Zallocchi Marisa, Igarreta Pilar, Calvo Juan Carlos, Reboucas Nancy Amaral, Damasco María Christina
Department of Biochemistry, FCEN, U.B.A, Buenos Aires, Argentina.
Med Sci Monit. 2003 Feb;9(2):BR85-90.
Previously we showed that corticosterone and aldosterone increased proton fluxes in proximal tubule, by micropuncture and stationary microperfusion. Since the Na+/H+ exchanger is responsible for the main proximal proton secretion, we have now evaluated the effects aldosterone on Na+/H+ exchange activity in brush border vesicles. In order to evaluate the mechanism of action of glucocorticoids and mineralocorticoids, we studied the comparative effects of corticosterone and aldosterone on the abundance of NHE3 and NHE2 isoforms.
MATERIAL/METHODS: We isolated renal brush border vesicles from rats by differential centrifugation in sham-operated, adrenalectomized, and adrenalectomized-aldosterone treated (ADX + aldosterone) animals. We measured the kinetics of H+ transport in response to increasing concentrations of Sodium Gluconate by fluorimetry using acridine orange. For Na+/H+ exchanger abundance we used Western blot analysis of brush border proteins in the above groups and in adrenalectomized-corticosterone treated rats.
The Vmax in adrenalectomized animals was 22,162+/-1828 fluorescence units/min; in sham animals, 37,020+/-2722; and in ADX + aldosterone, 42,344+/-3044 (p<0.01 adrenalectomized vs others). No differences in Km were observed. Adrenalectomy decreased NHE3 abundance over Sham by 32% without modifying NHE2. Corticosterone-replacement enhanced NHE3 abundance by 76% and failed to increase NHE2. Aldosterone enhanced NHE2 abundance by 75% and did not increase NHE3.
Mineralocorticoids enhance Na+/H+ exchange activity by increasing NHE2 abundance; glucocorticoids, by increasing NHE3 abundance.
此前我们通过微穿刺和固定微灌注技术发现，皮质酮和醛固酮可增加近端小管中的质子通量。由于钠氢交换体是近端小管主要的质子分泌途径，我们现在评估了醛固酮对刷状缘小泡中钠氢交换活性的影响。为了评估糖皮质激素和盐皮质激素的作用机制，我们研究了皮质酮和醛固酮对NHE3和NHE2亚型丰度的比较影响。
材料/方法：我们通过差速离心法从假手术、肾上腺切除和肾上腺切除后用醛固酮处理（ADX + 醛固酮）的大鼠中分离出肾刷状缘小泡。我们使用吖啶橙通过荧光法测量了随着葡萄糖酸钠浓度增加时氢离子转运的动力学。对于钠氢交换体丰度，我们对上述组以及肾上腺切除后用皮质酮处理的大鼠的刷状缘蛋白进行了蛋白质免疫印迹分析。
肾上腺切除动物的最大反应速度（Vmax）为22,162±1828荧光单位/分钟；假手术动物为37,020±2722；ADX + 醛固酮组为42,344±3044（肾上腺切除组与其他组相比，p<0.01）。未观察到米氏常数（Km）的差异。与假手术组相比，肾上腺切除使NHE3丰度降低了32%，而NHE2未改变。皮质酮替代使NHE3丰度增加了76%，但未增加NHE2。醛固酮使NHE2丰度增加了75%，但未增加NHE3。
盐皮质激素通过增加NHE2丰度来增强钠氢交换活性；糖皮质激素则通过增加NHE3丰度来增强。
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