Nitric oxide enhances extracellular ATP induced Ca2+ oscillation in HeLa cells

• Overexpression of two differentially-located eNOS in HeLa cells.
• Ca2+ oscillations induced by ATP were enhanced in eNOS overexpressing cells.
• More significant Ca2+ oscillations were observed in ER overexpressed eNOS cells.
• eNOS overexpression induced hyper-phosphorylation of PLB.
• Increased ER Ca2+ load was observed in eNOS overexpressed cells.

Calcium (Ca2+) oscillations play a central role in varieties of cellular processes including fertilization and immune response, but controversy over the regulation mechanisms still exists. It has been known that nitric oxide (NO) dependently regulates Ca2+ signaling in most physiopathological processes. Previous study indicated that eNOS translocation during some pathological process influences intracellular Ca2+ homeostasis. In this study, we investigated the role and mechanism of NO on Ca2+ release by overexpressing eNOS in cytoplasm (Cyto-eNOS) and endoplasmic reticulum (ER-eNOS) of HeLa cells. We found that the properties of Ca2+ release were altered by the overexpression of eNOS. The amplitude and frequency of extracellular ATP (eATP)-induced Ca2+ oscillation were enhanced in both Cyto-eNOS and ER-eNOS cells, respectively. Especially, the enhancement of the amplitude and frequency of the Ca2+ oscillation was much more significant in the ER-eNOS cells than that of Cyto-eNOS cells. Further study indicated that this effect was abrogated by NO inhibitor, l-NAME, suggesting it was not an artificial result induced by ER stress. Furthermore, an up-regulated phosphorylation of phospholamban (PLB) was observed and the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) function was activated followed by the significant increase in the ER Ca2+ load. Taken together, we revealed a novel regulatory mechanism of Ca2+ oscillation.