Contribution of sustained Ca2+ elevation for nitric oxide production in endothelial cells and subsequent modulation of Ca2+ transient in vascular smooth muscle cells in coculture

To elucidate the intracellular Ca2+ (Ca-i(2+)) transient responsible for nitric oxide (NO) production in endothelial cells (ECs) and the subsequent Ca-i(2+) reduction in vascular smooth muscle cells (VSMCs), we administrated four agonists with different Ca-i(2+)-mobilizing mechanisms for both cells in iso- or coculture. We monitored the Ca-i(2+) of both cells by two-dimensional fura-2 imaging, simultaneously measuring NO production as NO2-. The order of potency of the agonists in terms of the peak Ca-i(2+) in ECs was bradykinin (100 nM > ATP (10 mu M) > ionomycin (50 nM) > thapsigargin (1 mu M). In contrast, the order in reference to both the extent of Ca-i(2+) reduction in cocultured VSMCs and the elevation in NO production over the level of basal release in ECs completely matched and was ranked as thapsigargin > ionomycin > ATP > bradykinin. Treatment by N-G-monomethyl-L-arginine monoacetate but not indomethacin or glybenclamide restored the Ca-i(2+), response in cocultured VSMCs to the isoculture level. In ECs, when the Ca2+ influx was blocked by Ni2+ or by chelating extracellular Ca2+, all four agonists markedly decreased NO production, the half decay time of the Ca-i(2+) degenerating phase, and the area under the Ca-i(2+) curve. The amount of produced NO hyperbolically correlated to the half decay time and the area under the Ca-i(2+) curve but not to the Ca-i(2+) peak level. Thus, the sustained elevation of Ca-i(2+) in ECs, mainly a result of Ca2+ influx, determines the active NO production and subsequent Ca-i(2+) reduction in adjacent VSMCs. Furthermore, L-arginine but not D-arginine or L-lysine at high dose (5 mM) without agonist enhanced the NO production, weakly reduced the Ca-i(2+) in ECs, and markedly decreased the Ca-i(2+) in VSMCs, demonstrating the autocrine and paracrine effects of NO (Shin, W. S., Sasaki, T., Kato, M., Hara, K., Seko, A., Yang, W. D., Shimamoto, N., Sugimoto, T., and Toyo-oka, T. (1992) J. Biol. Chem. 267, 20377-20382).