Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The guanylnucleotide specificity of muscarinic acetylcholine receptor (MR) inhibitory coupling to cardiac adenylate cyclase (AC) was investigated under low MgCl2 (i.e., 0.5 mM) conditions. In purified cardiac sarcolemma, carbachol maximally inhibited AC activity 60% in the presence of GTP. Carbachol-dependent inhibition in the presence of guanosine 5'-O-(3-thiotriphosphate (GTP gamma S) or guanylylimidodiphosphate [Gpp(NH)p] was of lesser magnitude (i.e., 30%) and was evident only during short incubation periods. Of greater interest, carbachol maximally inhibited AC activity in the presence of GDP and guanosine 5'-O-(2-thiodiphosphate (GDP beta S) by 35 and 60%, respectively. Control studies ruled out transphosphorylation of GDP and GDP beta S by nucleoside diphosphate kinase or guanylnucleoside triphosphate contamination as reasons for the inhibitory effects of GDP and GDP beta S. Furthermore, isoproterenol stimulated AC in the presence of GTP, GTP gamma S, and Gpp(NH)p but not in the presence of GDP or GDP beta S. Therefore, GDP and GDP beta S may serve as agonists on MR-activated Gi but not on beta-adrenergic receptor-activated Gs in these membranes. Time course studies revealed that carbachol-dependent inhibition of AC in the presence of either GTP or GDP occurred without a detectable lag period, and this inhibition was rapidly reversed by atropine. In contrast, a 1-2-min lag time was required for carbachol- and GDP beta S-dependent inhibition of AC to occur, and inhibition, once developed, was only partially and slowly reversed by atropine. Preincubation of sarcolemma with carbachol and GDP beta S, in the absence of ATP or under nonphosphorylating conditions, eliminated the lag time for inhibition of AC activity. Although it is unlikely that GDP and GDP beta S have physiological relevance of MR-Gi-AC coupling, these studies provide unique insights into this coupling mechanism in cardiac membranes.
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PMID:Guanylnucleotide specificity for muscarinic receptor inhibitory coupling to cardiac adenylate cyclase. 131 Jan 41

The muscarinic receptor system involved in pepsinogen secretion from isolated guinea pig gastric chief cells was investigated by evaluating the effect of muscarinic receptor antagonists on carbamylcholine (CCh)-stimulated chief cell responses. CCh stimulated the hydrolysis of polyphosphoinositide in chief cells at the same concentrations as it stimulated pepsinogen secretion. Each of five different muscarinic receptor antagonists, atropine, pirenzepine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), AF-DX116 and scopolamine, inhibited both pepsinogen secretion and inositol phosphate accumulation stimulated by graded concentrations of CCh. The pA2 values of the antagonists calculated from data on inositol phosphate accumulation and pepsinogen secretion (atropine = scopolamine = 4-DAMP greater than pirenzepine greater than AF-DX116) suggest that the muscarinic acetylcholine receptor in gastric chief cells is the M3 subtype. On the other hand, CCh did not affect the adenylate cyclase/cAMP signaling pathway in gastric chief cells. All pA2 values of the antagonists were also in agreement with the Ki values determined by [3H]NMS binding to chief cells. Furthermore, GTP gamma S reduced [3H]acetylcholine binding to chief cell membranes in a concentration-dependent manner. The present study, therefore, suggests that muscarinic M3 receptors, which may be coupled to a G protein, mediate pepsinogen secretion, probably by activation of the polyphosphoinositide second messenger system in guinea pig gastric chief cells.
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PMID:M3 muscarinic receptors mediate pepsinogen secretion via polyphosphoinositide hydrolysis in guinea pig gastric chief cells. 132 64

The muscarinic acetylcholine receptor (mAChR) is an integral membrane protein that transduces stimulus to effectors through the activation of guanine nucleotide-binding (G) proteins. Four or more subtypes of mAChR were detected in various tissues, and their primary structures were elucidated by cloning and sequence analysis of complementary DNA. Functional differences between them existed when they were expressed in clonal culture cells. mAChRI (m1) and mAChRIII (m3) preferentially activated phosphoinositide (PI) hydrolysis and opened Ca(2+)-activated K+ channels followed by closure of the M (K+)-currents, while such current activities were rarely evoked by mAChRII (m2)- and mAChRIV (m4)-transformed cells. Although it has been reported that mAChRII and mAChRIV inhibited adenylate cyclase, there was little or no such inhibition by mAChRI and mAChRIII. It is known that heart and neuronal mAChR modulate voltage-sensitive Ca2+ currents, but which species of mAChR subtypes are involved has been poorly understood. Recently we identified that endogenous mAChRIV and exogenous mAChRII expressed in NG108-15 neuroblastoma-glioma hybrid cells, but not mAChRI and mAChRIII, efficiently depressed high-threshold Ca2+ currents in a pertussis toxin-sensitive manner.
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PMID:[Coupling of muscarinic acetylcholine receptors, m1/m3 and m2/m4, to phosphoinositide metabolism and Ca2+ channels in DNA-transfected NG108-15 cells]. 172 Jul 57

The cloning and functional expression of five mammalian muscarinic acetylcholine receptor genes (m1-m5) has revealed that m1, m3, and m5 primarily couple to stimulation of phosphoinositide (PI) turnover, whereas m2 and m4 are strongly linked to inhibition of adenylate cyclase, albeit not exclusively. To identify the structural domains responsible for this functional specificity, cDNAs encoding chimeric m2/m3 receptors were constructed. The abilities of these receptors to mediate stimulation of PI hydrolysis and inhibition of prostaglandin E2-stimulated cAMP accumulation, as well as the pertussis toxin (PTX) sensitivity of these responses, were examined after stable expression in mouse A9 L cells. Substitution of the putative third cytoplasmic loop (i3) of m2 with the corresponding m3 sequence resulted in a chimeric receptor that, similar to m3, stimulated PI breakdown by a PTX-insensitive mechanism but did not inhibit adenylate cyclase. Conversely, a chimeric m3 receptor containing the i3 domain of m2 showed the same functional profile as m2 (i.e., inhibition of adenylate cyclase and weak stimulation of PI turnover by a PTX-sensitive mechanism), indicating that the i3 loop is sufficient to determine coupling selectivity. Similarly, exchange of a short N-terminal segment of i3 (16 or 17 amino acids) between m2 and m3 resulted in chimeric receptors that gained the ability to mediate the functional responses of the wild-type receptor from which the segment was derived, although with substantially reduced efficiency. However, the chimeric m2 receptor containing the 17-amino acid m3 sequence in the N-terminal portion of i3 retained its ability to inhibit adenylate cyclase. Carbachol binding studies involving the use of the GTP analog 5'-guanylyl imidodiphosphate and PTX-pretreated cells generally correlated well with the functional findings. Our data indicate that the N-terminal portion of i3 is a sufficient but not the exclusive determinant of coupling selectivity displayed by the various muscarinic receptors.
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PMID:Delineation of muscarinic receptor domains conferring selectivity of coupling to guanine nucleotide-binding proteins and second messengers. 217 67

The muscarinic acetylcholine receptor-mediated inhibition of adenylate cyclase was studied in slices of guinea-pig cerebral cortex under normal and depolarizing conditions. Carbachol (1 mM) inhibited basal and isoproterenol (50 microM)-stimulated cyclic AMP (cAMP) accumulation by 20% and 25%, respectively, in normal Krebs-Ringer bicarbonate buffer solution (KRB). High-K+ medium (42 mM K+) increased cAMP accumulation to 330% of the basal level and abolished the inhibitory effect of carbachol. It also abolished the effect of morphine, an agonist of opioid receptors. Low-Ca2+ KRB or the presence of the Ca2+ channel blocker nifedipine, counteracted the effect of high K+ and restored the inhibitory effect of carbachol on the cAMP level. Pretreatment of slices with W-7 or trifluoperazine, two calmodulin antagonists, had the same effect as low Ca2+ or nifedipine on high-K(+)-stimulated cAMP accumulation and caused reappearance of the inhibitory effects of carbachol and morphine. On the contrary, H-7, an inhibitor of protein kinase C, and neomycin, an inhibitor of phospholipase C, had no significant effect on high-K(+)-induced phenomena and did not restore the effect of carbachol. These data suggest that the Ca2(+)-calmodulin system activated by membrane depolarization regulates the cAMP level directly and also by affecting the receptor-mediated process in nerve cells.
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PMID:Disappearance in high-K+ medium of receptor-mediated inhibition of adenylate cyclase in guinea-pig cortical slices. 217 2

The cardiac m2 muscarinic acetylcholine receptor (mAChR) is a sialoglycosylated transmembrane protein which has three potential sites for N-glycosylation (namely, Asn2, Asn3, and Asn6). To investigate the role of N-linked oligosaccharide(s) in the expression and function of the receptor, we constructed glycosylation-defective mutant receptor genes in which the three asparagine codons were substituted by codons for either aspartate (Asp2,3,6), lysine (Lys2,3,6), or glutamine (Gln2,3,6). The glycosylation-defective and wild-type receptor genes were stably expressed in Chinese hamster ovary cells. Binding experiments with the membrane-permeable radioligand [3H]quinuclidinyl-benzilate and the membrane-impermeable radioligand [3H]N-methylscopolamine revealed that the Asp2,3,6, Gln2,3,6, and wild-type receptors were located exclusively on the cell surface and expressed in similar numbers. The Lys2,3,6 mutant receptor was expressed at a relatively low level and was therefore not included in subsequent experiments. Wheat germ agglutinin-Sepharose chromatography and sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis demonstrated that the wild-type receptor, but not the Asp2,3,6 and Gln2,3,6 mutant receptors were N-glycosylated. The Asp2,3,6 and Gln2,3,6 mutant receptors had the same affinities for mAChR ligands as wild-type receptors. The time courses for degradation of the Asp2,3,6, Gln2,3,6, and wild-type receptors were also similar. In vivo functional analysis of the ability of the glycosylation mutant receptors to inhibit forskolin-stimulated cAMP accumulation revealed that maximal inhibition of adenylate cyclase activity was similar in the mutant and wild-type receptors. The Asp2,3,6 mutant receptor had an unaltered IC50 value for carbachol while the IC50 value of the Gln2,3,6 mutant receptor was 2-fold higher than that of the wild-type receptor. These results indicate that N-glycosylation of the m2 mAChR is not required for cell surface localization or ligand binding and does not confer increased stability against receptor degradation. Furthermore, N-glycosylation of the m2 mAChR is not required for functional coupling of the m2 mAChR to inhibition of adenylate cyclase.
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PMID:Site-directed mutagenesis of the m2 muscarinic acetylcholine receptor. Analysis of the role of N-glycosylation in receptor expression and function. 224 95

A method for the simultaneous visualization of neurotransmitter receptor binding sites and immunohistochemically characterized cells is described using in vitro sections of rat striatum. The striatum provides a rich neurochemical environment in which to examine muscarinic acetylcholine receptor relations to peptide-reactive somata, or to assess the cellular locale of the dopamine receptor linked to adenylate cyclase activation. Different modifications of the procedure are employed to determine the localization pattern of 3H-radioligands with slow or rapid receptor dissociation times. The present technique is compatible for use with any combination of immunohistochemical antibody/radioligand probe that can be studied on fresh-frozen, slide-mounted tissue sections.
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PMID:Neurotransmitter receptor autoradiography in immunohistochemically identified neurons. 241 10

1. Secretion of [3H]acetylcholine was studied in the guinea-pig ileum longitudinal muscle-myenteric plexus preparation. The transmitter stores of the cholinergic nerves were labelled by pre-incubation with [3H]choline. The preparation was mounted in an organ bath and superfused with Tyrode solution containing hemicholinium-3 and eserine. [3H]Acetylcholine secretion was evoked by electrical stimulation (0.5 Hz, 150 shocks). 2. 8-Bromo cyclic AMP, the adenylate cyclase activator forskolin, and the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine enhanced the [3H]acetylcholine secretion in a concentration-dependent manner. The values of 'maximal enhancement' calculated were similar, viz. 200-300% of control. 3. 8-Bromo cyclic GMP reduced the [3H]acetylcholine secretion. 4. The 'maximal enhancement' of 3-isobutyl-1-methylxanthine was not altered by the presence of forskolin (25 mumol/l) suggesting a common mechanism of action, i.e. elevation of endogenous cyclic AMP levels. 5. The muscarinic acetylcholine receptor antagonist atropine enhanced the [3H]acetylcholine secretion with a 'maximal enhancement' of 506% of control. Presence of neither forskolin (25 mumol/l) nor 3-isobutyl-1-methylxanthine (5 mmol/l) altered the 'maximal enhancement' for atropine. 6. In contrast, atropine (1 mumol/l) and 4-aminopyridine (0.5 mmol/l) additively enhanced the [3H]acetylcholine secretion. 7. The results suggest that neuronal cyclic AMP may be involved in muscarinic acetylcholine receptor-mediated control of [3H]acetylcholine secretion in guinea-pig ileum myenteric plexus.
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PMID:Interaction of forskolin with the effect of atropine on [3H]acetylcholine secretion in guinea-pig ileum myenteric plexus. 245 81

The m1 muscarinic acetylcholine receptor gene was transfected into and stably expressed in A9 L cells. The muscarinic receptor agonist, carbachol, stimulated inositol phosphate generation, arachidonic acid release, and cAMP accumulation in these cells. Carbachol stimulated arachidonic acid and inositol phosphate release with similar potencies, while cAMP generation required a higher concentration. Studies were performed to determine if the carbachol-stimulated cAMP accumulation was due to direct coupling of the m1 muscarinic receptor to adenylate cyclase via a GTP binding protein or mediated by other second messengers. Carbachol failed to stimulate adenylate cyclase activity in A9 L cell membranes, whereas prostaglandin E2 did, suggesting indirect stimulation. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated arachidonic acid release yet inhibited cAMP accumulation in response to carbachol. PMA also inhibited inositol phosphate release in response to carbachol, suggesting that activation of phospholipase C might be involved in cAMP accumulation. PMA did not inhibit prostaglandin E2-, cholera toxin-, or forskolin-stimulated cAMP accumulation. The phospholipase A2 inhibitor eicosatetraenoic acid and the cyclooxygenase inhibitors indomethacin and naproxen had no effect on carbachol-stimulated cAMP accumulation. Carbachol-stimulated cAMP accumulation was inhibited with TMB-8, an inhibitor of intracellular calcium release, and W7, a calmodulin antagonist. These observations suggest that carbachol-stimulated cAMP accumulation does not occur through direct m1 muscarinic receptor coupling or through the release of arachidonic acid and its metabolites, but is mediated through the activation of phospholipase C. The generation of cytosolic calcium via inositol 1,4,5-trisphosphate and subsequent activation of calmodulin by m1 muscarinic receptor stimulation of phospholipase C appears to generate the accumulation of cAMP.
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PMID:A transfected m1 muscarinic acetylcholine receptor stimulates adenylate cyclase via phosphatidylinositol hydrolysis. 255 56

A genomic clone encoding the gene for the mouse M1 muscarinic acetylcholine receptor has been isolated, placed under the control of the zinc-inducible mouse metallothionein promoter, and transfected into mouse Y1 adrenal cells. The receptor concentration was about 300 fmol/mg membrane protein in the absence of zinc and could be increased to 4000 fmol/mg membrane protein in the presence of increasing concentrations of zinc. The receptor expressed in zinc-induced cells exhibits the high affinity binding for quinuclidinyl benzilate, atropine, and pirenzepine expected of the M1 muscarinic receptor. The M1 receptor when expressed in Y1 or L cells is physiologically active, as measured by agonist-dependent stimulation of phosphatidylinositol metabolism, but does not inhibit forskolin stimulation of cAMP accumulation. In contrast, a cloned M2 muscarinic receptor when expressed in Y1 cells is able to inhibit forskolin stimulation of cAMP accumulation, but is unable to stimulate phosphatidylinositol metabolism. The stimulation of phosphatidylinositol metabolism mediated by the M1 receptor was not altered by prior treatment of Y1 cells with concentrations of islet-activating protein sufficient to eliminate M2 receptor-mediated inhibition of adenylate cyclase. The cloned M1 receptor gene thus exhibits both the pharmacological and physiological properties expected of the M1 muscarinic acetylcholine receptor. In addition, these results indicate that different subtypes of the muscarinic receptor are coupled to different physiological responses.
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PMID:Isolation, sequence, and functional expression of the mouse M1 muscarinic acetylcholine receptor gene. 284 36


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