Intracellular pH in single motile cells.

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Abstract
Cytoplasmic pH in single living specimens of Chaos carolinensis is determined microfluorometrically by measuring the ratio of fluorescence intensity of microinjected fluorescein-thiocarbamyl (FTC)-ovalbumin at 2 different excitation wavelengths. The probe is evenly distributed throughout and confined to the cytoplasm, and the fluorescence intensity ratio depends only upon pH. It is independent of pathlength, concentration of probe, divalent cations and ionic strength. Ratios are calibrated with a standard curve generated in situ by adjusting internal pH of FTC-ovalbumin-containing amoebae with weak acid and weak base or by injection of strong buffers. With this technique the average cytoplasmic pH of freely moving amoebae is 6.75 (SD .+-. 0.3). The pH of a given spot relative to the morphology of a moving amoeba remains fairly constant (.+-. 0.05 U), whereas the pH of 2 different spots in the same cell may differ by as much as 0.4 U, and average pH in different amoebae ranges from 6.3 to 7.4, with a suggestion of clustering about pH 6.5 and 6.8. During wound healing there is a local, transient drop in pH (as great as 0.35 U) at the wound site upon puncture, proportional in extent to the degree of damage. Comparison of tails and advancing pseudopod tips reveals no significant difference in cytoplasmic pH at this level of spatial (50 .mu.m diameter spot) and temporal (1.3 s) resolution. Fluctuations in intracellular pH and/or intracellular free Ca2+ may be involved in regulation of cytoplasmic structure and contractility.