Weber M, Lauer N, Mülsch A, Kojda G
Institut für Pharmakologie und Klinische Pharmakologie, Medizinische Einrichtungen, Heinrich-Heine-Universität, Düsseldorf, Germany.
Free Radic Biol Med. 2001 Dec 1;31(11):1360-7. doi: 10.1016/s0891-5849(01)00706-7.
Soluble guanylyl cyclase (sGC) is a key enzyme of the *NO/cGMP pathway. Many cardiovascular disorders are associated with reduced NO-mediated effects, while vascular superoxide (O(2)(-)) production is increased. Both radicals rapidly react to peroxynitrite. We investigated whether peroxynitrite affects the activity and protein expression of sGC in intact vascular preparations. Catalytic sGC activity and expression of the sGC-beta(1) subunit was measured by conversion of radiolabeled GTP and western blot, respectively, using cytosolic extracts from rat aorta that had been incubated for 4 h with NO/O(2)(-) systems (devoid of free *NO) generating either 0.13 microM or 7.5 microM peroxynitrite/min. Incubation of rat aorta with 0.13 microM peroxynitrite/min had no effect. In striking contrast, incubation with 7.5 microM peroxynitrite/min resulted in a shift of the concentration-response curve obtained with a NO donor (p =.0004) and a reduction of maximal specific activity from 3579 +/- 495 to 2422 +/- 265 pmol cGMP/mg/min (p =.036). The expression of the sGC-beta(1) subunit was unchanged. Exposure of aorta to the O(2)(-) component had no effect, while exposure to the *NO-component reduced sGC expression to 58.8 +/- 7% (p <.001) and maximal sGC activity from 4041 +/- 992 to 1429 +/- 491 pmol cGMP/mg/min (p =.031). These data suggest that continuous generation of extracellular peroxynitrite might interfere with the *NO/cGMP signaling in vascular cells.
可溶性鸟苷酸环化酶(sGC)是NO/cGMP信号通路的关键酶。许多心血管疾病都与NO介导的效应减弱有关,同时血管超氧化物(O₂⁻)生成增加。这两种自由基会迅速反应生成过氧亚硝酸盐。我们研究了过氧亚硝酸盐是否会影响完整血管制剂中sGC的活性和蛋白表达。分别使用大鼠主动脉的胞质提取物,通过放射性标记的GTP转化和蛋白质印迹法,测量催化性sGC活性和sGC-β₁亚基的表达,该提取物已与产生0.13μM或7.5μM过氧亚硝酸盐/分钟的NO/O₂⁻系统(无游离NO)孵育4小时。用0.13μM过氧亚硝酸盐/分钟孵育大鼠主动脉没有影响。与之形成鲜明对比的是,用7.5μM过氧亚硝酸盐/分钟孵育导致用NO供体获得的浓度-反应曲线发生偏移(p = 0.0004),最大比活性从3579±495降至2422±265 pmol cGMP/mg/分钟(p = 0.036)。sGC-β₁亚基的表达没有变化。将主动脉暴露于O₂⁻成分没有影响,而暴露于NO成分会使sGC表达降至58.8±7%(p < 0.001),最大sGC活性从4041±992降至1429±491 pmol cGMP/mg/分钟(p = 0.031)。这些数据表明,细胞外过氧亚硝酸盐的持续生成可能会干扰血管细胞中的NO/cGMP信号传导。