Wei Y, Lu M, Wang W H
Department of Pharmacology, New York Medical College, Valhalla, NY 10595, USA.
Am J Physiol Cell Physiol. 2001 Apr;280(4):C920-8. doi: 10.1152/ajpcell.2001.280.4.C920.
We investigated the effect of inhibiting Na+-K+-ATPase on the basolateral 18-pS K+ channel in the cortical collecting duct (CCD) of the rat kidney. Inhibiting Na+-K+-ATPase with strophanthidin decreased the activity of the 18-pS K+ channel and increased the intracellular Ca2+ to 420 nM. Removal of extracellular Ca2+ abolished the effect of strophanthidin. When intracellular Ca2+ was raised with 5 microM ionomycin or A-23187 to 300, 400, and 500 nM, the activity of the 18-pS K+ channel in cell-attached patches fell by 40, 85, and 96%, respectively. To explore the mechanism of Ca2+-induced inhibition, the effect of 400 nM Ca2+ on channel activity was studied in the presence of calphostin C, an inhibitor of protein kinase C, or KN-93 and KN-62, inhibitors of calmodulin-dependent kinase II. Addition of calphostin C or KN-93 or KN-62 failed to block the inhibitory effect of high concentrations of Ca2+ . This suggested that the inhibitory effect of high concentrations of Ca2+ was not mediated by protein kinase C or calmodulin-dependent kinase II pathways. To examine the possibility that the inhibitory effect of high concentrations of Ca2+ was mediated by the interaction of nitric oxide with superoxide, we investigated the effect of 400 nM Ca2+ on channel activity in the presence of 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron) or N(omega)-nitro-L-arginine methyl ester. Pretreatment of the tubules with 4,5-dihydroxy-1,3-benzenedisulfonic acid or N(omega)-nitro-L-arginine methyl ester completely abolished the inhibitory effect of 400 nM Ca2+ on channel activity. Moreover, application of 4,5-dihydroxy-1,3-benzenedisulfonic acid reversed the inhibitory effect of strophanthidin. We conclude that the effect of inhibiting Na+-K+-ATPase is mediated by intracellular Ca2+ and the inhibitory effect of high concentrations of Ca2+ is the result of interaction of nitric oxide with superoxide.
我们研究了抑制钠钾ATP酶对大鼠肾皮质集合管（CCD）基底外侧18-pS钾通道的影响。用毒毛花苷抑制钠钾ATP酶可降低18-pS钾通道的活性，并使细胞内钙离子浓度升高至420 nM。去除细胞外钙离子可消除毒毛花苷的作用。当用5 microM离子霉素或A-23187将细胞内钙离子浓度升高至300、400和500 nM时，细胞贴附式膜片上18-pS钾通道的活性分别下降了40%、85%和96%。为了探究钙离子诱导抑制的机制，在存在蛋白激酶C抑制剂钙调蛋白C或钙调素依赖性激酶II抑制剂KN-93和KN-62的情况下，研究了400 nM钙离子对通道活性的影响。添加钙调蛋白C或KN-93或KN-62未能阻断高浓度钙离子的抑制作用。这表明高浓度钙离子的抑制作用不是由蛋白激酶C或钙调素依赖性激酶II途径介导的。为了检验高浓度钙离子的抑制作用是否由一氧化氮与超氧化物的相互作用介导，我们研究了在存在4,5-二羟基-1,3-苯二磺酸（Tiron）或N（ω）-硝基-L-精氨酸甲酯的情况下，400 nM钙离子对通道活性的影响。用4,5-二羟基-1,3-苯二磺酸或N（ω）-硝基-L-精氨酸甲酯预处理肾小管可完全消除400 nM钙离子对通道活性的抑制作用。此外，应用4,5-二羟基-1,3-苯二磺酸可逆转毒毛花苷的抑制作用。我们得出结论，抑制钠钾ATP酶的作用是由细胞内钙离子介导的，高浓度钙离子的抑制作用是一氧化氮与超氧化物相互作用的结果。
