Epac1 mediates protein kinase A-independent mechanism of forskolin-activated intestinal chloride secretion.
Epac1 mediates protein kinase A-independent mechanism of forskolin-activated intestinal chloride secretion.
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DOI：
10.1085/jgp.200910339
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发表时间：
2010-01
期刊：
影响因子：
--
通讯作者：
Tse CM
中科院分区：
文献类型：
--
作者：
Hoque KM;Woodward OM;van Rossum DB;Zachos NC;Chen L;Leung GP;Guggino WB;Guggino SE;Tse CM
环磷酸腺苷(CAMP)和细胞内钙离子([−]i)均可刺激肠道Cl-cAMP的分泌。最近的研究表明，蛋白激酶A(PKA)和cAMP直接激活的交换蛋白(EPAC)是cAMP的下游靶标。因此，我们检测了PKA和EPAC是否都参与了佛司可林/cAMP刺激的Cl-−的分泌。用人小肠T84细胞和小鼠小肠测定激动剂刺激的Cl-−分泌的短路电流。−抑制剂H89(1µM)和1，2-双(邻氨基苯氧基)-乙烷-N，N，N‘，N’-四乙酸四乙酰氧甲酯(BAPTA-AM；25µM)的相加作用可完全抑制FSK刺激的Cl-PKA分泌。FSK和EPAC激动剂8-PCPT-2‘-O-Me-cAMP(50µM)均可使完整或基底侧膜通透性的T84细胞和小鼠回肠片[Ca~(2+)]i升高，激活RAS相关蛋白2，并诱导Cl-−的分泌。8-PCPT-2‘-O-Me-cAMP的作用可被BAPTA-AM完全消除，但不能被H89所消除。相反，沉默Epac1的T84细胞对FSK的ISC反应降低，这种反应可被H89完全抑制，但不能被磷脂酶C抑制剂U73122或BAPTA-AM抑制。8-PCPT-2‘-O-Me-cAMP对Cl-−分泌的刺激作用不能被囊性纤维化跨膜电导阻滞剂和格列本脲所阻断，提示cAMP不参与囊性纤维化跨膜电导通道。8-PCPT-2‘-O-Me-cAMP对中国仓鼠卵巢细胞瞬时表达人CFTR通道的全细胞膜片钳记录的CFTR电流无影响，证实了这一点。此外，对安装在Ussing小室中的T84细胞依赖于Epac1的Cl-−电导的生物物理特征表明，该电导是超极化激活的、向内整流的，并显示出CL-−>；BR−>；i−通透性序列。这些结果提示，EPAC-Rap-PLC-[Ca~(2+)]i信号通路参与了cAMP刺激的Cl-−的分泌，而cAMP刺激的Cl-−的分泌是通过一种新的、以前未被描述的Cl-DNA通道来实现的。
Intestinal Cl− secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca2+]i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl− secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (Isc) measurement in response to agonist-stimulated Cl− secretion. FSK-stimulated Cl− secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 µM), and the [Ca2+]i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 µM). Both FSK and the Epac activator 8-pCPT-2’-O-Me-cAMP (50 µM) elevated [Ca2+]i, activated Ras-related protein 2, and induced Cl− secretion in intact or basolateral membrane–permeabilized T84 cells and mouse ileal sheets. The effects of 8-pCPT-2’-O-Me-cAMP were completely abolished by BAPTA-AM, but not by H89. In contrast, T84 cells with silenced Epac1 had a reduced Isc response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2’-O-Me-cAMP on Cl− secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2’-O-Me-cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl− conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl−>Br−>I− permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca2+]i signaling pathway is involved in cAMP-stimulated Cl− secretion, which is carried by a novel, previously undescribed Cl− channel.
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影响因子：
3.5
作者：
Doolen, S;Zahniser, NR
通讯作者：
Zahniser, NR
影响因子：
2.1
作者：
Barrett, Kim E.
通讯作者：
Barrett, Kim E.
DOI：
10.1073/pnas.88.14.6003
发表时间：
1991-07-01
影响因子：
11.1
作者：
ANDERSON, MP;WELSH, MJ
通讯作者：
WELSH, MJ
影响因子：
--
作者：
DENNING, GM;CLARK, RA;WELSH, MJ
通讯作者：
WELSH, MJ
DOI：
10.1152/ajpcell.2000.279.6.c1787
发表时间：
2000-12-01
影响因子：
5.5
作者：
Gyömörey, K;Yeger, H;Bear, CE
通讯作者：
Bear, CE