INTRACELLULAR PH MEASUREMENTS IN EHRLICH ASCITES TUMOR-CELLS UTILIZING SPECTROSCOPIC PROBES GENERATED INSITU

The uncharged, colorless molecule fluorescein diacetate diffuses into Ehrlich ascites tumor cells at neutral pH, where intracellular esterases release the chromophore fluorescein. The negatively charged dye is retained by the cell, permitting the intracellular pH to be estimated from the shape of the pH-dependent absorption spectrum. The diacetate derivative of 6-carboxyfluorescein may be used similarly and has the additional advantage of a slower rate of leakage out of the cell but requires incubation at pH 6.2 to facilitate initial entry into the cell. After removal of external dye by centrifugation, 80-92% of the remaining dye is unresponsive to external pH changes. Calibration of the intracellular fluorescein spectra is obtained by equilibration of the internal and external pH with nigericin in K+ buffers. Results of intracellular pH measurements by this method are in good agreement with those obtained by measuring the distribution ratio of the weak acid 5,5-dimethyl[2-14C]oxazolidine-2,4-dione, under a variety of metabolic conditions. Besides the accurate estimation of intracellular pH, the method permits the kinetics of intracellular pH changes as small as 0.01 to be followed. Intracellular fluorescein reports pH changes occurring in both the cytoplasmic and the mitochondrial compartments, whereas 6-carboxyfluorescein reports only the cytoplasmic compartment. At equivalent concentrations, nigericin is more effective than valinomycin plus the proto-nophore 1799 in dissipating plasmalemma pH gradients. Either is effective at lower concentrations in dissipating mitochondrial pH gradients. Addition of glucose to Ehrlich ascites cells results in a transient acidification of the cytoplasm in close correspondence to the intracellular lactate levels. The transient acidification can be explained by the initial rapid rate of glycolysis exceeding the rate of lactate export. © 1979, American Chemical Society. All rights reserved.