Williams J P, Headrick J P
Department of Physiology and Pharmacology, School of Molecular Sciences, James Cook University of North Queensland, Townsville, Australia.
Biochim Biophys Acta. 1996 Aug 7;1276(1):71-9. doi: 10.1016/0005-2728(96)00036-9.
Free cytosolic concentrations of ATP, PCr, ADP and 5'-AMP, and the cytosolic [ATP]/[ADP].[Pi] 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/beta-NTP or PCr/gamma-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 mumol.g-1 dry wt, respectively), whereas 31P-NMR-determined [ATP] (18.6 +/- 3.2 mumol.g-1 dry wt) was only 71% of the total tissue concentration (26.1 +/- 1.7 mumol.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].[Pi], was much lower in isolated perfused hearts (30 mM-1) vs. in situ myocardium (approximately 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].[Pi] from in vivo 31P-NMR data.
利用31P-核磁共振波谱法测定了离体和原位大鼠心脏中ATP、磷酸肌酸（PCr）、ADP和5'-AMP的游离胞质浓度，以及胞质[ATP]/[ADP].[Pi]比值。通过对冷冻钳夹、高氯酸提取的组织进行HPLC分析来测定组织总代谢物浓度。在原位心肌中，根据31P-核磁共振数据（使用PCr/β-NTP或PCr/γ-NTP）测定的PCr/ATP比值为2.7±0.2，而根据组织总浓度测定的值为1.9±0.1（P<0.01）。31P-核磁共振测定的和组织总[PCr]高度一致（分别为49.6±8.4和49.5±1.0μmol·g-1干重），而31P-核磁共振测定的[ATP]（18.6±3.2μmol·g-1干重）仅为组织总浓度（26.1±1.7μmol·g-1干重，P<0.01）的71%。以葡萄糖为底物对大鼠心脏进行离体和Langendorff灌注，降低了组织总[ATP]和[PCr]，31P-核磁共振测定的PCr/ATP比值降至1.5±0.1。该值与组织总比值1.4±0.1吻合良好，且灌注心脏中31P-核磁共振测定的和组织总[PCr]及[ATP]值高度一致。向灌注液中添加丙酮酸后，由于[PCr]升高，31P-核磁共振测定的PCr/ATP比值升至1.7±0.1，且核磁共振测定的和组织总[PCr]及[ATP]值仍高度一致。游离胞质[ADP]（根据肌酸激酶平衡）为组织总ADP的5%，游离胞质[5'-AMP]（根据腺苷酸激酶平衡）占组织总5'-AMP的0.2 - 0.3%。以[ATP]/[ADP].[Pi]为指标的生物能状态，在离体灌注心脏中（30 mM-1）远低于原位心肌（约150 mM-1）。总之，我们观察到在高能量的原位心肌中，组织总PCr/ATP与31P-核磁共振测定的PCr/ATP之间存在显著的不均衡，但在离体灌注心脏中并非如此。这似乎是由于原位存在一个约占组织总ATP 29%的核磁共振不可见ATP区室。此外，在灌注和原位心肌中，超过95%的ADP和超过99%的5'-AMP以结合形式存在。这些观察结果的生理学意义尚不清楚。然而，31P-核磁共振可见的和组织总[ATP]之间的显著差异，在根据体内31P-核磁共振数据常规估算游离胞质[ADP]、[5'-AMP]和[ATP]/[ADP].[Pi]时会引入显著误差。
