Measurements of intracellular pH in Necturus antral mucosa by microelectrode technique

Intracellular pH (pH(i)) was measured in the surface epithelial cells of Necturus antrum using pH-sensitive intracellular microelectrodes. Electrodes were prepared by filling 10- to 20MΩ resistance glass microelectrodes with a H⁺ ion-selective exchange resin and calibrated before use in solutions of known pH 4.0-8.0. The electrode response (n = 15) was linear ( r = 0.93; P < 0.001) with a slope of 52.1 ± 2.3 mV/pH unit. Antral mucosa was mounted in a modified Ussing chamber and pH(i) was determined from the difference between the potentials recorded by intracellular H⁺-selective and conventional microelectrodes. These measurements of pH(i) were validated by examining the response of the intracellular microelectrodes to 1) depolarization of the cell membrane produced by K⁺ substitution for Na⁺, and 2) alkalinization and acidification of pH(i) produced by NH⁺₄ substitution for Na⁺ in the bathing solutions. In tissues bathed with HCO^-₃-Ringer solution (pH 7.0), the mean pH(i) was 7.34 ± 0.02 with a range from 7.24 to 7.43. In N-2-hydroxymethylpiperazine-N'-ethanesulfonic acid (HEPES)-Ringer solution (pH 7.0), pH(i) was reduced to 7.02 ± 0.05 (P < 0.01). Acidification of the luminal solution to pH 6.8 with CO₂ produced a 0.22 ± 0.04 pH unit fall in pH(i) (P < 0.001). In contrast, acidification to pH 4.0 with HCl had no significant effects on pH(i). These findings indicate that HCO^-₃ may play an important role in pH(i) regulation in this tissue. In addition, they suggest that, in contrast to CO₂, the surface epithelial cells of Necturus antrum are relatively resistant to acidification by luminal HCl.