Intracellular pH regulates superoxide production by the macula densa.
Intracellular pH regulates superoxide production by the macula densa.
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DOI：
10.1152/ajprenal.90204.2008
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发表时间：
2008-09
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通讯作者：
Ruisheng Liu;O. Carretero;Yilin Ren;Hong Wang;J. Garvin
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作者：
Ruisheng Liu;O. Carretero;Yilin Ren;Hong Wang;J. Garvin
我们假设，在小管肾小球反馈过程中，黄斑致密细胞内pH值（pH(i)）升高会增强NAD(P)H氧化酶产生O(2)（-）。显微解剖的兔粗升肢与完整的黄斑致密，插管和灌注生理盐水。当腔内NaCl从10 mM切换到80 mM时，O(2)（-）产量从0.53 +/- 0.09增加到2.62 +/- 0.54 U/min （P < 0.01）。为了确定抑制Na/H交换是否会阻断O(2)（-）的产生，我们使用了二甲基酰胺（DMA）来阻断Na/H交换。在DMA存在的情况下，NaCl诱导的O(2)（-）产量降低了40%。为了研究pH(i)对完整黄斑致密细胞中O(2)（-）的影响，我们测量了O(2)(-)，同时通过调节腔内pH改变pH(i)。当黄斑致密细胞灌注80 mM NaCl，并将灌注液的pH值切换到6.8、7.4和8.0时，O(2)（-）的产生显著增强，但在10 mM NaCl下则没有。为了确定O(2)（-）的来源，我们使用了NAD(P)H氧化酶抑制剂罗布麻碱。在apocynin （10(-5) M）存在的情况下，由pH(i)升高诱导的O(2)（-）的产生被阻断。最后，我们通过黄斑密度测量了O(2)（-）生产的最佳pH值，发现O(2)（-）生产的最佳细胞外pH值为7.7，最佳pH(i)约为8。我们发现，当管腔灌注80 mM（而不是10 mM） NaCl时，pH(i)升高可促进NAD(P)H氧化酶产生O(2)(-)，且O(2)（-）的产生对pH敏感，最佳pH(i)为8。
We hypothesized that elevated macula densa intracellular pH (pH(i)) during tubuloglomerular feedback enhances O(2)(-) production from NAD(P)H oxidase. Microdissected thick ascending limbs from rabbits with intact macula densa were cannulated and perfused with physiological saline. When luminal NaCl was switched from 10 to 80 mM, O(2)(-) production increased from 0.53 +/- 0.09 to 2.62 +/- 0.54 U/min (P < 0.01). To determine whether inhibiting the Na/H exchanger blocks O(2)(-) production, we used dimethyl amiloride (DMA) to block Na/H exchange. In the presence of DMA, O(2)(-) production induced by NaCl was blunted by 40%. To study the effect of pH(i) on O(2)(-) in intact macula densa cells, we measured O(2)(-) while pH(i) was changed by adjusting luminal pH. When the macula densa was perfused with 80 mM NaCl and the pH of the perfusate was switched to 6.8, 7.4, and 8.0, O(2)(-) production was significantly enhanced, but not at 10 mM NaCl. To ascertain the source of O(2)(-), we used the NAD(P)H oxidase inhibitor apocynin. In the presence of apocynin (10(-5) M), O(2)(-) production induced by elevating pH(i) was blocked. Finally, we measured the optimum pH for O(2)(-) production by the macula densa and found optimum extracellular pH is at 7.7 and optimum pH(i) is approximately 8 for O(2)(-) production. We found that elevated pH(i) enhances O(2)(-) production from NAD(P)H oxidase induced by increasing luminal NaCl when the lumen is perfused with 80 mM NaCl, not 10 mM, and O(2)(-) production is pH sensitive, with an optimum pH(i) of 8.