Lerouet Dominique, Jafarian-Tehrani Mehrnaz, Louin Gaëlle, Palmier Bruno, Bonnefont-Rousselot Dominique, Plotkine Michel, Margaill Isabelle
Laboratoire de Pharmacologie (UPRES EA 2510), Université René Descartes, 4 avenue de l'Observatoire, 75006 Paris, France.
Exp Neurol. 2005 Sep;195(1):218-28. doi: 10.1016/j.expneurol.2005.05.001.
Calcium-independent nitric oxide synthase (NOS) activity has been reported in ischemic brains and usually attributed to the inducible isoform, iNOS. Because calcium-independent mechanisms have recently been shown to regulate the constitutive calcium-dependent NOS, we proposed to confirm the presence of iNOS activity in our model of transient focal cerebral ischemia in rats. Our initial results showed that, in our model, ischemia induced an important increase in brain calcium concentration. Consequently, the determination of calcium-independent NOS activity required a higher concentration of calcium chelator than classically used in the NOS assay. In these conditions, calcium-independent NOS activity was not observed after ischemia. Moreover, our ischemia was associated with neither iNOS protein expression, measured by Western blotting, nor increased NO production, evaluated by its metabolites (nitrate/nitrite). Our results demonstrate that iNOS activity may be overestimated due to increased brain calcium concentration in ischemic conditions and also that iNOS is not systematically induced after cerebral ischemia.
缺血性脑内已报道存在不依赖钙的一氧化氮合酶（NOS）活性，通常认为这归因于诱导型同工酶iNOS。由于最近已表明不依赖钙的机制可调节组成型钙依赖性NOS，我们建议在大鼠短暂性局灶性脑缺血模型中证实iNOS活性的存在。我们的初步结果显示，在我们的模型中，缺血导致脑钙浓度显著升高。因此，测定不依赖钙的NOS活性需要比NOS检测中传统使用的更高浓度的钙螯合剂。在这些条件下，缺血后未观察到不依赖钙的NOS活性。此外，通过蛋白质印迹法测定，我们的缺血既未伴有iNOS蛋白表达，通过其代谢产物（硝酸盐/亚硝酸盐）评估也未出现一氧化氮生成增加。我们的结果表明，在缺血情况下，由于脑钙浓度升高，iNOS活性可能被高估，并且脑缺血后iNOS并非都会被诱导产生。
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