Silva Guillermo B, Ortiz Pablo A, Hong Nancy J, Garvin Jeffrey L
Division of Hypertension and Vascular Research, Henry Ford Hospital, 2799 W Grand Blvd, Detroit, MI 48202, USA.
Hypertension. 2006 Sep;48(3):467-72. doi: 10.1161/01.HYP.0000236646.83354.51. Epub 2006 Aug 7.
Abnormal production of superoxide (O(2)(-)) contributes to hypertension, in part because of its effects on the kidney. The thick ascending limb absorbs 20% to 30% of the filtered load of NaCl. O(2)(-) stimulates NaCl absorption by the thick ascending limb by enhancing Na(+)/K(+)/2Cl(-) cotransporter activity; however, the signaling mechanism is unknown. We hypothesized that O(2)(-) stimulates NaCl absorption by activating protein kinase C (PKC). To test this, we measured the effect of O(2)(-) on: (1) Cl(-) absorption in the presence and absence of PKC inhibitors, (2) total PKC activity, and (3) activation of specific PKC isoforms. Isolated perfused medullary thick ascending limbs were exposed to O(2)(-) generated by xanthine oxidase (1 mU/mL) and hypoxanthine (0.5 mmol/L). O(2)(-) increased Cl(-) absorption by 42% (from 76.2+/-3.6 to 108.2+/-11.9 pmol/min per millimeter; n=5; P<0.05). After treatment with the general PKC inhibitor staurosporine (10 nmol/L), O(2)(-) did not stimulate Cl(-) absorption (Delta-5.7+/-8.6%; n=6). In thick ascending limb suspensions, O(2)(-) increased total PKC activity by 33% (from 66+/-11 to 88+/-12 mU/mg protein; n=5; P<0.05) and increased PKC-alpha and PKC-delta activity by 1.75- and 0.37-fold, respectively. The PKC-alpha/beta-selective inhibitor Gö976 (100 nmol/L) blocked the ability of O(2)(-) to stimulate Cl(-) absorption by isolated perfused medullary thick ascending limbs (Delta4.5+/-15.0%; n=5). The role of PKC-delta could not be studied because of cell necrosis caused by the selective inhibitor rottlerin. We conclude that PKC-alpha is required for O(2)(-)-stimulated NaCl absorption in the thick ascending limb.
超氧化物（O₂⁻）生成异常会导致高血压，部分原因是其对肾脏的影响。髓袢升支粗段重吸收20%至30%的滤过氯化钠负荷。O₂⁻通过增强Na⁺/K⁺/2Cl⁻协同转运体活性刺激髓袢升支粗段对NaCl的重吸收；然而，其信号传导机制尚不清楚。我们推测O₂⁻通过激活蛋白激酶C（PKC）刺激NaCl重吸收。为验证这一点，我们测量了O₂⁻对以下方面的影响：（1）在有和没有PKC抑制剂存在时的Cl⁻重吸收；（2）总PKC活性；（3）特定PKC同工型的激活。将分离灌注的髓袢升支粗段暴露于黄嘌呤氧化酶（1 mU/mL）和次黄嘌呤（0.5 mmol/L）产生的O₂⁻中。O₂⁻使Cl⁻重吸收增加42%（从76.2±3.6增加到108.2±11.9 pmol/分钟·毫米；n = 5；P<0.05）。用通用PKC抑制剂星形孢菌素（10 nmol/L）处理后，O₂⁻不再刺激Cl⁻重吸收（变化-5.7±8.6%；n = 6）。在髓袢升支粗段悬浮液中，O₂⁻使总PKC活性增加33%（从66±11增加到88±12 mU/mg蛋白质；n = 5；P<0.05），并使PKC-α和PKC-δ活性分别增加1.75倍和0.37倍。PKC-α/β选择性抑制剂Gö976（100 nmol/L）阻断了O₂⁻刺激分离灌注的髓袢升支粗段Cl⁻重吸收的能力（变化4.5±15.0%；n = 5）。由于选择性抑制剂rottlerin导致细胞坏死，无法研究PKC-δ的作用。我们得出结论，PKC-α是O₂⁻刺激髓袢升支粗段NaCl重吸收所必需的。
