Robinson Mary A, Baumgardner James E, Good Virginia P, Otto Cynthia M
Department of Clinical Studies-Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Am J Physiol Cell Physiol. 2008 Apr;294(4):C1079-87. doi: 10.1152/ajpcell.00469.2007. Epub 2008 Feb 13.
Nitric oxide (NO) production by inducible NO synthase (iNOS) is dependent on O(2) availability. The duration and degree of hypoxia that limit NO production are poorly defined in cultured cells. To investigate short-term O(2)-mediated regulation of NO production, we used a novel forced convection cell culture system to rapidly (response time of 1.6 s) and accurately (+/-1 Torr) deliver specific O(2) tensions (from <1 to 157 Torr) directly to a monolayer of LPS- and IFNgamma-stimulated RAW 264.7 cells while simultaneously measuring NO production via an electrochemical probe. Decreased O(2) availability rapidly (<or=30 s) and reversibly decreased NO production with an apparent K(m)O(2) of 22 (SD 6) Torr (31 microM) and a V(max) of 4.9 (SD 0.4) nmol min(-1) 10(-6) cells. To explore potential mechanisms of decreased NO production during hypoxia, we investigated O(2)-dependent changes in iNOS protein concentration, iNOS dimerization, and cellular NO consumption. iNOS protein concentration was not affected (P = 0.895). iNOS dimerization appeared to be biphasic [6 Torr (P = 0.008) and 157 Torr (P = 0.258) >36 Torr], but it did not predict NO production. NO consumption was minimal at high O(2) and NO tensions and negligible at low O(2) and NO tensions. These results are consistent with O(2) substrate limitation as a regulatory mechanism during brief hypoxic exposure. The rapid and reversible effects of physiological and pathophysiological O(2) tensions suggest that O(2) tension has the potential to regulate NO production in vivo.
诱导型一氧化氮合酶（iNOS）产生一氧化氮（NO）取决于氧气（O₂）的可利用性。在培养细胞中，限制NO产生的缺氧持续时间和程度尚不明确。为了研究短期O₂介导的对NO产生的调节作用，我们使用了一种新型的强制对流细胞培养系统，以快速（响应时间为1.6秒）且精确（±1托）地将特定的O₂张力（从<1到157托）直接输送到经脂多糖（LPS）和γ干扰素（IFNγ）刺激的RAW 264.7细胞单层，同时通过电化学探针测量NO的产生。O₂可利用性降低会迅速（≤30秒）且可逆地降低NO产生，其表观米氏常数（K(m)O₂）为22（标准差6）托（31微摩尔），最大反应速度（V(max)）为4.9（标准差0.4）纳摩尔·分钟⁻¹·10⁻⁶个细胞。为了探究缺氧时NO产生减少的潜在机制，我们研究了O₂依赖性的iNOS蛋白浓度、iNOS二聚化以及细胞对NO的消耗变化。iNOS蛋白浓度未受影响（P = 0.895）。iNOS二聚化似乎呈双相性[6托（P = 0.008）和157托（P = 0.258）>36托]，但它并不能预测NO的产生。在高O₂和NO张力下，NO消耗极少，而在低O₂和NO张力下可忽略不计。这些结果与在短暂缺氧暴露期间O₂底物限制作为一种调节机制相一致。生理和病理生理O₂张力的快速且可逆作用表明，O₂张力有可能在体内调节NO的产生。
