Abstract
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).
Highlights
Endothelial cells (ECs)1 modulate the contractility of underlying vascular smooth muscle cells (VSMCs), secreting several vasoconstrictors and vasorelaxants [1, 2]
There is a lack of information regarding the amount of nitric oxide (NO) produced in ECs and its action to reduce the Ca2ϩi levels in neighboring VSMCs
After the administration of ATP, the level of NO2Ϫ gradually elevated from 10.8 Ϯ 0.4 to 13.5 Ϯ 0.8 ng/ml/106 cells (p Ͻ 0.01) at 3 min; adding BK to the medium resulted in a rise from 10.7 Ϯ 0.6 to 12.4 Ϯ 0.8 ng/ml/106 cells at 3 min (p Ͻ 0.05, Fig. 2A)
Summary
Endothelial cells (ECs) modulate the contractility of underlying vascular smooth muscle cells (VSMCs), secreting several vasoconstrictors and vasorelaxants [1, 2]. The principle endothelium-derived relaxing factors have been identified as nitric oxide (NO) [3] and prostacyclin. Both are regulated by intracellular calcium ions (Ca2ϩi) [4]. There is a lack of information regarding the amount of NO produced in ECs and its action to reduce the Ca2ϩi levels in neighboring VSMCs. The Ca2ϩi in VSMCs is crucial, because it is a primary factor in the regulation of muscle contractility [8]. To determine which component of the Ca2ϩi transients of ECs is most significant in indicating the production of NO and to monitor the biological action of NO on VSMCs, we simultaneously measured the stable NO metabolite, nitrite (NO2Ϫ), in the medium and the Ca2ϩi of both ECs and VSMCs in coculture by two-dimensional image analysis. We report here a unique communication between the Ca2ϩi in ECs and the Ca2ϩi in VSMCs mainly mediated by NO
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