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

The permeability of biological membranes to the rapidly penetrating co mpound ammonia is extremely difficult to study due to the lack of read ily available radionuclides. N-14 and N-15 saturation transfer nuclear magnetic resonance (NMR) experiments were used to measure the erythro cyte membrane permeability of ammonia under equilibrium exchange condi tions. When N-14 spectra from erythrocytes suspended in NH4Cl solution were obtained in the presence of the extracellular shift reagent dysp rosium tripolyphosphate, intracellular and extracellular ammonia signa ls were readily resolved. Comparison with N-15 spectra from erythrocyt e 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 permeabilitie s, indicating no loss of saturation transfer due to quadrupolar relaxa tion 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 r ange of pH 6-8 as the fraction of free NH3 increased. However, diffusi on through unstirred layers became increasingly rate Limiting. The per meability of the unstirred layers (1.1 +/- 0.45 x 10(-3) cm/s) was con siderably 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 ev idence for NH4+ influx over the time domain of these experiments was f ound.