Functional and molecular identification of a novel chloride conductance in canine colonic smooth muscle
Functional and molecular identification of a novel chloride conductance in canine colonic smooth muscle
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DOI：
10.1152/ajpcell.1998.275.4.c940
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发表时间：
1998-10-01
影响因子：
5.5
通讯作者：
Sanders, KM
中科院分区：
文献类型：
--
作者：
Dick, GM;Bradley, EK;Sanders, KM
肿胀激活或体积敏感的氯电流存在于多种细胞类型中，并在其功能中发挥多种作用。然而，通常缺乏通道的分子表征。最近，心肌细胞中负责肿胀激活Cl-电流的分子实体已被确定为ClC-3。我们研究的目的是利用分子生物学和电生理学技术确定犬结肠平滑肌细胞中是否存在这样的通道，如果存在，则表征其功能和分子特性。我们假设 ClC-3 存在于结肠平滑肌中，并以类似于从心脏克隆的分子实体的方式进行调节。事实上，ClC-3 基因在结肠肌细胞中表达，如在分离细胞上进行的逆转录酶聚合酶链反应所证明的。通过将细胞外渗透压从 300 mosM 降低至 250 mosM 激活的电流是向外整流的并且依赖于 Cl-梯度。当 Cl- 被 I- 或 Br- 取代时，电流强度增加并在更负的电位下反转。他莫昔芬（[Z]-1-[对二甲氨基乙氧基苯基]-1,2-二苯基-1-丁烯；10μM）和DIDS（100μM）抑制电流，而25μM尼氟酸、10μM尼卡地平和Ca2+去除没有效果。 1 mM 细胞外 ATP 以电压依赖性方式抑制电流。 Cl-电流也受蛋白激酶 C 调节，佛波醇 12,13-二丁酸酯 (300 nM) 降低 Cl-电流强度，而氯化白屈菜红碱 (30 μM) 在等渗条件下激活它。我们的研究结果表明，结肠肌细胞中存在由低渗溶液激活的电流，并且可能由 ClC-3 介导。此外，我们认为 ClC-3 可能是在对细胞施加物理压力的条件下控制结肠平滑肌去极化和收缩的重要机制。
Swelling-activated or volume-sensitive Cl- currents are found in numerous cell types and play a variety of roles in their function; however, molecular characterization of the channels is generally lacking. Recently, the molecular entity responsible for swelling-activated Cl- current in cardiac myocytes has been identified as ClC-3. The goal of our study was to determine whether such a channel exists in smooth muscle cells of the canine colon using both molecular biological and electrophysiological techniques and, if present, to characterize its functional and molecular properties. We hypothesized that ClC-3 is present in colonic smooth muscle and is regulated in a manner similar to the molecular entity cloned from heart. Indeed, the ClC-3 gene was expressed in colonic myocytes, as demonstrated by reverse transcriptase polymerase chain reaction performed on isolated cells. The current activated by decreasing extracellular osmolarity from 300 to 250 mosM was outwardly rectifying and dependent on the Cl- gradient. Current magnitude increased and reversed at more negative potentials when Cl- was replaced by I- or Br-. Tamoxifen ([Z]-1-[p-dimethylaminoethoxy-phenyl]-1,2-diphenyl -1-butene; 10 mu M) and DIDS (100 mu M) inhibited the current, whereas 25 mu M niflumic acid, 10 mu M nicardipine, and Ca2+ removal had no effect. Current was inhibited by 1 mM extracellular ATP in a voltage-dependent manner. Cl- current was also regulated by protein kinase C, as phorbol 12,13-dibutyrate (300 nM) decreased Cl- current magnitude, while chelerythrine chloride (30 mu M) activated it under isotonic conditions. Our findings indicate that a current activated by hypotonic solution is present in colonic myocytes and is likely mediated by ClC-3. Furthermore, we suggest that the ClC-3 may be an important mechanism controlling depolarization and contraction of colonic smooth muscle under conditions that impose physical stress on the cells.