AMMONIA PERMEABILITY OF ERYTHROCYTE-MEMBRANE STUDIED BY N-14 AND N-15 SATURATION-TRANSFER NMR-SPECTROSCOPY

The permeability of biological membranes to the rapidly penetrating compound ammonia is extremely difficult to study due to the lack of readily available radionuclides. N-14 and N-15 saturation transfer nuclear magnetic resonance (NMR) experiments were used to measure the erythrocyte membrane permeability of ammonia under equilibrium exchange conditions. When N-14 spectra from erythrocytes suspended in NH4Cl solution were obtained in the presence of the extracellular shift reagent dysprosium tripolyphosphate, intracellular and extracellular ammonia signals were readily resolved. Comparison with N-15 spectra from erythrocyte suspensions containing (NH4Cl)-N-15 revealed that the intracellular [N-14]ammonia signals were 100% NMR visible. N-14 and N-15 saturation transfer NMR experiments showed similar influx rates and permeabilities, indicating no loss of saturation transfer due to quadrupolar relaxation of N-14 nuclei upon membrane passage. Ammonia influx was directly proportional to concentration (0.39 +/- 0.012 fmol . cell(-1). s(-1). 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 NH3 increased. However, diffusion through unstirred layers became increasingly rate Limiting. The permeability of the unstirred layers (1.1 +/- 0.45 x 10(-3) cm/s) was considerably lower than that of NH3 (0.21 +/- 0.014 cm/s). The Arrhenius activation energy for NH3 permeability was 49.5 +/- 11.8 kJ/mol. No evidence for NH4+ influx over the time domain of these experiments was found.