Measurements of intracellular pH in Necturus antral mucosa by microelectrode technique

Intracellular pH (pHi) was measured in the surface epithelial cells of Necturus antrum using pH-sensitive intracellular microelectrodes. Electrodes were prepared by filling 10- to 20-M omega 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 less than 0.001) with a slope of 52.1 +/- 2.3 mV/pH unit. Antral mucosa was mounted in a modified Ussing chamber and pHi was determined from the difference between the potentials recorded by intracellular H+-selective and conventional microelectrodes. These measurements of pHi 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 pHi produced by NH+4 substitution for Na+ in the bathing solutions. In tissues bathed with HCO-3-Ringer solution (pH 7.0), the mean pHi 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), pHi was reduced to 7.02 +/- 0.05 (P less than 0.01). Acidification of the luminal solution to pH 6.8 with CO2 produced a 0.22 +/- 0.04-pH unit fall in pHi (P less than 0.001). In contrast, acidification to pH 4.0 with HCl had no significant effects on pHi. These findings indicate that HCO-3 may play an important role in pHi 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.