Ling B N, Eaton D C
Department of Medicine, Emory University School of Medicine, Atlanta, Georgia.
Kidney Int. 1993 Nov;44(5):974-84. doi: 10.1038/ki.1993.339.
We used the cell-attached patch clamp configuration to examine the effect of basolateral cyclosporin A (CsA) exposure on low conductance K+ channels found in the principal cell apical membrane of rabbit cortical collecting tubule (CCT) primary cultures. Baseline K+ channel activity, measured as mean NPo (number of channels x open probability), was 2.7 +/- 1.1 (N = 29). NPo fell by 69% (0.84 +/- 0.32; N = 32) in cultures pretreated with 500 ng/ml CsA for 30 minutes prior to patching. Chelation of intracellular [Ca2+]i (10 mM BAPTA/AM; N = 8) or removal of extracellular Ca2+ (N = 9), but not prevention of [Ca2+]i store release (10 microM TMB-8; N = 7), abolished CsA-induced inhibition. This suggested that CsA effects were mediated by an initial rise in [Ca2+]i via Ca2+ influx. Either 25 nM AVP (N = 10) or 0.25 microM thapsigargin (N = 8) (causing IP3-dependent and -independent release of [Ca2+]i stores, respectively) augmented, while 25 pM (N = 6) or 250 pM AVP (N = 8) reversed CSA-induced channel inhibition. Apical membrane protein kinase C (PKC) activation with 0.1 microM phorbol ester, PMA (N = 8) or 10 microM synthetic diacylglycerol, OAG (N = 7), mimicked (mean NPo = 0.99 +/- 0.40) the inhibitory effect of CsA. Apical PKC inhibition by prolonged apical exposure to PMA (N = 10) or 100 microM D-sphingosine (N = 6) blocked CsA's effect. Cyclic AMP increasing maneuvers, 10 microM forskolin (N = 5) or 0.5 mM db-cAMP (N = 8), stimulated basal K+ channel activity in the absence of CsA.
(1) basolateral exposure to CsA inhibits the activity of apical membrane 13 pS channels responsible for physiologic K+ secretion in rabbit CCT principal cells. (2) The inhibition is mediated by changes in intracellular Ca2+ and activation of apical PKC. (3) Pharmacologic AVP (nM) augments CsA-induced inhibition by releasing intracellular Ca2+ stores; more physiologic AVP (pM) attenuates channel inhibition, probably through cAMP generation. (4) Inhibition of apical secretory K+ channels by CsA likely contributes to decreased kaliuresis and clinical hyperkalemia observed in patients on CsA therapy.
我们采用细胞贴附式膜片钳配置，研究基底外侧暴露环孢素A（CsA）对兔皮质集合管（CCT）原代培养主细胞顶端膜中低电导钾通道的影响。以平均NPo（通道数量×开放概率）衡量的基线钾通道活性为2.7±1.1（N = 29）。在进行膜片钳记录前，用500 ng/ml CsA预处理30分钟的培养物中，NPo下降了69%（0.84±0.32；N = 32）。螯合细胞内[Ca2+]i（10 mM BAPTA/AM；N = 8）或去除细胞外Ca2+（N = 9），但不是阻止[Ca2+]i储存释放（10 μM TMB - 8；N = 7），消除了CsA诱导的抑制作用。这表明CsA的作用是通过Ca2+内流导致的[Ca2+]i初始升高介导的。25 nM血管升压素（AVP）（N = 10）或0.25 μM毒胡萝卜素（N = 8）（分别导致[Ca2+]i储存的IP3依赖性和非依赖性释放）增强了CsA诱导的通道抑制，而25 pM（N = 6）或250 pM AVP（N = 8）则逆转了CsA诱导的通道抑制。用0.1 μM佛波酯PMA（N = 8）或10 μM合成二酰甘油OAG（N = 7）激活顶端膜蛋白激酶C（PKC），模拟了（平均NPo = 0.99±0.40）CsA的抑制作用。通过长时间顶端暴露于PMA（N = 10）或100 μM D - 鞘氨醇（N = 6）抑制顶端PKC，阻断了CsA的作用。环磷酸腺苷（cAMP）增加的操作，10 μM福斯高林（N = 5）或0.5 mM二丁酰环磷腺苷（db - cAMP）（N = 8），在没有CsA的情况下刺激了基础钾通道活性。
（1）基底外侧暴露于CsA会抑制兔CCT主细胞中负责生理性钾分泌的顶端膜13 pS通道的活性。（2）这种抑制是由细胞内Ca2+的变化和顶端PKC的激活介导的。（3）药理学浓度的AVP（纳摩尔）通过释放细胞内Ca2+储存增强了CsA诱导的抑制作用；更生理性浓度的AVP（皮摩尔）减弱了通道抑制，可能是通过cAMP的产生。（4）CsA对顶端分泌性钾通道的抑制可能导致接受CsA治疗的患者尿钾减少和临床高钾血症。
