Wang Y, Shin W S, Kawaguchi H, Inukai M, Kato M, Sakamoto A, Uehara Y, Miyamoto M, Shimamoto N, Korenaga R, Ando J, Toyo-oka T
Second Department of Internal Medicine, Health Service Center, University of Tokyo, Tokyo 113, Japan.
J Biol Chem. 1996 Mar 8;271(10):5647-55. doi: 10.1074/jbc.271.10.5647.
To elucidate the intracellular Ca2+ (Ca2+i ) transient responsible for nitric oxide (NO) production in endothelial cells (ECs) and the subsequent Ca2+i reduction in vascular smooth muscle cells (VSMCs), we administrated four agonists with different Ca2+i-mobilizing mechanisms for both cells in iso- or coculture. We monitored the Ca2+i 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 Ca2+i in ECs was bradykinin (100 nM) > ATP (10 microM) > ionomycin (50 nM) > thapsigargin (1 microM). In contrast, the order in reference to both the extent of Ca2+i 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 NG-monomethyl-L-arginine monoacetate but not indomethacin or glybenclamide restored the Ca2+i 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 Ca2+i degenerating phase, and the area under the Ca2+i curve. The amount of produced NO hyperbolically correlated to the half decay time and the area under the Ca2+i curve but not to the Ca2+i peak level. Thus, the sustained elevation of Ca2+i in ECs, mainly a result of Ca2+ influx, determines the active NO production and subsequent Ca2+i 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 Ca2+i in ECs, and markedly decreased the Ca2+i 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).
为了阐明负责内皮细胞（ECs）中一氧化氮（NO）产生以及随后血管平滑肌细胞（VSMCs）中细胞内Ca2+（Ca2+i）瞬变减少的机制，我们在同培养或共培养中对两种细胞施用了四种具有不同Ca2+i动员机制的激动剂。我们通过二维fura-2成像监测两种细胞的Ca2+i，同时测量作为NO2- 的NO产生。就ECs中Ca2+i峰值而言，激动剂的效力顺序为缓激肽（100 nM）>ATP（10 μM）>离子霉素（50 nM）>毒胡萝卜素（1 μM）。相比之下，关于共培养的VSMCs中Ca2+i减少程度以及ECs中NO产生相对于基础释放水平升高的顺序完全匹配，排名为毒胡萝卜素>离子霉素>ATP>缓激肽。用NG-单甲基-L-精氨酸单乙酸盐而非吲哚美辛或格列本脲处理可将共培养的VSMCs中的Ca2+i反应恢复到同培养水平。在ECs中，当Ca2+内流被Ni2+或通过螯合细胞外Ca2+阻断时，所有四种激动剂均显著降低NO产生、Ca2+i退化期的半衰期以及Ca2+i曲线下面积。产生的NO量与半衰期以及Ca2+i曲线下面积呈双曲线相关，但与Ca2+i峰值水平无关。因此，ECs中Ca2+i的持续升高（主要是Ca2+内流的结果）决定了活性NO的产生以及相邻VSMCs中随后的Ca2+i减少。此外，在无激动剂的情况下，高剂量（5 mM）的L-精氨酸而非D-精氨酸或L-赖氨酸可增强NO产生，微弱降低ECs中的Ca2+i，并显著降低VSMCs中的Ca2+i，证明了NO的自分泌和旁分泌作用（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）。
