Natural Alkaloid Murrayafoline-A Sensitizes Ca2+ Release Sites via Adenylate Cyclase and Protein Kinase C Pathway in Rat Ventricular Myocytes

Abstract

A naturally found alkaloid murrayafoline-A (Mu-A, 1-methoxy-3-methylcarbazole) acutely increases ventricle cell contractility partly via enhancement of L-type Ca2+ current. In the present study,we explored the effects of Mu-A on elementary Ca2+ release events (“Ca2+ sparks”) in cardiac myocytes and possible molecular mechanisms underlying the effect of Mu-A. Rapid two-dimensional confocal Ca2+ imaging and image analysis were used to assess spontaneous Ca2+ sparks in single adult rat ventricular myocytes. The frequency of Ca2+ sparks was dose-dependently increased by Mu-A. The duration and the size of single sparks were also significantly increased by Mu-A. The sarcoplasmic reticulum (SR) Ca2+ loading, measured as caffeine-induced Ca2+ transients, was increased by Mu-A. Neither SR Ca2+ clamping nor removal of external Ca2+ and Na+ affects the Mu-A-mediated enhancement in Ca2+ sparks. In contrast, prolongation and enlargement of individual Ca2+ sparks in Mu-A were attenuated by matching SR Ca2+ content to the control level. Higher propensity of Ca2+ sparks in the presence of Mu-A was not suppressed by the protein kinase A inhibitor, KT5720, or by the calmodulin-dependent protein kinase II inhibitor, KN93, whereas it was strongly reduced by the inhibitors of adenylate cyclase (MDL-12,330A) and protein kinase C (GF109203X). Our data suggest that Mu-A sensitizes ryanodine receptor (RyR) Ca2+ release channels via specialized signaling pathway involving adenylate cyclase and protein kinase C, and that larger SR Ca2+ loading in Mu-A may also play a role in enhanced Ca2+ release through the RyRs.