Functional coupling between ryanodine receptors, mitochondria and Ca2+ ATPases in rat submandibular acinar cells

Agonist stimulation of exocrine cells leads to the generation of intracellular Ca2+ signals driven by inositol 1,4,5-trisphosphate receptors (IP3Rs) that rapidly become global due to propagation throughout the cell. In many types of excitable cells the intracellular Ca2+ signal is propagated by a mechanism of Ca2+-induced Ca2+ release (CICR), mediated by ryanodine receptors (RyRs). Expression of RyRs in salivary gland cells has been demonstrated immunocytochemically although their functional role is not clear. We used microfluorimetry to measure Ca2+ signals in the cytoplasm, in the endoplasmic reticulum (ER) and in mitochondria. In permeabilized acinar cells caffeine induced a dose-dependent, transient decrease of Ca2+ concentration in the endoplasmic reticulum ([Ca2+]ER). This decrease was inhibited by ryanodine but was insensitive to heparin. Application of caffeine, however, did not elevate cytosolic Ca2+ concentration ([Ca2+]i) suggesting fast local buffering of Ca2+ released through RyRs. Indeed, activation of RyRs produced a robust mitochondrial Ca2+ transient that was prevented by addition of Ca2+ chelator BAPTA but not EGTA. When mitochondrial Ca2+ uptake was blocked, activation of RyRs evoked only a non-transient increase in [Ca2+]i and substantially smaller Ca2+ release from the ER. Upon simultaneous inhibition of mitochondrial Ca2+ uptake and either plasmalemmal or ER Ca2+ ATPase, activation of RyRs caused a transient rise in [Ca2+]i. Collectively, our data suggest that Ca2+ released through RyRs is mostly “tunnelled” to mitochondria, while Ca2+ ATPases are responsible for the fast initial sequestration of Ca2+. Ca2+ uptake by mitochondria is critical for maintaining continuous CICR. A complex interplay between RyRs, mitochondria and Ca2+ ATPases is accomplished through strategic positioning of mitochondria close to both Ca2+ release sites in the ER and Ca2+ pumping sites of the plasmalemma and the ER.