Mayer B, Pfeiffer S, Schrammel A, Koesling D, Schmidt K, Brunner F
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria.
J Biol Chem. 1998 Feb 6;273(6):3264-70. doi: 10.1074/jbc.273.6.3264.
Nitric oxide (NO), a physiologically important activator of soluble guanylyl cyclase (sGC), is synthesized from L-arginine and O2 in a reaction catalyzed by NO synthases (NOS). Previous studies with purified NOS failed to detect formation of free NO, presumably due to a fast inactivation of NO by simultaneously produced superoxide (O-2). To characterize the products involved in NOS-induced sGC activation, we measured the formation of cyclic 3',5'-guanosine monophosphate (cGMP) by purified sGC incubated in the absence and presence of GSH (1 mM) with drugs releasing different NO-related species or with purified neuronal NOS. Basal sGC activity was 0.04 +/- 0.01 and 0.19 +/- 0.06 micromol of cGMP x mg-1 x min-1 without and with 1 mM GSH, respectively. The NO donor DEA/NO activated sGC in a GSH-independent manner. Peroxynitrite had no effect in the absence of GSH but significantly stimulated the enzyme in the presence of the thiol (3.45 +/- 0.60 micromol of cGMP x mg-1 x min-1). The NO/O-2 donor SIN-1 caused only a slight accumulation of cGMP in the absence of GSH but was almost as effective as DEA/NO in the presence of the thiol. The profile of sGC activation by Ca2+/calmodulin-activated NOS resembled that of SIN-1; at a maximally active concentration of 200 ng/0.1 ml, NOS increased sGC activity to 1.22 +/- 0.12 and 8.51 +/- 0.88 micromol of cGMP x mg-1 x min-1 in the absence and presence of GSH, respectively. The product of NOS and GSH was identified as the thionitrite GSNO, which activated sGC through Cu+-catalyzed release of free NO. In contrast to S-nitrosation by peroxynitrite, the novel NO/O-2-triggered pathway was very efficient (25-45% GSNO) and insensitive to CO2. Cu+-specific chelators inhibited bradykinin-induced cGMP release from rat isolated hearts but did not interfere with the direct activation of cardiac sGC, suggesting that thionitrites may occur as intermediates of NO/cGMP signaling in mammalian tissues.
一氧化氮（NO）是可溶性鸟苷酸环化酶（sGC）的一种重要生理激活剂，它由L-精氨酸和O₂在一氧化氮合酶（NOS）催化的反应中合成。先前对纯化的NOS的研究未能检测到游离NO的形成，推测是由于同时产生的超氧化物（O₂⁻）使NO快速失活。为了表征参与NOS诱导的sGC激活的产物，我们在不存在和存在谷胱甘肽（1 mM）的情况下，用释放不同NO相关物质的药物或纯化的神经元NOS孵育纯化的sGC，测量环磷酸鸟苷（cGMP）的形成。在不存在和存在1 mM谷胱甘肽的情况下，基础sGC活性分别为0.04±0.01和0.19±0.06 μmol cGMP·mg⁻¹·min⁻¹。NO供体DEA/NO以不依赖谷胱甘肽的方式激活sGC。在不存在谷胱甘肽的情况下，过氧亚硝酸盐没有作用，但在存在硫醇的情况下显著刺激了该酶（3.45±0.60 μmol cGMP·mg⁻¹·min⁻¹）。NO/O₂供体SIN-1在不存在谷胱甘肽的情况下仅导致cGMP略有积累，但在存在硫醇的情况下几乎与DEA/NO一样有效。Ca²⁺/钙调蛋白激活的NOS对sGC的激活模式类似于SIN-1；在最大活性浓度为200 ng/0.1 ml时，在不存在和存在谷胱甘肽的情况下，NOS分别将sGC活性提高到1.22±0.12和8.51±0.88 μmol cGMP·mg⁻¹·min⁻¹。NOS和谷胱甘肽的产物被鉴定为亚硝基硫醇GSNO，它通过Cu⁺催化释放游离NO来激活sGC。与过氧亚硝酸盐的S-亚硝化作用相反，这种新的NO/O₂触发途径非常有效（25 - 45% GSNO）且对CO₂不敏感。Cu⁺特异性螯合剂抑制缓激肽诱导的大鼠离体心脏中cGMP的释放，但不干扰心脏sGC的直接激活，这表明亚硝基硫醇可能作为哺乳动物组织中NO/cGMP信号传导的中间体出现。
