Feranchak A P, Roman R M, Schwiebert E M, Fitz J G
Departments of Pediatrics and Medicine, Children's Hospital and the University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
J Biol Chem. 1998 Jun 12;273(24):14906-11. doi: 10.1074/jbc.273.24.14906.
Regulated changes in cell volume represent a signal that modulates a broad range of cell and organ functions. In HTC hepatoma cells, increases in volume are coupled to membrane ion permeability through a pathway involving (i) ATP efflux, (ii) autocrine stimulation of P2 receptors, and (iii) increases in anion permeability and Cl- efflux, contributing to recovery of volume toward basal values. Based on recent evidence that cell volume increases also stimulate phosphoinositide kinases, the purpose of these studies was to determine if phosphatidylinositol 3-kinase (PI 3-kinase) modulates these pathways. Exposure of cells to hypotonic buffer (20 or 40% less NaCl) caused an initial increase in cell volume and stimulated a rapid increase in ATP release. Subsequent opening of Cl- channels was followed by recovery of cell volume toward basal values, despite the continuous presence of hypotonic buffer. Inhibition of PI 3-kinase with wortmannin (Ki = 3 nM) significantly inhibited both the rate of volume recovery and activation of Cl- currents; similar results were obtained with LY294002 (10 microM). Additionally, current activation was inhibited by intracellular dialysis with antibodies specific for the 110-kDa catalytic subunit of PI 3-kinase. Since release of ATP is a critical element in the volume-regulatory pathway, the role of PI 3-kinase on volume-stimulated ATP release was assessed. Both wortmannin and LY294002 decreased basal and volume-stimulated ATP permeability but had no effect on the current response to exogenous ATP (10 microM). These findings indicate that PI 3-kinase plays a significant role in regulation of cell volume and suggest that the effects are mediated in part through modulation of cellular ATP release.
细胞体积的调节性变化代表一种信号，可调节多种细胞和器官功能。在HTC肝癌细胞中，体积增加通过以下途径与膜离子通透性相关联：（i）ATP外流；（ii）自分泌刺激P2受体；（iii）阴离子通透性增加和Cl-外流，有助于使体积恢复至基础值。基于最近细胞体积增加也刺激磷酸肌醇激酶的证据，这些研究的目的是确定磷脂酰肌醇3激酶（PI 3激酶）是否调节这些途径。将细胞暴露于低渗缓冲液（NaCl减少20%或40%）导致细胞体积最初增加，并刺激ATP释放迅速增加。随后Cl-通道开放，尽管持续存在低渗缓冲液，细胞体积仍恢复至基础值。用渥曼青霉素（Ki = 3 nM）抑制PI 3激酶可显著抑制体积恢复速率和Cl-电流激活；LY294002（10 microM）也得到类似结果。此外，用针对PI 3激酶110 kDa催化亚基的特异性抗体进行细胞内透析可抑制电流激活。由于ATP释放是体积调节途径中的关键因素，因此评估了PI 3激酶对体积刺激的ATP释放的作用。渥曼青霉素和LY294002均降低基础和体积刺激的ATP通透性，但对外源ATP（10 microM）的电流反应无影响。这些发现表明PI 3激酶在细胞体积调节中起重要作用，并提示其作用部分通过调节细胞ATP释放介导。
