Control of respiration by products of ATP hydrolysis was examined in the in situ rat heart using a purpose-built nuclear magnetic resonance (NMR) coil. The in situ ratio of phosphocreatine to ATP concentrations ([PCr]/[ATP]) was 2.30 +/- 0.05, free Mg2+ concentration ([Mg2+]) was 0.57 mM, and cytosolic pH was 7.35 +/- 0.03 (n = 7). Basal inorganic phosphate concentration ([Pi]) was below NMR detection but was estimated to be 0.83 mM. The [ATP]/[ADP] [Pi] ratio, free ADP concentration ([ADP]), and free energy of ATP hydrolyses (delta GATP) were calculated to be 700,000 +/- 78,000 M-1, 18 +/- 3 microM, and -63.93 +/- 0.33 kJ/mol in situ, respectively (n = 7). In contrast, in the Langendorff perfused rat heart [ATP]/[ADP] [Pi] was only 76,140 +/- 12,830 M-1, [ADP] was 65 +/- 9 microM, and delta GATP was -59.92 +/- 0.48 kJ/mol (n = 7), all indicative of a lower energy state in vitro. Epinephrine infusion in situ (0.9 microgram.min-1.kg-1) increased the rate-pressure product 2.05-fold. During stimulation [ATP] was stable at 97 +/- 3% signal intensity, [PCr] declined by 25%, and [Pi] increased to 1.83 mM. Cytosolic pH was 7.27 +/- 0.01 and [Mg2+] was 0.64 +/- 0.05 mM. [PCr]/[ATP] declined to 1.83 +/- 0.13, and [ATP]/[ADP] [Pi] fell to 108,000 +/- 15,000 M-1. delta GATP only fell marginally to -59.56 +/- 0.49 kJ/mol. Free [ADP] increased threefold to 55 +/- 10 microM. Infusion of 2.8 +/- 0.5 microgram.min-1.kg-1 epinephrine increased the rate-pressure product 2.7-fold, further reduced [ATP]/[ADP] [Pi] (5% of basal), and elevated [ADP] more than fourfold without changing [ATP]. We conclude that the in situ heart is highly energetic compared with isolated perfused hearts and operates at a different metabolic "set-point." Because free [ADP] and [Pi] in situ approximate apparent Michaelis constants for mitochondrial respiration in vitro and increase with increased cardiac work, we conclude that each fulfills the criteria for the kinetic control of O2 consumption in the in situ rat myocardium.
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