Jin Yan, Wang Zhijian, Zhang Yan, Yang Baofeng, Wang Wen-Hui
Department of Pharmacology, Harbin Medical University, Harbin, China.
Am J Physiol Renal Physiol. 2007 Oct;293(4):F1299-307. doi: 10.1152/ajprenal.00293.2007. Epub 2007 Aug 8.
We used the patch-clamp technique and Western blot analysis to explore the effect of PGE(2) on ROMK-like small-conductance K (SK) channels and Ca(2+)-activated big-conductance K channels (BK) in the cortical collecting duct (CCD). Application of 10 microM PGE(2) inhibited SK and BK channels in the CCD. Moreover, either inhibition of PKC or blocking mitogen-activated protein kinase (MAPK), P38 and ERK, abolished the effect of PGE(2) on SK channels in the CCD. The effect of PGE(2) on SK channels was completely blocked in the presence of SC-51089, a specific EP1 receptor antagonist, and mimicked by application of sulprostone, an agonist for EP1 and EP3 receptors. To determine whether PGE(2) stimulates the phosphorylation of P38 and ERK, we treated mouse CCD cells (M-1) with PGE(2). Application of PGE(2) significantly stimulated the phosphorylation of P38 and ERK within 5 min. The dose-response curve of PGE(2) effect shows that 1, 5, and 10 microM PGE(2) increased the phosphorylation of P38 and ERK by 20-21, 50-80, and 80-100%, respectively. The stimulatory effect of PGE(2) on MAPK phosphorylation was not affected by indomethacin but abolished by inhibition of PKC. This suggests that the effect of PGE(2) on MAPK phosphorylation is PKC dependent. Also, the expression of cyclooxygenase II and PGE(2) concentration in renal cortex and outer medulla was significantly higher in rats fed a K-deficient diet than those on a normal-K diet. We conclude that PGE(2) inhibits SK and BK channels and that there is an effect of PGE(2) on SK channels in the CCD through activation of EP1 receptor and MAPK pathways. Also, high concentrations of PGE(2) induced by K restriction may be partially responsible for increasing MAPK activity during K restriction.
我们运用膜片钳技术和蛋白质免疫印迹分析，探究前列腺素E2（PGE(2)）对皮质集合管（CCD）中类ROMK小电导钾（SK）通道和钙激活大电导钾通道（BK）的影响。施加10微摩尔/升的PGE(2)可抑制CCD中的SK和BK通道。此外，抑制蛋白激酶C（PKC）或阻断丝裂原活化蛋白激酶（MAPK）、P38和细胞外信号调节激酶（ERK），均可消除PGE(2)对CCD中SK通道的影响。在存在特异性EP1受体拮抗剂SC-51089的情况下，PGE(2)对SK通道的作用完全被阻断，而EP1和EP3受体激动剂舒前列素的应用则可模拟该作用。为确定PGE(2)是否刺激P38和ERK的磷酸化，我们用PGE(2)处理小鼠CCD细胞（M-1）。施加PGE(2)在5分钟内显著刺激了P38和ERK的磷酸化。PGE(2)作用的剂量反应曲线表明，1、5和10微摩尔/升的PGE(2)分别使P38和ERK的磷酸化增加20 - 21%、50 - 80%和80 - 100%。PGE(2)对MAPK磷酸化的刺激作用不受吲哚美辛影响，但可被PKC抑制所消除。这表明PGE(2)对MAPK磷酸化的作用依赖于PKC。此外，低钾饮食喂养的大鼠肾皮质和外髓中环氧合酶II的表达及PGE(2)浓度显著高于正常钾饮食的大鼠。我们得出结论，PGE(2)抑制SK和BK通道，且PGE(2)通过激活EP1受体和MAPK途径对CCD中的SK通道产生影响。此外，钾限制诱导的高浓度PGE(2)可能部分导致钾限制期间MAPK活性增加。
