No abstract

Addition of ATP to medium surrounding intact, transformed 3T3 cells activates the formation of aqueous channels in the plasma membrane. This results in efflux of nucleotide pools and ions and entry into the cytosol of charged, phosphorylated species. In such permeabilized cells, glycolysis is totally dependent on the external addition of glucose, inorganic phosphate, ADP, K+, Mg2+ and NAD+ which restore lactic acid formation to levels found in untreated cells. As expected, such reconstitution of glycolytic activity is found to restore intracellular ATP levels. This is accompanied by sealing of the membrane channels so that efflux of nucleotide pools ceases. Pyruvate, a substrate for mitochondrial ATP synthesis, when provided along with ADP and inorganic phosphate also produces sealing of the membrane channels. On the other hand, reactivation of pentose phosphate shunt activity, which does not lead to ATP synthesis, does not induce restoration of the membrane permeability barrier. Furthermore, compounds which lower the internal ATP pool prevent sealing, and also render the plasma membrane more sensitive to external ATP (Rozengurt and Heppel, '79). Sealing of aqueous channels following restoration of the internal ATP pool is associated with phosphorylation of the inner membrane surface, and is unaffected by inhibitors of protein synthesis, microfilament or microtubular assembly. These results indicate the probable role of intracellular ATP in the restoration and/or maintenance of an active membrane barrier against efflux of small molecules and ions in transformed 3T3 cells.

Addition of ATP to medium surrounding intact, transformed 3T3 cells causes the formation of aqueous channels in the plasma membrane. This effect of extracellular ATP is sharply dependent on the pH and temperature of the incubation medium, and is inhibited by low levels of La3+ or ruthenium red; inhibition is also obtained with concentrations of Mg2+ ions that exceed a ratio of Mg/ATP of one. The effect of ATP on membrane channel formation is unaffected by chelators of metal ions or by prior modification of the cell surface with various surface-active enzymes or sulfhydryl reagents. Under conditions which favor aqueous channel formation, incubation of intact 3T6 cells with ATP (gamma-32P) leads to phosphorylation of two membrane components with apparent molecular weight of 40,000 (40K) and 110,000 (110K) daltons; the 110K component which is unaffected by trypsin under normal conditions is rendered trypsin-sensitive by the phosphorylation reaction, probably as a result of a conformational change. Conditions which inhibit aqueous channel formation also inhibit phosphorylation of the 110K protein and decrease the labeling of the 40K component. These results indicate the probable role of cell surface phosphorylation, involving one or both of these components, in the formation of aqueous channels in transformed 3T3 cells. Aqueous channel formation by extracellular ATP is not associated with gross unfolding of the cell surface as revealed by lactoperoxidase-catalyzed iodination of the 3T6 cell surface.

Exogenous ATP has been shown earlier to activate a permeability change in transformed 3T3 cultures leading to massive efflux of the acid-soluble pools. This leads to reduction of the basal rate of glycolysis to a very low level so that glycolysis becomes almost totally dependent on the addition to the medium of glucose, inorganic phosphate and ADP in order to restore the rate to that of untreated cells. No such depression of glycolysis is observed in untreated transformed cells or in ATP-treated normal 3T3 cells. In such permeabilized cultures, phosphorylated intermediates such as glucose-6-phosphate and fructose-1,6-diphosphate can serve as effective substrates for lactic acid formation. ATP treatment of cultured cells also allows molecules as big as NADP to enter the cells and participate in the pentose phosphate shunt pathway. This ability to temporarily and differentially render transformed cells permeable allows a review of several aspects of cellular metabolism and biosynthesis in the intact cell where the cellular organization is maintained. Furthermore, it deserves serious consideration as a means to achieve differential cytotoxicity of transformed cells by chemotherapeutic agents which, on their own, are indiscriminate in their action.

Various agents alter mammalian cells so that they rapidly become nonspecifically permeable to substances that ordinarily do not penetrate intact cells. Thus, toluene renders liver cells permeable to nucleotides and macromolecules. Tween 80 and Tween 60 act on similar fashion, and the effect is reversible. Dextran sulfate reversibly alters the permeability of Ehrlich ascites tumor cells, which offers a tool for studying the control of macromolecular syntheses and other processes. Brief exposure to external ATP alters the permeability of certain transformed mouse cells but not of untransformed cells. The effect of ATP is rapidly reversible.