myo-inositol 3,4,5,6-tetrakisphosphate inhibits an apical calcium-activated chloride conductance in polarized monolayers of a cystic fibrosis cell line.
myo-inositol 3,4,5,6-tetrakisphosphate inhibits an apical calcium-activated chloride conductance in polarized monolayers of a cystic fibrosis cell line.
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DOI：
10.1074/jbc.m002316200
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发表时间：
2000-09
期刊：
影响因子：
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通讯作者：
M. Carew;X. Yang;C. Schultz;S. Shears
中科院分区：
文献类型：
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作者：
M. Carew;X. Yang;C. Schultz;S. Shears
肌醇3,4,5,6-四磷酸(Ins（3,4,5,6)P(4)）是否抑制顶端Ca（2+）激活的Cl（-）传导（CaCC）？我们使用在极化单层中生长的人CFPAC-1胰腺炎细胞来研究这个问题。细胞Ins(3,4,5,6)P(4)水平对嘌呤能受体的激活非常敏感，在加入激动剂1分钟内上升3倍。因此，细胞内的Ins(3,4,5,6)P(4)水平通过细胞渗透的生物激活类似物1,2-二- o-丁基肌醇3,4,5,6-四akisphosphate octakis(acetoxymethyl)ester (Bt(2)Ins （3,4,5,6)P(4)/AM）培养而特异性升高，而不受受体激活。后者抑制Ca（2+）激活的Cl（-）分泌60%。接下来，我们使用制霉菌素选择性地使基底侧膜渗透到单价阴离子和阳离子中，从而防止该膜在电化学上主导离子通过顶膜的运动。因此，我们在原位研究了顶端Cl（-）通道的自主调节。通过顶膜的Cl（-）通量的性质与CaCC的预期一致：尼氟酸敏感性，向外整流，I（-）的渗透性比Cl（-）高2倍。在接受制氨抑素治疗后，我们用嘌呤能激动剂或Bt(2)Ins(3,4,5,6)P(4)/AM提高细胞内Ins(3,4,5,6)P(4)的水平。两种方案都抑制Ca（2+）激活的Cl（-）分泌（高达70%）。这些研究首次证明，在极化单层的生理学相关背景下，存在一个顶端，Ins(3,4,5,6)P(4)抑制CaCC。
Does inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P(4)) inhibit apical Ca(2+)-activated Cl(-) conductance (CaCC)? We studied this question using human CFPAC-1 pancreatoma cells grown in polarized monolayers. Cellular Ins(3,4,5,6)P(4) levels were acutely sensitive to purinergic receptor activation, rising 3-fold within 1 min of agonist addition. Intracellular Ins(3,4,5,6)P(4) levels were therefore specifically elevated, independently of receptor activation, by incubating cells with a cell-permeant bioactivable analogue, 1,2-di-O-butyl-myo-inositol 3,4,5,6-tetrakisphosphate octakis(acetoxymethyl)ester (Bt(2)Ins (3,4,5,6)P(4)/AM). The latter inhibited Ca(2+)-activated Cl(-) secretion by 60%. We next used nystatin to selectively permeabilize the basolateral membrane to monovalent anions and cations, thereby preventing this membrane from electrochemically dominating ion movements through the apical membrane. Thus, we studied autonomous regulation of apical Cl(-) channels in situ. The properties of Cl(-) flux across the apical membrane were those expected of CaCC: niflumic acid sensitivity, outward rectification, and 2-fold greater permeability of I(-) over Cl(-). Following nystatin-treatment, we elevated intracellular levels of Ins(3,4,5,6)P(4) with either purinergic agonists or with Bt(2)Ins(3,4,5,6)P(4)/AM. Both protocols inhibited Ca(2+)-activated Cl(-) secretion (up to 70%). These studies provide the first demonstration that, in a physiologically relevant context of a polarized monolayer, there is an apical, Ins(3,4,5,6)P(4)-inhibited CaCC.