Regulation of intracellular pH in sea urchin eggs by medium containing both weak acid and base

To establish a method of pHi regulation and to understand the pH regulation mechanism in the cell, we investigated the pHi response of unfertilized or fertilized eggs of sea urchin, applying sea water containing both weak permeant acid, acetic acid and/or base, ammonia, to eggs. Pyranine was employed as a pH indicator to measure intracellular pH (pHi) by microfluorometry. The unfertilized/fertilized eggs had a pHi of 6.80/7.34 and 6.81/7.32 for Schaphechinus mirabilis and Hemicentrotus pulcherrimus, respectively. With the addition of both acetic acid and ammonia to the media, pHi changed linearly against extracellular pH (pHo) between 6-8 and was almost equal to pHo at the concentration of 20 mM acetate and ammonia. This mixed application was proved to be available for regulating pHi at the desired value within a wide range involving the original pHi by a single solution system. pHi after the treatment was dependent on various factors, such as the concentration of the weak acid and base, the pHi before the treatment, and pH buffering power in the cytoplasm. The latter was estimated to be 43 mM and 58 mM in unfertilized and fertilized eggs, respectively, from the measurement of pHi change induced by microinjecting a HEPES solution, assuming that the pH buffering power is caused by phosphate.