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The coupling of opioid receptor-like (ORL1) receptors to adenylyl cyclase has been investigated in specific layers of the rat main olfactory bulb. Membranes prepared from the olfactory nerve-glomerular layer (ON-G layer), external plexiform layer (EP layer) and granule cell layer (GR layer) displayed specific binding sites for [(3)H]-nociceptin/orphanin FQ ([(3)H]Noc/OFQ). In each layer, the presence of high-and low-affinity binding sites, with K(D) values in the picomolar and nanomolar range, respectively, was detected. The binding of [(3)H]Noc/OFQ was displaced by unlabelled Noc/OFQ, but not by opioid antagonists. In each layer, Noc/OFQ significantly stimulated [(35)S]GTPgammaS binding with nanomolar potencies. In ON-G layer, Noc/OFQ inhibited basal adenylyl cyclase activity and the enzyme stimulations by corticotropin releasing hormone (CRH), Ca(2+)/calmodulin (Ca(2+)/CaM) and forskolin (FSK). In EP layer, Noc/OFQ inhibited Ca(2+)/CaM-and FSK-stimulated enzyme activities. Conversely, in GR layer the peptide stimulated basal cyclase activity and potentiated the enzyme activation by CRH. The Noc/OFQ stimulation was counteracted by the GDP-bound form of the alpha subunit of transducin and was mimicked by transducin betagamma subunits. In the same tissue layer, Ca(2+)/CaM-and FSK-stimulated enzyme activities were inhibited. Naloxone failed to antagonize all the actions of Noc/OFQ. Western blot and RT-PCR analysis revealed the expression of Ca(2+)-insensitive and -sensitive adenylyl cyclases in the three layers. These results demonstrate that in rat main olfactory bulb ORL1 receptors can differentially affect distinct forms of adenylyl cyclase in a layer specific manner.

A number of studies have shown that activation of gamma-aminobutyric acid(B) (GABA(B)) receptors potentiates neurotransmitter-induced accumulation of cyclic AMP in brain slices, but the mechanisms involved in the facilitatory effect have not been fully elucidated. In the present study, we showed that in membranes of rat frontal cortex the GABA(B) receptor agonist (-)baclofen increased basal adenylyl cyclase activity and potentiated the maximal enzyme stimulation elicited by corticotropin-releasing hormone (CRH). The less active enantiomer (+)baclofen had no effect on cyclic AMP formation, whereas the natural agonist GABA mimicked the stimulatory action of (-)baclofen. In radioligand-binding experiments, the affinity and maximal binding capacity of (125)I-Tyr-CRH was not affected by (-)baclofen. The GABA(B) receptor antagonist CGP 55845A competitively counteracted the (-)baclofen potentiation of CRH-stimulated adenylyl cyclase activity with a pA(2) value of 6.70. Moreover, both (-)baclofen and GABA, but not (+)baclofen, caused a concentration-dependent stimulation of [(35)S]GTP gamma S binding to membrane G-proteins. The intracerebral injection of pertussis toxin significantly reduced the facilitatory effects of (-)baclofen on both basal and CRH-stimulated adenylyl cyclase activities. Moreover, membrane incubation with the GDP-bound form of the alpha subunit of transducin, a scavenger of G protein beta gamma subunits, blocked the stimulatory effects of (-)baclofen. The data indicate that in rat frontal cortex activation of GABA(B) receptors potentiates the CRH stimulation of adenylyl cyclase activity through a mechanism involving the beta gamma subunits of the pertussis toxin-sensitive G protein G(i)/G(o).

Previous studies have shown that GABA(B) receptors facilitate cyclic AMP formation in brain slices likely through an indirect mechanism involving intracellular second messengers. In the present study, we have investigated whether a positive coupling of GABA(B) receptors to adenylyl cyclase could be detected in a cell-free preparation of rat olfactory bulb, a brain region where other Gi/Go-coupled neurotransmitter receptors have been found to stimulate the cyclase activity. The GABA(B) receptor agonist (-)-baclofen significantly increased basal adenylyl cyclase activity in membranes of the granule cell and external plexiform layers, but not in the olfactory nerve-glomerular layer. The adenylyl cyclase stimulation was therefore examined in granule cell layer membranes. The (-)-baclofen stimulation (pD2=4.53) was mimicked by 3-aminopropylphosphinic acid (pD2=4.60) and GABA (pD2=3.56), but not by (+)-baclofen, 3-aminopropylphosphonic acid, muscimol and isoguvacine. The stimulatory effect was counteracted by the GABA(B) receptor antagonists CGP 35348 (pA2=4.31), CGP 55845 A (pA2=7.0) and 2-hydroxysaclofen (pKi=4.22). Phaclofen (1 mM) was inactive. The (-)-baclofen stimulation was not affected by quinacrine, indomethacin, nordihydroguaiaretic acid and staurosporine, but was completely prevented by pertussis toxin and significantly reduced by the alpha subunit of transducin, a betagamma scavenger. The betagamma subunits of transducin stimulated the cyclase activity and this effect was not additive with that produced by (-)-baclofen. In the external plexiform and granule cell layers, but not in the olfactory nerve-glomerular layer, (-)-baclofen enhanced the adenylyl cyclase stimulation elicited by the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) 38. Conversely, the adenylyl cyclase activity stimulated by either forskolin or Ca2+/calmodulin-(Ca2+/CaM) was inhibited by (-)-baclofen in all the olfactory bulb layers examined. These data demonstrate that in specific layers of rat olfactory bulb activation of GABA(B) receptors enhances basal and neurotransmitter-stimulated adenylyl cyclase activities by a mechanism involving betagamma subunits of Gi/Go. This positive coupling is associated with a widespread inhibitory effect on forskolin- and Ca2+/CaM-stimulated cyclic AMP formation.

By using acetylcholine-induced stimulation of [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding to membrane G proteins as a functional assay of the cloned human m1-m4 muscarinic receptor subtypes stably expressed in Chinese hamster ovary cells, muscarinic toxin 3 (MT3) was found to block the m4 receptor with a potency (pA2 = 8.33) much higher than those displayed at the m1 (pA2 = 6.78), m3 (pA2 = 6.3), and m2 (pA26.3) subtypes. In N1E-115 cells, which have been reported to express m4 receptors coupled to inhibition of cAMP, MT3 potently antagonized the carbachol-induced inhibition of adenylyl cyclase with a pA2 of 8. 81 and displayed monophasic inhibitory curves. Unexpectedly, in NG108-15 cells, known to express only m4 receptors, MT3 counteracted the carbachol inhibition of adenylyl cyclase with a lower potency (pA2 = 7.60) and showed a biphasic inhibitory curve, suggesting the participation of both m4 and m2 receptors. This possibility was supported by radioligand binding data showing that MT3 failed to completely displace the binding of [3H]N-methylscopolamine to NG108-15 cell membranes and by reverse transcription-polymerase chain reaction analysis, revealing the presence of mRNAs for both m4 and m2 receptor subtypes. These data demonstrate that MT3 possesses a high functional receptor selectivity for both the cloned and native m4 receptors and that in cell systems containing m4 and m2 receptors coupled to a common response, the toxin constitutes a powerful tool to resolve the relative contribution by each receptor subtype.

In membranes of the rat frontal cortex, acetylcholine (ACh) and other cholinergic agonists were found to potentiate the stimulation of adenylyl cyclase activity elicited by corticotropin-releasing hormone (CRH). Oxotremorine-M, carbachol and methacholine were as effective as ACh, whereas oxotremorine and arecoline were much less effective. The facilitating effect of Ach was potently blocked by the M1 antagonists R-trihexyphenidyl, telenzepine and pirenzepine and by the M3 antagonists hexahydro-sila-difenidol and p-fluorohexahydro-sila-difenidol, whereas the M2 and M4 antagonists himbacine, methoctramine, AF-DX 116 and AQ-RA 741 were less potent. The mamba venom toxin MT-1, which binds with high affinity to M1 receptors, was also a potent blocker. The pharmacological profile of the muscarinic potentiation of CRH receptor activity was markedly different from that displayed by the muscarinic inhibition of forskolin-stimulated adenylyl cyclase, which could be detected in the same membrane preparations. Moreover, the intracerebral injection of pertussis toxin impaired the muscarinic inhibition of cyclic AMP formation and reduced the Ach stimulation of [35S]GTPgammaS binding to membrane G proteins but failed to affect the facilitating effect on CRH receptor activity. The latter response was also insensitive to the phospholipase C inhibitor U-73122, the protein kinase inhibitor staurosporine and to the inhibitors of arachidonic acid metabolism indomethacin and nordihydroguaiaretic acid. These data demonstrate that in the rat frontal cortex, muscarinic receptors of the M1 subtype potentiate CRH transmission by interacting with pertussis toxin-insensitive G proteins.

We investigated the identity of the G protein mediating the muscarinic stimulation of adenylyl cyclase in rat olfactory bulb membranes by examining the sensitivity of this response to selective anti-G protein antisera. Preincubation of tissue membranes with the antisera AS/7 (anti-Gi1/2 alpha), EC/2 (anti-Gi3 alpha/Go alpha), and GO/1 (anti-G(o) alpha) but not with the antiserum QL (anti-Gq/11 alpha) significantly attenuated the carbachol-stimulated adenylyl cyclase activity. These antisera had no effect on the enzyme activity stimulated by the beta-adrenergic agonist L-isoproterenol. On the other hand, the anti-Gs alpha antiserum RM/1 markedly depressed both carbachol- and L-isoproterenol-stimulated adenylyl cyclase activities. This antiserum also reduced the basal enzyme activity to a similar extent. However, different than the anti-Gi/Go antisera, the RM/1 antiserum failed to affect the carbachol-stimulated [35S]guanosine 5'-O-(3-thiotriphosphate) binding to membrane G proteins, whereas it curtailed the [35S]guanosine 5'-O-(3-thiotriphosphate) binding stimulated by pituitary adenylate cyclase-activating peptide. Exposure to either pertussis toxin or the anti-Go alpha antiserum 9072 but not to cholera toxin or the anti-Gs alpha antiserum 1191 reduced the high-affinity binding of oxotremorine M to muscarinic receptors. Moreover, the labeling of a 45-kDa protein catalyzed by cholera toxin was markedly stimulated by pituitary adenylate cyclase-activating peptide but not by carbachol. These data indicate that in rat olfactory bulb membranes, muscarinic receptors interact with both Gi and Go and that these G proteins mediate the stimulation of adenylyl cyclase. Although this response appears to require Gs activity, no evidence was found for the direct coupling of muscarinic receptors to Gs.

A number of recent investigations have proposed the existence of two pharmacologically distinct delta opioid receptor subtypes, named delta 1 and delta 2. In the present study, we have investigated the involvement of the two delta receptors in the opioid stimulation of adenylyl cyclase activity in rat olfactory bulb. In addition, we have conducted a similar investigation in rat striatum, where delta agonists are known to inhibit cyclic AMP formation. Both (D-Ala2, Glu4) deltorphin (DELT), a delta 2 agonist, and [D-Pen2, D-Pen5] enkephalin (DPDPE), a delta 1 agonist, stimulated adenylyl cyclase activity in rat olfactory bulb in a concentration-dependent manner, DELT being 25-fold more potent than DPDPE. The selective delta 2 antagonist naltriben counteracted the stimulatory effects of both agonists with a potency about 10-fold higher than that of the selective delta 1 antagonist 7-benzylidenenaltrexone. Moreover, pretreatment of olfactory bulb membranes with the nonequilibrium antagonist naltrindole 5'-isothiocyanate, which irreversibly blocks the delta 2 subtype, reduced the stimulatory effects of both DELT and DPDPE, whereas pretreatment with [D-Ala2, Leu5, Cys6]enkephalin, which binds covalently to delta 1 receptors, failed to affect the response to the agonists. Similar results were obtained in rat striatum. These data indicate that delta opioid receptors coupled to either stimulation or inhibition of adenylyl cyclase in two different brain areas predominantly belong to the delta 2 subtype.

We have investigated the pharmacological profile of the opioid stimulation of adenylate cyclase activity in rat olfactory bulb, in order to identify the opioid receptor subtype(s) involved in this response. The synthetic delta-selective agonists (D-Ala2)deltorphin I, (2-D-penicillamine,5-D-penicillamine)-enkephalin, and (D-Ser-Leu5-enkephalyl)-threonine were effective stimulators of the enzyme activity, with EC50 values of 6.7, 420, and 63 nM, respectively. A significant increase was also observed with the mu-selective agonists (N-methyl-Phe3,D-Pro4)-morphiceptin, dermorphin, and (D-Ala2-N-methyl-Phe4-Gly-ol5)-enkephalin (DAGO). The latter two agonists displayed biphasic concentration-response curves, with high affinity components accounting for 75-80% of the maximal responses. The kappa-selective agonists U-50,488 and U-69,593 were ineffective, whereas (D-Ala2)dynorphin A-1-11, dynorphin A, dynorphin A-1-13, and dynorphin A-1-6 acted with a rank order of potency consistent with their affinity for delta receptors. The stimulatory responses of Leu-enkephalin, beta-endorphin, dynorphin A, and delta-selective agonists were counteracted by naltrindole with pA2 values of 9.39-8.93, whereas naloxone was less potent (pA2 = 8.17-7.59). The kappa-selective antagonist norbinaltorphimine was the least potent. The inhibition by naltrindole and naloxone of DAGO stimulation showed biphasic curves, with 90% of the response being antagonized more potently by naloxone than by naltrindole. These results demonstrate that delta- and mu- but not kappa-opioid receptor subtypes stimulate basal adenylate cyclase activity in rat olfactory bulb.

In rat olfactory bulb, stimulation of muscarinic receptors activates adenylate cyclase. In the present study we have examined a variety of muscarinic receptor stimulants to characterize the agonist profile of this response. Analysis of agonist concentration-response curves revealed the following rank order of potency: oxotremorine-M greater than oxotremorine greater than BM5 greater than acetylcholine greater than carbachol = methacholine greater than (+/-)muscarine greater than arecoline greater than pilocarpine greater than RS 86 greater than McN-A-343 greater than bethanechol. Acetylcholine, oxotremorine-M, carbachol, (+/-)muscarine and metacholine behaved as full agonists, whereas the other stimulants displayed lower efficacies. The slope values of the concentration-response curves were close or equal to 1, except those of the carbachol and pilocarpine curves, which showed values significantly lower than 1. Moreover, the slope of the pilocarpine curve was differentially changed by the M1 antagonist pirenzepine and the M2 antagonist AF-DX 116. The agonist profile of the muscarinic stimulation of adenylate cyclase in the olfactory bulb correlated well with that exhibited by the muscarinic inhibition of the enzyme activity in the striatum, suggesting that the two responses are mediated by a similar receptor subtype. Sodium ion modulated the agonist affinity for both adenylate cyclase-coupled receptor systems.

The pharmacological profile of the muscarinic stimulation of adenylate cyclase activity in rat olfactory bulb was investigated by using a number of antagonists with different selectivity for the muscarinic receptor subtypes. Schild analysis showed that this response was counteracted with the following rank order of potency: R(-)quinuclidinyl benzilate greater than atropine greater than RS(+/-)-3-quinuclidinyl xanthene-9-carboxylate hemioxalate greater than S(+)quinuclidinyl benzylate greater than 4-diphenylacetoxy-N-methylpiperidine methbromide greater than secoverine greater than methoctramine greater than dicyclomine greater than hexahydrosiladifenidol greater than p-fluorohexahydro-sila-difenidol greater than AF-DX 116 greater than pirenzepine. In the heart and corpus striatum, the antagonist rank orders of potency in counteracting the muscarinic inhibition of adenylate cyclase activity were consistent with the involvement of homogeneous populations of M2 and M3 receptors, respectively. Conversely, in the olfactory bulb, the pirenzepine inhibition curve of acetylcholine-stimulated enzyme activity was biphasic, with a major component of the response being inhibited with low affinity. These results indicate that more than one receptor subtype may participate in the stimulation of adenylate cyclase in rat olfactory bulb. The predominant receptor involved appears to be different from the M1, M2 and M3 subtypes and pharmacologically equivalent to the m4 gene product.

In homogenate of rat olfactory bulb, the opioid receptor agonists beta-endorphin, Leu-enkephalin, and dynorphin A stimulated adenylate cyclase activity in a concentration-dependent manner, with half-maximal effects displayed at 22, 63, and 176 nM, respectively. The maximal stimulation of the enzyme activity corresponded to about a 40% increase of basal activity for all three peptides. Naloxone antagonized the stimulation of beta-endorphin, Leu-enkephalin, and dynorphin A, with pA2 values of 8.0, 7.7, and 8.1, respectively. Kinetic analysis performed with Leu-enkephalin showed that the opioid peptide increased the Vmax of the enzyme, without changing the Km for the substrate Mg-ATP. Moreover, the opioid stimulation was associated with a significant increase of the affinity of the enzyme for Mg2+ activation and occurred in membranes incubated in a Ca2(+)-free medium. Addition of exogenous GTP at micromolar concentrations was absolutely necessary for the detection of the opioid effect. Treatment of olfactory bulbs with cholera toxin did not alter the stimulation of adenylate cyclase by Leu-enkephalin. However, the opioid stimulation disappeared in membranes obtained from bulbs injected with pertussis toxin. These results demonstrate the presence in the brain of a new functional class of opiate receptors coupled to stimulation of adenylate cyclase via a transduction mechanism that is Ca2+ independent and seems to involve a pertussis toxin-sensitive GTP-binding protein.

The dopamine receptor adenylate cyclase complex of a rat striatal membrane preparation became more responsive to dopamine following the injection of 6-hydroxydopamine (6-OHDA) into the median forebrain bundle or following the subcutaneous implantation of morphine pellets. Moreover, the membrane cyclase system was more responsive to activation by GTP, guanyl-5'-yl-imidodiphosphate and Mn-ATP. These observations suggest that both 6-OHDA and morphine induce similar biochemical changes in striatum and that the increased responsiveness arises, in part, from modification of the nucleotide regulatory and/or catalytic components of adenylate cyclase.

Acetylcholine inhibits, by 30-40%, the basal adenylate cyclase activity of purified synaptic plasma membranes prepared from rat striatum (EC50 = 3 microM). Cholinergic receptor agonists inhibit this cyclase activity with the following rank order of potency: oxtremorine greater than acetylcholine greater than arecoline greater than methacholine greater than or equal to muscarine greater than or equal to carbachol greater than bethanechol. Nicotine fails to inhibit the cyclase, and d-tubocurarine fails to inhibit the action of cholinergic drugs. In contrast, atropine and scopolamine antagonize the effect of acetylcholine. The enzyme inhibition elicited by acetylcholine requires the presence of GTP, and disappears after intrastriatal injection of kainic acid. From these results, we infer that striatal adenylate cyclase can be modulated by muscarinic receptors.

In GH3 pituitary cell homogenates, acetylcholine (ACh) (IC50 200 nM) inhibits adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in a concentration- and GTP-dependent manner. Maximal inhibition was obtained with 10 microM ACh and corresponded to approximately a 50% decrease in basal enzyme activity. ACh inhibition is antagonized by atropine and is mimicked by muscarinic receptor agonists, but not by nicotine. ACh reduces the adenylate cyclase stimulation by vasoactive intestinal peptide (VIP), without changing its EC50. In intact GH3 cells, ACh decreases the cyclic AMP content and the rate of prolactin release in a concentration-dependent manner. When the cells are simultaneously exposed to VIP and ACh, the VIP-induced increases in cyclic AMP accumulation and prolactin release are reduced by 80% and 40%, respectively. The potency of VIP is not significantly changed by the presence of ACh, and vice versa.

In the presence of SCH 23390, a potent blocker of D1 dopamine receptors, dopamine inhibits adenylate cyclase activity of synaptic plasma membranes isolated from rat striatum. Maximal inhibition corresponds to a 20-25% decrease of basal enzyme activity and is reached with 100 microM dopamine. The apparent IC50 of dopamine is 2.5 microM. The inhibitory effect of dopamine is mimicked by various dopamine receptor agonists with the following rank order of potency: (-)-propylnorapomorphine greater than or equal to bromocriptine greater than (+/-)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene = (-)-apomorphine greater than dopamine greater than LY 171555 greater than l-noradrenaline greater than l-phenylephrine. Clonidine and l-isoproterenol are inactive at 100 microM. Bromocriptine and LY 171555, two agents which stimulate selectively D2 receptors, inhibit striatal adenylate cyclase activity in the absence of SCH 23390. However, bromocriptine behaves like a partial agonist. A variety of neuroleptic drugs antagonize the dopamine inhibition with a rank order of potency which qualitatively correlates with their relative affinity for D2 receptors. l-Sulpiride (EC50 = 210 nM) and (+)-butaclamol (EC50 = 130 nM) are severalfold more potent than d-sulpiride (EC50 = 5 microM) and (-)-butaclamol (EC50 = 10 microM). The inhibitory effect of dopamine on striatal adenylate cyclase activity is dependent on the presence of GTP, with half-maximal inhibition occurring at 1 microM GTP. In the absence of SCH 23390, dopamine stimulates adenylate cyclase activity, reaching a maximum at 1 microM GTP. At higher concentrations of the nucleotide, the dopamine-stimulated enzyme activity decreases, and this decline is antagonized by the D2 receptor blocker l-sulpiride. Guanyl-5'-yl imidodiphosphate, a stable analogue of GTP, has a biphasic effect on the striatal adenylate cyclase activity, inhibiting at low concentration (from 1 to 100 nM) and stimulating at higher concentrations. Selective activation of D2 receptors by LY 171555 does not increase the extent of enzyme inhibition elicited by guanyl-5'-yl imidodiphosphate. Sodium chloride amplifies the inhibition of striatal adenylate cyclase activity by LY 171555 and reduces the potency of the D2 agonist by a factor of 4. The dopamine-inhibited enzyme activity is lost following intrastriatal injection of kainic acid. The results indicate that in rat striatum dopamine inhibits adenylate cyclase activity by acting on postsynaptic dopamine receptors with pharmacological properties of D2 type.

The stimulation of GTP hydrolysis has been proposed as a mechanism by which hormones inhibit receptor-coupled adenylate cyclase activity. The present study attempts to verify whether this mechanism is also operative in transmitter-mediated receptor-coupled attenuation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] located in synaptic plasma membrane preparations. As a model, we used the inhibition of adenylate cyclase activity by muscarinic receptor activation in rat striatum. This striatal preparation contains high-affinity GTPase (EC 3.6.1-) activity which is stimulated when the recognition site for muscarinic agonists is occupied. Acetylcholine (ACh), but not nicotine, increases the Vmax of the high-affinity GTPase, and the stimulatory effect is antagonized by atropine but not by d-tubocurarine. The rank order of potency of various cholinergic agonists to stimulate GTPase correlates with their ability to inhibit adenylate cyclase activity of striatal membranes. Pre-exposure of striatal membranes to guanosine-5'-O-(3-thiotriphosphate) causes a parallel decrease in the basal and ACh-stimulated GTPase activities and in the ACh-induced inhibition of adenylate cyclase. Treatment of the membranes with cholera toxin does not affect the ACh-stimulated GTPase activity but amplifies the extent of adenylate cyclase inhibition elicited by the cholinergic agonist. These results indicate that the stimulation of a high-affinity GTPase parallels the inhibitory coupling of central muscarinic receptors to adenylate cyclase.