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

Arginine vasopressin (AVP) induces an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) with an oscillatory pattern in isolated perfused kidney inner medullary collecting duct (IMCD). The AVP-induced Ca²⁺ 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 Ca²⁺ mobilization in mpkCCD cells, and utilized mpkCCD as a model to investigate the Epac-induced intracellular and intra-organellar Ca²⁺ mobilization. Ca²⁺ mobilization in cytosol, endoplasmic reticulum lumen, and mitochondrial matrix were monitored with a Ca²⁺ sensitive fluorescent probe and site-specific Ca²⁺ 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 (IP₃Rs) both triggered increase of [Ca²⁺]_i and Ca²⁺ oscillations in mpkCCD cells as reported previously in IMCD. The cell permeant Epac-specific cAMP analog Me-cAMP/AM also caused a robust Ca²⁺ mobilization and oscillations in mpkCCD cells. Using biosensors to monitor endoplasmic reticulum (ER) luminal Ca²⁺ and mitochondrial matrix Ca²⁺, Me-cAMP/AM not only triggered Ca²⁺ release from ER into cytoplasm, but also shuttled Ca²⁺ from ER into mitochondria. The Epac-agonist induced synchronized Ca²⁺ spikes in cytosol and mitochondrial matrix, with concomitant declines in ER luminal Ca²⁺. Me-cAMP/AM also effectively triggered store-operated Ca²⁺ entry (SOCE), suggesting that Epac-agonist is capable of depleting ER Ca²⁺ stores. These Epac-induced intracellular and inter-organelle Ca²⁺ signals were mimicked by the RyR agonist 4-CMC, but they were distinctly different from IP₃R activation. The present study hence demonstrated that mpkCCD cells retain all reported features of Ca²⁺ mobilization observed in isolated perfused IMCD. It further revealed information on the dynamics of Epac-induced RyR-dependent Ca²⁺ signaling and ER-mitochondrial Ca²⁺ transfer. ER-mitochondrial Ca²⁺ 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 Ca²⁺ regulates cytosolic Ca²⁺ signals, inter-organellar Ca²⁺ signaling, and renal tubular functions.