Swelling-induced, CFTR-independent ATP release from a human epithelial cell line -: Lack of correlation with volume-sensitive Cl- channels
Swelling-induced, CFTR-independent ATP release from a human epithelial cell line -: Lack of correlation with volume-sensitive Cl- channels
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DOI：
10.1085/jgp.114.4.525
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发表时间：
1999-10-01
影响因子：
3.8
通讯作者：
Okada, Y
中科院分区：
文献类型：
--
作者：
Hazama, A;Shimizu, T;Okada, Y
为了研究肿胀诱导的ATP释放途径和体积敏感性Cl（-）通道之间的可能关系，我们测量了在人上皮细胞系Intestine 407中渗透性肿胀和全细胞体积敏感性Cl（-）电流时释放的ATP的细胞外浓度，Intestine 407缺乏囊性纤维化跨膜传导调节因子（CFTR）的表达。在低渗激发（56-80%渗透压摩尔浓度）后几分钟内，通过胰蛋白酶/荧光素酶试验观察到ATP的显著释放。一种羧酸盐类似物Cl（-）通道阻滞剂; 5-硝基-2-（3-苯丙基氨基）-苯甲酸酯，以浓度依赖性方式抑制ATP释放，半最大抑制浓度为6.3 μ M。然而，溶胀诱导的STP释放不受芪衍生物Cl（-）通道阻断剂4-乙酰氨基-4 '-异硫氰酸芪（100 μ M）的影响。格列本脲（500 μ M）和花生四烯酸（100 μ M）已知可阻断体积敏感性外向整流（VSOR）Cl（-）通道，但在抑制肿胀诱导的ATP释放方面也无效。Gd（3+）是牵张激活通道的阻断剂，可浓度依赖性地抑制肿胀诱导的ATP释放，而三价镧系元素则不能抑制VSOR Cl（-）电流。在渗透性溶胀时，通过使用PCl 2细胞中表达的P2 X（2）受体的生物传感器技术，发现细胞表面附近的局部ATP浓度达到类似于13 μ M。我们已经提出了抑制肠407细胞肿胀诱导的STP释放的抗体。早期用抗体处理几乎完全抑制肿胀诱导的STP释放，而VSOR Cl（-）通道的活性不受抗体预处理的影响。综合以上结果，我们得出以下结论：第一，在CFTR缺失的人上皮细胞系中，渗透性肿胀诱导ATP释放，并使细胞表面ATP浓度增加超过10 μ M，这足以刺激嘌呤能受体;第二，ATP释放途径不同于体积敏感性外向整流Cl（-）通道的孔;第三，ATP的释放不是激活Cl（-）通道的先决条件。
To examine a possible relation between the swelling-induced ATP release pathway and the volume-sensitive Cl(-) channel, we measured the extracellular concentration of ATP released upon osmotic swelling and whole-cell volume-sensitive Cl(-) currents in a human epithelial cell line, Intestine 407, which lacks expression of cystic fibrosis transmembrane conductance regulator (CFTR). Significant release of ATP was observed within several minutes after a hypotonic challenge (56-80% osmolality) by the luciferin/luciferase assay. A carboxylate analogue Cl(-) channel blocker; 5-nitro-2-(3-phenylpropylamino)-benzoate, suppressed ATP release in a concentration-dependent manner with a half-maximal inhibition concentration of 6.3 mu M. However, swelling-induced STP release was not affected by a stilbene-derivative Cl(-) channel blocker, 4-acetamido-4'-isothiocyanostilbene at 100 mu M. Glibenclamide (500 mu M) and arachidonic acid (100 mu M), which are known to block volume-sensitive outwardly rectifying (VSOR) Cl(-) channels, were also ineffective in inhibiting the swelling-induced ATP release. Gd(3+), a putative blocker of stretch-activated channels, inhibited swelling-induced ATP release in a concentration-dependent manner, whereas the trivalent lanthanide failed to inhibit VSOR Cl(-) currents. Upon osmotic swelling, the local ATP concentration in the immediate vicinity of the cell surface was found to reach similar to 13 mu M by a biosensor technique using P2X(2) receptors expressed in PCl2 cells. We have raised antibodies that inhibit swelling-induced STP release from Intestine 407 cells. Earlier treatment with the antibodies almost completely suppressed swelling-induced STP release whereas the activity of VSOR Cl(-) channel was not affected by pretreatment with the antibodies. Taking the above results together, the following conclusions were reached: first, in a CFTR-lacking human epithelial cell line, osmotic swelling induces ATP release and increases the cell surface ATP concentration over 10 mu M, which is high enough to stimulate purinergic receptors; second, the pathway of ATP release is distinct from the pore of the volume-sensitive outwardly rectifying Cl(-) channel; and third, the ATP release is not a prerequisite to activation of the Cl(-) channel.