Stimulation of cyclic AMP production by the Caenorhabditis elegans muscarinic acetylcholine receptor GAR-3 in Chinese hamster ovary cells.
Among the three G-protein-linked acetylcholine receptors (GARs) in Caenorhabditis elegans (C. elegans), GAR-3 is structurally and pharmacologically most similar to mammalian muscarinic acetylcholine receptors (mAChRs). Using Chinese hamster ovary (CHO) cells stably expressing GAR-3b, the major alternatively spliced isoform of GAR-3, we observed that carbachol stimulated cyclic AMP (cAMP) production in a dose- and time-dependent manner. The stimulating effect of carbachol was abolished by atropine, a muscarinic antagonist, indicating that the cAMP production is specifically mediated by GAR-3b. When the cells were treated with BAPTA-AM and EGTA, which reduce the cytosolic Ca(2+) level, carbachol-stimulated cAMP accumulation was inhibited by approximately 56%. Inhibition of protein kinase C (PKC) by chronic treatment with phorbol 12-myristate 13-acetate (PMA) or by GF109203X decreased carbachol-stimulated cAMP production by as much as 68%. It thus appears that Ca(2+) and PKC are critically involved in GAR-3b-mediated cAMP formation. We also observed that carbachol-stimulated cAMP production was further enhanced by pertussis toxin (PTX) treatment. This observation indicates that GAR-3b couples to a PTX-sensitive G protein, presumably Gi, to attenuate the cAMP accumulation. Taken together, our data show that GAR-3b stimulates cAMP production in CHO cells and suggest that GAR-3b couples to both stimulatory and inhibitory pathways to modulate the intracellular cAMP level.
Park
Yang-Seo
YS
School of Life Sciences, Chungbuk National University, Cheongju 361-763, Republic of Korea.
Cho
Tae-Ju
TJ
Cho
Nam Jeong
NJ
D000818:Animals; D016466:CHO Cells; D017173:Caenorhabditis elegans / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism*; D002118:Calcium / Q000502:physiology; D002217:Carbachol / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000096:biosynthesis*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D018721:Muscarinic Agonists / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology; D037342:Pertussis Toxin / Q000494:pharmacology; D020033:Protein Isoforms / Q000096:biosynthesis; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D011976:Receptors, Muscarinic / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D029742:Caenorhabditis elegans Proteins; C477876:GAR-3 protein, C elegans; D007211:Indoles; D008301:Maleimides; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D020033:Protein Isoforms; D011976:Receptors, Muscarinic; D002217:Carbachol; D000242:Cyclic AMP; D037342:Pertussis Toxin; D011493:Protein Kinase C; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium