Mechanisms of cystic fibrosis transmembrane conductance regulator activation by S-nitrosoglutathione
Mechanisms of cystic fibrosis transmembrane conductance regulator activation by S-nitrosoglutathione
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DOI：
10.1074/jbc.m513231200
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发表时间：
2006-04-07
影响因子：
4.8
通讯作者：
Matalon, S
中科院分区：
文献类型：
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作者：
Chen, L;Patel, RP;Matalon, S
我们研究了S-亚硝基谷胱甘肽（GSNO）改变囊性纤维化跨膜传导调节因子（CFTR）介导的跨Calu-3细胞（一种广泛使用的人气道腺浆细胞模型）的氯（Cl-）分泌的机制。将在空气-液体界面下生长的Calu-3细胞的汇合单层安装在用于测量氯离子短路电流（I-sc）和跨上皮电阻（R-t）的Ussing室中。将GSNO加入到这些室的顶端室中导致I-sc的显著和持续增加，IC 50为3.2 +/- 1 μ M（平均值+/- 1 S.E.; n = 6）。加入格列本脲或用可溶性鸟苷酸环化酶抑制剂1H-（1，2，4）-恶二唑并[4，3-a]喹喔啉-1-酮预处理Calu-3细胞完全阻止了GSNO诱导的I-sc增加。相反，BAY 41-2272（sGC刺激剂）以剂量-反应方式增加I-sc。在含有Calu-3单层的Ussing室的顶室中加入两种磷酸酶（PP 2A 1，PP 2A 2），可逆转GSNO增加的I-sc。在GSNO之前或之后将氧合肌红蛋白（oxy-Mb，300 μ M）添加到尤辛室的顶部隔室中，可以完全阻止或立即逆转I-SC的增加。然而，较低浓度的氧合肌红蛋白（1-10 μ M）足以清除培养基中的NO（通过使用ISO-NO计直接测量尤辛室中的NO来评估）部分降低I-SC。Oxy-Mb没有逆转加入GSNO和半胱氨酸（50 μ M）后I-sc的增加。这些发现表明，GSNO刺激Cl分泌通过cGMP依赖性和cGMP非依赖性机制。
We investigated the mechanisms by which S-nitrosoglutathione (GSNO) alters cystic fibrosis transmembrane conductance regulator (CFTR) mediated chloride (Cl-) secretion across Calu-3 cells, an extensively used model of human airway gland serous cells. Confluent monolayers of Calu-3 cells, grown under an air-liquid interface, were mounted in Ussing chambers for the measurements of chloride short circuit current (I-sc) and trans-epithelial resistance (R-t). Addition of GSNO into the apical compartment of these chambers resulted in significant and sustained increase of I-sc with an IC50 of 3.2 +/- 1 mu M (mean +/- 1 S.E.; n = 6). Addition of either glibenclamide or pre-treatment of Calu-3 cells with the soluble guanylate cyclase inhibitor 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one totally prevented the GSNO-induced increase of I-sc. Conversely, BAY 41-2272, a sGC stimulator, increased I-sc in a dose-response fashion. The GSNO increase of I-sc was reversed by addition of two phosphatases (PP2A1, PP2A2) into the apical compartment of Ussing chambers containing Calu-3 monolayers. Oxy-myoglobin (oxy-Mb, 300 mu M) added into the apical compartment of Ussing chambers either prior or after GSNO either completely prevented or immediately reversed the increase of I-sc. However, smaller concentrations of oxy-Mb (1-10 mu M), sufficient to scavenge NO in the medium (as assessed by direct measurement of NO in the Ussing chamber using an ISO-NO meter) decreased I-sc partially. Oxy-Mb did not reverse the increase of I-sc following addition of GSNO and cysteine (50 mu M). These findings indicate that GSNO stimulates Cl secretion via both cGMP-dependent and cGMP-independent mechanisms.