Schlüter Torsten, Steinbach Antje C, Steffen Anja, Rettig Rainer, Grisk Olaf
Department of Physiology, University of Greifswald, Greifswalder Street 11c, D17495 Greifswald, Karlsburg, Germany.
Cardiovasc Res. 2008 Nov 1;80(2):271-9. doi: 10.1093/cvr/cvn185. Epub 2008 Jul 2.
The present study was designed to test the hypothesis that NADPH oxidase inhibition with apocynin would lower blood pressure and improve endothelial function in spontaneously hypertensive rats (SHRs). Although apocyin effectively dilated arterial segments in vitro, it failed to lower blood pressure or improve endothelial function. Further experiments were performed in normotensive rats and in NADPH oxidase subunit knock-out mice to test if apocynin-induced vasodilation depends on NADPH oxidase inhibition at all.
SHRs were treated with apocynin orally or i.v. Arterial pressure was recorded directly. Rat and mouse arterial function was investigated in vitro by small vessel wire myography. NADPH oxidase activity was measured in human granulocytes and in rat vascular preparations. Rho kinase activity was determined by Western blot analysis. Apocynin did not reduce arterial pressure acutely in SHR when given at 50, 100, or 150 mg kg(-1) day(-1) orally over 1-week intervals or when given i.v. Apocynin potently inhibited granulocyte NADPH oxidase but not vascular NADPH-oxidase-dependent oxygen radical formation unless exogenous peroxidase was added to vascular preparations. Apocynin dilated rat intrarenal and coronary arteries independently of pharmacological interventions that reduce vascular superoxide radical abundance and actions. Aortic rings from p47phox(-/-) mice were more sensitive to apocynin-induced dilation than wild-type aortic rings. Rho kinase inhibition reduced or prevented the inhibitory effect of apocynin on agonist-induced vasoconstriction and apocynin inhibited the phosphorylation of Rho kinase substrates.
Apocynin per se does not inhibit vascular NADPH-oxidase-dependent superoxide formation. Its in vitro vasodilator actions are not due to NADPH oxidase inhibition but may be explained at least in part by inhibition of Rho kinase activity. The discrepancy between apocynin-induced vasodilation in vitro and the failure of apocynin to lower arterial pressure in SHR suggests opposing effects on arterial pressure-regulating systems in vivo. Its use as a pharmacological tool to investigate vascular NADPH oxidase should be discontinued.
本研究旨在验证阿朴吗啡抑制NADPH氧化酶可降低自发性高血压大鼠（SHR）血压并改善其内皮功能这一假说。尽管阿朴吗啡在体外能有效扩张动脉节段，但它未能降低血压或改善内皮功能。我们在正常血压大鼠和NADPH氧化酶亚基基因敲除小鼠中进行了进一步实验，以检验阿朴吗啡诱导的血管舒张是否完全依赖于NADPH氧化酶的抑制作用。
给SHR口服或静脉注射阿朴吗啡。直接记录动脉血压。通过小血管线肌描记法在体外研究大鼠和小鼠的动脉功能。在人粒细胞和大鼠血管标本中测量NADPH氧化酶活性。通过蛋白质印迹分析测定Rho激酶活性。当以50、100或150 mg·kg⁻¹·天⁻¹的剂量口服给药1周或静脉注射时，阿朴吗啡并未使SHR的动脉血压急性降低。阿朴吗啡能有效抑制粒细胞NADPH氧化酶，但不能抑制血管中依赖NADPH氧化酶的氧自由基生成，除非向血管标本中添加外源性过氧化物酶。阿朴吗啡可使大鼠肾内动脉和冠状动脉舒张，且与降低血管超氧阴离子自由基丰度及作用的药理学干预无关。p47phox基因敲除小鼠的主动脉环对阿朴吗啡诱导的舒张比野生型主动脉环更敏感。Rho激酶抑制可降低或阻止阿朴吗啡对激动剂诱导的血管收缩的抑制作用，且阿朴吗啡可抑制Rho激酶底物的磷酸化。
阿朴吗啡本身并不抑制血管中依赖NADPH氧化酶的超氧阴离子生成。其体外血管舒张作用并非由于抑制NADPH氧化酶，而是至少部分可由抑制Rho激酶活性来解释。阿朴吗啡在体外诱导血管舒张与在SHR中未能降低动脉血压之间的差异表明其对体内动脉血压调节系统有相反作用。应停止将其用作研究血管NADPH氧化酶的药理学工具。
