Regulation of ATP-sensitive K+ channels by protein kinase C in murine colonic myocytes
Regulation of ATP-sensitive K+ channels by protein kinase C in murine colonic myocytes
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DOI：
10.1152/ajpcell.2001.281.3.c857
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发表时间：
2001-09-01
影响因子：
5.5
通讯作者：
Sanders, KM
中科院分区：
文献类型：
--
作者：
Jun, JY;Kong, ID;Sanders, KM
应用膜片钳技术研究了小鼠结肠肌细胞ATP敏感性K ~+（K-ATP）电流的调节。吡那地尔（10（-5）M）在透析细胞中存在高外部K+（90 mM）时，在-80 mV的保持电位下激活内向电流。格列本脲（10（-5）M）抑制吡那地尔激活电流。佛波醇12，13-二丁酸酯（PDBu; 2 × 10（-7）M）抑制吡那地尔激活电流。4-α-佛波酯（5 × 10（-7）M）是PDBu的一种无活性形式，对吡那地尔激活电流没有影响。在细胞贴附贴片中，吡那地尔增加了K-ATP通道的开放概率，PDBu抑制了K-ATP通道的开放。当用白屈菜红碱（10（-6）M）或钙磷抑制素C（10（-7）M）预处理细胞时，PDBu对吡那地尔激活的全细胞电流的抑制显著降低。在用穿孔膜片技术研究的细胞中，PDBu也抑制吡那地尔激活电流，白屈菜红碱（10（-6）M）可降低这种抑制作用。乙酰胆碱（ACh; 10（-5）M）抑制吡那地尔激活电流，用钙磷蛋白0（10（-7）M）预孵育细胞可降低ACh的作用。用蛋白激酶C亚型（PKC β）抗体透析的细胞对吡那地尔有正常反应，但PDBu和ACh对K-ATP的作用在这些细胞中被阻断。免疫荧光和Western印迹显示，在小鼠近端结肠的完整肌肉和分离的平滑肌细胞中表达PKC β。这些数据表明，PKC调节KATP在结肠肌细胞和ACh对K-ATP的影响主要是由PKC介导的。PKC β是调节小鼠结肠肌细胞K-ATP的主要同工酶。
We investigated the regulation of ATP-sensitive K+ (K-ATP) currents in murine colonic myocytes with patch-clamp techniques. Pinacidil (10(-5) M) activated inward currents in the presence of high external K+ (90 mM) at a holding potential of -80 mV in dialyzed cells. Glibenclamide (10(-5) M) suppressed pinacidil-activated current. Phorbol 12,13-dibutyrate (PDBu; 2 x 10(-7) M) inhibited pinacidil-activated current. 4-alpha -Phorbol ester (5 x 10(-7) M), an inactive form of PDBu, had no effect on pinacidil-activated current. In cell-attached patches, the open probability of K-ATP channels was increased by pinacidil, and PDBu suppressed openings of K-ATP channels. When cells were pretreated with chelerythrine (10(-6) M) or calphostin C (10(-7) M), inhibition of the pinacidil-activated whole cell currents by PDBu was significantly reduced. In cells studied with the perforated patch technique, PDBu also inhibited pinacidil-activated current, and this inhibition was reduced by chelerythrine (10(-6) M). Acetylcholine (ACh; 10(-5) M) inhibited pinacidil-activated currents, and preincubation of cells with calphostin 0 (10(-7) M) decreased the effect of ACh. Cells dialyzed with protein kinase C epsilon -isoform (PKC epsilon) antibody had normal responses to pinacidil, but the effects of PDBu and ACh on K-ATP were blocked in these cells. Immunofluorescence and Western blots showed expression of PKC epsilon in intact muscles and isolated smooth muscle cells of the murine proximal colon. These data suggest that PKC regulates KATP in colonic muscle cells and that the effects of ACh on K-ATP are largely mediated by PKC. PKC epsilon appears to be the major isozyme that regulates K-ATP in murine colonic myocytes.