Concentration-Dependent Effects on Intracellular and Surface pH of Exposing Xenopus oocytes to Solutions Containing NH3/NH4+

Others have shown that exposing oocytes to high levels of NH3/NH4+(10-20 mM) causes a paradoxical fall in intracellular pH (pH(i)), whereas low levels (e. g., 0.5 mM) cause little pHi change. Here we monitored pHi and extracellular surface pH (pH(S)) while exposing oocytes to 5 or 0.5 mM NH3/NH4+. We confirm that 5 mM NH3/NH4+ causes a paradoxical pHi fall (-Delta pH(i) congruent to 0.2), but also observe an abrupt pHS fall (-Delta pH(S) congruent to 0.2)-indicative of NH3 influx-followed by a slow decay. Reducing [NH3/NH4+] to 0.5 mM minimizes pHi changes but maintains pHS changes at a reduced magnitude. Expressing AmtB (bacterial Rh homologue) exaggerates -Delta pH(S) at both NH3/NH4+ levels. During removal of 0.5 or 5 mM NH3/NH4+, failure of pHS to markedly overshoot bulk extracellular pH implies little NH3 efflux and, thus, little free cytosolic NH3/NH4+. A new analysis of the effects of NH3 vs. NH4+ fluxes on pH(S) and pH(i) indicates that (a) NH3 rather than NH4+ fluxes dominate pHi and pH(S) changes and (b) oocytes dispose of most incoming NH3. NMR studies of oocytes exposed to N-15-labeled NH3/NH4+ show no significant formation of glutamine but substantial NH3/NH4+ accumulation in what is likely an acid intracellular compartment. In conclusion, parallel measurements of pHi and pHS demonstrate that NH3 flows across the plasma membrane and provide new insights into how a protein molecule in the plasma membrane AmtB-enhances the flux of a gas across a biological membrane.