Stimulation of P2Y receptors activates c-fos gene expression and inhibits DNA synthesis in cultured cardiac fibroblasts

Objectives: The aims of this study were to determine (1) whether neonatal rat cardiac fibroblasts (CAFB) express P2Y receptors; (2) whether CAFB respond to extracellular ATP by inducing expression of c-fos mRNA; and (3) whether extracellular ATP modulates norepinephrine (NE)-stimulated cell growth in CAFB. Methods: Expression of P2Y₁ and P2Y₂ receptors and induction of c-fos were examined by Northern blot analysis. CAFB growth was assessed by measuring [³H]thymidine incorporation and DNA content. P2Y receptor pharmacology was studied using various ATP analogues. Results: Northern blot analysis of polyA enriched RNA confirmed that at least 2 subtypes of P2Y receptors (P2Y₁ and P2Y₂) are expressed in cultured CAFB. Extracellular ATP induced the expression of c-fos mRNA through a pathway that was sensitive to inhibitors of protein kinase C (PKC), but not to inhibitors of intracellular Ca²⁺ signaling. Extracellular ATP inhibited the NE- stimulated increases in DNA content and in [³H]thymidine incorporation into DNA. Whereas the potency order for stimulation of c-fos expression was ATP = UTP > ADP > adenosine, the potency order to inhibit the NE-induced increase of [³H]thymidine incorporation into DNA was ATP > ADP > UTP > adenosine. Conclusions: These data demonstrate that CAFB express both P2Y₁ and P2Y₂ receptor mRNA and that CAFB respond to P2Y receptor stimulation by induction of c-fos and inhibition of DNA synthesis. These findings suggest that the effects of ATP on [³H]thymidine incorporation into DNA and on expression of c-fos mRNA are exerted via distinct P2Y receptor subtypes.