Endothelin-1 (ET1) is a vasoactive peptide that stimulates hypertrophy of vascular smooth muscle cells (VSMC) through diverse signaling pathways mediated by G q /G i /G 13 heterotrimeric G proteins. We have found that ET1 stimulates the activity of cAMP-dependent protein kinase (PKA) in VSMC as profoundly as the G s -linked -adrenergic agonist, isoproterenol (ISO), but in a transient manner. PKA activation by ET1 was mediated by type-A ET1 receptors (ETA) and recruited an autocrine signaling mechanism distinct from that of ISO, involving G i -coupled ␥ subunits of heterotrimeric G proteins, extracellular signal-regulated kinases ERK1/2, cyclooxygenase COX-1 (but not COX-2) and prostacyclin receptors. In the functional studies, inhibition of PKA or COX-1 attenuated ET1-induced VSMC hypertrophy, suggesting the positive role of PKA in this response to ET1. Furthermore, we found that ET1 stimulates a G␥-mediated, PKA-dependent phosphorylation and inactivation of glycogen synthase kinase-3 (GSK3), an enzyme that regulates cell growth. Together, this study describes that (i) PKA can be transiently activated by G i -coupled agonists such as ET1 by an autocrine mechanism involving G␥/calcium/ERK/COX-1/prostacyclin signaling, and (ii) this PKA activation promotes VSMC hypertrophy, at least in part, through PKA-dependent phosphorylation and inhibition of GSK3.
Endothelin-1 (ET1)2 is a vasoconstrictor peptide implicated in embryonic development, cardiovascular homeostasis, and the pathogenesis of several cardiovascular disorders, such as pulmonary hypertension, heart failure, atherosclerosis, and restenosis (1). Physiological effects of ET1 are largely mediated by vascular smooth muscle cells (VSMC) that respond to ET1 by contraction (2) and hypertrophy (3). Two types of ET1 receptors, termed ET A and ET B , have been cloned (4, 5), and they belong to a family of G protein-coupled receptors, whose signaling is mediated by heterotrimeric G proteins (6, 7). ET A receptors are expressed primarily on arterial vessels, whereas ET B receptors predominate on the low pressure side of circulation (8). ET A receptors (the focus of this study) are coupled to G q/11 , G i , and G 12/13 classes of G proteins that mediate the contractile and growth promoting effects of ET1 in VSMC through activation of diverse signaling molecules including phospholipase C (PLC) (9), intracellular Ca 2ϩ (10, 11), protein kinase C (12), and the extracellular signal-regulated kinase (ERK) (13).We have previously shown that ET1 stimulates activation of protein kinase A (14), an enzyme commonly induced via G scoupled receptors through the production of cAMP, and is thought to regulate VSMC growth. Given that ET A receptors are not coupled directly to G s proteins, we sought to determine the signaling mechanism of PKA activation by ET1 the functional significance of PKA activation by ET1 in regulation of VSMC growth.
EXPERIMENTAL PROCEDURESVSMC Culture and DNA Transfection-The rat aortic smooth muscle cells RASMC were obtained by enzymatic dissociation of...