Cardiac-directed adenylyl cyclase expression improves heart function in murine cardiomyopathy.
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BACKGROUND: We tested the hypothesis that increased cardiac myocyte adenylyl cyclase (AC) content increases cardiac function and response to catecholamines in cardiomyopathy. METHODS AND RESULTS: Transgenic mice with cardiac-directed expression of AC type VI (ACVI) were crossbred with mice with cardiomyopathy induced by cardiac-directed Gq expression. Gq mice had dilated left ventricles, reduced heart function, decreased cardiac responsiveness to catecholamine stimulation, and impaired beta-adrenergic receptor (betaAR)-dependent and AC-dependent cAMP production. Gq/AC mice showed improved basal cardiac function in vivo (P=0.01) and ex vivo (P<0.0005). When stimulated through the betaAR, cardiac responsiveness was increased (P=0.02), and cardiac myocytes showed increased cAMP production in response to isoproterenol (P=0.03) and forskolin (P<0.0001). CONCLUSIONS: Increasing myocardial ACVI content in cardiomyopathy restores cAMP-generating capacity and improves cardiac function and responsiveness to betaAR stimulation. (+info)
Presynaptic serotonergic inhibition of GABAergic synaptic transmission in mechanically dissociated rat basolateral amygdala neurons.
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1. The basolateral amygdala (ABL) nuclei contribute to the process of anxiety. GABAergic transmission is critical in these nuclei and serotonergic inputs from dorsal raphe nuclei also significantly regulate GABA release. In mechanically dissociated rat ABL neurons, spontaneous miniature inhibitory postsynaptic currents (mIPSCs) arising from attached GABAergic presynaptic nerve terminals were recorded with the nystatin-perforated patch method and pharmacological isolation. 2. 5-HT reversibly reduced the GABAergic mIPSC frequency without affecting the mean amplitude. The serotonergic effect was mimicked by the 5-HT1A specific agonist 8-OH DPAT (8-hydroxy-2-(di-n-propylamino)tetralin) and blocked by the 5-HT1A antagonist spiperone. 3. The GTP-binding protein inhibitor N-ethylmaleimide removed the serotonergic inhibition of mIPSC frequency. In either K+-free or Ca2+-free external solution, 5-HT could inhibit mIPSC frequency. 4. High K+ stimulation increased mIPSC frequency and 8-OH DPAT inhibited this increase even in the presence of Cd2+. 5. Forskolin, an activator of adenylyl cyclase (AC), significantly increased synaptic GABA release frequency. Pretreatment with forskolin prevented the serotonergic inhibition of mIPSC frequency in both the standard and high K+ external solution. 6. Ruthenium Red (RR), an agent facilitating the secretory process in a Ca2+-independent manner, increased synaptic GABA release. 5-HT also suppressed RR-facilitated mIPSC frequency. 7. We conclude that 5-HT inhibits GABAergic mIPSCs by inactivating the AC-cAMP signal transduction pathway via a G-protein-coupled 5-HT1A receptor and this intracellular pathway directly acts on the GABA-releasing process independent of K+ and Ca2+ channels in the presynaptic nerve terminals. (+info)
Adenylyl cyclase, a coincidence detector for nitric oxide.
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Nitric oxide (NO) donors inhibit hormone- and forskolin-stimulated adenylyl cyclase activity in purified plasma membrane preparations from N18TG2 neuroblastoma cells. Northern blot analyses indicate that the predominant isoform of adenylyl cyclase in N18TG2 cells is the type VI. Our experiments eliminate all the known regulatory proteins for this isoform as possible targets of NO. NO decreases the Vmax of the enzyme without altering the Km for ATP. Occupancy of the substrate-binding site protects the enzyme from the inhibitory effects of NO, suggesting that the conformation of the enzyme determines its sensitivity. The inhibition is reversed by reducing agents, implicating a Cys residue(s) as the target for nitric oxide and an S-nitrosylation as the underlying modification. These findings implicate NO as a novel cellular regulator of the type VI isoform of adenylyl cyclase. (+info)
Two cytoplasmic loops of the glucagon receptor are required to elevate cAMP or intracellular calcium.
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The glucagon receptor is a member of a distinct class of G protein-coupled receptors (GPCRs) sharing little amino acid sequence homology with the larger rhodopsin-like GPCR family. To identify the components of the glucagon receptor necessary for G-protein coupling, we replaced sequentially all or part of each intracellular loop (i1, i2, and i3) and the C-terminal tail of the glucagon receptor with the 11 amino acids comprising the first intracellular loop of the D4 dopamine receptor. When expressed in transiently transfected COS-1 cells, the mutant receptors fell into two different groups with respect to hormone-mediated signaling. The first group included the loop i1 mutants, which bound glucagon and signaled normally. The second group comprised the loop i2 and i3 chimeras, which caused no detectable adenylyl cyclase activation in COS-1 cells. However, when expressed in HEK 293T cells, the loop i2 or i3 chimeras caused very small glucagon-mediated increases in cAMP levels and intracellular calcium concentrations, with EC50 values nearly 100-fold higher than those measured for wild-type receptor. Replacement of both loops i2 and i3 simultaneously was required to completely abolish G protein signaling as measured by both cAMP accumulation and calcium flux assays. These results show that the i2 and i3 loops play a role in glucagon receptor signaling, consistent with recent models for the mechanism of activation of G proteins by rhodopsin-like GPCRs. (+info)
Inhibition of human platelet adenylate cyclase activity by adrenaline, thrombin and collagen: analysis and reinterpretation of experimental data.
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Mathematical models based on the current understanding of stimulation and inhibition of adenylate cyclase (AC) activity have been developed and used to analyse experimental data [Farndale, Winkler, Martin and Barnes (1992) Biochem. J. 282, 2532] describing the inhibition of human platelet AC by collagen, thrombin and adrenaline. Here it has been demonstrated that neither affinities of receptors specific for adrenaline or thrombin nor the activity of cAMP phosphodiesterase are affected by collagen. Both collagen and thrombin at high doses act as effective inhibitors of AC activity. Inhibition of AC activity by collagen proceeds via two parallel pathways; the same is true for thrombin at moderate concentrations, and the two ligands act independently. The G-protein-dependence of these pathways is distinct from that mediating inhibition of AC activity by adrenaline, i.e. Gi2. Convergence of the inhibitory pathways takes place at the catalytic subunit of AC. (+info)
A cytoskeletal localizing domain in the cyclase-associated protein, CAP/Srv2p, regulates access to a distant SH3-binding site.
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In the yeast, Saccharomyces cerevisiae, adenylyl cyclase consists of a 200-kDa catalytic subunit (CYR1) and a 70-kDa subunit (CAP/SRV2). CAP/Srv2p assists the small G protein Ras to activate adenylyl cyclase. CAP also regulates the cytoskeleton through an actin sequestering activity and is directed to cortical actin patches by a proline-rich SH3-binding site (P2). In this report we analyze the role of the actin cytoskeleton in Ras/cAMP signaling. Two alleles of CAP, L16P(Srv2) and R19T (SupC), first isolated in genetic screens for mutants that attenuate cAMP levels, reduced adenylyl cyclase binding, and cortical actin patch localization. A third mutation, L27F, also failed to localize but showed no loss of either cAMP signaling or adenylyl cyclase binding. However, all three N-terminal mutations reduced CAP-CAP multimer formation and SH3 domain binding, although the SH3-binding site is about 350 amino acids away. Finally, disruption of the actin cytoskeleton with latrunculin-A did not affect the cAMP phenotypes of the hyperactive Ras2(Val19) allele. These data identify a novel region of CAP that controls access to the SH3-binding site and demonstrate that cytoskeletal localization of CAP or an intact cytoskeleton per se is not necessary for cAMP signaling. (+info)
Isoproterenol, forskolin, and cAMP-induced nitric oxide production in pig ciliary processes.
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PURPOSE: To investigate whether isoproterenol and forskolin, two adenylylcyclase activators, or 8-bromo-cAMP, an adenosine 3',5'-cyclic monophosphate (cAMP) analog, increase nitric oxide (NO) production in isolated porcine ciliary processes. METHODS: Nitrite (an NO metabolite) was measured (Griess reaction) before and 2 hours after exposure to 0.1 to 100 microM isoproterenol (a beta-adrenoreceptor agonist), 0.01 to 100 microM forskolin, or 0.1 to 1000 microM 8-bromo-cAMP. Some experiments were conducted in the presence of 0.5 mM N(G)-nitro-L-arginine methyl ester (L-NAME; a nitric oxide synthase [NOS] inhibitor), 10 microM propranolol (a beta-adrenoreceptor antagonist), or 1 microM KT 5720 (a cAMP-dependent protein kinase inhibitor). cAMP production was also measured (by immunoassay). RESULTS: Nitrite production was increased by isoproterenol (maximum, 10 microM: 164%; P < 0.001), forskolin (maximum, 10 microM: 254%; P < 0.001), and 8-bromo-cAMP (maximum, 100 microM: 184%; P < 0.001), an effect prevented by L-NAME (P < 0.05-0.001). Propranolol inhibited only isoproterenol-induced (10 microM) nitrite production (P < 0.05), whereas KT 5720 (P < 0.05) inhibited isoproterenol- (10 microM) and 8-bromo-cAMP-induced (10 microM) nitrite production. Furthermore, cAMP production evoked by isoproterenol (10 microM, P < 0.05) but not by forskolin (10 microM, P < 0.001) was inhibited by propranolol (P < 0.05). CONCLUSIONS: In isolated porcine ciliary processes, drugs activating adenylylcyclase or mimicking cAMP increase the production of NO by a mechanism that appears to involve both a cAMP-dependent protein kinase and NOS. (+info)
Participation of ABH glycoconjugates in the secretory response to Escherichia coli heat-labile toxin in rabbit intestine.
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The ability of membrane ABH blood group-active glycoconjugates to act as receptors of the heat-labile enterotoxin of Escherichia coli (LTh) was studied in vitro and in vivo when GM1 was blocked by the cholera toxin B subunit. Rabbits were classified as AB or H based on intestinal ABH-antigenic activities. Brush border membranes from AB rabbits contained 4 times more LTh binding sites than the H ones. LTh interaction could be inhibited by lectins that recognize ABH determinants. LTh induced a similar dose-dependent secretory response in ligated ileal loops of both types of animals. Anti-AB antibodies and Ulex europaeus I lectin could significantly reduce the fluid accumulation in AB and H rabbits, respectively. LTh caused adenylate cyclase activation even when GM1 was blocked, and this effect was abolished by the addition of specific ABH ligands. These results suggest that ABH glycoconjugates are involved in the host secretory response to LTh in rabbit intestine. (+info)