HAMSTER ALPHA(1B)-ADRENERGIC RECEPTOR DIRECTLY ACTIVATES G(S) IN THE TRANSFECTED CHINESE-HAMSTER OVARY CELLS

A prototypic Ca2+-mobilizing hormone receptor, alpha(1)-adrenergic receptor (alpha(1)AR), stimulates cAMP accumulation. The mechanism underlying this phenomenon was previously suggested to be secondary to phosphatidylinositol hydrolysis-protein kinase C activation in some cells. We transfected Chinese hamster ovary (CHO)-K1 cells with hamster alpha(1B)AR cDNA and isolated cells stably expressing alpha(1B)AR (CHO alpha(1B) cells). We investigated the molecular mechanism underlying the alpha(1)AR-mediated cAMP production in the CHO alpha(1B) cells. Norepinephrine (NE) stimulated intracellular calcium mobilization and cAMP production through alpha(1B)AR. Pretreatment with a phospholipase C inhibitor, U-73,122 (10 mu M), abolished the NE-induced intracellular calcium response, whereas it did not affect the NE-stimulated cAMP production. Treatment with various agents (protein kinase C inhibitors, calcium ionophore, cyclo-oxygenase inhibitor, or pertussis toxin) had little effect on the NE-induced cAMP production. The parent CHO and CHO alpha(1B) cells contained similar amounts of G(s alpha) (42 and 45 kDa, respectively), as detected with immunoblot analysis, and exhibited similar extents of cAMP synthesis with cholera toxin and forskolin. Adenylyl cyclase activity in the CHO alpha(1B) cell membranes was also enhanced by NE. Furthermore, incubation of CHO alpha(1B) cell membranes with antiserum directed against the carboxyl-terminal portion of G(s alpha) inhibited the NE-stimulated adenylyl cyclase activity. Taken together, the results indicate that the alpha(1B)AR-mediated cAMP synthesis in CHO alpha(1B) cells reflects direct stimulation of G(s)-adenylyl cyclase. Therefore, the alpha(1)AR-stimulated cAMP production observed in some native tissues may involve the multiple mechanisms of the direct activation of G(s)-adenylyl cyclase and a secondary effect through activation of phosphatidylinositol hydrolysis.