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

Concentration-Dependent Effects on Intracellular and Surface pH of Exposing Xenopus oocytes to... Others have shown that exposing oocytes to high levels of $$ {\text{NH}}_{ 3} /{\text{NH}}_{ 4}{}^{ + } $$ (10–20 mM) causes a paradoxical fall in intracellular pH (pHi), whereas low levels (e.g., 0.5 mM) cause little pHi change. Here we monitored pHi and extracellular surface pH (pHS) while exposing oocytes to 5 or 0.5 mM NH3/NH4 +. We confirm that 5 mM $$ {\text{NH}}_{ 3} /{\text{NH}}_{ 4}{}^{ + } $$ causes a paradoxical pHi fall (−ΔpHi ≅ 0.2), but also observe an abrupt pHS fall (−ΔpHS ≅ 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 −ΔpHS at both $$ {\text{NH}}_{ 3} /{\text{NH}}_{ 4}{}^{ + } $$ 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 pHS and pHi indicates that (a) NH3 rather than NH4 + fluxes dominate pHi and pHS changes and (b) oocytes dispose of most incoming NH3. NMR studies of oocytes exposed to 15N-labeled $$ {\text{NH}}_{ 3} /{\text{NH}}_{ 4}{}^{ + } $$ show no significant formation of glutamine but substantial $$ {\text{NH}}_{ 3} /{\text{NH}}_{ 4}{}^{ + } $$ 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

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

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Publisher
Springer-Verlag
Copyright
Copyright © 2009 by Springer Science+Business Media, LLC
Subject
Life Sciences; Human Physiology ; Biochemistry, general
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s00232-009-9155-7
Publisher site
See Article on Publisher Site

Abstract

Others have shown that exposing oocytes to high levels of $$ {\text{NH}}_{ 3} /{\text{NH}}_{ 4}{}^{ + } $$ (10–20 mM) causes a paradoxical fall in intracellular pH (pHi), whereas low levels (e.g., 0.5 mM) cause little pHi change. Here we monitored pHi and extracellular surface pH (pHS) while exposing oocytes to 5 or 0.5 mM NH3/NH4 +. We confirm that 5 mM $$ {\text{NH}}_{ 3} /{\text{NH}}_{ 4}{}^{ + } $$ causes a paradoxical pHi fall (−ΔpHi ≅ 0.2), but also observe an abrupt pHS fall (−ΔpHS ≅ 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 −ΔpHS at both $$ {\text{NH}}_{ 3} /{\text{NH}}_{ 4}{}^{ + } $$ 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 pHS and pHi indicates that (a) NH3 rather than NH4 + fluxes dominate pHi and pHS changes and (b) oocytes dispose of most incoming NH3. NMR studies of oocytes exposed to 15N-labeled $$ {\text{NH}}_{ 3} /{\text{NH}}_{ 4}{}^{ + } $$ show no significant formation of glutamine but substantial $$ {\text{NH}}_{ 3} /{\text{NH}}_{ 4}{}^{ + } $$ 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.

Journal

The Journal of Membrane BiologySpringer Journals

Published: Feb 26, 2009

References

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