Regulation of cardiac fibroblast collagen synthesis by adenosine: roles for Epac and PI3K

Departments of 1 Medicine and 2 Pharmacology and 3 Biomedical Sciences PhD Program, Univeristy of California, San Diego School of Medicine, La Jolla, California Submitted 2 June 2008 ; accepted in final form 3 March 2009 Rat cardiac fibroblasts (CF) express multiple adenosine (ADO) receptors. Pharmacological evidence suggests that activation of A 2 receptors may inhibit collagen synthesis via adenylyl cyclase-induced elevation of cellular cAMP. We have characterized the signaling pathways involved in ADO-mediated inhibition of collagen synthesis in primary cultures of adult rat CF. ANG II stimulates collagen production in these cells. Coincubation with agents that elevate cellular cAMP [the ADO agonist, 5'- N -ethylcarboxamidoadensoine (NECA), and forskolin] inhibited the stimulatory effects of ANG II. However, direct stimulators and inhibitors of protein kinase A (PKA) did not alter ANG II-induced collagen synthesis, indicating that PKA does not mediate the inhibitory effects of NECA. Inhibitors of AMP-kinase (AMPK) and extracellular signal-regulated kinase 1/2 (ERK1/2) do not alter NECA-inhibited collagen synthesis. However, activation of exchange factor directly activated by cAMP (Epac) mimicked the effects of NECA on ANG II-stimulated collagen synthesis. Inhibition of phosphoinositol-3 kinase (PI3K) reduced the inhibitory effects of NECA on ANG II-induced collagen synthesis, suggesting that NECA acts via PI3K. Furthermore, inhibition of PI3K also relieved the inhibitory effect of Epac activation on ANG II-stimulated collagen synthesis. Thus it appears that ADO activates the A 2 R-G s -adenylyl cyclase pathway and that the resultant cAMP reduces collagen synthesis via a PKA-independent, Epac-dependent pathway that feeds through PI3K. A 2 receptors; Epac; phosphoinositol-3 kinase; cardiac collagen deposition Address for reprint requests and other correspondence: F. Villarreal, 9500 Gilman Dr., 0613J, BSB 4028, La Jolla, CA 92093 (e-mail: fvillarr{at}ucsd.edu )