Department of Neuroscience, Cell Biology & Physiology, Wright State University Boonshoft School of Medicine, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA.
Exp Physiol. 2011 Mar;96(3):305-16. doi: 10.1113/expphysiol.2010.055038. Epub 2010 Dec 17.
Adrenaline activates transient Cl(-) secretion and sustained K(+) secretion across isolated distal colonic mucosa of guinea-pigs. The Ca(2+)-activated Cl(-) channel inhibitor CaCCinh-A01 (30 μm) significantly reduced electrogenic K(+) secretion, detected as short-circuit current (I(sc)). This inhibition supported the cell model for K(+) secretion in which basolateral membrane Cl(-) channels provide an exit pathway for Cl(-) entering the cell via Na(+)-K(+)-2Cl(-) cotransporters. CaCCinh-A01 inhibited both I(sc) and transepithelial conductance in a concentration-dependent manner (IC(50) = 6.3 μm). Another Cl(-) channel inhibitor, GlyH-101, also reduced sustained adrenaline-activated I(sc) (IC(50) = 9.4 μm). Adrenaline activated whole-cell Cl(-) current in isolated intact colonic crypts, confirmed by ion substitution. This adrenaline-activated whole-cell Cl(-) current was also inhibited by CaCCinh-A01 or GlyH-101. In contrast to K(+) secretion, CaCCinh-A01 augmented the electrogenic Cl(-) secretion activated by adrenaline as well as that activated by prostaglandin E(2). Synergistic Cl(-) secretion activated by cholinergic/prostaglandin E(2) stimulation was insensitive to CaCCinh-A01. Colonic expression of the Ca(2+)-activated Cl(-) channel protein Tmem16A was supported by RT-PCR detection of Tmem16A mRNA, by immunoblot with a Tmem16A antibody, and by detection of immunofluorescence in lateral membranes of epithelial cells. Alternative splices of Tmem16A were detected for exons that are involved in channel activation. Inhibition of K(+) secretion and augmentation of Cl(-) secretion by CaCCinh-A01 support a common colonic cell model for these two ion secretory processes, such that activation of basolateral membrane Cl(-) channels contributes to the production of electrogenic K(+) secretion and limits the rate of Cl(-) secretion. Maximal physiological Cl(-) secretion occurs only for synergistic activation mechanisms that close these basolateral membrane Cl(-) channels.
肾上腺素激活豚鼠离体远端结肠黏膜的瞬时 Cl(-)分泌和持续的 K(+)分泌。Ca(2+)激活 Cl(-)通道抑制剂 CaCCinh-A01（30μm）显著减少了电致 K(+)分泌，表现为短路电流（I(sc)）。这种抑制作用支持了 K(+)分泌的细胞模型，即基底外侧膜 Cl(-)通道为通过 Na(+)-K(+)-2Cl(-)共转运体进入细胞的 Cl(-)提供了一个出口途径。CaCCinh-A01 以浓度依赖的方式抑制 I(sc)和跨上皮电导（IC(50)=6.3μm）。另一种 Cl(-)通道抑制剂 GlyH-101 也降低了持续肾上腺素激活的 I(sc)（IC(50)=9.4μm）。肾上腺素在分离的完整结肠隐窝中激活了全细胞 Cl(-)电流，这通过离子替代得到了证实。这种肾上腺素激活的全细胞 Cl(-)电流也被 CaCCinh-A01 或 GlyH-101 抑制。与 K(+)分泌相反，CaCCinh-A01 增强了肾上腺素以及前列腺素 E(2)激活的电致 Cl(-)分泌。胆碱能/前列腺素 E(2)刺激激活的协同 Cl(-)分泌对 CaCCinh-A01 不敏感。RT-PCR 检测 Tmem16A mRNA、Tmem16A 抗体的免疫印迹和上皮细胞侧膜的免疫荧光检测均支持 RT-PCR 检测 Tmem16A mRNA、Tmem16A 抗体的免疫印迹和上皮细胞侧膜的免疫荧光检测均支持 Tmem16A 基因的结肠表达。Tmem16A 的替代剪接检测到了涉及通道激活的外显子。CaCCinh-A01 对 K(+)分泌的抑制和 Cl(-)分泌的增强支持这两种离子分泌过程的共同结肠细胞模型，即基底外侧膜 Cl(-)通道的激活有助于产生电致性 K(+)分泌，并限制 Cl(-)分泌的速率。只有在这些基底外侧膜 Cl(-)通道关闭的协同激活机制下，才会发生最大的生理性 Cl(-)分泌。
