A novel signaling pathway of ADP-ribosyl cyclase activation by angiotensin II in adult rat cardiomyocytes

Abstract

ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca2+-mobilizing second messenger, cADP-ribose (cADPR), from NAD+. In this study, we investigated the molecular basis of ADPR-cyclase activation in the ANG II signaling pathway and cellular responses in adult rat cardiomyocytes. The results showed that ANG II generated biphasic intracellular Ca2+concentration increases that include a rapid transient Ca2+elevation via inositol trisphosphate (IP3) receptor and sustained Ca2+rise via the activation of L-type Ca2+channel and opening of ryanodine receptor. ANG II-induced sustained Ca2+rise was blocked by a cADPR antagonistic analog, 8-bromo-cADPR, indicating that sustained Ca2+rise is mediated by cADPR. Supporting the notion, ADPR-cyclase activity and cADPR production by ANG II were increased in a time-dependent manner. Application of pharmacological inhibitors and immunological analyses revealed that cADPR formation was activated by sequential activation of Src, phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt), phospholipase C (PLC)-γ1, and IP3-mediated Ca2+signal. Inhibitors of these signaling molecules not only completely abolished the ANG II-induced Ca2+signals but also inhibited cADPR formation. Application of the cADPR antagonist and inhibitors of upstream signaling molecules of ADPR-cyclase inhibited ANG II-stimulated hypertrophic responses, which include nuclear translocation of Ca2+/calcineurin-dependent nuclear factor of activated T cells 3, protein expression of transforming growth factor-β1, and incorporation of [3H]leucine in cardiomyocytes. Taken together, these findings suggest that activation of ADPR-cyclase by ANG II entails a novel signaling pathway involving sequential activation of Src, PI 3-kinase/Akt, and PLC-γ1/IP3and that the activation of ADPR-cyclase can lead to cardiac hypertrophy.