Role of cytoplasmic ATP in the restoration and maintenance of a membrane permeability barrier in transformed mammalian cells

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.

Ca2+ homeostasis and cytotoxicity in isolated hepatocytes: Studies with extracellular adenosine 5′‐triphosphate

“…This is further supported by the finding presented in this report that no changes in the endogenous ATP concentration postulated as the cause of membrane damage in previous reports (35,36) occurred during extracellular ATP treatment. Moreover, we have recently performed experiments with the dye ethidium bromide, which becomes fluorescent upon binding to nucleic acids and has been employed by other investigators as a marker of plasma membrane permeability (37,38).…”

Control of membrane permeability by external and internal ATP in 3T6 cells grown in serum-free medium.

“…Some recent evidence (2,4,21,22,26,27) is consistent with the involvement of membrane phosphorylation in the control of membrane permeability. We speculated that a critical protein regulates the passive permeability of the cell membrane and that such a protein can be phosphorylated at both the inner and outer surfaces of the plasma membrane (2).…”

Cellular responses to external ATP which precede an increase in nucleotide permeability in transformed cells

“…Compounds which lower intracellular levels of ATP, such as uncouplers, energy-transfer inhibitors, or inhibitors of respiration, enable 25-50 pM external ATP to permeabilize 3T6 cells (Dicker et al, 1980;Rozengurt and Heppel, 1979). Addition of glucose elevates intracellular ATP and prevents this synergistic effect, supporting the interpretation that membrane permeability in transformed cells is controlled by both internal and external levels of ATP (Makan, 1979).Previous work has shown that efflux of Rb+ is an early effect of external ATP (Rozengurt et al, 1977;Dicker et al, 1980) and that the efflux of nucleotides occurs after a lag period of 3-5 min a t 37°C (Rozengurt and Heppel, 1975;Rozengurt et al, 1977). Since the earliest detectable effect of external ATP may provide a clue as to the mechanism of ATP-dependent permeabilization, ion fluxes and changes in intracellular nucleotide levels that might occur during the lag period were investigated.…”