Al-Nakkash L, Hwang T C
Department of Physiology, Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO-65211, USA.
Pflugers Arch. 1999 Mar;437(4):553-61. doi: 10.1007/s004240050817.
The cAMP-dependent activation of the cystic fibrosis transmembrane conductance regulator (CFTR) and its modulation through inhibition of phosphodiesterases (PDE) were studied with the cell-attached patch-clamp technique in Calu-3 cells (expressing endogenous CFTR) and NIH3T3 cells [expressing either wild-type (Wt)-CFTR or DeltaF508-CFTR]. In Calu-3 cells, CFTR current was augmented by increasing concentrations of 8-(4-chlorophenylthio)-adenosine 3', 5'-cyclic monophosphate (CPT-cAMP) and reached a saturating level at >/=60 microM. Varying the forskolin concentration also modulated CFTR activity; 10 microM was maximally effective since supplemental application of 200 microM CPT-cAMP had no additional effect. Activation of CFTR by increasing the cAMP concentration occurs through an increase of the NPo (product of the number of functional channels and the open probability) since the single-channel amplitude remains unchanged. In Calu-3 and NIH3T3-Wt cells, PDE inhibitors, milrinone (100 microM), 8-cyclopentyl-1, 3-dipropylxanthine (CPX, 25 microM), and 3-isobutyl-1-methylxanthine (IBMX, 200 microM), did not enhance CFTR current initially activated with 10 microM forskolin, but each potentiated CFTR activity elicited with a submaximal forskolin concentration (e.g., 100 nM) and prolonged the deactivation of CFTR channel current upon removal of forskolin. Millimolar IBMX increased the NPo of both Wt- and DeltaF508-CFTR even under maximal cAMP stimulation. Quantitatively, these effects of millimolar IBMX on NPo approximate those of genistein, which potentiates the cAMP-dependent CFTR activity via a mechanism that does not involve increases in cellular cAMP. Thus, depending on the concentration, PDE inhibitors may affect CFTR through different mechanisms.
运用细胞贴附式膜片钳技术，在Calu-3细胞（表达内源性囊性纤维化跨膜传导调节因子，CFTR）和NIH3T3细胞[表达野生型（Wt）-CFTR或DeltaF508-CFTR]中，研究了环磷酸腺苷（cAMP）依赖性激活囊性纤维化跨膜传导调节因子（CFTR）及其通过抑制磷酸二酯酶（PDE）进行的调节。在Calu-3细胞中，随着8-（4-氯苯硫基）-腺苷3'，5'-环磷酸酯（CPT-cAMP）浓度增加，CFTR电流增强，且在≥60微摩尔时达到饱和水平。改变福斯高林浓度也可调节CFTR活性；10微摩尔最为有效，因为补充200微摩尔CPT-cAMP没有额外作用。由于单通道幅度保持不变，通过增加cAMP浓度激活CFTR是通过增加NPo（功能性通道数量与开放概率的乘积）实现的。在Calu-3和NIH3T3-Wt细胞中，PDE抑制剂米力农（100微摩尔）、8-环戊基-1,3-二丙基黄嘌呤（CPX，25微摩尔）和3-异丁基-1-甲基黄嘌呤（IBMX，200微摩尔），最初不会增强用10微摩尔福斯高林激活的CFTR电流，但每种抑制剂都会增强用次最大福斯高林浓度（如100纳摩尔）引发的CFTR活性，并在去除福斯高林后延长CFTR通道电流的失活时间。毫摩尔浓度的IBMX即使在最大cAMP刺激下也会增加Wt-CFTR和DeltaF508-CFTR的NPo。从数量上看，毫摩尔浓度的IBMX对NPo的这些影响与金雀异黄素的影响相近，金雀异黄素通过一种不涉及细胞内cAMP增加的机制增强cAMP依赖性CFTR活性。因此，根据浓度不同，PDE抑制剂可能通过不同机制影响CFTR。
