Abstract
In a model liver cell line, recovery from swelling is mediated by a sensitive autocrine pathway involving conductive release of ATP, P2 receptor stimulation, and opening of membrane Cl- channels (Wang, Y., Roman, R. M., Lidofsky, S. D., and Fitz, J. G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12020-12025). However, the mechanisms coupling changes in cell volume to ATP release are not known. Based on evidence that certain ATP-binding cassette (ABC) proteins may function as ATP channels or channel regulators, we evaluated the potential role of ABC proteins by comparing ATP release and volume regulation in rat HTC and HTC-R hepatoma cells, the latter of which overexpress Mdr proteins. In both cell types, Cl- current activation (ICl-swell) and volume recovery following swelling were dependent on conductive ATP efflux. The rate of volume recovery was approximately 6-fold faster in HTC-R cells compared with HTC cells. This effect is likely due to enhanced ABC protein-dependent ATP release since (i) ICl-swell and cell volume recovery were eliminated by inhibition of P-glycoprotein transport (20 microM verapamil and 15 microM cyclosporin A); (ii) swelling-induced Cl- current density was similar in both cell types (approximately -50 pA/pF; not significant); and (iii) ATP conductance measured by whole-cell techniques was increased approximately 3-fold in HTC-R cells compared with HTC cells. Moreover, HTC-R cells exhibited enhanced survival during hypotonic stress. By modulating ATP release, hepatic ABC proteins may play a key role in the cellular pathways coupling changes in cell volume to ion permeability and secretion.
Highlights
Extracellular ATP is a potent signaling factor that modulates a variety of cellular functions through the activation of P2 purinergic receptors in the plasma membrane
Based on evidence that certain ATPbinding cassette (ABC) proteins may function as ATP channels or channel regulators, we evaluated the potential role of ATP-binding cassette (ABC) proteins by comparing ATP release and volume regulation in rat HTC and HTC-R hepatoma cells, the latter of which overexpress Mdr proteins
Findings in rat HTC hepatoma cells were compared with those in a selected population of HTC cells (HTC-R) that overexpress both endogenous and novel Mdr proteins [11]. These studies demonstrate that inhibition of P-glycoprotein transport prevents recovery from swelling and that overexpression of Mdr proteins is associated with enhanced ATP release, volume recovery, and cell survival during hypotonic stress
Summary
Materials—The purinergic agonists used were ATP, UTP, and ATP␥S, and the purinergic antagonists were suramin and reactive blue 2. Glycocholic acid methyl ester was synthesized as described previously [11] and added to the HTC-R cell medium at concentrations of 300 – 650 M. Swelling was induced by switching to a buffer containing 20% less NaCl. The standard pipette (intracellular) solution contained 130 mM KCl, 10 mM NaCl, 2 mM MgCl2, 10 mM HEPES/KOH (pH ϳ7.2), and free Ca2ϩ adjusted to ϳ100 nM (0.5 mM CaCl2 and 1 mM EGTA) with a total ClϪ concentration of 145 mM [16]. To enhance measurements of ATP transport, whole-cell recordings were performed with high ATP concentrations in both the bath and pipette solutions including 100 mM MgATP, 5 mM MgCl2, and 10 mM HEPES/Tris-OH (pH 7.4) as described previously [1, 4, 5]. Statistical comparisons were made using the paired or unpaired t test as indicated, and p Ͻ 0.05 was considered significant
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