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Adenylate cyclase (EC 4.6.1.1) type 9 (AC9) activity has been shown to be inhibited by PMA activation of novel protein kinase C (nPKC) isoforms. In the current study the effect on AC9 activity of activating PKC in physiological relevant manner was examined. Contrary to the anticipated inhibitory effect of activating PKCs through Gq-coupled receptors, activation of transiently expressed Gq-coupled serotonin 5-HT2A or muscarinic M5 receptors resulted in the potentiation of isoproterenol-stimulated cyclic AMP accumulation in HEK293 cells stably expressing AC9 (HEK-AC9). Consistent with Gq-mediated activation of PKC, the addition of the PKC inhibitor bisindolylmaleimide further potentiated isoproterenol-stimulated cyclic AMP accumulation. Expression of a constitutively active mutant of Galphaq in HEK-AC9 cells also produced an enhancement in basal and isoproterenol-stimulated cyclic AMP accumulation. We also examined the role of Galphaq-mediated release of intracellular Ca2+ on the observed potentiation of AC9 activity, by depleting intracellular Ca2+ stores with thapsigargin. In Ca2+-depleted HEK-AC9 cells, activation of transiently expressed M5 receptors resulted in inhibition of isoproterenol-stimulated cyclic AMP accumulation that was blocked by bisindolylmaleimide, indicating that M5 potentiation of AC9 activity requires Ca2+. This prompted us to examine the effects of the calmodulin antagonist W7 and the Ca2+/calmodulin-dependent kinase II (CaMK II) inhibitor KN-93. Pretreating cells with W7 and KN-93 significantly inhibited M5-mediated potentiation of isoproterenol-stimulated cyclic AMP accumulation in HEK-AC9 cells, suggesting that Galphaq potentiation of AC9 activity involves Ca2+/calmodulin and CaMK II. This data provides evidence for Ca2+-mediated potentiation of AC9 activity.

Nine membrane-bound members of the mammalian adenylate cyclase family have been identified. The least characterized and most divergent in sequence of the nine adenylate cyclase isoforms is AC9. Stimulation by Galpha(s) and inhibition by Ca2+/calcineurin are two modes of regulation that have been reported for AC9. We explored the possibility of additional modes of regulation of human AC9. We now report that quinpirole activation of the inhibitory G protein-coupled D2L dopamine receptor inhibits Galpha(s) stimulation of AC9 by approximately 50%. The effects of quinpirole were reversed by the D2 antagonist spiperone and by pertussis toxin pretreatment. We also report the first evidence for regulation of AC9 by protein kinase C (PKC). Specifically, phorbol ester activation of PKC significantly attenuated (approximately 50%) Galpha(s)-stimulated AC9 activity. The effect of PKC activation on AC9 was reversed by the PKC inhibitor bisindolylmaleimide. Galpha(s)-stimulated cyclic accumulation was reduced more by simultaneous addition of both quinpirole and phorbol 12-myristate 13-acetate than by either drug alone. Additional studies investigated the role of glycosylation on AC9 activity. The results show that blocking glycosylation of AC9 significantly attenuates Galpha(s) stimulation. In contrast, the ability of PKC and Galpha(i/o) to negatively regulate AC9 did not seem to be affected by the glycosylation state of AC9. These observations demonstrate the diverse regulatory features of AC9 and the ability of AC9 to integrate multiple signals.

Rationale: There is substantial evidence that lisuride can produce effects linked to 5-HT(1A) receptor occupancy. Nevertheless, this action has generally been ignored in the mechanism of action of lisuride, in favor of an exclusive role for dopamine receptors in considering its antiparkinsonian effects, or an exclusive role of 5-HT(2A/2C) receptor activation in hallucinogenesis. These conclusions are surprising when one considers that the potent interaction of lisuride with 5-HT(1A) receptors has been demonstrated in several different laboratories and that activation of 5-HT(1A) and 5-HT(1B) receptors can modulate dopaminergically mediated responses., Objective: The lack of full substitution of lisuride for lysergic acid diethylamide (LSD) in drug discrimination experiments and induction of a pronounced 5-HT syndrome by this compound at relatively low doses convinced us to execute two series of experiments that might explain the primary mechanism responsible for lisuride-mediated biological effects and its paradoxical classification as a dopamine agonist in the literature., Results: In drug discrimination studies, lisuride fully mimicked the 5-HT(1A) agonist LY 293284, only partially substituted for LSD and DOI, and failed to substitute for (+)-amphetamine. Lisuride produced a significant dose-related increase in flat body posture, forepaw treading, and lower-lip retraction which reflect a modulation of behavior by action at central 5-HT(1A) receptors. Only pMPPI [4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridynyl-benzamide hydrochloride], a selective 5-HT(1A) antagonist, was effective in inhibiting all 5-HT syndrome behaviors produced by lisuride, whereas pMPPI was without effect on any behavior induced by LSD. Lisuride dose dependently decreased body temperature in rats with a potency similar to that of the selective 5-HT(1A) agonist LY 293284. The hypothermic effect of lisuride was prevented by pre-injection of pMPPI, but not by ketanserin or haloperidol., Conclusion: We have demonstrated that the behavioral effects of low doses of lisuride are clearly mediated by stimulation of 5-HT(1A) receptors.

A homology model of the dopamine D2 receptor was constructed based on the crystal structure of rhodopsin. A putative sodium-binding pocket identified in an earlier model (PDB ) was revised. It is now defined by Asn-419 backbone oxygen at the apex of a pyramid and Asp-80, Ser-121, Asn-419, and Ser-420 at each vertex of the planar base. Asn-423 stabilizes this pocket through hydrogen bonds to two of these residues. Highly conserved Asn-52 is positioned near the sodium pocket, where it hydrogen-bonds with Asp-80 and the backbone carbonyl of Ser-420. Mutation of three of these residues, Asn-52 in helix 1, Ser-121 in helix 3, and Ser-420 in helix 7, profoundly altered the properties of the receptor. Mutants in which Asn-52 was replaced with Ala or Leu or Ser-121 was replaced with Leu exhibited no detectable binding of radioligands, although receptor immunoreactivity in the membrane was similar to that in cells expressing the wild-type D2L receptor. A mutant in which Asn-52 was replaced with Gln, preserving hydrogen-bonding capability, was similar to D2L in affinity for ligands and ability to inhibit cAMP accumulation. Mutants in which either Ser-121 or Ser-420 was replaced with Ala or Asn had decreased affinity for agonists (Ser-121), but increased affinity for the antagonists haloperidol and clozapine. Interestingly, the affinity of these Ser-121 and Ser-420 mutants for substituted benzamide antagonists showed little or no dependence on sodium, consistent with our hypothesis that Ser-121 and Ser-420 contribute to the formation of a sodium-binding pocket.

Persistent activation of Galpha(i/o)-coupled receptors results in an enhanced responsiveness of drug-stimulated adenylate cyclase activity through an unknown mechanism. This agonist-induced heterologous sensitization of drug-stimulated cyclic AMP accumulation has been proposed to be a mechanism by which cells adapt to prolonged Galpha(i/o) activation. Heterologous sensitization was examined in human embryonic kidney 293 cells stably expressing D(2L) dopamine receptors in combination with recombinant isoforms of adenylate cyclase. The ability of each isoform to be differentially regulated by G protein subunits and other signaling intermediates allowed us to identify potential mechanisms that are involved in heterologous sensitization of adenylate cyclase. We now report that both short- and long-term activation of D(2L) dopamine receptors resulted in a marked degree of sensitization of ACI, ACII, ACV, and ACIX, but not ACVIII. The effects of agonist treatment on ACI, ACII, and ACVIII appeared to be dependent upon the ability of these adenylate cyclase isoforms to synergistically respond to selective activators in the presence of activated Galpha(s). Sensitization of ACV was characterized by enhanced cyclic AMP accumulation following Galpha(s) or forskolin stimulation. Furthermore, agonist pretreatment enhanced the basal levels of cyclic AMP accumulation in ACV/D(2L) cells, an effect that was not observed with the other adenylate cyclase isoforms. ACIX, which has no known activators other than Galpha(s), showed robust agonist-induced sensitization of isoproterenol-stimulated cyclic AMP accumulation. In summary, heterologous sensitization appeared to be related to the ability of each adenylate cyclase isoform to be modulated by Galpha(s).

Adenylate cyclase activity in NS20Y cells expressing D2L dopamine receptors was examined following chronic treatment with norepinephrine and epinephrine. Initial acute experiments revealed that both norepinephrine and epinephrine inhibited forskolin-stimulated cyclic AMP accumulation via D2 receptors. Furthermore, chronic 18 h activation of D2 dopamine receptors by norepinephrine or epinephrine induced a marked increase (>10-fold) in subsequent forskolin-stimulated cyclic AMP accumulation. This heterologous sensitization of adenylate cyclase activity was blocked by D2 dopamine receptor antagonists and by pertussis toxin pretreatment. In contrast, concurrent activation of Galpha(s) or adenylate cyclase did not appear to alter noradrenergic agonist-induced sensitization.

A series of fluorinated analogues of the hallucinogenic tryptamines N,N-diethyltryptamine (DET), 4-hydroxy-N,N-dimethyltryptamine (4-OH-DMT, psilocin), and 5-methoxy-DMT was synthesized to investigate possible explanations for the inactivity of 6-fluoro-DET as a hallucinogen and to determine the effects of fluorination on the molecular recognition and activation of these compounds at serotonin receptor subtypes. The target compounds were evaluated using in vivo behavioral assays for hallucinogen-like and 5-HT(1A) agonist activity and in vitro radioligand competition assays for their affinity at 5-HT(2A), 5-HT(2C), and 5-HT(1A) receptor sites. Functional activity at the 5-HT(2A) receptor was determined for all compounds. In addition, for some compounds functional activity was determined at the 5-HT(1A) receptor. Hallucinogen-like activity, evaluated in the two-lever drug discrimination paradigm using LSD-trained rats, was attenuated or abolished for all of the fluorinated analogues. One of the tryptamines, 4-fluoro-5-methoxy-DMT (6), displayed high 5-HT(1A) agonist activity, with potency greater than that of the 5-HT(1A) agonist 8-OH-DPAT. The ED(50) of 6 in the two-lever drug discrimination paradigm using rats trained to discriminate the 5-HT(1A) agonist LY293284 was 0.17 micromol/kg, and the K(i) at [(3)H]8-OH-DPAT-labeled 5-HT(1A) receptors was 0.23 nM. The results indicate that fluorination of hallucinogenic tryptamines generally has little effect on 5-HT(2A/2C) receptor affinity or intrinsic activity. Affinity at the 5-HT(1A) receptor was reduced, however, in all but one example, and all of the compounds tested were full agonists but with reduced functional potency at this serotonin receptor subtype. The one notable exception was 4-fluoro-5-methoxy-DMT (6), which had markedly enhanced 5-HT(1A) receptor affinity and functional potency. Although it is generally considered that hallucinogenic activity results from 5-HT(2A) receptor activation, the present results suggest a possible role for involvement of the 5-HT(1A) receptor with tryptamines.

1. Decline in beta-adrenoceptor (beta-AR)-mediated function occurs with increasing age, as well as in multiple disease conditions. The mechanisms responsible for this decline include alterations in beta-AR itself, beta-AR coupling proteins, such as G-proteins, or other beta-AR-linked proteins, such as G-protein receptor kinases and/or phosphatases. 2. The present study examines the physiological effects of in vitro transfer of constitutively activated G alpha s (G alpha s-Q227L) to both cultured vascular smooth muscle cells (VSMC) and whole aortic tissue of 6-month-old (adult) animals via a replication-deficient Herpes simplex virus (HSV) vector. These studies were conducted to provide a model for future examination of the role of G alpha s in the age-related decline in beta-AR-mediated vasorelaxation. 3. Gene transfer was confirmed by western blotting for specific proteins. Aortic tissue infected with HSV-G alpha s-Q227L had reduced phenylephrine-induced contraction and enhanced isoproterenol-stimulated vasorelaxation. Infection of cultured VSMC with HSV-G alpha s-Q227L increased both basal- and isoproterenol-stimulated cAMP accumulation, whereas forskolin-stimulated cAMP production was unchanged. 4. These results implicate G alpha s as a target for further investigation in age-related changes in vascular reactivity and support the use of viral-mediated gene transfer as an effective tool to study adrenergic signal transduction and physiology in vascular tissue.

D2L dopamine receptor activation results in rapid inhibition and delayed heterologous sensitization of adenylate cyclase in several host cell types. The D2L dopamine receptor was stably transfected into NS20Y neuroblastoma cells to examine inhibition and sensitization in a neuronal cell environment and to identify the particular G-proteins involved. Acute activation of D2L receptors with the selective D2 agonist quinpirole inhibited forskolin-stimulated cAMP accumulation, whereas prolonged incubation (2 hr) with quinpirole resulted in heterologous sensitization (more than twofold) of forskolin-stimulated cAMP accumulation in NS20Y-D2L cells. To unambiguously identify the pertussis toxin (PTX)-sensitive G-proteins responsible for inhibition and sensitization, we used viral-mediated gene delivery to assess the ability of genetically engineered PTX-resistant G-proteins (Galphai1*, Galphai2*, Galphai3*, and Galphao*) to rescue both responses after PTX treatment. The expression and function of individual recombinant G-proteins was confirmed with Western blotting and inhibition of GTPgammaS-stimulated adenylate cyclase, respectively. To assess the specificity of D2L-Galpha coupling, cells were infected with herpes simplex virus (HSV) recombinants expressing individual PTX-resistant G-protein alpha subunits and treated with PTX, and quinpirole-induced responses were measured. Infection of NS20Y-D2L cells with HSV-Galphao* rescued both inhibition and sensitization in PTX-treated cells, whereas infection with HSV-Galphai1*, HSV-Galphai2*, or HSV-Galphai3* failed to rescue either response. In summary, the current study provides strong evidence that the D2L dopamine receptor couples to Galphao in neuronal cells, and that this coupling is responsible for both the acute and subacute effects of D2 receptor activation on adenylate cyclase activity.