Extracellular ATP reversibly increases the plasma membrane permeability of transformed mouse fibroblasts to large...

Extracellular ATP increases the plasma membrane permeability of transformed mouse fibroblasts (3T6 cells) to fluorescein isothiocyanate-dextrans (4 and 9 kDa) and [3H]inulin (5.2 kDa). This increase in permeability to large macromolecules occurred 20 to 30 min after ATP addition, subsequent to an increase in permeability to small macromolecules (< 1 kDa). Permeability changes to small and large macromolecules were modulated in the same manner by pH, temperature, and ATP concentration. Likewise, conditions previously used to reseal 3T6 cells after ATP treatments that enabled the entry of molecules < 1 kDa also resealed cells that had been treated with ATP for 20 to 30 min. Resealed cells propagated at the same rate as control cells, although many cells became detached from the culture dish. Aequorin, a 20-kDa Ca(2+)-sensitive photoprotein, was sealed inside ATP-treated 3T6 cells and then used to quantitate changes in the concentration of cytoplasmic free Ca2+. Both ATP and ionomycin, a calcium ionophore, increased aequorin luminescence in 3T6 cells, indicative of an elevation in levels of cytoplasmic free Ca2+. The time course for uptake of macromolecules (4-20 kDa) was similar to the time course of lactic dehydrogenase (150 kDa) release from 3T6 cells treated with ATP, suggesting that the procedure may be generally applicable for incorporation of high molecular weight macromolecules into viable, ATP-sensitive cells.