31 P NMR and enzymatic analysis of cytosolic phosphocreatine, ATP, P i and intracellular pH in the isolated working perfused rat heart

Hearts from fed male Wistar rats (200–350 g) were perfused at low and high workloads with P i ‐free Krebs‐Henseleit medium containing either 10 m M glucose or 10 m M glucose plus 15mU/mL insulin. The intracellular pH by 31 P NMR ranged between 6.99 and 7.02 and agreed to within 0.1 pH unit of estimates calculated using enzymatically determined total tissue HCO 3 − /CO 2 contents. At high work, where the tissue contents of phosphocreatine (PCr) and ATP were determined on the same heart as NMR areas ( n = 16), the proportionality factors, defined as the 31 P NMR area units divided by the total enzymatically determined tissue content (area units/μmol/g dry wt), were 112±8 for PCr, 99±4 for γ‐ATP, 138±9 for α‐ATP and 100±4 for β‐ATP. These values were normalized by taking β‐ATP as 100 area units/μmol/g dry wt. Since the proportionality factor for PCr and γ‐ and β‐ATP were not statistically different (p < 0.05), it was concluded that each was equally visible by 31 P NMR and that no significant breakdown of PCr occurred during freezing or tissue acid extraction procedures. The cytosolic P i estimated from NMR in glucose plus insulin perfused hearts at low and high work was 4.92±0.67 and 6.33±0.42 μmol/g dry wt. Using the near‐equilibrium expression of K CK /K G+G and the metabolite levels in heart extracts, the calculated cytosolic P i was 13.08±1.83 and 16.17±3.08 μmol/g dry wt, respectively. The cytosolic NMR P i in the glucose hearts was 8.42±1.0 and 8.42±0.75 μmol/g dry wt at low and high work and 12.08±1.58 and 27.20±4.20±μmol/g dry wt from near‐equilibrium estimates. The total tissue P i measured enzymatically on freeze‐clamped hearts ranged from 18.0 to 26.42 μmol/g dry wt. The validity of using both the 31 P NMR and the near‐equilibrium method for estimating cytosolic P i in the heart was discussed.