cAMP activates an ATP-conductive pathway in cultured shark rectal gland cells
cAMP activates an ATP-conductive pathway in cultured shark rectal gland cells
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DOI：
10.1152/ajpcell.1997.272.2.c466
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发表时间：
1997-02-01
影响因子：
5.5
通讯作者：
Ausiello, DA
中科院分区：
文献类型：
--
作者：
Cantiello, HF;Jackson, GR;Ausiello, DA
ATP转运和释放到细胞外环境的分子机制在很大程度上是未知的。为探讨鲨鱼直肠腺细胞是否存在内源性三磷酸腺苷传导通路(S)，将膜片钳技术应用于鲨鱼直肠腺细胞的原代培养。用细胞内三羟甲基氨基甲烷(Tris)或含镁离子的三磷酸腺苷盐(200 mM标称三磷酸腺苷)和280 mM氯化钠溶液获得全细胞电流。对于镁三磷酸腺苷的外移，基础电流显示出相当大的三磷酸腺苷通透性。3‘，5’-环磷酸腺苷(CAMP)刺激显著增加全细胞电导(细胞内Tris-ATP或MgATP)。在cAMP激活后，也观察到对称的全细胞ATP电流，这与作为主要电荷载体的ATP相一致。CAMP-通道阻断剂4，4‘-二异硫氰基二苯乙烯-2，2’-二磺酸、二苯胺-2-羧酸盐和菲-9-羧酸对cAMP诱导的ATP电流不敏感，但易被硝苯地平(400mM)和格列本脲(400mM)阻断。通过对自发性和蛋白激酶A激活后的自发性和蛋白激酶A激活后的MgATP或Tris-ATP电流的单通道分析，进一步评估了电扩散ATP运动的性质。单通道ATP电流可被硝苯地平或格列本脲抑制。因此，SRG细胞在cAMP刺激前后都表达内源性的ATP通透通路。SRG细胞的电扩散运动可能在细胞内ATP向细胞外环境的运输和输送中发挥重要作用，这可能有助于协调该细胞模型中上皮细胞分泌反应的动力学。
The molecular mechanisms associated with ATP transport and release into the extracellular milieu are largely unknown. To assess the presence of endogenous ATP-conductive pathway(s) in shark rectal gland (SRG) cells, patch-clamp techniques were applied to primary cultures of SRG cells. Whole cell currents were obtained with either intracellular tris(hydroxymethyl)-aminomethane (Tris) or Mg2+ salts of ATP (200 mM nominal ATP) and 280 mM NaCl bathing solution. Basal currents showed a sizable ATP permeability for outward movement of MgATP. Adenosine 3',5'-cyclic monophosphate (cAMP) stimulation significantly increased the whole cell conductance (with either intracellular Tris-ATP or MgATP). Symmetrical whole cell ATP currents were also observed after cAMP activation, thus consistent with ATP as the main charge carrier. The cAMP-inducible ATP currents were insensitive to the Cl- channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, diphenylamine-2-carboxylate, and anthracene-9-carboxylic acid but were readily blocked by nifedipine (400 mu M) and glibenclamide (400 mu M). The nature of the electrodiffusional ATP movement was further assessed by single-channel analysis of either MgATP or Tris-ATP currents ill excised inside-out patches, both spontaneous and after activation with protein kinase A. Single-channel ATP currents were inhibited by either nifedipine or glibenclamide. Thus SRG cells express endogenous ATP-permeable pathways both before and after cAMP stimulation. Electrodiffusional ATP movement by SRG cells may play a significant role in the transport and delivery of cellular ATP to the extracellular milieu, which may help coordinate the dynamics of the epithelial secretory response in this cell model.