Regulation of vascular smooth muscle cell function by adenylyl cyclase isoforms

Abstract

Adenylyl cyclases (AC), a family of enzymes that catalyze the synthesis of cyclic AMP, are critical regulators of cellular functions. Regulation patterns differ between the AC isoforms, however the consequences of activation of any individual AC isoform (e.g., to catalyze the synthesis of cAMP) were thought to be common to all isoforms. To examine this hypothesis we studied the effect of adenoviral gene transfer of representative AC isoforms AC I, II, V and VI on whole cell cAMP activity, regulation of vascular smooth muscle cell proliferation, isoproterenol (ISO)-mediated arborization responses (an index of cytoskeletal re-organization) and protein kinase A (PKA) activity Although all AC isoforms when expressed in smooth muscle cells comparably enhanced forskolin (FSK)-stimulated cAMP synthesis (186–208% of the AC activity in GFP-infected cells), only AC I-infected cells demonstrated an enhancement of FSK-stimulated inhibition of proliferation (19±3% of GFP, p<0.01). Only gene transfer of AC VI enhanced ISO-mediated arborization (155±16% of GFP, p<0.05). To further examine the contribution of AC VI to the arborization response, we utilized a “knockdown” approach based on expression of a truncated-AC VI dominant negative mutant (TM1T). TM1T expression significantly reduced FSK-stimulated AC activation (79±8% of GFP alone) and ISO-mediated arborization (54±6% of GFP alone). This reduction in arborization was recovered by the co-expression of AC isoform VI (84±10%). Lastly, FSK-stimulated PKA activity was significantly reduced (61±5% of GFP) by AC I expression and significantly enhanced (132±8% of GFP) by AC VI expression. In aggregate these data support a previously unappreciated role for specific AC isoforms in directing the effect of elevation of intracellular cAMP in the regulation of vascular function. Supported by CIHR.