title: Coupling of expressed alpha 1B- and alpha 1D-adrenergic receptor to multiple signaling pathways is both G protein and cell type specific.
abstract: alpha 1-Adrenergic receptors (ARs) are members of the G protein-coupled receptor superfamily. alpha 1-AR subtypes mediate the effects of the sympathetic nervous system, especially those involved in cardiac homeostasis. To investigate signal transduction by a novel subtype (alpha 1D), which we recently cloned, and to compare it with that by the previously characterized alpha 1B-AR, we assessed the ability of each subtype to activate polyphosphoinositide (PI) metabolism, cAMP accumulation, and arachidonic acid release in Chinese hamster ovary (CHO) and COS-1 cells expressing these subtypes after stable or transient transfection, respectively. In COS-1 and CHO cells, both the alpha 1D- and alpha 1B-AR were found to couple to PI hydrolysis through a pertussis toxin-insensitive G protein. Both alpha 1-AR subtypes also increased intracellular cAMP by an indirect mechanism, although this effect was observed only in COS-1 cells and not in CHO cells. Interestingly, alpha 1-AR-stimulated arachidonic acid release was also demonstrated for both subtypes in COS-1 cells. This release was mediated through phospholipase A2 activation and involved a pertussis toxin-sensitive G protein. alpha 1-AR-stimulated arachidonic acid release was dependent upon extracellular calcium and was inhibited by 1 microM nifedipine. Inhibitors of protein kinase C, phospholipase C, and diacylglycerol lipase did not alter alpha 1-AR-stimulated release of arachidonic acid. These findings indicate that in COS-1 cells alpha 1-AR-stimulated arachidonic acid release is most likely coupled to dihydropyridine-sensitive L-type calcium channels via a pertussis toxin-sensitive G protein. The influx of extracellular calcium then stimulates phospholipase A2 to release arachidonic acid. alpha 1-AR-stimulated arachidonic acid release could also be demonstrated in CHO cells and was pertussis toxin sensitive but nifedipine insensitive. These cells were also unresponsive to Bay K8644, indicating a lack of voltage-sensitive calcium channels in CHO cells. Nevertheless, alpha 1-AR activation increased intracellular Ca2+ levels, as assessed by fura-2 fluorescence studies. Neomycin blocked both alpha 1-AR-stimulated PI hydrolysis and increases in intracellular Ca2+ levels but did not inhibit the increase in arachidonic acid release. Taken together, these data indicate that in CHO cells alpha 1-ARs can couple directly to phospholipase A2 activation via a pertussis toxin-sensitive pathway. Thus, in these model systems we demonstrate for the first time that a single alpha 1-AR subtype can activate multiple distinct signal transduction pathways, in which receptor-effector coupling is modulated by distinct G proteins.
authors.affiliation: Department of Cardiovascular Biology, Cleveland Clinic Foundation, Ohio 44195.
authors.forename: D M
authors.initials: DM
authors.lastname: Perez
authors.forename: M B
authors.initials: MB
authors.lastname: DeYoung
authors.forename: R M
authors.initials: RM
authors.lastname: Graham
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D016466:CHO Cells / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000502:physiology*; D006868:Hydrolysis; D007295:Inositol Phosphates / Q000096:biosynthesis / Q000378:metabolism; D008954:Models, Biological; D037342:Pertussis Toxin; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics / Q000502:physiology*; D015290:Second Messenger Systems / Q000502:physiology; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D007295:Inositol Phosphates; D018340:Receptors, Adrenergic, alpha-1; D010566:Virulence Factors, Bordetella; D016718:Arachidonic Acid; D000242:Cyclic AMP; D037342:Pertussis Toxin; D010741:Phospholipases A; D054467:Phospholipases A2; D019204:GTP-Binding Proteins; D002118:Calcium