Signaling Pathway for Endothelin-1- and Phenylephrine-Induced cAMP Response Element Binding Protein Activation in Rat Ventricular Myocytes: Role of Inositol 1,4,5-Trisphosphate Receptors and CaMKII

Abstract

Background/Aims: Endothelin-1 (ET-1) and the α<Sub>1</Sub>-adrenoceptor agonist phenylephrine (PE) activate cAMP response element binding protein (CREB), a transcription factor implicated in cardiac hypertrophy. The signaling pathway involved in CREB activation by these hypertrophic stimuli is poorly understood. We examined signaling pathways for ET-1- or PE-induced cardiac CREB activation. Methods: Western blotting was performed with pharmacological and genetic interventions in rat ventricular myocytes. Results: ET-1 and PE increased CREB phosphorylation, which was inhibited by blockade of phospholipase C, the extracellular-signal-regulated kinase 1/2 (ERK1/2) pathway, protein kinase C (PKC) or Ca²⁺-calmodulin-dependent protein kinase II (CaMKII). Intracellular Ca²⁺ buffering decreased ET-1- and PE-induced CREB phosphorylation by ≥80%. Sarcoplasmic reticulum Ca²⁺ pump inhibitor, inositol 1,4,5-trisphosphate receptor (IP<Sub>3</Sub>R) blockers, or type 2 IP<Sub>3</Sub>R (IP<Sub>3</Sub>R2) knock-out abolished ET-1- or PE-induced CREB phosphorylation. ET-1 and PE increased phosphorylation of CaMKII and ERK1/2, which was eliminated by IP<Sub>3</Sub>R blockade/knock-out or PKC inhibition. Activation of CaMKII, but not ERK1/2, by these agonists was sensitive to Ca²⁺ buffering or to Gö6976, the inhibitor of Ca²⁺-dependent PKC and protein kinase D (PKD). Conclusion: CREB phosphorylation by ET-1 and PE may be mainly mediated by IP<Sub>3</Sub>R2/Ca²⁺-PKC-PKD-CaMKII signaling with a minor contribution by ERK1/2, linked to IP<Sub>3</Sub>R2 and Ca²⁺-independent PKC, in ventricular myocytes.