Devor D C, Frizzell R A
Department of Cell Biology and Physiology, University of Pittsburgh, Pennsylvania 15261, USA. dd2+@pitt.edu
Am J Physiol. 1998 Jan;274(1):C138-48. doi: 10.1152/ajpcell.1998.274.1.C138.
The Cl- secretory response of colonic cells to Ca(2+)-mediated agonists is transient despite a sustained elevation of intracellular Ca2+. We evaluated the effects of second messengers proposed to limit Ca(2+)-mediated Cl- secretion on the basolateral membrane, Ca(2+)-dependent K+ channel (Kca) in colonic secretory cells, T84. Neither protein kinase C (PKC) nor inositol tetrakisphosphate (1,3,4,5 or 3,4,5,6 form) affected Kca in excised inside-out patches. In contrast, arachidonic acid (AA; 3 microM) potently inhibited Kca, reducing NP0, the product of number of channels and channel open probability, by 95%. The apparent inhibition constant for this AA effect was 425 nM. AA inhibited Kca in the presence of both indomethacin and nordihydroguaiaretic acid, blockers of the cyclooxygenase and lipoxygenase pathways. In the presence of albumin, the effect of AA on Kca was reversed. A similar effect of AA was observed on Kca during outside-out recording. We determined also the effect of the cis-unsaturated fatty acid linoleate, the trans-unsaturated fatty acid elaidate, and the saturated fatty acid myristate. At 3 microM, all of these fatty acids inhibited Kca, reducing NP0 by 72-86%. Finally, the effect of the cytosolic phospholipase A2 inhibitor arachidonyltrifluoromethyl ketone (AACOCF3) on the carbachol-induced short-circuit current (Isc) response was determined. In the presence of AACOCF3, the peak carbachol-induced Isc response was increased approximately 2.5-fold. Our results suggest that AA generation induced by Ca(2+)-mediated agonists may contribute to the dissociation observed between the rise in intracellular Ca2+ evoked by these agonists and the associated Cl- secretory response.
尽管细胞内Ca2+持续升高，但结肠细胞对Ca(2+)介导的激动剂的Cl-分泌反应是短暂的。我们评估了被认为可限制Ca(2+)介导的Cl-分泌的第二信使对结肠分泌细胞T84基底外侧膜上Ca(2+)依赖性K+通道（Kca）的影响。蛋白激酶C（PKC）和肌醇四磷酸（1,3,4,5或3,4,5,6形式）均未影响外翻式膜片钳记录中的Kca。相反，花生四烯酸（AA；3 microM）强烈抑制Kca，使通道数量与通道开放概率的乘积NP0降低95%。这种AA效应的表观抑制常数为425 nM。在环氧化酶和脂氧合酶途径的阻滞剂吲哚美辛和去甲二氢愈创木酸存在的情况下，AA仍抑制Kca。在白蛋白存在时，AA对Kca的作用被逆转。在膜片外翻式记录过程中，观察到AA对Kca有类似作用。我们还确定了顺式不饱和脂肪酸亚油酸、反式不饱和脂肪酸反油酸和饱和脂肪酸肉豆蔻酸的作用。在3 microM时，所有这些脂肪酸均抑制Kca，使NP0降低72 - 86%。最后，测定了胞质磷脂酶A2抑制剂花生四烯酰三氟甲基酮（AACOCF3）对卡巴胆碱诱导的短路电流（Isc）反应的影响。在AACOCF3存在时，卡巴胆碱诱导的Isc反应峰值增加约2.5倍。我们的结果表明，Ca(2+)介导的激动剂诱导产生的AA可能导致了这些激动剂引起的细胞内Ca2+升高与相关的Cl-分泌反应之间的解离。
