Ou Jingsong, Fontana Jason T, Ou Zhijun, Jones Deron W, Ackerman Allan W, Oldham Keith T, Yu Jun, Sessa William C, Pritchard Kirkwood A
Division of Pediatric Surgery, Department of Surgery, Cardiovascular Center, Medical College of Wisconsin, Milwaukee 53226, USA.
Am J Physiol Heart Circ Physiol. 2004 Feb;286(2):H561-9. doi: 10.1152/ajpheart.00736.2003. Epub 2003 Oct 9.
An increase in the association of heat shock protein 90 (HSP90) with endothelial nitric oxide (NO) synthase (eNOS) is well recognized for increasing NO (NO*) production. Despite the progress in this field, the mechanisms by which HSP90 modulates eNOS remain unclear due, in part, to the fact that geldanamycin (GA) redox cycles to generate superoxide anion (O(2)(-) and the fact that inhibiting HSP90 with GA or radicicol (RAD) destabilizes tyrosine kinases that rely on the chaperone for maturation. In this report, we determine the extent to which these side effects alter vascular and endothelial cell function in physiologically relevant systems and in cultured endothelial cells. Vascular endothelial growth factor (VEGF)-stimulated vascular permeability, as measured by Evans blue leakage in the ears of male Swiss mice in vivo, and acetylcholine-induced vasodilation of isolated, pressurized mandibular arterioles from male C57BL6 mice ex vivo were attenuated by N(omega)-nitro-L-arginine methyl ester (L-NAME), GA, and RAD. Z-1[N-(2-aminoethyl)-N-(2-ammonoethyl)amino]diazen-1-ium-1,2-dioate (DETA-NONOate), a slow releasing NO. donor, increased vasodilation of arterioles pretreated with GA, RAD, and L-NAME equally well except at 10(-5) M, the highest concentration used, where vasodilation was greater in pressurized arterioles treated with L-NAME than in arterioles pretreated with GA or RAD alone. Both GA and RAD reduced NO release from stimulated endothelial cell cultures and increased O(2)(-) production in the endothelium of isolated aortas by an L-NAME-inhibitable mechanism. Pretreatment with RAD increased stimulated O(2)(-) production from eNOS, whereas pretreatment with genistein (GE), a broad-spectrum tyrosine kinase inhibitor, did not; however, pretreatment with GE + RAD resulted in a super-induced state of uncoupled eNOS activity upon stimulation. These data suggest that the tyrosine kinases, either directly or indirectly, and HSP90-dependent signaling pathways act in concert to suppress uncoupled eNOS activity.
热休克蛋白90(HSP90)与内皮型一氧化氮合酶(eNOS)结合增加可显著提高一氧化氮(NO*)的生成,这一点已得到广泛认可。尽管该领域取得了进展,但HSP90调节eNOS的机制仍不清楚,部分原因是格尔德霉素(GA)发生氧化还原循环生成超氧阴离子(O(2)(-),以及用GA或雷迪西科尔(RAD)抑制HSP90会使依赖伴侣蛋白成熟的酪氨酸激酶不稳定。在本报告中,我们确定了这些副作用在生理相关系统和培养的内皮细胞中改变血管和内皮细胞功能的程度。通过体内雄性瑞士小鼠耳部伊文思蓝渗漏测量的血管内皮生长因子(VEGF)刺激的血管通透性,以及通过体外雄性C57BL6小鼠分离的加压下颌小动脉中乙酰胆碱诱导的血管舒张,均被N(ω)-硝基-L-精氨酸甲酯(L-NAME)、GA和RAD减弱。Z-1[N-(2-氨乙基)-N-(2-氨乙基)氨基]重氮-1,2-二醇盐(DETA-NONOate),一种缓释NO供体,同样能很好地增加用GA、RAD和L-NAME预处理的小动脉的血管舒张,除了在10(-5) M(使用的最高浓度)时,用L-NAME处理的加压小动脉中的血管舒张大于单独用GA或RAD预处理的小动脉。GA和RAD均通过L-NAME可抑制的机制减少刺激的内皮细胞培养物中NO的释放,并增加分离主动脉内皮中O(2)(-)的产生。用RAD预处理增加了eNOS刺激产生的O(2)(-),而用广谱酪氨酸激酶抑制剂染料木黄酮(GE)预处理则没有;然而,用GE + RAD预处理在刺激后导致eNOS活性解偶联的超诱导状态。这些数据表明,酪氨酸激酶直接或间接与HSP90依赖性信号通路协同作用以抑制解偶联的eNOS活性。