyclase activity by (-)adrenaline was studied in ten heroin addicts during spontaneous withdrawal. Both basal and forskolin-stimulated enzyme activities were increased during heroin withdrawal and the inhibitory effect induced by (-)adrenaline was potentiated with parallel shifts to the left of the concentration-effect curves. The number of binding sites for [3H](-)adrenaline in platelet membranes was also increased during withdrawal. Treatment with clonidine markedly attenuated the inhibitory effect induced by (-)adrenaline on platelet adenylate cyclase activity. The results indicate that the heroin withdrawal syndrome induces supersensitivity of the platelet alpha 2-adrenoceptor-adenylate cyclase system.

Functional recovery of D 1 dopamine receptor-mediated stimulation of rat striatal adenylate cyclase activity following irreversible receptor modification by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ): Evidence for spare receptors

We report here the functional relationship between the time-dependent recovery of [3H]SCH 23390-labeled D1 dopamine receptors and the D1 receptor-mediated stimulation of rat striatal adenylate cyclase activity following irreversible receptor modification by in vivo administration of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. Initial decreases in receptor density (-93%) and receptor-mediated enzyme activity (-78%) were accomplished without concomitant changes in guanosine triphosphate or forskolin-stimulated enzyme activity. The percentage of maximal D1 receptor-mediated enzyme activity was significantly greater than that of D1 receptor density at all recovery times. Dopamine-stimulated enzyme activity returned to control values by day 4, although D1 receptor density remained significantly below control levels at this time. No differences in the EC50's for dopamine stimulation of enzyme activity were observed at any of the recovery times. These data demonstrate that the stoichiometric relationship between the recovering D1 dopamine receptors and D1 receptor-mediated enzyme activity is not one to one, providing evidence for the presence of 'spare' D1 dopamine receptors in rat striatum.

Alterations of rat cardiac adenylate cyclase activity with age

Basal, 5'-guanylimidodiphosphate, GTP-, NaF-, forskolin-, D,L-isoproterenol-, glucagon- and secretin-stimulated adenylate cyclase activities were investigated in cardiac membranes from young adult (6 month old), old (20 month old) and senescent (24 month old) Sprague Dawley rats. The only significant difference between old and young adult rats was a 43% decrease of the glucagon-stimulated enzyme activity. In senescent rats compared to young adult rats, we observed a 23% decrease in forskolin-stimulated enzyme activity, a more severe (-73%) decrease in glucagon-stimulated adenylate cyclase activity and a decrease (-38%) of the response to secretin. The response to the beta-adrenoreceptor agonist D,L-isoproterenol was unaffected. These results suggest an alteration with age in the vicinity of the catalytic unit of adenylate cyclase and a selective decrease of functional glucagon and secretin receptors.

Forskolin potentiates the stimulation of rat striatal adenylate cyclase mediated by D-1 dopamine receptors, guanine nucleotides, and sodium fluoride.

We report here that forskolin acts in a synergistic manner with dopaminergic agonists, guanine nucleotides, or sodium fluoride to potentiate the stimulation of rat striatal adenylate cyclase mediated by these reagents. In the presence of 100 microM GTP, 100 microM guanyl-5'-yl imidodiphosphate [Gpp(NH)p], or 10 mM NaF, there is a greater than additive increase in forskolin-stimulated enzyme activity as well as a concomitant decrease (two- to fourfold) in the EC50 value for forskolin stimulation of striatal enzyme activity. In the presence of various concentrations of forskolin (10 nM-100 microM), the stimulation of adenylate cyclase elicited by GTP, Gpp(NH)p, and NaF is potentiated 194-1,825%, 122-1,141%, and 208-938%, respectively, compared with the stimulation by these agents above basal activity in the absence of forskolin. With respect to 3,4-dihydroxyphenylethylamine (dopamine) receptor-mediated stimulation of striatal enzyme activity, the stimulation of enzyme activity by dopaminergic agonists, in the absence or presence of forskolin, was GTP-dependent and could be antagonized by the selective D-1 antagonist SCH23390 (100 nM), indicating that these effects are mediated by D-1 dopamine receptors. In the presence of 100 microM GTP, forskolin at various concentrations markedly potentiates the stimulation elicited by submaximal as well as a maximally effective concentrations of dopamine (100 microM) and SKF38393 (1 microM). At higher concentrations of forskolin (10-100 microM) the stimulation elicited by the partial agonist SKF38393 is comparable to that of the full agonist dopamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of human erythrocyte guanine nucleotide-binding regulatory protein on parathyroid hormone-responsive adenylate cyclase from canine renal cortex.

We studied the effects of the guanine nucleotide-binding regulatory protein (Gs) from human erythrocytes on PTH-responsive adenylate cyclase from partially purified membranes of canine renal cortex (CRC). Extracts of erythrocyte membranes, containing soluble Gs, was obtained by treatment with a detergent (Lubrol PX). Gs did not stimulate adenylate cyclase activity by itself, but amplified the response of adenylate cyclase in CRC membranes to both synthetic bovine PTH-(1-34) [bPTH-(1-34)] and to the hydrolysis-resistant GTP analog 5'-guanylimido-diphosphate [Gpp(NH)p]. Gs increased PTH stimulation of adenylate cyclase activity in both the presence and absence of Gpp(NH)p. In the absence of Gpp(NH)p, the potentiating effect of Gs occurred only when the concentration of bPTH-(1-34) was greater than 10 ng/ml. bPTH-(1-34), Gpp(NH)p, and Gs each enhanced the catalytic activity of adenylate cyclase when added separately or in combination by increasing the apparent maximum velocity (Vmax) of the enzyme without altering the apparent Km for MgATP. The effect of Gs on CRC membrane adenylate cyclase activity in the presence of NaF (10 mM) and forskolin (100 microM) was also examined. NaF- and forskolin-stimulated enzyme activities were significantly increased by Gs in both the presence and absence of Gpp(NH)p (100 microM). Analysis of double reciprocal plots of substrate concentration and enzyme activity revealed that NaF and forskolin increased the Vmax of the catalytic activity and did not alter the apparent Km of the enzyme for MgATP. These data support the role of Gs as a regulator of the response of adenylate cyclase to hormones, guanyl nucleotides, NaF, and forskolin. Our studies address the relative functional stoichiometry between Gs and catalytic unit present in CRC membranes and suggest that the CRC adenylate cyclase system must contain insufficient Gs to couple with all available catalytic units. These results are consistent with the possibility that deficiency of Gs impairs hormonal stimulation by diminishing the apparent Vmax of the catalytic unit and does not alter the apparent affinity of the enzyme for MgATP.