Glutamate Inhibits Adenylate Cyclase Activity in Dispersed Rat Hippocampal Cells Directly via an N-Methyl-d-Aspartate-Like Metabotropic Receptor

Three major subtypes of glutamate receptors that are coupled to cation channels--N-methyl-D-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors--are known as ionotropic receptors in the mammalian CNS. Recently, an additional subtype that is coupled to GTP binding proteins and stimulates (or inhibits) metabolism of phosphoinositides has been proposed as a metabotropic receptor. Incubation of dispersed hippocampal cells from adult rats with glutamate or NMDA decreased forskolin-stimulated cyclic AMP (cAMP) accumulation; half-maximal effects were obtained with 5.6 +/- 2.2 and 6.4 +/- 2.3 microM, respectively. Kainate and quisqualate were less potent. The effect of glutamate was antagonized by 2,3-diaminopropionate and 2-amino-5-phosphonovalerate, NMDA/glutamate receptor antagonists, but not by 0.5 microM Joro spider toxin, a specific blocker of the AMPA receptor. The inhibitory effect of glutamate on cAMP formation was not blocked by 2 microM tetrodotoxin or by the absence of Ca2+. In hippocampal membranes, glutamate, similar to carbachol, inhibited adenylate cyclase activity in a GTP-dependent manner. These findings suggest that the glutamate inhibition of adenylate cyclase is direct and is not due to a result of the release of other neurotransmitters. The effect of glutamate on cAMP accumulation was observed in an assay medium containing 0.7 mM MgCl2, which is known to inhibit both ionotropic NMDA receptor/channels in the hippocampus and metabotropic NMDA receptors in the cerebellum. The inhibitory effect of glutamate was abolished by pertussis toxin treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opiate-Inhibited Adenylate Cyclase in Rat Brain Membranes Depleted of Gs-Stimulated Adenylate Cyclase

Opiate agonists inhibit adenylate cyclase in brain membranes, but under normal conditions the maximal inhibition is small (10-15%). When rat brain membranes were preincubated at pH 4.5, washed, and then assayed for adenylate cyclase at pH 7.4, stimulation of activity by agents (fluoride, guanylyl-5'-imidodiphosphate, cholera toxin) that act through the stimulatory GTP-binding coupling protein (Gs) protein was lost. At the same time, inhibition of basal adenylate cyclase by opiate agonists was increased to a maximum of 30-40%. Opiate inhibition was maximal at low magnesium concentrations (less than 5 mM), required guanine nucleotides, and decreased the Vmax, not Km, of the enzyme. Incubation of membranes with pertussis toxin lowered the apparent affinity for agonists in inhibiting activity. The delta opioid agonists were more potent than mu agonists, and the Ke values for naloxone in blocking agonist inhibition were similar for both mu and delta agonists (50-90 nM). These results suggest that inhibition of adenylate cyclase in brain is not mediated by mu opiate receptors, but whether classic high-affinity delta and kappa receptors are involved with this enzyme cannot be confirmed by these experiments.     

Characterization of Adenylate Cyclase Purified from Rat Brain by Hydrophobic Chromatography

Abstract. Hydrophobic chromatography of detergent-solubilized rat brain adenylate cyclase on dodecyl-Sepharose produced a species that was soluble in the absence of detergent and could be manipulated like a conventional hydrophilic protein. Sevenfold purification was achieved by this technique. Further purification could then be effected by affinity chromatography on ATP-Sepharose. The purified enzyme was no longer sensitive to fluoride or guanyl nucleotides. No interaction of brain adenylate cyclase was observed with immobilized triazinyl dyes such as Cibacron Blue 3GA nor with concanavalin A-Sepharose. The molecular weight of the fluoride-activated catalytic complex in a freeze-dried membrane preparation was estimated to be 133,000 by irradiation inactivation.     

Amitriptyline-Mediated Inhibition of Neurite Outgrowth from Chick Embryonic Cerebral Explants Involves a Reduction in Adenylate Cyclase Activity

We have previously shown that amitriptyline, a tricyclic antidepressant, inhibited neurite outgrowth from chick embryonic cerebral explants, and that dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthine, or theophylline can enhance neurite outgrowth from embryonic olfactory explants. In the present study, we examined the mechanism(s) underlying amitriptyline-mediated inhibition of neurite outgrowth by studying the effects of amitriptyline on adenylate cyclase activity and cyclic AMP levels. In cultured chick embryonic cerebral explants, dibutyryl cyclic AMP or theophylline, but not dibutyryl cyclic GMP, enhanced neurite outgrowth and partially reduced the inhibitory effects of amitriptyline on neurite outgrowth. Explants treated with amitriptyline for 2 days showed decreased cyclic AMP levels that significantly correlated with the degree of neurite outgrowth. Amitriptyline inhibited both basal and forskolin-stimulated adenylate cyclase activity in vitro, but only in the presence of GTP. Taken together, these data suggest that amitriptyline inhibits the activity of adenylate cyclase via a GTP-dependent mechanism, and that the subsequent decrease in cyclic AMP level may be involved in amitriptyline-mediated inhibition of neurite outgrowth.     

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.     

Nerve Growth Factor Affects Cyclic AMP Metabolism, but Not by Directly Stimulating Adenylate Cyclase Activity

Published experiments both support and contradict the hypothesis that nerve growth factor (NGF) can regulate adenylate cyclase activity. Using a sensitive assay that measures the conversion of [2-3H]adenine to [3H]cyclic AMP, we have shown that NGF alone cannot measurably stimulate cyclic AMP production, whereas the adenosine analog phenylisopropyladenosine (PIA) stimulates adenylate cyclase 20-fold over basal activity. NGF potentiates the capacity of both PIA and cholera toxin to stimulate cyclic AMP accumulation at all concentrations tested. This potentiation occurs at the earliest measurable times and does not require RNA synthesis. Therefore, we conclude that cyclase activation alone does not account for the effect of NGF on cyclic AMP accumulation and we discuss possible mechanisms.     

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.     

In Vivo Evidence that GABAB Receptors Are Negatively Coupled to Adenylate Cyclase in Rat Striatum

Abstract: The characteristics of the cerebral GABA_B receptor/cyclic AMP (cAMP)-generating system were investigated using the in vivo microdialysis technique in freely moving rats. Addition of forskolin, an activator of adenylate cyclase, to perfusate for 20 min resulted in a dose-dependent increase of cAMP efflux from the striatum. Pre- and coinfusions of baclofen for 80 min had no effect on the basal efflux of cAMP from the striatum but induced a significant decrease of forskolin (10 µ M )-stimulated cAMP efflux from the striatum in a dose-dependent manner. SKF 97541 (100 µ M ), a GABA_B receptor agonist, and GABA (50 µ M ) also decreased forskolin-induced cAMP efflux from the striatum. Coinfusion of CGP 54626A (100 µ M ), a GABA_B receptor antagonist, counteracted the effect of baclofen on the forskolin-stimulated cAMP efflux. In contrast, the isoproterenol (5 m M )-induced increase of cAMP efflux from the striatum was significantly enhanced by pre- and coinfusions with baclofen. These results suggest that this test system using in vivo microdialysis may be useful for examining the effect of drugs on the GABA_B receptor-linked cAMP-generating system in vivo.     

Calcium Dependence of Vasoactive Intestinal Peptide-Stimulated Cyclic AMP Accumulation in Rat Hippocampal Slices

Previous work has shown that incubation of hippocampal slices in medium without added calcium markedly attenuates the capacity of vasoactive intestinal peptide (VIP) to elevate cyclic AMP levels. The present studies examined the mechanism that confers calcium dependence on VIP stimulation of cyclic AMP accumulation in hippocampal slices. Calcium dependence was apparent immediately on slice preparation and was reversible only if calcium ions were added back very early during slice incubation (within 5 min). The cyclic AMP response to VIP was not abolished by preincubating slices in 100 microM adenosine, suggesting that calcium-dependent, VIP-induced release of adenosine does not mediate VIP elevation of cyclic AMP. VIP-stimulated cyclic AMP accumulation was not decreased by agents that block calcium influx (verapamil, nifedipine, magnesium ions), or by calmodulin antagonists (trifluoperazine, calmidozolium). In fact both verapamil (100 microM) and magnesium (14 mM) augmented VIP stimulation of cyclic AMP generation. Incubation of slices with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX) did not affect VIP activation of cyclic AMP accumulation if slices were incubated without added calcium, but MIX did enhance VIP elevation of cyclic AMP content in slices incubated with calcium. Thus calcium dependence of the cyclic AMP response to VIP in hippocampal slices is unlikely to result from VIP-dependent calcium influx, from interactions with calmodulin, or from calcium-inhibited phosphodiesterase(s).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Elevation of Intracellular Cyclic AMP and Stimulation of Adenylate Cyclase Activity by Vasoactive Intestinal Peptide and Glucaeon in the Retinal Pigment Epithelium

Both vasoactive intestinal peptide (VIP) and glucagon rapidly elevated cyclic AMP levels in embryonic chick retinal pigment epithelium (RPE), in culture as well as in freshly dissected tissue. In cultured cells, the half-maximal activities of VIP and glucagon were 5 X 10(-8) M and 3 X 10(-8) M, respectively. After 3 min of reaction, VIP elevated intracellular cyclic AMP by 100-fold; elevation with glucagon was up to 10-fold. Both neuropeptides stimulated adenylate cyclase activity in RPE membranes. Glucagon showed a half-maximal activity of 1 X 10(-8) M. VIP remained more effective than glucagon in stimulating adenylate cyclase activity, but the dose-response curve was shifted to a higher concentration range when compared to that of the VIP-elevated intracellular cyclic AMP.     

Distribution of Protein I, a Synapse-Specific Phosphoprotein, and Adenylate Cyclase in the Rat Spinal Cord

The longitudinal and transverse distributions of the synapse-specific phosphoprotein Protein I and adenylate cyclase in the rat spinal cord were studied. Protein I was found to be enriched in all cervical and midlumbar (L3-L5) segments, and sparse in midthoracic and sacral segments. Adenylate cyclase activity was high in all cervical and lumbosacral segments, and low in mid-thoracic segments. Cross sectionally, both Protein I and adenylate cyclase were more enriched in the dorsal half than in the ventral half in the various segments studied. The similar topographical distributions of Protein I and adenylate cyclase in the spinal cord support the idea that adenylate cyclase may be intimately associated with Protein I in the nervous system, and could thereby regulate the state of in vivo phosphorylation of Protein I through formation of cyclic AMP.     

Studies of the Regulation of Basal Adenylate Cyclase Activity by Membrane Polyunsaturated Fatty Acids in Cultured Neuroblastoma

The role of membrane polyunsaturated fatty acids (PUFAs) in the regulation of basal adenylate cyclase activity was examined in intact N1E-115 neuroblastoma cells. Addition of linoleic acid (50 microM) to the culture medium for 48 h resulted in a significant increase in phospholipid PUFA content and in a two- to fivefold increase in basal accumulation of cyclic AMP (cAMP). Both phenomena were reversed on removal of linoleate from the medium. PUFA enrichment stimulated cell proliferation by approximately 20% without altering the relative proportion of cellular protein. The supplemented cells synthesized significantly larger amounts of prostaglandin (PG) E and D than did the controls; however, blockade of PG synthesis by indomethacin or ibuprofen did not alter cAMP formation. Supplemented cells contained higher levels of malondialdehyde (MDA) than did controls, and MDA formation was reduced by coculture with alpha-tocopherol; however, its inclusion in the medium did not affect cAMP accumulation. Linoleate-supplemented cells responded to cyclase-activating agonists to the same extent as did control cells. Responses to inhibitory agonists (e.g., isoproterenol and carbamylcholine) were altered, but not to a sufficient extent to account for the PUFA-dependent increases in basal adenylate cyclase activity.     

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.     

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.     

Effects of Lead on Adenylate Cyclase Activity in Rat Cerebral Cortex

Lead decreased in a dose dependent manner the basal AC activity in membranes of rat cerebral cortex (IC₅₀ = 2.5 ± 0.1 M). In membranes preincubated under basal conditions, AC activity was stimulated by approximately two and fourfold by 10 M Gpp(NH)p or forskolin, respectively. Under basal conditions, lead (3 M) inhibited enzyme activity up to 50%, but was not able to inhibit the Gpp(NH)p- or the forskolin-stimulated AC activity. However, in membranes preincubated with Gpp(NH)p (10 M), lead (3 M) had no significant effect on enzyme activity, but it partly blocked the stimulation of AC activity elicited by forskolin (10 M). In membranes preincubated with 10 M lead, the addition of 10 M Gpp(NH)p or forskolin in the incubation medium did not stimulate AC activity. However, when added together in the incubation medium Gpp(NH)p + forskolin produced an increase in enzyme activity. In membranes preincubated with 10 M lead + 10 M Gpp(NH)p, Gpp(NH)p (10 M) or forskolin (10 M) added alone or in combination to the incubation medium did not stimulate AC activity. Moreover, under these latter conditions lead had no further effect on enzyme activity. These results indicate that lead may interact with G-proteins and with the catalytic subunit of cerebral cortical AC to produce inhibition of the enzyme activity.     

Presence of Corticotropin-Releasing Factor-Stimulated Adenylate Cyclase Activity in Rat Retina

Corticotropin-releasing factor (CRF) stimulates rat retinal adenylate cyclase activity in a concentration-dependent manner. The half-maximal effect is obtained at 50 nM CRF and the maximal stimulation corresponds to approximately 90% increase of basal enzyme activity. The CRF effect is counteracted by the CRF antagonist alpha-helical CRF 9-41 with a Ki value of 40 nM. Other CRF-like peptides such as sauvagine and urotensin I are as effective as CRF with a rank order of potency of urotensin I greater than or equal to sauvagine greater than CRF. The sauvagine and urotensin I effects are not additive with that elicited by CRF. Moreover, the CRF stimulation is not additive with the increase of enzyme activity produced by vasoactive intestinal peptide or dopamine. The CRF effect is independent of the concentration of free Ca2+, is optimal at 5-10 mM MgCl2, and requires GTP. The results indicate that rat retinal adenylate cyclase is modulated by CRF via a receptor-mediated mechanism.     

Regulation of Calmodulin- and Dopamine-Stimulated Adenylate Cyclase Activities by Light in Bovine Retina

Abstract: Neural retina from most species contains 3,4-dihydroxyphenylethylamine (dopamine) receptors coupled to stimulation of adenylate cyclase activity. It has been demonstrated that release of dopamine from its neurons and subsequent occupation of dopamine receptors is increased by light. In this study, we have shown that adenylate cyclase activity in bovine retina is highly responsive to the endogenous Ca²⁺-binding protein, cal-modulin, and that calmodulin can increase dopamine-sen-sitive adenylate cyclase activity in bovine retina. We further demonstrate that both dopamine- and calmodulin-stimulated adenylate cyclase activities can be regulated by alterations in light. Bovine retinas were dissected from the eye under a low-intensity red safety light, defined as dark conditions, and incubated for 20 min in an oxygenated Krebs Henseleit buffer under either dark or light conditions. The retinas were then homogenized and adenylate cyclase activity measured in a paniculate fraction washed to deplete it of endogenous Ca²⁺ and calmodulin. Activation of adenylate cyclase activity by calmodulin, dopamine, and the nonhydrolyzable GTP analog, gua-nosine-5′-(β,γ-imido)triphosphate (GppNHp), was significantly (60%) greater in paniculate fractions from retinas that had been incubated under dark conditions as compared to those incubated under light conditions. Basal, Mn²⁺-, and GTP-stimulated adenylate cyclase activities were not altered by changes in lighting conditions. Calmodulin could increase the maximum stimulation of adenylate cyclase by dopamine in retinas incubated under either dark or light conditions, but the degree of its effect was greater in retinas incubated under light conditions. Activation of adenylate cyclase by calmodulin, dopamine, and GppNHp in paniculate fractions from retinas incubated under light conditions was indistinguishable from the activation obtained when retinas were incubated in the dark in the presence of exogenous dopamine. These results suggest that an increased release of dopamine occurs in light. The decreased response of adenylate cyclase to exogenous dopamine can then be explained by a subsequent down-regulation of dopamine receptor activity. The down-regulation of dopamine receptor activity can also regulate activation of adenylate cyclase by GppNHp and calmodulin. The results suggest that dopamine, calmodulin, and GppNHp are modulators of a common component of adenylate cyclase activity, and this component is regulated by light.     

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.     

Formation of a Persistent Inhibitory State of Brain Adenylate Cyclase by GTP Analogs

Addition of 10 microM guanyl-5'-ylimidodiphosphate at 30 degrees or 0 degree to guinea pig brain particulates instantaneously evoked nearly 50% inhibition of adenylate cyclase activity as determined after removal of the GTP analog by washing of the particulates. The inhibitory state, once formed, persisted for at least 60 min as long as the preparation was kept either in a medium devoid of the analog (0-30 degrees) or in its presence at 0 degree. During incubation at 30 degrees in the presence of the analog, however, the inhibited or nontreated enzyme showed a gradual increase in enzyme activity. Both the inhibitory and the activating effects of the analog were saturable, with a half-maximal concentration of about 1.0 microM, and were antagonized by simultaneous addition of GTP, GDP, and GMP (in decreasing order). The persistently inhibited enzyme enabled the detection of marked stimulation by norepinephrine and histamine, whereas these amines showed only marginal stimulation of the enzyme before treatment with the analog. Formation of such a persistent inhibitory state appears to be specific to brain cyclase.