COUPLING OF EXPRESSED ALPHA(1B)-ADRENERGIC AND ALPHA(1D)-ADRENERGIC RECEPTORS TO MULTIPLE SIGNALING PATHWAYS IS BOTH G-PROTEIN AND CELL-TYPE-SPECIFIC

Alpha1-Adrenergic receptors (ARs) are members of the G protein-coupled receptor superfamily. Alpha1-AR subtypes mediate the effects of the s ympathetic nervous system, especially those involved in cardiac homeos tasis. To investigate signal transduction by a novel subtype (alpha1D) , which we recently cloned, and to compare it with that by the previou sly characterized alpha1B-AR, we assessed the ability of each subtype to activate polyphosphoinositide (PI) metabolism, cAMP accumulation, a nd arachidonic acid release in Chinese hamster ovary (CHO) and COS-1 c ells expressing these subtypes after stable or transient transfection, respectively. In COS-1 and CHO cells, both the alpha1D- and alpha1B-A R were found to couple to PI hydrolysis through a pertussis toxin-inse nsitive G protein. Both alpha1-AR subtypes also increased intracellula r cAMP by an indirect mechanism, although this effect was observed onl y in COS-1 cells and not in CHO cells. Interestingly, alpha1-AR-stimul ated arachidonic acid release was also demonstrated for both subtypes in COS-1 cells. This release was mediated through phospholipase A2 act ivation and involved a pertussis toxin-sensitive G protein. Alpha1-AR- stimulated arachidonic acid release was dependent upon extracellular c alcium and was inhibited by 1 muM nifedipine. Inhibitors of protein ki nase C, phospholipase C, and diacylglycerol lipase did not alter alpha 1-AR-stimulated release of arachidonic acid. These findings indicate t hat in COS-1 cells alpha1-AR-stimulated arachidonic acid release is mo st likely coupled to dihydropyridine-sensitive L-type calcium channels via a pertussis toxin-sensitive G protein. The influx of extracellula r calcium then stimulates phospholipase A2 to release arachidonic acid . Alpha1-AR-stimulated arachidonic acid release could also be demonstr ated in CHO cells and was pertussis toxin sensitive but nifedipine ins ensitive. These cells were also unresponsive to Bay K8644, indicating a lack of voltage-sensitive calcium channels in CHO cells. Nevertheles s, alpha1-AR activation increased intracellular Ca2+ levels, as assess ed by fura-2 fluorescence studies. Neomycin blocked both alpha1-AR-sti mulated PI hydrolysis and increases in intracellular Ca2+ levels but d id not inhibit the increase in arachidonic acid release. Taken togethe r, these data indicate that in CHO cells alpha1-ARs can couple directl y to phospholipase A2 activation via a pertussis toxin-sensitive pathw ay. Thus, in these model systems we demonstrate for the first time tha t a single alpha1-AR subtype can activate multiple distinct signal tra nsduction pathways, in which receptor-effector coupling is modulated b y distinct G proteins.