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Abstract
HEPPEL,~ AND ILAN FRIEDBERG§ From the Imperial Cancer Research Fund, Lincoln’s Inn Fields, London, England External ATP produces an extensive change in the mem- brane permeability of transformed cultured mouse cells, leading to a depletion of intracellular pools labeled with 13Hluridine, L3H1adenosine, ““Rb, or Z-deoxy[“HIglucose. For example, when 3T6 (a cell line derived from 3T3 by spontaneous transformation), SV3T3 (3T3 cells transformed by simian vacuolating virus 40), and PY3T3 (3T3 cells trans- formed by polyoma virus) cells are exposed to as little as 0.2 mM ATP in buffered saline medium for several minutes, there results a 20- to 30-fold increase in rate of efflux of the acid-soluble uridine nucleotide pool. A much smaller stimu- lation (from 0- to 3-fold) is seen with resting or growing 3T3 cells or with secondary cultures of mouse embryo fibro- blasts. The increase in permeability produced by ATP is highly specific; it was not obtained with GTP, CTP, UTP, ADP, inorganic phosphate, and various chelating agents. The effect of ATP in increasing permeability is pa-depend- ent and occurs much faster at 37.5” than at 20”. Stimulation of efflux persists after removal of ATP and the enhanced rate of efflux is the same at 20” as at 37.5”. The effect of ATP is seen in various buffered saline media. When the solutions are removed and replaced with Dul- becco’s modified Eagle’s serum-free medium (Smith, J. D., Freeman, G., Vogt, D., and Dulbecco, R. (1960) Virology 12, 185-196), the increased rate of efflux is quickly suppressed. The presence of bicarbonate, divalent cations, and a more neutral pH all contribute to restore a normal permeability barrier. Upon addition of serum, the acid-soluble pools are restored in 1 h and the process can be repeated. This treat- ment with ATP does not affect the subsequent appearance of the cultured cells or their rate of growth.
Highlights
External ATP produces an extensive change in the membrane permeability of transformed cultured mouse cells, leading to a depletion of intracellular pools labeled with 13Hluridine, L3H1adenosine, ““Rb, or Z-deoxy[“HIglucose. For example, when 3T6, SV3T3 (3T3 cells transformed by simian vacuolating virus 40), and PY3T3 (3T3 cells transformed by polyoma virus) cells are exposed to as little as 0.2 mM ATP in buffered saline medium for several minutes, there results a 20- to 30-fold increase in rate of efflux of the acid-soluble uridine nucleotide pool
The purpose of the present paper is to describe the effect of exogenous ATP on the membrane permeability for metabolites and ions present inside the cell
I The abbreviations used are: 3T6, a cell line derived from 3T3 by spontaneous transformation; SV3T3, 3T3 cells transformed by simian vacuolating virus 40; PY3T3, 3T3 cells transformed by polyoma virus; Hepes, 4-(2.hydroxyethyll-1-piperazineethanesulfonic acid; EGTA, ethylene glycol bis(p-aminoethyl ether)N,N’-tetraacetic acid; Medium A, 0.1 M Tris/HCl, 0.05 M NaCl, 50 ELM CaCl, 5.0 mg/
Summary
External ATP produces an extensive change in the membrane permeability of transformed cultured mouse cells, leading to a depletion of intracellular pools labeled with 13Hluridine, L3H1adenosine, ““Rb, or Z-deoxy[“HIglucose. For example, when 3T6 (a cell line derived from 3T3 by spontaneous transformation), SV3T3 (3T3 cells transformed by simian vacuolating virus 40), and PY3T3 (3T3 cells transformed by polyoma virus) cells are exposed to as little as 0.2 mM ATP in buffered saline medium for several minutes, there results a 20- to 30-fold increase in rate of efflux of the acid-soluble uridine nucleotide pool. The acid-soluble pools are restored in 1 h and the process can be repeated This treatment with ATP does not affect the subsequent appearance of the cultured cells or their rate of growth. The effect was specific for ATP and was not found for other nucleoside triphosphates, nor was it obtained by the use of EDTA, inorganic pyrophosphate, or other chelating agents This effect of exogenous ATP was observed for 3T6,’ SV3T3, and PY3T3 cells, but not for 3T3 cells nor for mouse embryo secondary cultures. It was rapidly reversible, especially in the presence of glutamine. When this work was published, we had no evidence for an effect of external ATP on the membrane permeability for naturally occurring metabolites
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