Ares Gustavo R
Hypertension and Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital , Detroit, Michigan.
Am J Physiol Renal Physiol. 2019 May 1;316(5):F838-F846. doi: 10.1152/ajprenal.00287.2018. Epub 2019 Feb 27.
The thick ascending limb of Henle's loop (TAL) reabsorbs NaCl via the apical Na-K-2Cl cotransporter (NKCC2). NKCC2 activity is regulated by surface NKCC2 levels. The second messenger cGMP decreases NKCC2 activity by decreasing surface NKCC2 levels. We found that surface NKCC2 undergoes constitutive degradation. Therefore, we hypothesized that cGMP decreases NKCC2 levels by increasing NKCC2 ubiquitination and proteasomal degradation. We measured surface NKCC2 levels by biotinylation of surface proteins, immunoprecipitation of NKCC2, and ubiquitin in TALs. First, we found that inhibition of proteasomal degradation blunts the cGMP-dependent decrease in surface NKCC2 levels [vehicle: 100%, db-cGMP (500 µM): 70.3 ± 9.8%, MG132 (20 µM): 97.7 ± 5.0%, and db-cGMP + MG132: 103.3 ± 3.4%, n = 5, P < 0.05]. We then found that cGMP decreased the internalized NKCC2 pool and that this effect was prevented by inhibition of the proteasome but not the lysosome. Finally, we found that NKCC2 is constitutively ubiquitinated in TALs and that cGMP enhances the rate of NKCC2 ubiquitination [vehicle: 59 ± 14% and db-cGMP (500 µM): 111 ± 25%, n = 5, P < 0.05]. We conclude that NKCC2 is constitutively ubiquitinated and that cGMP stimulates NKCC2 ubiquitination and proteasomal degradation. Our data suggest that the cGMP-induced NKCC2 ubiquitination and degradation may contribute to the cGMP-induced decrease of the NKCC2-dependent NaCl reabsorption in TALs.
亨氏袢厚升支（TAL）通过顶端的钠-钾-2氯协同转运蛋白（NKCC2）重吸收氯化钠。NKCC2的活性受其表面水平的调节。第二信使环磷酸鸟苷（cGMP）通过降低表面NKCC2水平来降低NKCC2活性。我们发现表面NKCC2会发生组成型降解。因此，我们推测cGMP通过增加NKCC2的泛素化和蛋白酶体降解来降低NKCC2水平。我们通过对表面蛋白进行生物素化、免疫沉淀TAL中的NKCC2和泛素来测量表面NKCC2水平。首先，我们发现抑制蛋白酶体降解可减弱cGMP依赖性的表面NKCC2水平降低[溶剂：100%，8-溴环磷酸鸟苷（db-cGMP，500 μM）：70.3±9.8%，MG132（20 μM）：97.7±5.0%，db-cGMP + MG132：103.3±3.4%，n = 5，P < 0.05]。然后我们发现cGMP减少了内化的NKCC2库，并且这种作用可通过抑制蛋白酶体而非溶酶体来阻止。最后，我们发现NKCC2在TAL中会发生组成型泛素化，并且cGMP可提高NKCC2的泛素化速率[溶剂：59±14%，db-cGMP（500 μM）：111±25%，n = 5，P < 0.05]。我们得出结论，NKCC2会发生组成型泛素化，并且cGMP刺激NKCC2的泛素化和蛋白酶体降解。我们的数据表明，cGMP诱导的NKCC2泛素化和降解可能导致cGMP诱导的TAL中依赖NKCC2的氯化钠重吸收减少。
