Haque Mohammed Ziaul, Ortiz Pablo A
Department of Internal Medicine, Hypertension and Vascular Research, Henry Ford Hospital , Detroit, Michigan.
Am J Physiol Renal Physiol. 2019 Jul 1;317(1):F99-F106. doi: 10.1152/ajprenal.00232.2018. Epub 2019 May 15.
The apical Na-K-2Cl cotransporter (NKCC2) mediates NaCl reabsorption by the thick ascending limb (TAL). The free radical superoxide ( ) stimulates TAL NaCl absorption by enhancing NKCC2 activity. In contrast, nitric oxide (NO) scavenges and inhibits NKCC2. NKCC2 activity depends on the number of NKCC2 transporters in the TAL apical membrane and its phosphorylation. We hypothesized that stimulates NKCC2 activity by enhancing apical surface NKCC2 expression. We measured surface NKCC2 expression in rat TALs by surface biotinylation and Western blot analysis. Treatment of TALs with produced by exogenous xanthine oxidase (1 mU/ml) and hypoxanthine (500 µM) stimulated surface NKCC2 expression by ~18 ± 5% ( < 0.05). -stimulated surface NKCC2 expression was blocked by the scavenger tempol (50 µM). Scavenging HO with 100 U/ml catalase did not block the stimulatory effect of xanthine oxidase-hypoxanthine (22 ± 8% increase from control, < 0.05). Inhibition of endogenous NO production with -nitro-l-arginine methyl ester enhanced surface NKCC2 expression by 21 ± 6% and, when added together with xanthine oxidase-hypoxanthine, increased surface NKCC2 by 41 ± 10% ( < 0.05). Scavenging with superoxide dismutase (300 U/ml) decreased this stimulatory effect by 60% (39 ± 4% to 15 ± 10%, < 0.05). Protein kinase C inhibition with Gö-6976 (100 nM) blocked -stimulated surface NKCC2 expression ( < 0.05). did not affect NKCC2 phosphorylation at Thr or its upstream kinases STE20/SPS1-related proline/alanine-rich kinase-oxidative stress-responsive kinase 1. We conclude that increases surface NKCC2 expression by stimulating protein kinase C and that this effect is blunted by endogenous NO. -stimulated apical trafficking of NKCC2 may be involved in the enhanced surface NKCC2 expression observed in Dahl salt-sensitive rats.
顶端钠-钾-2氯协同转运蛋白（NKCC2）介导髓袢升支粗段（TAL）对氯化钠的重吸收。自由基超氧化物（ ）通过增强NKCC2活性来刺激TAL对氯化钠的吸收。相反，一氧化氮（NO）可清除超氧化物并抑制NKCC2。NKCC2的活性取决于TAL顶端膜上NKCC2转运蛋白的数量及其磷酸化状态。我们推测超氧化物通过增强顶端表面NKCC2的表达来刺激NKCC2活性。我们通过表面生物素化和蛋白质印迹分析来测定大鼠TAL中表面NKCC2的表达。用外源性黄嘌呤氧化酶（1 mU/ml）和次黄嘌呤（500 µM）产生的超氧化物处理TAL，可使表面NKCC2的表达增加约18±5%（P<0.05）。超氧化物刺激的表面NKCC2表达被超氧化物清除剂tempol（50 µM）阻断。用100 U/ml过氧化氢酶清除过氧化氢并不阻断黄嘌呤氧化酶-次黄嘌呤的刺激作用（较对照组增加22±8%，P<0.05）。用L-硝基精氨酸甲酯抑制内源性NO生成可使表面NKCC2表达增加21±6%，当与黄嘌呤氧化酶-次黄嘌呤一起添加时，表面NKCC2增加41±10%（P<0.05）。用超氧化物歧化酶（300 U/ml）清除超氧化物可使这种刺激作用降低60%（从39±4%降至15±10%，P<0.05）。用Gö-6976（100 nM）抑制蛋白激酶C可阻断超氧化物刺激的表面NKCC2表达（P<0.05）。超氧化物不影响NKCC2在苏氨酸位点的磷酸化或其上游激酶STE20/SPS1相关富含脯氨酸/丙氨酸激酶-氧化应激反应激酶1。我们得出结论，超氧化物通过刺激蛋白激酶C增加表面NKCC2的表达，并且这种作用被内源性NO减弱。超氧化物刺激的NKCC2顶端转运可能参与了在 Dahl 盐敏感大鼠中观察到的表面NKCC2表达增强。
