Differences in nucleotide compartmentation and energy state in isolated and in situ rat heart: Assessment by 31P-NMR spectroscopy

Abstract

Free cytosolic concentrations of ATP, PCr, ADP and 5′-AMP, and the cytosolic [ATP]/[ADP].[P i] ratio, were determined in isolated and in situ rat hearts using 31P-NMR spectroscopy. Total tissue metabolite concentrations were determined by HPLC analysis of freeze-clamped, perchloric acid-extracted tissue. In in situ myocardium the PCr/ATP ratio was 2.7 ± 0.2 determined from 31P-NMR data (using either PCr/β-NTP or PCr/γ-NTP), and 1.9 ± 0.1 ( P < 0.01) determined from total tissue concentrations. 31P-NMR-determined and total tissue [PCr] were in excellent agreement (49.6 ± 8.4 and 49.5 ± 1.0 μmol.g −1 dry wt, respectively), whereas 31P-NMR-determined [ATP] (18.6 ± 3.2 μmol.g−1 dry wt) was only 71% of the total tissue concentration (26.1 ± 1.7 μmol.g −1 dry wt, P < 0.01). Isolation and Langendorff perfusion of rat hearts with glucose as substrate reduced total tissue [ATP] and [PCr] and the 31P-NMR-determined PCr/ATP ratio fell to 1.5 ± 0.1. This value agreed well with the total tissue ratio of 1.4 ± 0.1, and there was excellent agreement between 31P-NMR-determined and total tissue [PCr] and [ATP] values in the perfused heart. Addition of pyruvate to perfusate increased the 31P-NMR-determined PCr/ATP ratio to 1.7 ± 0.1 due to elevated [PCr], and there remained excellent agreement between NMR-determined and total tissue [PCr] and [ATP] values. Free cytosolic [ADP] (from the creatine kinase equilibrium) was 5% of total tissue ADP, and free cytosolic [5′-AMP] (from the adenylate kinase equilibrium) ranged from 0.2–0.3% of total tissue 5′-AMP. Bioenergetic state, indexed by [ATP]/[ADP].[P i], was much lower in isolated perfused hearts (30 mM − 1) vs. in situ myocardium (∼ 150 mM − 1). In summary, we observe a substantial disproportionality between total tissue PCr/ATP and 31P-NMR-determined PCr/ATP in highly energised in situ myocardium but not in isolated perfused hearts. This appears due to an NMR invisible ATP compartment approximating 29% of total tissue ATP in situ. Additionally, more than 95% of ADP and more than 99% of 5′-AMP exist in bound forms in perfused and in situ myocardium. The physiological significance of these observations is unclear. However, substantial differences between 31P-NMR visible and total tissue [ATP] introduces significant errors in conventional estimation of free cytosolic [ADP], [5′-AMP] and [ATP]/[ADP].[P i] from in vivo 31P-NMR data.