Pharmacological Preconditioning Using Diazoxide Regulates Store-Operated Ca Channels in Adult Rat Cardiomyocytes.

Voltage-dependent Ca channels and store-operated Ca channels (SOCs) are the major routes of Ca entry into mammalian cells. Previously, we reported that pharmacological preconditioning (PPC) leads to a decrease in the amplitude of L-type calcium channel current in the heart. In this study, we examined PPC-associated changes in SOC function. We measured adult cardiomyocyte membrane currents using the whole-cell patch-clamp technique, and we evaluated reactive oxygen species (ROS) production and intracellular Ca levels in cardiomyocytes using fluorescent probes. Diazoxide (Dzx) and thapsigargin (Tg) were used to induce PPC and to deplete internal stores of Ca, respectively. Ca store depletion generated inward currents with strong rectification, which were suppressed by the SOC blocker GSK-7975-A. These currents were completely abolished by PPC, an effect that could be countered with 5-hydroxydecanoate (5-HD; a selective mitochondrial ATP-sensitive K channel blocker), an intracellular mitochondrial energizing solution, or Ni [a blocker of sodium-calcium exchanger (NCX)]. Buffering of ROS and intracellular Ca also prevented PPC effects on SOC currents. Refilling of intracellular stores was largely suppressed by PPC, as determined by measuring intracellular Ca with a fluorescent Ca indicator. These results indicate that influx of Ca through SOCs is inhibited by their ROS and Ca-dependent inactivation during PPC and that NCX is a likely source of PPC-inactivating Ca. We further showed that NCX associates with Orai1. Down-regulation of SOCs by PPC may play a role in cardioprotection following ischemia-reperfusion.