31P-nuclear magnetic resonance analysis of perfused single frog skins

Perfusion of single frog skins has produced stability of the 31P-nuclear magnetic resonance (NMR) spectra over periods as long as 8 h at room temperature. With this approach, relatively large phosphocreatine (PCr) signals were recorded for each of the 12 frog skins studied. The ratio of the concentration of PCr to ATP was estimated to be 0.76 +/- 0.07, a value an order of magnitude larger than that previously reported. Comparison of the ratio of the intracellular concentrations of inorganic phosphate (Pi) to ATP determined in the present NMR study with that previously estimated by chemical analysis suggests that little intracellular Pi is likely to be immobilized. Inclusion of methylphosphonate in the perfusing solutions permitted simultaneous determination of extracellular and intracellular pH from the NMR spectra alone. The methylphosphonate has been found to be nontoxic. At an extracellular pH of 7.72 +/- 0.04, the intracellular pH was 7.47 +/- 0.06.