Abstract

α₁-Adrenergic receptors mediate mitogenic responses and increase intracellular free Ca²⁺([Ca²⁺]_i) in vascular smooth muscle cells. Induction of c-fos is a critical early event in cell growth; expression of this gene is regulated by a number of signaling pathways including Ca²⁺. We wondered whether Ca²⁺ signaling plays a critical role in the induction of c-fos gene by α₁-adrenergic receptors. Using stably transfected rat-1 fibroblasts, we confirmed that PE induced c-fos mRNA expression in a time- and dose-dependent manner, and also increased [Ca²⁺]_i (measured with Fura-2 AM). These responses were blocked by the α₁-adrenergic receptor antagonist doxazosin. Both intracellular Ca²⁺ chelation (using BAPTA/AM) and extracellular Ca²⁺ depletion (using EGTA) significantly inhibited PE-induced c-fosexpression by α_1A and α_1B receptors. Brief (1-min) stimulation of α_1A and α_1Breceptors with PE did not maximally induce c-fosexpression, suggesting that a sustained increase in [Ca²⁺]_i due to Ca²⁺ influx is required. The calmodulin (CaM) antagonists, R24571, W7, and trifluoperazine, but not the CaM-dependent protein kinases inhibitor KN-62, significantly inhibited c-fos induction by α_1A and α_1B receptors. Neither inhibition of protein kinase C nor inhibition of adenylyl cyclase modified c-fos induction by PE. These results suggest that α₁-adrenergic receptor-induced c-fosexpression in rat-1 cells is dependent on a Ca²⁺/CaM-associated pathway.