O'Connor Paul M, Lu Limin, Liang Mingyu, Cowley Allen W
Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53202, USA.
Hypertension. 2009 Aug;54(2):248-54. doi: 10.1161/HYPERTENSIONAHA.109.134692. Epub 2009 Jun 29.
It has been reported previously that H(+) efflux via the Na(+)/H(+) exchange stimulates NAD(P)H oxidase-dependent superoxide (O(2)(.-)) production in medullary thick ascending limb. We have demonstrated recently that N-methyl-amiloride-sensitive O(2)(.-) production is enhanced in the thick ascending limb of Dahl salt-sensitive (SS) rats, suggesting that H(+) efflux through Na(+)/H(+) exchangers may promote renal oxidative stress and the development of hypertension in these animals. In the current study we demonstrate, using selective and potent inhibitors, that inhibition of Na(+)/H(+) exchange does not mediate the ability of N-methyl-amiloride to inhibit thick ascending limb O(2)(.-) production. To determine the mechanism of action of N-methyl-amiloride, we examined H(+) efflux and O(2)(.-) production in SS and SS.13(BN) thick ascending limbs of prehypertensive, 0.4% NaCl-fed rats. Tissue strips containing the medullary thick ascending limb were isolated from male SS and salt-resistant consomic SS.13(BN) rats, loaded with either dihydroethedium or 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, and imaged in a heated tissue bath. In Na(+)-replete media, activation of Na(+)/H(+) exchange using an NH(4)Cl prepulse did not stimulate thick ascending limb O(2)(.-) production. In Na(+)-free media containing BaCl(2) in which Na(+)/H(+) activity was inhibited, an NH(4)Cl prepulse stimulated O(2)(.-) production in medullary thick ascending limb renal tubular segments. This response was enhanced in medullary thick ascending limb of SS rats (slope Deltaethidium/Deltadihydroethedium=0.029+/-0.004) compared with SS.13(BN) rats (slope=0.010+/-0.004; P<0.04) and could be inhibited by N-methyl-amiloride (slope=0.005+/-0.002 and 0.006+/-0.002 for SS and SS.13(BN), respectively). We concluded that only H(+) efflux through a specific, as-yet-unidentified, amiloride-sensitive H(+) channel promotes O(2)(.-) production in the medullary thick ascending limb and that this channel is upregulated in SS rats.
先前已有报道称，通过钠氢交换体的氢离子外流可刺激髓袢升支粗段中依赖烟酰胺腺嘌呤二核苷酸磷酸氧化酶的超氧阴离子（O₂⁻）生成。我们最近证明，在 Dahl 盐敏感（SS）大鼠的髓袢升支粗段中，对氨氯地平敏感的 O₂⁻生成增加，这表明通过钠氢交换体的氢离子外流可能会促进这些动物的肾脏氧化应激和高血压的发展。在本研究中，我们使用选择性强效抑制剂证明，抑制钠氢交换并不能介导氨氯地平抑制髓袢升支粗段 O₂⁻生成的能力。为了确定氨氯地平的作用机制，我们检测了喂食 0.4%氯化钠的高血压前期大鼠的 SS 和 SS.13(BN)髓袢升支粗段中的氢离子外流和 O₂⁻生成。从雄性 SS 大鼠和耐盐近交系 SS.13(BN)大鼠中分离出含有髓袢升支粗段的组织条，用二氢乙啶或 2',7'-双（2-羧乙基）-5-（和-6）-羧基荧光素乙酰氧基甲酯进行负载，并在加热的组织浴中成像。在富含钠的培养基中，使用氯化铵预脉冲激活钠氢交换并不能刺激髓袢升支粗段的 O₂⁻生成。在含有氯化钡的无钠培养基中，钠氢交换活性受到抑制，氯化铵预脉冲可刺激髓袢升支粗段肾小管节段的 O₂⁻生成。与 SS.13(BN)大鼠（斜率 = 0.010±0.004；P<0.04）相比，SS 大鼠髓袢升支粗段的这种反应增强（斜率 Δ乙啶/Δ二氢乙啶 = 0.029±0.004），并且可被氨氯地平抑制（SS 和 SS.13(BN)的斜率分别为 0.005±0.002 和 0.006±0.002）。我们得出结论，只有通过一种特定的、尚未明确的、对氨氯地平敏感的氢离子通道的氢离子外流才能促进髓袢升支粗段的 O₂⁻生成，并且该通道在 SS 大鼠中上调。
