Stewart L C, Deslauriers R, Kupriyanov V V
Institute for Biodiagnostics, National Research Council, Winnipeg, MB, Canada.
J Mol Cell Cardiol. 1994 Oct;26(10):1377-92. doi: 10.1006/jmcc.1994.1156.
In order to assess the relationship between cytosolic [ATP] or [ATP]/[ADP] and the intracellular Na+ concentration ([Na+]i), we have used the phosphate trap 2-deoxy-D-glucose (DG) to alter the high energy phosphate levels in rat cardiomyocytes. Pyruvate-perfused rat hearts were treated with 2 mM DG in the presence of 10IU/l of insulin for 28 min, followed by perfusion with DG without insulin for 60 min. The DG + insulin treatment resulted in dramatic changes in the 31P NMR spectra: phosphocreatine (PCr) and total ATP decreased (to 15 and 35%, respectively) and deoxyglucose-6-phosphate accumulated, with little change in either inorganic phosphate or intracellular pH. These changes corresponded to a decrease in cytoplasmic [ATP] (from 7.6 to 1.8 mM), [ATP]/[ADP] (from 494 to 24) and ATP affinity [A(ATP), by 8.9 kJ/mol] and an increase in [ADP] (five-fold) and free [Mg2+] (two-fold). Subsequent perfusion with DG--insulin resulted in slow recovery of PCr, [ATP]/[ADP] and A(ATP) such that the "low energy" state lasted an additional 16 min during which ATP remained low and constant. There were no detectable changes in the intracellular Na+ content as assessed by shift reagent-aided 23Na NMR at the end of DG + insulin treatment (98 +/- 18%, 28-36 min of the protocol). In addition, there was no change in the Rb+ influx rate as measured by 87Rb NMR at the beginning of insulin washout which was achieved by replacing 20% of the KCl with RbCl ([K+] = 3.76 mM, [Rb+] = 0.94 mM). During DG + insulin treatment the pressure-rate product (PRP) decreased by half and was restored upon insulin washout to 80% of its initial value both in the presence and in the absence of the shift reagent [5 mM Dy (triethylenetetraminehexaacetate)3-]. These data imply that unfavorable thermodynamic [low A(ATP)] and kinetic (low [ATP] and [ATP]/[ADP]) conditions induced by DG treatment do not inhibit Na+, K(+)-ATPase activity. We speculate that during anoxia when changes in [ATP]/[ADP] are comparable to those induced by DG treatment, the observed increase in [Na+]i is not due to inhibition of the Na+ pump by reduced [ATP] or [ATP]/[ADP].
为了评估胞质[ATP]或[ATP]/[ADP]与细胞内Na⁺浓度（[Na⁺]i）之间的关系，我们使用磷酸阱2-脱氧-D-葡萄糖（DG）来改变大鼠心肌细胞中的高能磷酸水平。用丙酮酸灌注的大鼠心脏在10IU/l胰岛素存在的情况下用2mM DG处理28分钟，然后在无胰岛素的情况下用DG灌注60分钟。DG +胰岛素处理导致³¹P NMR光谱发生显著变化：磷酸肌酸（PCr）和总ATP减少（分别降至15%和35%），6-磷酸脱氧葡萄糖积累，无机磷酸或细胞内pH几乎没有变化。这些变化对应于细胞质[ATP]降低（从7.6mM降至1.8mM）、[ATP]/[ADP]降低（从494降至24）以及ATP亲和力[A(ATP)，降低8.9kJ/mol]，同时[ADP]增加（五倍）和游离[Mg²⁺]增加（两倍）。随后用DG -胰岛素灌注导致PCr、[ATP]/[ADP]和A(ATP)缓慢恢复，使得“低能量”状态又持续了16分钟，在此期间ATP保持低水平且恒定。在DG +胰岛素处理结束时（方案的28 - 36分钟，98±18%），通过位移试剂辅助的²³Na NMR评估，细胞内Na⁺含量没有可检测到的变化。此外，在胰岛素洗脱开始时，通过用RbCl替代20%的KCl（[K⁺]=3.76mM，[Rb⁺]=0.94mM）测量的⁸⁷Rb NMR显示Rb⁺流入速率没有变化。在DG +胰岛素处理期间，压力 - 速率乘积（PRP）降低了一半，并且在胰岛素洗脱后恢复到其初始值的80%，无论是否存在位移试剂[5mM三乙撑四胺六乙酸镝（Dy(triethylenetetraminehexaacetate)₃ - ）]。这些数据表明，DG处理诱导的不利热力学[低A(ATP)]和动力学（低[ATP]和[ATP]/[ADP]）条件不会抑制Na⁺，K⁺ - ATP酶活性。我们推测在缺氧期间，当[ATP]/[ADP]的变化与DG处理诱导的变化相当时，观察到的[Na⁺]i增加不是由于[ATP]或[ATP]/[ADP]降低对Na⁺泵的抑制。
