Na+/Ca2+ exchanger overexpression impairs calcium signaling in fibroblasts:: Inhibition of the [Ca2+] increase at the cell periphery and retardation of cell adhesion

We examined the Ca2+ handling property and cell function of CCL39 fibroblasts highly overexpressing the cardiac isoform (NCX1) of Na+/Ca2+ exchanger. In NCX1 transfectants in 146 mM Na+, ionomycin, alpha-thrombin or thapsigargin only produced a small transient increase in [Ca2+](i) compared to the large increase seen in control cells, although resting [Ca2+](i) was not significantly different between these cells. In Na+-free medium, in contrast, the [Ca2+](i) responses in NCX1 transfectants and control cells stimulated with these agents were not different, indicating that the Ca2+ content of the intracellular store(s) does not decrease on NCX1 transfection. The expression levels of the endo plasmic reticulum and plasma membrane Ca2+-ATPases, and thrombin- or serum-stimulated cell growth were not altered in NCX1 transfectants, The latter finding suggests that Ca2+ signaling in the nucleus is not impaired appreciably On fluorescence imaging and confocal microscopy, we found that [Ca2+] did not increase in the peripheral cytoplasm of these cells treated with a-thrombin in Na+-containing medium. In these NCX1 transfectants, activation of the plasma membrane Ca2+-activated K+ channels by thrombin or ionomycin was markedly suppressed, and the integrin-mediated adhesion to substrate was significantly delayed compared with control cells. NCX1-overexpressing CCL39 cells thus seem to be a good model with which we can study the Ca2+-regulated membrane processes under physiologically relevant conditions.