The following study was undertaken to elucidate the cytoskeletal phenotype of neonatal rat cardiac fibroblasts (NNCF) and the signaling pathways coupled to beta-adrenergic receptor stimulated DNA synthesis. The cytoskeletal proteins vimentin, and smooth muscle alpha-actin were detected in NNCF, suggestive of a myofibroblast phenotype. Isoproterenol (ISO) treatment stimulated (3)H-thymidine uptake, and concomitantly increased intracellular cyclic AMP levels. However, cyclic AMP-elevating agents markedly decreased DNA synthesis. Coincident with growth, ISO-stimulated phosphatidylinositol 3-kinase (PI3-K) activity, and the PI3-K inhibitor LY294002 abrogated enzyme activity, and DNA synthesis. Unexpectedly, the serine/threonine kinase protein kinase Balpha (PKBalpha), a putative downstream target of PI3-K, was dephosphorylated following ISO treatment. Despite PKBalpha inactivation, the phosphorylation of its putative downstream target, the pro-apoptotic enzyme glycogen synthase kinase-3alpha was significantly increased in response to ISO. These latter effects of ISO were mimicked by the cyclic AMP-elevating agent forskolin. Lastly, ISO treatment increased p70 ribosomal S6 kinase (p70S6K) phosphorylation, as reflected by an upward electrophoretic mobility shift. The pretreatment with rapamycin abrogated the ISO-mediated mobility shift of p70S6K, and DNA synthesis. Collectively, these data demonstrate that NNCF express a myofibroblast phenotype, and beta-adrenergic agonists promote DNA synthesis via a PI3-K-dependent pathway involving p70S6K. Although unable to suppress ISO-stimulated DNA synthesis, cyclic AMP can influence specific downstream targets of PI3-K highlighting a novel crosstalk between these signaling pathways.
Copyright 2003 Wiley-Liss, Inc.