Endoplasmic reticulum (ER) and lysosomes are physiologically active, physically and functionally connected intracellular Ca2+ stores. In this study we investigated agonist-triggered Ca2+ release from these two stores in mouse microvascular endothelial bEND.3 cells. Addition of nigericin to discharge lysosomal Ca2+ did not affect endoplasmic reticulum Ca2+ release induced by cyclopiazonic acid (CPA) and vice versa, suggesting lysosomes and ER were separate Ca2+ stores whose Ca2+ content was not readily reduced by depletion of the counterpart. ATP triggered Ca2+ release was partially inhibited by Ned-19 (lysosomal two-pore channel inhibitor) or xestospongin C (inositol 1,4,5-trisphosphate receptor-channel inhibitor), suggesting ATP mobilized Ca2+ from both ER and lysosomes. Whilst ATP-triggered Ca2+ release did not affect subsequent CPA- or nigericin-induced Ca2+ discharge, pretreatment with either CPA or nigericin abolished subsequent ATP-triggered Ca2+ release. Thus, the empty state of ER suppressed lysosomal Ca2+ release elicited by ATP, and vice versa, the empty state of lysosome inhibited ATP triggered Ca2+ release from ER. These data suggest cross-talk of the two organelles on the Ca2+ filling state to regulate agonist-stimulated Ca2+ release of each other.

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