Hypotonic treatment evokes biphasic ATP release across the basolateral membrane of cultured renal epithelia (A6)
Hypotonic treatment evokes biphasic ATP release across the basolateral membrane of cultured renal epithelia (A6)
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DOI：
10.1113/jphysiol.2002.026641
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发表时间：
2002-12-01
影响因子：
5.5
通讯作者：
Van Driessche, W
中科院分区：
文献类型：
--
作者：
Jans, D;Srinivas, SP;Van Driessche, W
在肾 A6 上皮细胞中，急性低渗休克通过对 P2 受体拮抗剂苏拉明敏感的机制引起细胞内 Ca2+ 浓度短暂增加 ([Ca2+](i)，仅应用于基底外侧边界。通过用荧光素-荧光素酶 (LL) 发光检查跨基底外侧膜的 ATP 释放，进一步表征了这一发现。安装了在渗透性支持物上培养的偏振上皮单层。我们开发了 LL 脉冲方案来确定基底外侧隔室中 ATP 释放 (R-ATP) 的速率，因此，在短时间内（90 秒）重复中断基底外侧边界，以测量 R-ATP 作为 LL 发光检测到的 ATP 含量初始上升的斜率。 fmol min(-1)。基底外侧渗透压从 260 突然降低至 I A 140 mosmol (kg H2O)(-1) R-ATP 迅速达到峰值 1.89 +/- 0.11 pmol min(-1)（R-ATP（峰值）升高 R ATP，随后达到 0.51 +/- 0.07 pmol min-' (RAP'Tap)。 R eTp 和 RAPTP 值随着稀释程度的增加而增加。同样，在基底外侧渗透压逐渐稀释至 140 mosmol (kg H2O)(-1) 后，记录到稳定的 ATP 释放（在没有细胞肿胀的情况下）。因此，稳定的 ATP 释放与膜拉伸无关，但可能是由细胞体积调节过程中细胞内离子强度的降低引起的。对响应外源 ATP 和基底外侧渗透压降低的峰值 [Ca2+](i) 增加的剂量反应曲线的独立测定表明，模拟渗透压降低所需的外源 ATP 浓度与 R-ATP(峰值) 呈线性相关。 ATP 释放和快速 Ca2+ 瞬变也通过从基底外侧灌注液中去除 Cl- 来抑制这两种现象得到证实。这些数据与低渗期间，基底外侧 ATP 释放激活嘌呤能受体的观点一致，这是低渗休克期间 [Ca2+](i) 苏拉明敏感升高的基础。
In renal A6 epithelia, an acute hypotonic shock evokes a transient increase in the intracellular Ca2+ concentration ([Ca2+](i) through a mechanism that is sensitive to the P2 receptor antagonist suramin, applied to the basolateral border only. This finding has been further characterized by examining ATP release across the basolateral membrane with luciferin-luciferase (LL) luminescence. Polarized epithelial monolayers, cultured on permeable supports were mounted in an Ussing-type chamber. We developed a LL pulse protocol to determine the rate of ATP release (R-ATP) in the basolateral compartment. Therefore, the perfusion at the basolateral border was repetitively interrupted during brief periods (90 s) to measure R-ATP as the slope of the initial rise in ATP content detected by LL luminescence. Under isosmotic conditions, 1 mul of A6 cells released ATP at a rate of 66 +/- 8 fmol min(-1). A sudden reduction of the basolateral osmolality from 260 to I A 140 mosmol (kg H2O)(-1) R-ATP rapidly to a peak value of 1.89 +/- 0.11 pmol min(-1) (R-ATP(peak) elevated R ATP followed by a plateau phase reaching 0.51 +/- 0.07 pmol min-' (RAP'Tap). Both R eTp and RAPTP values increased with the degree of dilution. The magnitude of R-ATP(plat) remained constant as long as the hyposmolality was maintained. Similarly, a steady ATP release of 0.78 +/- 0.08 pmol min(-1) was recorded after gradual dilution of the basolateral osmolality to 140 mosmol (kg H2O)(-1). This R-ATP value, induced in the absence of cell swelling, is comparable to RVTATPplat. Therefore, the steady ATP release is unrelated to membrane stretching, but possibly caused by the reduction of intracellular ionic strength during cell volume regulation. Independent determinations of dose-response curves for peak [Ca2+](i) increase in response to exogenous ATP and basolateral hyposmolality demonstrated that the exogenous ATP concentration, required to mimic the osmotic reduction, peak [Ca2+] (i) was linearly correlated with R-ATP(peak). The link between the ATP release and the fast Ca2+ transient was also demonstrated by the depression of both phenomena by Cl- removal from the basolateral perfusate. The data are consistent with the notion that during hypotonicity, basolateral ATP release activates purinergic receptors, which underlies the suramin-sensitive rise of [Ca2+](i) during the hyposmotic shock.