Merlin D, Guo X, Laboisse C L, Hopfer U
Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA.
Am J Physiol. 1995 Jun;268(6 Pt 1):C1503-11. doi: 10.1152/ajpcell.1995.268.6.C1503.
The mechanism of regulated Cl- secretion was evaluated in the mucin-secreting cell line HT29-Cl.16E by transepithelial electrophysiology and fura 2 measurements of cytosolic Ca2+. Carbachol by itself was a weak secretagogue, but augmented adenosine 3',5'-cyclic monophosphate (cAMP)-mediated secretion more than twofold, consistent with activation of a rate-limiting K+ conductance. To characterize this conductance, monolayers were apically permeabilized with amphotericin B. At least two types of K+ conductances were identified. One type was activated by elevated cytosolic cAMP levels and inhibited by Ba2+ (inhibitor constant 0.3 mM) in the basolateral solution but was not affected by quinidine or elevated cytosolic Ca2+. The other type was activated by carbachol via cytosolic Ca2+ and was partially inhibited by quinidine (60% inhibition by 2.5 mM quinidine) but was not affected by Ba2+ up to 1 mM. Both conductances appear to be involved in active, transepithelial Cl- secretion in intact monolayers but under different conditions because 1) the cAMP-stimulated short-circuit current (Isc) can be partially inhibited by 1 mM Ba2+ (50%) but not quinidine, 2) the Ba2+ inhibition does not affect the carbachol-induced increase in Isc in cells with elevated cAMP levels, and 3) the carbachol-dependent Isc can be inhibited by quinidine. Therefore, the contribution of the cAMP-dependent K+ conductance appears important for maintaining the membrane potential and therewith Cl- secretion when cAMP is the only messenger for secretion signals, whereas the Ca(2+)-dependent K+ conductance is responsible for the carbachol-stimulated increase in Isc.
通过跨上皮电生理学和用fura 2测量胞质Ca2+，在分泌粘蛋白的细胞系HT29-Cl.16E中评估了调节性Cl-分泌的机制。卡巴胆碱本身是一种弱促分泌剂，但能使腺苷3',5'-环磷酸（cAMP）介导的分泌增加两倍以上，这与限速K+电导的激活一致。为了表征这种电导，用两性霉素B使单层细胞的顶端通透。鉴定出至少两种类型的K+电导。一种类型由胞质cAMP水平升高激活，并在基底外侧溶液中被Ba2+（抑制常数0.3 mM）抑制，但不受奎尼丁或胞质Ca2+升高的影响。另一种类型由卡巴胆碱通过胞质Ca2+激活，并被奎尼丁部分抑制（2.5 mM奎尼丁抑制60%），但在高达1 mM的Ba2+作用下不受影响。两种电导似乎都参与完整单层细胞中活跃的跨上皮Cl-分泌，但在不同条件下，因为：1）cAMP刺激的短路电流（Isc）可被1 mM Ba2+（50%）部分抑制，但不受奎尼丁抑制；2）Ba2+抑制不影响cAMP水平升高的细胞中卡巴胆碱诱导的Isc增加；3）卡巴胆碱依赖性Isc可被奎尼丁抑制。因此，当cAMP是分泌信号的唯一信使时，cAMP依赖性K+电导对于维持膜电位以及Cl-分泌似乎很重要，而Ca(2+)依赖性K+电导则负责卡巴胆碱刺激的Isc增加。
