Objective To clarify the signaling mechanisms underlying angiotensin Ⅱ biphasic regulation of renal proximal Na+-HCO3- transport. Methods Different concentration Ang Ⅱ to the responses of Na+-HCO3- cotransporter (NBC) activity in isolated proximal tubules, with or without ATR, MAPK, cPLA2α, P450 blockade was compared in wild-type and Ang Ⅱ type 1a receptor (AT1aR)-deficient mice. The phospholipase of ERK was examined by Western blotting. AT1aR mRNA was examined by RT-PCR from kidney proximal tubules. Results (1)In isolated wild-type mouse, renal proximal tubules showed biphasic effects of Ang Ⅱ on NBC activity. Low concentration Ang Ⅱ (10-10 mol/L) increased NBC activity, but high concentration Ang Ⅱ (10-6 mol/L) decreased NBC activity. Olmcsartan (AT1 antagonist) blocked both stimnlatory and inhibitory effects of Ang Ⅱ on NBC activity, but PD98059 (mitogen-activated protein kinase inhibitor) blocked only the stimulatory effect of low concentration Ang Ⅱ ( 10-10 mol/L). (2)In AT1aR-deficient mice, only the stimulatory effect by high concentration of Ang Ⅱ (10-6 mol/L) was observed, which was blocked by olmesartan and PD98059. (3)In wild-type mice, pharmacological blockade of cPLA2 or P450 converted the inhibition effect by high concentration Ang Ⅱ (10-6 mol/L) to the stimulation, which was blocked by olmesanan and PD98059. These results indicated that the extracellular sigual-regulated kinase (ERK) activation via AT1 mediated only the stimulatory effect of Ang Ⅱ, while the cPLA2α/P450 activation via AT1 mediated the inhibitory effect of Ang Ⅱ independently of ERK. The analysis of ERK phosphorylation by Ang Ⅱ also supported a view that the cPLA2α/P450 pathway worked to suppress the ERK activation. Conclusions Ang Ⅱ activates ERK and cPLA2α with different concentration dependency via AT1. The balance between ERK and cPLA2α activities determines the final responses to Ang Ⅱ in intact proximal tubules.%目的 探讨血管紧张素Ⅱ(Ang Ⅱ)在肾近曲小管Na+-HCO3-转运中的作用及细胞外信号调节激酶(ERK)、胞质磷脂酶A2α(cPLA2α)通路对其调节的机制.方法 从野生小鼠和血管紧张素1α型受体(AT1aR)基因缺陷小鼠分离新鲜单根肾近曲小管,在不同浓度Ang Ⅱ(10-10、10-8、10-6 mol/L)及AT1、AT2受体阻滞剂或促分裂原活化蛋白激酶(MAPK)、cPLA2、P450抑制剂存在下对比Na+-HCO3-离子转运活动度变化.Western印迹方法测定ERK磷酸化(p-ERK).RT-PCR测定AT1bR在两种小鼠肾小管中的表达.结果 (1)在野生小鼠,低浓度Ang Ⅱ(10-10mol/L)刺激Na+-HCO3-转运并被AT1受体阻滞剂及MAPK阻滞剂PD98059阻滞;而高浓度Ans Ⅱ(10-6mol/L)抑制Na+-HCO3-的转运,被AT1受体阻滞剂阻滞,但PD98059对其无阻滞作用.显示AngⅡ在肾脏近曲小管双向性调节Na++HCO3-转运,ERK通路仅参与低浓度AngⅡ的刺激作用.(2)在AT1aR基因缺陷小鼠.只有高浓度AngⅡ(10-6mol/L)能刺激Na+-HCO3-转运,并被AT1受体阻滞剂及PD98059阻滞.显示在AT1aR缺乏时,AT1bR起到部分代偿作用,RT-PCR也证实AT1bR在肾小管的存在.(3)在野生小鼠,cPLA2阻滞剂或P450阻滞剂存在下,所有浓度Ang Ⅱ均显示刺激作用,并被AT1受体阻滞剂及PD98059阻滞.Western印迹检测也证实上述结论.这显示经由AT1受体,低浓度Ang Ⅱ通过ERK通路仅参与刺激肾近曲小管Na+-HCO3-离子转运作用,而高浓度AngⅡ经cPLA2α-P450通路抑制Na+-HCO3-离子转运,cPLA2α-P450通路同时也参与了抑制ERK的激活作用.结论 不同浓度Ang Ⅱ经由AT1受体介导了ERK和cPLA2α通路的平衡,从而决定AngⅡ调节在肾近曲小管水和钠的重吸收.
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