K-Opiate Agonists Inhibit Adenylate Cyclase and Produce Heterologous Desensitization in Rat Spinal Cord

The nature of the opiate modulation of adenylate cyclase following acute and chronic agonist exposure has been investigated in rat spinal cord. Using membranes of both adult rat spinal cord and spinal cord-dorsal root ganglion cocultures, we found that kappa-opiate receptors are negatively coupled to adenylate cyclase. The kappa-opiate agonists (e.g., U50488) inhibit significantly and dose-dependently the basal and the forskolin-stimulated cyclase activities, whereas mu and delta agonists are ineffective. The regulatory action is stereospecific and requires the presence of GTP. EGTA treatment of the plasma membranes abolished the effect of kappa-opiate agonists on the basal cyclase activity, and this inhibitory effect could not be restored by subsequent addition of Ca2+. The EGTA treatment did not affect the kappa agonist inhibition of the forskolin-stimulated cyclase. The results also show that following chronic exposure of cultured cells to etorphine or U50488, there is a loss of kappa agonist inhibition of the cyclase. Moreover, this desensitization process appears to be heterologous, because alpha 2-adrenergic agonists (e.g., clonidine or norepinephrine) and the muscarinic agonist (carbachol) exhibited significantly lower potency for inhibiting cyclase activity when compared to untreated cultures. This pattern of heterologous desensitization suggests that chronic exposure to kappa opiates leads to alterations in postreceptor regulatory components, possibly GTP-binding proteins.     

Opioid-Inhibited Adenylyl Cyclase in Rat Brain Membranes: Lack of Correlation with High-Affinity Opioid Receptor Binding Sites

Opioid agonists bind to GTP-binding (G-protein)-coupled receptors to inhibit adenylyl cyclase. To explore the relationship between opioid receptor binding sites and opioid-inhibited adenylyl cyclase, membranes from rat striatum were incubated with agents that block opioid receptor binding. These agents included irreversible opioid agonists (oxymorphone-p-nitrophenylhydrazone), irreversible antagonists [naloxonazine, beta-funaltrexamine, and beta-chlornaltrexamine (beta-CNA)], and phospholipase A2. After preincubation with these agents, the same membranes were assayed for high-affinity opioid receptor binding [3H-labeled D-alanine-4-N-methylphenylalanine-5-glycine-ol-enkephalin (mu), 3H-labeled 2-D-serine-5-L-leucine-6-L-threonine enkephalin (delta), and [3H]ethylketocylazocine (EKC) sites] and opioid-inhibited adenylyl cyclase. Although most agents produced persistent blockade in binding of ligands to high-affinity mu, delta, and EKC sites, no change in opioid-inhibited adenylyl cyclase was detected. In most treated membranes, both the IC50 and the maximal inhibition of adenylyl cyclase by opioid agonists were identical to values in untreated membranes. Only beta-CNA blocked opioid-inhibited adenylyl cyclase by decreasing maximal inhibition and increasing the IC50 of opioid agonists. This effect of beta-CNA was not due to nonspecific interactions with G(i), Gs, or the catalytic unit of adenylyl cyclase, as neither guanylylimidodiphosphate-inhibited, NaF-stimulated, nor forskolin-stimulated activity was altered by beta-CNA pretreatment. Phospholipase A2 decreased opioid-inhibited adenylyl cyclase only when the enzyme was incubated with brain membranes in the presence of NaCl and GTP. These results confirm that the receptors that inhibit adenylyl cyclase in brain do not correspond to the high-affinity mu, delta, or EKC sites identified in brain by traditional binding studies.     

Pertussis Toxin Attenuates 5-Hydroxytryptamine1A Receptor-Mediated Inhibition of Forskolin-Stimulated Adenylate Cyclase Activity in Rat Hippocampal Membranes

The inhibition of forskolin-stimulated adenylate cyclase activity by 5-hydroxytryptamine (5-HT) receptor agonists was measured in rat hippocampal membranes isolated from animals treated with vehicle or islet-activating protein (IAP; pertussis toxin). In vehicle-treated animals, 5-HT, 8-hydroxy-2-(di-n-propylamino)tetralin, buspirone, and gepirone were potent in inhibiting forskolin-stimulated adenylate cyclase activity with EC50 values of 60, 76, 376, and 530 nM, respectively. IAP treatment reduced by 30-55% the 5-HT1A agonist inhibition of adenylate cyclase activity via 5-HT1A receptors. The data indicate that the inhibitory guanine nucleotide-binding protein or Go (a similar GTP-binding protein of unknown function purified from brain) mediates the 5-HT1A agonist inhibition of hippocampal adenylate cyclase.     

Pertussis Toxin Attenuates D2 Inhibition and Enhances D1 Stimulation of Adenylate Cyclase by Dopamine in Rat Striatum

The response of adenylate cyclase to GTP and to dopamine (DA) was investigated in synaptic plasma membranes isolated from rat striatum injected with pertussis toxin, which inactivates the inhibitory guanine nucleotide-binding regulatory protein (Ni) of adenylate cyclase. Pertussis toxin treatment reverted the inhibitory effects on the enzyme activity elicited by micromolar concentrations of GTP and reduced by 50% the DA inhibition of cyclase activity via D2 receptors. The toxin treatment enhanced the net stimulation of enzyme activity by DA in the presence of micromolar concentrations of GTP. However, the stimulatory effect of the selective D1 receptor agonist SKF 38393 was not significantly affected. The data indicate that Ni mediates D2 inhibition of striatal adenylate cyclase and participates in the modulation of D1 stimulation of the enzyme activity by DA.     

Kappa opiate agonists inhibit Ca2+ influx in rat spinal cord-dorsal root ganglion cocultures. Involvement of a GTP-binding protein

The aim of the present study has been to characterize the regulation by opiates of 45Ca2+ influx in rat spinal cord-dorsal root ganglion cocultures. We have demonstrated that K+-induced depolarization, in the presence of the Ca2+ channel agonist Bay K8644, stimulated Ca2+ influx (3-4-fold) via the dihydropyridine class of voltage-dependent Ca2+ channels. While mu and delta opiates had no effect, kappa opiate agonists (e.g. U50488, dynorphin) profoundly depressed the stimulated Ca2+ influx (86% inhibition at 100 microM U50488). The kappa agonist action was stereospecific and could be reversed by the opiate antagonist naloxone. The inhibition produced by kappa agonists was greatly diminished following pertussis toxin treatment, and this effect was accompanied by toxin-induced ADP-ribosylation of a 40-41-kDa protein. This suggests that kappa opiate receptors are negatively coupled to voltage-dependent Ca2+ channels, via a pertussis toxin-sensitive GTP-binding protein. Basal 45Ca2+ uptake, stimulated by adenylate cyclase activators (forskolin and cholera toxin), was potently inhibited by kappa opiates suggesting that, under conditions of neurohormonal stimulation of adenylate cyclase, kappa receptors are coupled to Ca2+ channels indirectly via the adenylate cyclase complex. In addition, cAMP-independent coupling pathways may also be involved.     

Involvement of Both Inhibitory and Stimulatory Guanine Nucleotide Binding Proteins in the Expression of Chronic Opiate Regulation of Adenylate Cyclase Activity in NG108-15 Cells

Abstract: Chronic etorphine treatment of neuroblastoma × glioma NG108-15 cells results in both an increase in adenylate cyclase activity (upon addition of the opiate antagonist naloxone) as well as an homologous desensitization of the opiate receptor. The continued ability of opiate agonists to regulate adenylate cyclase activity following opiate receptor desensitization can be understood by proposing that the catalytic subunit of adenylate cyclase in NG108-15 cells is under tonic regulation by both guanine nucleotide regulatory (N_i) and stimulatory (N_s) components. Inactivation of N_i by pertussis toxin (PT) treatment resulted in elevated adenylate cyclase activities comparable to those observed in control cells following chronic opiate treatment. This increased enzymatic activity could not be further induced by PT treatment of cells exposed to opiate previously. In addition, procedures that prevented receptor-mediated activation of N_s, i.e., treatment with NaF or desensitization of the stimulatory receptors (prostaglandin E₁, adenosine) eliminated the increase in adenylate cyclase activity induced by naloxone following chronic opiate exposure. Hence, the increase in enzymatic activity observed following chronic opiate treatment may be due to a loss in tonic inhibitory regulation of adenylate cyclase mediated through N_i resulting in the unimpeded expression of N_s activity. This tonic inhibition of adenylate cyclase activity is one of the multiple mechanisms by which N_i regulates adenylate cyclase in this cell line.     

Opiate Receptor-Mediated Inhibition of Adenylate Cyclase in Rat Striatal Plasma Membranes

Abstract: Plasma membranes from rat striatum contain adenylate cyclase activity that is subject to dual regulation by GTP. Low concentrations (up to 30 nM) of the nucleotide increase activity whereas higher concentrations evoke a steady decline in activity; such behavior characterizes dually regulated adenylate cyclase systems. The opiates, morphine sulfate and D-Ala-Met-enkephalin, produce naloxone-reversible inhibition of the enzyme that is dependent on "inhibitory concentrations" of GTP (above 50 nM). In the absence of GTP no inhibition is observed. Sodium ions decrease the inhibition of activity promoted by GTP alone, but amplify the degree of inhibition seen in the presence of the opiates and GTP. The potencies of the opiates in mediating these effects mirror their affinities for 8 opiate receptors in striatum. It is suggested that this action of the opiates may represent their primary action in striatum.     

Phorbol Esters Increase GTP-Dependent Adenylate Cyclase Activity in Rat Brain Striatal Membranes

Abstract: 4β-Phorbol 12-myristate 13-acetate (PMA), added to a lysed mitochondrial fraction of rat striatum, stimulates adenylate cyclase activity with an apparent time lag of ～30 s. Half-maximal and maximal enzyme stimulations are obtained with 8 and 200 nM PMA, respectively. The PMA stimulation is GTP dependent, reaching a maximum of ～60% at 50 μ.M GTP, and is associated with disappearance of the enzyme inhibition induced by micromolar concentrations of GTP. Enhancement of enzyme activity by cholera toxin and 3,4-dihydroxyphenylethylamine is amplified by PMA only at micromolar concentrations of GTP. PMA does not affect the enzyme stimulation by forskolin but reverses the inhibition of forskolin-stimulated enzyme by GTP. When guanyl-5′-yl-imidodiphosphate is substituted for GTP, PMA does not modify adenylate cyclase activity. Enzyme inhibition by acetylcholine, Leu-enkephalin, and R(-)N⁶-(2-phenylisopropyl)adenosine is magnified by PMA. Stimulation of adenylate cyclase by PMA is markedly reduced following EGTA treatment, is not observed when adenyl-5′-yl-imidodiphosphate is substituted for ATP as substrate for adenylate cyclase, and is enhanced by l-α-phosphatidyl-l-serine. Like PMA, 4β-phorbol 12,13-dibutyrate and 1-oleoyl-2-acetylglycerol stimulate striatal adenylate cyclase, whereas 4β-phorbol and 4β-phorbol 13-acetate are ineffective. The results indicate that phorbol esters increase striatal adenylate cyclase activity by reducing the GTP-induced inhibition of the enzyme, presumably as a result of protein kinase C activation.     

Dopaminergic Receptors Linked to Adenylate Cyclase in Human Cerebromicrovascular Endothelium

Cultured endothelium derived from three fractions of human cerebral microvessels was used to characterize dopamine (DA) receptors linked to adenylate cyclase activity. DA or D1 agonist, (+/-)-SKF-82958 hydrobromide, stimulated endothelial cyclic AMP formation in a dose-dependent manner. The selective D1 antagonist, (+/-)SCH-23390, inhibited in a dose-dependent manner the production of cyclic AMP induced by DA. The affinity for the D1 receptor appeared to be greater in endothelium derived from large and small microvessels than from capillaries. Cholera toxin ADP-ribosylation of Gs proteins abolished the DA stimulatory effect on endothelial adenylate cyclase, whereas pertussis toxin ADP-ribosylation enhanced the DA-inducible formation, indicating the presence of both D1 and D2 receptors. Agonists of alpha 1-adrenergic receptors (phenylephrine, 6-fluoronorepinephrine) or serotonin (5-HT), which stimulated the production of cyclic AMP, had no additive effect on DA-stimulated cyclic AMP formation. Incubation of these agents with DA produced the same or lower levels of cyclic AMP as compared to that formed by DA alone. The effect of alpha 1-adrenergic agonists or 5-HT on DA production of cyclic AMP was partially prevented by the D2 antagonist, S(-)-sulpiride, or ketanserin (5-HT2 greater than alpha 1 greater than H1 antagonists), respectively. These findings represent the first demonstration of D1- (stimulatory) and D2- (inhibitory) receptors linked to adenylate cyclase in microvascular endothelium derived from human brain. The data also indicate that dopaminergic receptors can interact with either alpha 1-adrenergic or or 5-HT receptors in endothelium on the adenylate cyclase level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sodium Regulation of Hormone-Sensitive Adenylate Cyclase

Abstract

The influence of sodium was studied on hormone and guanine nucleotide-induced stimulation and inhibition of adenylate cyclase and on ß-adrenoceptor binding in various membrane systems. Sodium exerted almost identical effects on stimulation and inhibition of adenylate cyclase by various stimulatory and inhibitory hormones in all of the systems studied. The potencies of the hormones and of GTP to increase or to decrease the enzyme activity were reduced by sodium ions, without changing the maximal degree of adenylate cyclase stimulation or inhibition. Stimulation and inhibition of adenylate cyclase by the stable GTP analog, GTPγS, was affected in an identical manner by sodium, causing a retardation in the onset without a change in final stimulation or inhibition by the analog. Similar to the well-known reduction in α₂-adrenoceptor affinity for agonists, sodium also reduced the apparent affinity of ß-ad-renoceptors for the agonist, isoproterenol. It is concluded that sodium exerts identical effects on N_s and N_i, inhibiting the activation process of these two coupling components of the adenylate cyclase.     

μ-Opioid Receptors and Not K-Opioid Receptors Are Coupled to the Adenylate Cyclase in the Cerebellum

The putative regulatory effect of opioids on adenylate cyclase was investigated in two different preparations containing, respectively, two different populations of opioid receptors: the rabbit cerebellum (greater than 75% mu-opioid receptors) and the guinea pig cerebellum (greater than 80% kappa-opioid receptors). In the mu-preparation, but not in the kappa-preparation, opioids inhibited the basal and the forskolin-stimulated adenylate cyclase activity in a dose-dependent manner and stereospecifically. The inhibition was in the 20-30% range, required the presence in the assay medium of Mg2+ and of GTP, but was independent of the presence of Na+. Pharmacological characterization of the inhibitory response in the rabbit cerebellum clearly showed that it was under the control of a mu-opioid binding site, with the effect being elicited by non-selective (etorphine and morphine) and mu-selective (Tyr-D-Ala-Gly-Me-Phe-Gly-ol) agonists, whereas delta- and kappa-selective agonists were almost totally ineffective. ADP ribosylation of inhibitory GTP-binding protein by pertussis toxin failed to block the inhibitory effect of opioids, and data presented suggest that this failure is likely to be the consequence of a limited access of the toxin to its substrate in rabbit cerebellum membranes.     

Modification of Gs-Stimulated Adenylate Cyclase in Brain Membranes by Low pH Pretreatment: Correlation with Altered Guanine Nucleotide Exchange

Pretreatment of rat brain membranes at pH 4.5 before assay at pH 7.4 modifies the function of GTP-binding proteins (G-proteins) by eliminating Gs-stimulated adenylate cyclase activity while increasing opiate-inhibited adenylate cyclase activity. To help characterize the molecular nature of the low pH effect, we labeled Gs and Gi alpha-subunits in both control and low pH-pretreated membranes with the GTP photoaffinity analog [32P]P3 (4-azidoanilido)-P1-5'-GTP ([32P]AAGTP). When membranes were preincubated with unlabeled AAGTP, a persistent inhibitory state of adenylate cyclase was produced, which was overcome in untreated membranes with high (greater than 1 microM) concentrations of guanylyl-5'-imidodiphosphate [Gpp(NH)p]. In low pH-pretreated membranes, this inhibition could not be overcome, and stimulation by Gpp(NH)p was eliminated. Maximal inhibition of adenylate cyclase achieved by incubation with AAGTP was not altered by low pH pretreatment. Incorporation of [32P]AAGTP into Gs (42 kilodaltons) or Gi/o (40 kilodaltons) was unaffected by low pH pretreatment; however, transfer of 32P from Gi/o to Gs, which occurs with low (10 nM) concentrations of Gpp(NH)p in untreated membranes, was severely retarded in low pH-pretreated membranes. Both the potency and efficacy of Gpp(NH)p in producing exchange of [32P]AAGTP from Gi/o to Gs were markedly reduced by low pH pretreatment. These results correlate the loss of Gs-stimulated adenylate cyclase with a loss of transfer of nucleotide from Gi/o to Gs alpha-subunits and suggest that the nucleotide exchange participates in the modulation of neuronal adenylate cyclase.     

Inhibitory Coupling of Hormone and Neurotransmitter Receptors to Adenylate Cyclase

Abstract

The last few years have provided evidence that beside the familiar and partially well characterized stimulation of the adenylate cyclase the enzyme is also subject to a hormone and neurotransmitter-induced inhibition serving as a general information-transfer system. Similar to hormonal stimulation, the coupling of the inhibitory hormone receptors to adenylate cyclase is mediated by a GTP-dependent process. In some membrane systems, the inhibitory coupling is additionally amplified by sodium ions. Inhibitory hormones stimulate a high affinity GTPase in plasma membranes. The observed correlation suggests that there is a close connection between the hormone-induced adenylate cyclase inhibition and GTPase stimulation. Several lines of evidence are presented suggesting that the guanine nucleotide-dependent component apparently involved in adenylate cyclase inhibition and apparently exhibiting GTPase activity is at least partially different from that involved in adenylate cyclase stimulation by hormones.     

Adenosine Inhibition of Calmodulin-Sensitive Adenylate Cyclase from Bovine Cerebral Cortex

Calmodulin (CaM)-sensitive adenylate cyclase has recently been purified extensively from bovine brain. In this study, the sensitivity of the CaM-sensitive adenylate cyclase to adenosine and adenosine analogs was examined. The highly purified enzyme preparation retained sensitivity to inhibition by adenosine and adenosine analogs with ribose ring modifications, but not to those with purine ring modifications. Adenosine inhibition of this enzyme was not dependent on GTP and was noncompetitive with respect to ATP. Enzyme that had been dissociated from functional guanine nucleotide binding protein interactions by gel filtration in the presence of the zwitterionic detergent 3-[3-(cholamidopropyl)-dimethylammonio]-propanesulfonate and Mn2+ retained sensitivity to adenosine inhibition. The Ki for adenosine inhibition of the CaM-sensitive adenylate cyclase was approximately 2.6 X 10(-4) M. 5'-Guanylylimidodiphosphate and CaM did not affect the Ki of 3'-deoxyadenosine for the enzyme, but the presence of Ca2+ in the millimolar range raised the Ki by a factor of 5. These results show that the CaM-sensitive form of adenylate cyclase from bovine brain is subject to adenosine inhibition, and strongly suggest that this inhibition is due to interaction of ligands with a purine-specific ("P") site located on the catalytic subunit of the enzyme.     

Adenylate cyclase from sea urchin eggs is positively and negatively regulated by D-1 and D-2 dopamine receptors

The adenylate cyclase present in membranes prepared from sea urchin eggs is sensitive to dopamine stimulation. The receptor sites coupled to sea urchin adenylate cyclase were characterized by means of specific agonists and antagonists. The D-1 dopamine agonist SKF-38393 was able to stimulate enzyme activity, while the two D-1 dopamine antagonists, SCH-23390 and SKF-83566, suppressed the stimulatory effect of dopamine. In addition, the D-2 dopamine agonists, PPHT and metergoline, brought about a dose-dependent inhibition of dopamine-stimulated adenylate cyclase activity. These data show that: (i) in sea urchin eggs adenylate cyclase is regulated by dopamine receptors; (ii) these receptors share characteristics with D-1 and D-2 dopamine receptors present in the mammalian brain.     

Ethanol Does Not Modify Opiate-Mediated Inhibition of Striatal Adenylate Cyclase

Ethanol increases the activity of "basal," guanine nucleotide- and dopamine-stimulated adenylate cyclase in mouse striatum. In contrast, ethanol, in vitro, did not modify the inhibition of striatal adenylate cyclase activity by opiates (morphine or [D-Ala2,D-Leu5] enkephalin). Following chronic in vivo ethanol treatment of mice, there was also no change in the character of opiate inhibition of striatal adenylate cyclase activity. Since ethanol, in vitro, does decrease striatal opiate receptor binding, the results suggest that the changes in affinity detected by ligand binding studies are not relevant for receptor-coupled adenylate cyclase activity, or that opiate receptor binding and opiate regulation of adenylate cyclase can be modulated independently. The selective effects of ethanol on systems that modulate adenylate cyclase activity may produce imbalances in neuronal function during in vivo ethanol exposure.     

Regulation of Adenosine-Sensitive Adenylate Cyclase from Rat Brain Striatum

An adenosine-sensitive adenylate cyclase has been characterized from rat brain striatum. In whole homogenates as well as in particulate fractions, N⁶-phenylisopropyl adenosine (PIA), 2-chloroadenosine, and adenosine N′-oxide were equipotent in stimulating adenylate cyclase. Although GTP inhibited basal as well as PIA-stimulated activity of whole homogenates, the enzyme showed an absolute dependency on GTP for stimulation by PIA, dopamine, epinephrine, and norepinephrine in a particulate fraction derived from discontinuous sucrose gradient centrifugation. Adenosine exerts two effects on this adenylate cyclase, stimulation at low concentrations and inhibition at high concentrations, suggesting the presence of two adenosine binding sites. The stimulation of adenylate cyclase by PIA was dependent on the concentration of Mg^2-. The degree of stimulation by PIA was greater at a low concentration of Mg²⁺, which suggests that stimulation by PIA was accompanied by increasing the apparent affinity for Mg²⁺. Activation of adenylate cyclase by PIA was blocked by theophylline or 3-isobutyl- 1-methylxanthine (IBMX). The pH optimum for basal or (PIA + GTP)-stimulated activities was broad, with a peak between 8.5 and 9.5. In the presence of GTP, stimulation by an optimal concentration of PIA was additive, with maximal stimulation by the catecholamines. Phospholipase A₂ treatment at a concentration of 1 U/ml for 5 min completely abolished the stimulatory effect of dopamine, whereas PIA-stimulated activity remained unaltered. These data suggest that rat brain striatum either has a single adenylate cyclase, which is stimulated by catecholamines and adenosine by distinct mechanisms, or has different cyclase populations, stimulated by either adenosine or catecholamines.     

Properties of Muscarinic-Stimulated Adenylate Cyclase Activity in Rat Olfactory Bulb

The muscarinic stimulation of adenylate cyclase activity in rat olfactory bulb was characterized, with the aim of elucidating the nature of the molecular mechanism involved. Carbachol (CCh) stimulated the enzyme activity in either crude or purified cell membrane preparations and increased cyclic AMP accumulation in miniprisms of olfactory bulb. The CCh stimulation of adenylate cyclase activity displayed a fast onset and was rapidly reversed by addition of atropine. The stimulation was associated with an increase in the apparent Vmax of the enzyme, with no change in the Km for Mg-ATP. The affinity of the enzyme for Mg2+ was enhanced by CCh. The muscarinic effect required GTP at concentrations higher than those needed for enzyme stimulation with either l-isoproterenol or vasoactive intestinal peptide. Moreover, contrary to the beta-adrenergic stimulation, the muscarinic effect disappeared when guanosine 5'-O-(3'-thiotriphosphate) was substituted for GTP. In vivo treatment of olfactory bulbs with pertussis toxin completely prevented the muscarinic stimulation of adenylate cyclase, whereas cholera toxin was without effect. These results indicate that in rat olfactory bulb muscarinic receptors increase adenylate cyclase activity by interacting with a pertussis toxin-sensitive GTP-binding protein different from the stimulatory GTP-binding protein.     

Pertussis Toxin-Catalyzed ADP-Ribosylation: Effects on the Coupling of Inhibitory Receptors to the Adenylate Cyclase System

Abstract

The adenylate cyclase system consists of stimulatory and inhibitory hormone and drug receptors coupled through different GTP-binding proteins to a catalytic unit, responsible for the synthesis of cAMP from ATP. Pertussis toxin blocks the effect of inhibitory agonists on the catalytic unit by enzymatically inactivating the inhibitory GTP-binding protein (G_i). Study of the inhibitory arm of the cyclase system has been facilitated by the dissection of the overall process of hormonal inhibition of cAMP formation into a series of reactions characteristic of the individual protein components of this complex system; pertussis toxin has proven to be a useful tool with which to study these individual reactions. Exposure of cells or membranes to pertussis toxin in the presence of NAD results in ADP-ribosylation of a 41,000 Da subunit of G_i. ADP-ribosylation of G_i has a number of effects on the overall and partial reactions of the cyclase system, including a loss of a) hormonal inhibition of cAMP formation, b) hormonal stimulation of GTPase and c) agonist-induced release of membrane-bound guanyl nucleotides. In addition, in toxin-treated membranes, the affinity of inhibitory receptors for agonist but not antagonist is decreased with no significant change in receptor number.     

Modulation by Monoamines of Somatostatin-Sensitive Adenylate Cyclase on Neuronal and Glial Cells from the Mouse Brain in Primary Cultures

Primary cultures of mouse embryonic neuronal or glial cells from the cerebral cortex, striatum, and mesencephalon were used to identify and determine the cellular localization of somatostatin receptors coupled to an adenylate cyclase. Somatostatin inhibited basal adenylate cyclase activity on neuronal but not on glial crude membranes in the three structures examined. The somatostatin-inhibitory effect on neuronal crude membranes was still observed in the presence of (-)-isoproterenol, 3,4-dihydroxyphenylethylamine (dopamine, DA), or 5-hydroxytryptamine (5-HT, serotonin) used at a concentration (10(-5) M) inducing maximal adenylate cyclase activation. In addition, in most cases biogenic amines modified the pattern of the somatostatin-inhibitory effect, triggering either an increase in the peptide apparent affinity for its receptors or an increase in the maximal reduction of adenylate cyclase activity or both. However, 5-HT did not modify the somatostatin-inhibitory response on striatal and cortical neuronal crude membranes. The changes in somatostatin-inhibitory responses were interpreted as a colocalization of the amine and the peptide receptors on subtypes of neuronal cell populations. Finally, somatostatin was shown to inhibit adenylate cyclase activity following its activation by (-)-isoproterenol on glial crude membranes of the striatum and the mesencephalon but not on those of the cerebral cortex.