Abstract-Adenosine inhibits growth of vascular smooth muscle cells. The goals of this study were to determine which adenosine receptor subtype mediates the antimitogenic effects of adenosine and to investigate the signal transduction mechanisms involved. In rat aortic vascular smooth muscle cells, platelet-derived growth factor-BB (PDGF-BB) (25 ng/mL) stimulated DNA synthesis ([ 3 H]thymidine incorporation), cellular proliferation (cell number), collagen synthesis ([ 3 H]proline incorporation), total protein synthesis ([ 3 H]leucine incorporation), and mitogen-activated protein (MAP) kinase activity. The adenosine receptor agonists 2-chloroadenosine and 5Ј-N-methylcarboxamidoadenosine, but not N 6 -cyclopentyladenosine or CGS21680, inhibited the growth effects of PDGF-BB, an agonist profile consistent with an A 2B receptor-mediated effect. The adenosine receptor antagonists KF17837 and 1,3-dipropyl-8-p-sulfophenylxanthine, but not 8-cyclopentyl-1,3-dipropylxanthine, blocked the growth-inhibitory effects of 2-chloroadenosine and 5Ј-N-methylcarboxamidoadenosine, an antagonist profile consistent with an A 2 receptor-mediated effect. Antisense, but not sense or scrambled, oligonucleotides to the A 2B receptor stimulated basal and PDGF-induced DNA synthesis, cell proliferation, and MAP kinase activity. Moreover, the growth-inhibitory effects of 2-chloroadenosine, 5Ј-N-methylcarboxamidoadenosine, and erythro-9-(2-hydroxy-3-nonyl) adenine plus iodotubericidin (inhibitors of adenosine deaminase and adenosine kinase, respectively) were abolished by antisense, but not scrambled or sense, oligonucleotides to the A 2B receptor. Our findings strongly support the hypothesis that adenosine causes inhibition of vascular smooth muscle cell growth by activating A 2B receptors coupled to inhibition of MAP kinase activity. Although the standard view is that A 1 and A 2A receptors are the most important with regard to adenosine-mediated cardioprotection, indirect evidence suggests that adenosine inhibits vascular SMC growth via activation of A 2 and more specifically A 2B receptors. 2-6 However, the inference that A 2B receptors mediate the growth-inhibiting effects of adenosine is weak because to date researchers do not have available agonists or antagonists selective for A 2B receptors. Moreover, recent data suggest that in contrast to SMCs, A 2 receptors induce growth in vascular endothelial cells 7,8 and stimulate mitogen-activated protein (MAP) kinase activity in several cell types. 9 -11 Blockade of A 2B receptor synthesis is an alternative approach to investigate the participation of A 2B receptors in adenosine-mediated inhibition of vascular SMC growth. Accordingly, in the present study we developed antisense oligonucleotides against rat A 2B receptors and used them to test the role of A 2B receptors in regulating SMC growth and MAP kinase activity. Methods SMC CulturesArterial SMCs were cultured as explants from the abdominal aortas, obtained from anesthetized (50 mg/kg IP injection of pentobarbital) Sprague-Dawley male r...