Distinct cAMP signaling pathways differentially regulate α_2c-adrenoceptor expression: Role in serum induction in human arteriolar smooth muscle cells

The physiological role of α₂-adrenoceptors (α₂-ARs) in cutaneous, arteriolar, vascular smooth muscle cells (VSMs) is to mediate cold-induced constriction. In VSMs cultured from human cutaneous arterioles, there is a selective increase in α_2C-AR expression after serum stimulation. In the present study, we examined the cellular mechanisms contributing to this response. Serum induction of α_2C-ARs was paralleled by increased expression, of cyclooxygenase-2 (COX-2), increased release of prostaglandins, and increased intracellular concentration of cAMP. Inhibition of COX-2 by acetyl salicylic acid (1 mM), NS-398 (5 μM), or celecoxib (3 μM) abolished the increase in cAMP and markedly reduced α_2C-AR induction in response to serum stimulation. The cAMP agonists, forskolin (10 μM), isoproterenol (10 μM), and cholera toxin (0.1 μg/ml) each dramatically increased expression of α_2C-ARs in human cutaneous VSMs. The A-kinase inhibitor H-89 (2 μM) inhibited phosphorylation of cAMP response element binding protein, but not the increase in α_2C-AR expression in response to these agonists. cAMP-dependent but A-kinase independent signaling can involve activation of guanine nucleotide exchange factors for the GTP-binding protein, Rap. Indeed, pull-down assays demonstrated Rap1 activation by serum and forskolin in VSM. Transient transfections using α_2C-AR promoter-luciferase reporter construct demonstrated that Rap1 increased reporter activity, whereas the A-kinase catalytic subunit decreased reporter activity. These results indicate that cAMP signaling can have dual effects in cutaneous VSMs:activation of α_2C-AR transcription mediated by Rap1 GTPase and suppression mediated by A-kinase. The former effect predominates in serum-stimulated VSMs leading to a COX-2, cAMP, and Rap 1-dependent increase in α_2C-AR expression. Such increased expression of α_2C-ARs may contribute to enhanced cold-induced vasoconstriction and Raynaud's phenomenon.