Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44120, USA.
Am J Physiol Cell Physiol. 2010 Feb;298(2):C386-96. doi: 10.1152/ajpcell.00430.2009. Epub 2009 Nov 11.
We previously reported that ATP release from 1321N1 human astrocytoma cells could be stimulated either by activation of G protein-coupled receptors (GPCR) or by hypotonic stress. Cheema et al. (Cheema TA, Ward CE, Fisher SK. J Pharmacol Exp Ther 315: 755-763, 2005) have demonstrated that thrombin activation of protease-activated receptor 1 (PAR1) in 1321N1 cells and primary astrocytes acts synergistically with hypotonic stress to gate the opening of volume-sensitive organic osmolyte and anion channels (VSOAC) and that hypertonic stress strongly inhibits PAR1 gating of VSOAC. We tested the hypothesis that a VSOAC-type permeability might comprise a GPCR-regulated pathway for ATP export by determining whether PAR1-sensitive ATP release from 1321N1 cells is similarly potentiated by hypotonicity but suppressed by hypertonic conditions. Strong hypotonic stress by itself elicited ATP release and positively modulated the response to thrombin. Thrombin-dependent ATP release was also potentiated by mild hypotonic stress that by itself did not stimulate ATP export. Notably, PAR1-sensitive ATP export was greatly inhibited in hypertonic medium. Neither the potency nor efficacy of thrombin as an activator of proximal PAR1 signaling was affected by hypotonicity or hypertonicity. 1,9-Dideoxyforskolin and carbenoxolone similarly attenuated PAR1-dependent ATP release and suppressed the PAR1-independent ATP elicited by strong hypotonic stress. Probenecid attenuated PAR1-stimulated ATP release under isotonic but not mild hypotonic conditions and had no effect on PAR1-independent release stimulated by strong hypotonicity. PAR1-dependent ATP export under all osmotic conditions required concurrent signaling by Ca(2+) mobilization and Rho-GTPase activation. In contrast, PAR1-independent ATP release triggered by strong hypotonicity required neither of these intracellular signals. Thus, we provide the new finding that GPCR-regulated ATP release from 1321N1 astrocytoma cells is remarkably sensitive to both positive and negative modulation by extracellular osmolarity. This supports a model wherein GPCR stimulation and osmotic stress converge on an ATP release pathway in astrocytes that exhibits several features of VSOAC-type channels.
我们之前报道过，1321N1 人星形细胞瘤细胞中的 ATP 释放可以通过激活 G 蛋白偶联受体（GPCR）或低渗应激来刺激。Cheema 等人（Cheema TA、Ward CE、Fisher SK。J Pharmacol Exp Ther 315：755-763，2005）已经证明，1321N1 细胞和原代星形胶质细胞中凝血酶激活蛋白酶激活受体 1（PAR1）与低渗应激协同作用，打开体积敏感的有机渗透物和阴离子通道（VSOAC），并且高渗应激强烈抑制 PAR1 门控 VSOAC。我们通过确定 PAR1 敏感的 1321N1 细胞中 ATP 释放是否同样受到低渗性增强但受到高渗条件抑制，来检验 VSOAC 型通透性是否构成 GPCR 调节的 ATP 外排途径的假说。强烈的低渗应激本身引发 ATP 释放，并正向调节对凝血酶的反应。低渗应激本身不会刺激 ATP 外排，但也增强了依赖于凝血酶的 ATP 释放。值得注意的是，PAR1 敏感的 ATP 外排在高渗培养基中受到很大抑制。低渗性或高渗性均不影响凝血酶作为近端 PAR1 信号激活剂的效力或功效。1,9-二脱氧佛司可林和 carbenoxolone 也同样减弱了 PAR1 依赖性 ATP 释放，并抑制了由强烈低渗应激引起的 PAR1 非依赖性 ATP 释放。Probenecid 在等渗条件下减弱了 PAR1 刺激的 ATP 释放，但在低渗条件下没有作用，并且对强低渗性刺激引起的 PAR1 非依赖性释放没有影响。在所有渗透条件下，PAR1 依赖性 ATP 外排都需要 Ca（2+）动员和 Rho-GTPase 激活的并发信号。相比之下，由强烈低渗应激触发的 PAR1 非依赖性 ATP 释放不需要这两种细胞内信号。因此，我们提供了新的发现，即来自 1321N1 星形细胞瘤的 GPCR 调节的 ATP 释放对细胞外渗透压的正调节和负调节非常敏感。这支持了这样一种模型，即 GPCR 刺激和渗透应激在星形胶质细胞中的 ATP 释放途径上汇聚，该途径具有 VSOAC 型通道的几个特征。
