Lu M, Zhu Y, Balazy M, Reddy K M, Falck J R, Wang W
Department of Pharmacology, New York Medical College, Valhalla 10595, USA.
J Gen Physiol. 1996 Dec;108(6):537-47. doi: 10.1085/jgp.108.6.537.
We have used the patch-clamp technique to study the effect of angiotensin II (AII) on the activity of the apical 70 pS K+ channel and used Na(+)-sensitive fluorescent dye (SBFI) to investigate the effect of AII on intracellular Na+ concentration (Na+i) in the thick ascending limb (TAL) of the rat kidney. Addition of 50 pM AII reversibly reduced NPo, a product of channel open probability (Po) and channel number (N), to 40% of the control value and reduced the Na+i by 26%. The AII (50 pM)-induced decrease in channel activity defined by NPo was partially reversed by addition of 5 microM 17-octadecynoic acid (17-ODYA), an agent which blocks the cytochrome P450 monooxygenase. The notion that P450 metabolites of arachidonic acid (AA) may mediate the inhibitory effect of AII was further suggested by experiments in which addition of 10 nM of 20-hydroxyeicosatetraenoic acid (20-HETE) blocked the channel activity in cell-attached patches in the presence of 17-ODYA. We have used gas chromatography mass spectrometry (GC/MS) to measure the production of 20-HETE, a major AA metabolite of the P450-dependent pathway in the TAL of the rat. Addition of 50 pM AII increased the production of 20-HETE to 260% of the control value, indicating that 20-HETE may be involved in mediating the effect of AII (50 pM). In contrast to the inhibitory effect of 50 pM AII, addition of 50-100 nM AII increased the channel activity to 270% of the control value and elevated the Na+i by 45%. The effect of AII on the activity of the 70 pS K+ channel was also observed in the presence of 5 microM 17-ODYA and 5 microM calphostin C, an inhibitor of protein kinase C. However, addition of 100 microM NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, abolished completely the AII (50-100 nM)-induced increase in channel activity and addition of an exogenous nitric oxide (NO) donor, S-nitroso-N-acetyl-penicillamine (SNAP), increased channel activity in the presence of L-NAME. These data suggest that the stimulatory effect of AII is mediated by NO. We conclude that AII has dual effects on the activity of the apical 70 pS K+ channel. The inhibitory effect of AII is mediated by P450-dependent metabolites whereas the stimulatory effect may be mediated via NO.
我们运用膜片钳技术研究血管紧张素II（AII）对顶端70 pS钾通道活性的影响，并使用对钠敏感的荧光染料（SBFI）来探究AII对大鼠肾脏髓袢升支粗段（TAL）细胞内钠浓度（Na+i）的影响。添加50 pM的AII可使通道开放概率（Po）与通道数量（N）的乘积NPo可逆性降低至对照值的40%，并使Na+i降低26%。添加5 microM 17-十八炔酸（17-ODYA）可部分逆转由50 pM AII诱导的、由NPo定义的通道活性降低，17-ODYA是一种可阻断细胞色素P450单加氧酶的试剂。花生四烯酸（AA）的P450代谢产物可能介导AII的抑制作用这一观点，在另外的实验中得到了进一步证实，即在存在17-ODYA的情况下，添加10 nM的20-羟基二十碳四烯酸（20-HETE）可阻断细胞贴附式膜片中的通道活性。我们使用气相色谱-质谱联用（GC/MS）技术来测定20-HETE的生成量，20-HETE是大鼠TAL中P450依赖途径的主要AA代谢产物。添加50 pM AII可使20-HETE的生成量增加至对照值的260%，表明20-HETE可能参与介导50 pM AII的作用。与5 pM AII的抑制作用相反，添加50 - 100 nM AII可使通道活性增加至对照值的270%，并使Na+i升高45%。在存在5 microM 17-ODYA和5 microM钙调蛋白C（一种蛋白激酶C抑制剂）的情况下，也观察到了AII对70 pS钾通道活性的影响。然而，添加100 microM NG-硝基-L-精氨酸甲酯（L-NAME，一种一氧化氮合酶抑制剂）可完全消除AII（50 - 100 nM）诱导的通道活性增加；而添加外源性一氧化氮（NO）供体S-亚硝基-N-乙酰青霉胺（SNAP），则可在存在L-NAME的情况下增加通道活性。这些数据表明，AII的刺激作用是由NO介导的。我们得出结论，AII对顶端70 pS钾通道的活性具有双重作用。AII的抑制作用由P450依赖的代谢产物介导，而刺激作用可能通过NO介导。
