31P NMR and enzymatic analysis of cytosolic phosphocreatine, ATP, Pi 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 Pi-free Krebs-Henseleit medium containing either 10 mM glucose or 10 mM glucose plus 15 mU/mL insulin. The intracellular pH by 31P NMR ranged between 6.99 and 7.02 and agreed to within 0.1 pH unit of estimates calculated using enzymatically determined total tissue HCO3-/CO2 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 31P NMR area units divided by the total enzymatically determined tissue content (area units/mumol/g dry wt), were 112 +/- 8 for PCr, 99 +/- 4 for gamma-ATP, 138 +/- 9 for alpha-ATP and 100 +/- 4 for beta-ATP. These values were normalized by taking beta-ATP as 100 area units/mumol/g dry wt. Since the proportionality factor for PCr and gamma- and beta-ATP were not statistically different (p less than 0.05), it was concluded that each was equally visible by 31P NMR and that no significant breakdown of PCr occurred during freezing or tissue acid extraction procedures. The cytosolic Pi estimated from NMR in glucose plus insulin perfused hearts at low and high work was 4.92 +/- 0.67 and 6.33 +/- 0.42 mumol/g dry wt. Using the near-equilibrium expression of KCK/KG + G and the metabolite levels in heart extracts, the calculated cytosolic Pi was 13.08 +/- 1.83 and 16.17 +/- 3.08 mumol/g dry wt, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)