Ammonia permeability of erythrocyte membrane studied by 14N and 15N saturation transfer NMR spectroscopy
Labotka, R.J.; Lundberg, P.; Kuchel, P.W.
American Journal of Physiology 268(3 Pt 1): C686-C699
1995
ISSN/ISBN: 0002-9513 PMID: 7900775 Document Number: 441587
The permeability of biological membranes to the rapidly penetrating compound ammonia is extremely difficult to study due to the lack of readily available radionuclides. 14N and 15N saturation transfer nuclear magnetic resonance (NMR) experiments were used to measure the erythrocyte membrane permeability of ammonia under equilibrium exchange conditions. When 1-4N spectra from erythrocytes suspended in NH-4Cl solution were obtained in the presence of the extracellular shift reagent dysprosium tripolyphosphate, intracellular and extracellular ammonia signals were readily resolved. Comparison with 15N spectra from erythrocyte suspensions containing 15NH-4Cl revealed that the intracellular (14N)ammonia signals were 100% NMR visible. 14N and 15N saturation transfer NMR experiments showed similar influx rates and permeabilities, indicating no loss of saturation transfer due to quadrupolar relaxation of 14N nuclei upon membrane passage. Ammonia influx was directly proportional to concentration (0.39 +- 0.012 fmol cntdot cell-1 cntdot s-1 cntdot mM-1 at pH 7.0) and not saturable, which is consistent with passive diffusion. Apparent ammonia permeability increased with pH over the range of pH 6-8 as the fraction of free NH-3 increased. However, diffusion through unstirred layers became increasingly rate limiting. The permeability of the unstirred layers (1.1 +- 0.45 times 10-3 cm/s) was considerably lower than that of NH-3 (0.21 +- 0.014 cm/s). The Arrhenius activation energy for NH-3 permeability was 49.5 +- 11.8 kJ/mol. No evidence for NH-4+ influx over the time domain of these experiments was found.