Epac induces ryanodine receptor-dependent intracellular and inter-organellar calcium mobilization in mpkCCD cells

Arginine vasopressin (AVP) induces an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) with an oscillatory pattern in isolated perfused kidney inner medullary collecting duct (IMCD). The AVP-induced Ca(2+) mobilization in inner medullary collecting ducts is essential for apical exocytosis and is mediated by the exchange protein directly activated by cyclic adenosine monophosphate (Epac). Murine principal kidney cortical collecting duct cells (mpkCCD) is the cell model used for transcriptomic and phosphoproteomic studies of AVP signaling in kidney collecting duct. The present study examined the characteristics of Ca(2+) mobilization in mpkCCD cells, and utilized mpkCCD as a model to investigate the Epac-induced intracellular and intra-organellar Ca(2+) mobilization. Ca(2+) mobilization in cytosol, endoplasmic reticulum lumen, and mitochondrial matrix were monitored with a Ca(2+) sensitive fluorescent probe and site-specific Ca(2+) sensitive biosensors. Fluorescence images of mpkCCD cells and isolated perfused inner medullary duct were collected with confocal microscopy. Cell permeant ligands of ryanodine receptors (RyRs) and inositol 1,4,5 trisphosphate receptors (IP(3)Rs) both triggered increase of [Ca(2+)](i) and Ca(2+) oscillations in mpkCCD cells as reported previously in IMCD. The cell permeant Epac-specific cAMP analog Me-cAMP/AM also caused a robust Ca(2+) mobilization and oscillations in mpkCCD cells. Using biosensors to monitor endoplasmic reticulum (ER) luminal Ca(2+) and mitochondrial matrix Ca(2+), Me-cAMP/AM not only triggered Ca(2+) release from ER into cytoplasm, but also shuttled Ca(2+) from ER into mitochondria. The Epac-agonist induced synchronized Ca(2+) spikes in cytosol and mitochondrial matrix, with concomitant declines in ER luminal Ca(2+). Me-cAMP/AM also effectively triggered store-operated Ca(2+) entry (SOCE), suggesting that Epac-agonist is capable of depleting ER Ca(2+) stores. These Epac-induced intracellular and inter-organelle Ca(2+) signals were mimicked by the RyR agonist 4-CMC, but they were distinctly different from IP(3)R activation. The present study hence demonstrated that mpkCCD cells retain all reported features of Ca(2+) mobilization observed in isolated perfused IMCD. It further revealed information on the dynamics of Epac-induced RyR-dependent Ca(2+) signaling and ER-mitochondrial Ca(2+) transfer. ER-mitochondrial Ca(2+) coupling may play a key role in the regulation of ATP and reactive oxygen species (ROS) production in the mitochondria along the nephron. Our data suggest that mpkCCD cells can serve as a renal cell model to address novel questions of how mitochondrial Ca(2+) regulates cytosolic Ca(2+) signals, inter-organellar Ca(2+) signaling, and renal tubular functions.