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J Biol Chem, Vol. 273, Issue 24, 14906-14911, June 12, 1998
From the Departments of Pediatrics and Medicine, Children's
Hospital and the University of Colorado Health Sciences Center, Denver,
Colorado 80262 and the § Department of Physiology and
Biophysics, University of Alabama, Birmingham, Alabama 35294
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 µM). 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 µM). 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.
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