Cytochrome P-450 metabolites mediate extracellular Ca2+-induced inhibition of apical K+ channels in the TAL
Cytochrome P-450 metabolites mediate extracellular Ca2+-induced inhibition of apical K+ channels in the TAL
复制标题
DOI：
10.1152/ajpcell.1996.271.1.c103
复制
发表时间：
1996-07-01
影响因子：
5.5
通讯作者：
Hebert, SC
中科院分区：
文献类型：
--
作者：
Wang, WH;Lu, M;Hebert, SC
应用膜片钳技术研究了细胞外Ca ~（2+）（Ca ~（2+））对离体大鼠肾劈裂粗升支（TAL）顶端70 pS钾通道（K ~+）活动的影响。将Ca-0（2+）从1.1 mM升高至5 mM，可在300 s内将细胞贴附贴片中70 pS K+通道的活性可逆性降低至对照值的16 +/- 2%。此外，50 μ M的新霉素模仿的影响，增加在Ca-0（2+）的通道活性在细胞附着的补丁和完全抑制通道活性。新霉素对细胞贴附贴片中通道活性的影响是间接的，因为在由内而外的贴片中，在70 pS K+通道上加入50 μ M新霉素仅降低通道电流的表观振幅，而不改变通道开放概率。我们进一步研究了蛋白激酶C（PKC）和花生四烯酸的细胞色素P-450依赖性代谢产物在介导Ca-0（2+）诱导的通道活性抑制中的作用。添加佛波醇12-肉豆蔻酸酯13-乙酸酯（2 μ M）可逆地阻断了细胞贴附贴片中的通道活性至对照值的4 +/- 1%，而75 nM calphostin C使通道活性增加了115 +/-10%。此外，添加1 nM外源性PKC可逆地和完全地抑制70 pS K+通道。然而，用钙磷蛋白C（75 nM）抑制PKC仅略微延长Ca-0（2+）对通道活性的作用的时间过程（370 +/- 40 s），并且未能消除5 mM Ca-0（2+）对细胞附着斑中通道活性的抑制作用，表明PKC不是Ca-0（2+）对通道活性的作用的主要原因。相反，5 mM Ca-0（2+）对顶端70 pS K+通道的影响完全废除时，TAL小管首次孵育的17-十八炔酸（5 μ M），含有解决方案，一个代理，具体块细胞色素P-450单加氧酶。总之，这些数据表明，Ca-0（2+）是顶端70 pS K+通道的重要调节剂，花生四烯酸的细胞色素P-450依赖性代谢产物参与介导这种抑制作用。
We used the patch-clamp technique to study the effect of extracellular Ca2+ (Ca-0(2+)) on the activity of the apical 70-pS K+ channel in the isolated split-open thick ascending limb (TAL) of the rat kidney. Raising Ca-0(2+) from 1.1 to 5 mM reversibly reduced the activity of the 70-pS K+ channel in cell-attached patches to 16 +/- 2% of the control value within 300 s. In addition, 50 mu M neomycin mimicked the effect of an increase in Ca-0(2+) on channel activity in cell-attached patches and completely inhibited channel activity. The effect of neomycin on the channel activity in cell-attached patches is an indirect effect, since addition of 50 mu M neomycin on the 70-pS K+ channel in inside-out patches reduced only the apparent amplitude of the channel current without changing channel open probability. We examined further the role of protein kinase C (PKC) and the cytochrome P-450-dependent metabolites of arachidonic acid in mediating the Ca-0(2+)-induced inhibition of channel activity. Addition of phorbol 12-myristate 13-acetate (2 mu M) reversibly blocked channel activity in cell-attached patches to 4 +/- 1% of the control value, whereas 75 nM calphostin C increased the channel activity by 115 +/- 10%. Moreover, addition of 1 nM exogenous PKC reversibly and completely inhibited the 70-pS K+ channel. However, inhibition of PKC with calphostin C (75 nM) only slightly prolonged the time course of the effect of Ca-0(2+) on channel activity (370 +/- 40 s) and failed to abolish the inhibitory effect of 5 mM Ca-0(2+) on channel activity in cell-attached patches, indicating that PKC was not mainly responsible for the effect of Ca-0(2+) on channel activity. In contrast, the effect of 5 mM Ca-0(2+) on the apical 70-pS K+ channel was completely abolished when TAL tubules were first incubated in the 17-octadecynoic acid (5 mu M)-containing solution, an agent that specifically blocks cytochrome P-450 monooxygenase. In conclusion, these data indicate that Ca-0(2+) is an important regulator of the apical 70-pS K+ channel and that a cytochrome P-450-dependent metabolite of arachidonic acid is involved in mediating this inhibitory effect.