Perturbation of cellular energy state in complete ischemia: Relationship to dissipative ion fluxes

Summary

Loss of cellular ion homeostasis during anoxia, with rapid downhill fluxes of K⁺, Ca²⁺, Na⁺ and Cl^-, is preceded by a slow rise in extracellular K⁺ concentration (K ⁺_e ), probably reflecting early activation of a K⁺ conductance. It has been proposed that this conductance is activated by either a rise in intracellular calcium concentration (Ca ²⁺_i ), or by a fall in ATP concentration. In a previous study from this laboratory (Folbergrová et al. 1990) we explored whether the early activation of a K⁺ conductance could be triggered by a rise in Ca ²⁺_i . To that end, labile metabolites and phosphorylase a, a calcium sensitive enzyme, were measured after 15, 30, 60 and 120 s of complete ischemia (“anoxia”). In the present study, we investigated whether brief anoxia is accompanied by changes in ATP/ADP ratio, or in the phosphate potential, which could cause activation of a K⁺ conductance. To provide information on this issue, we added a group with 45 s of anoxia to the previously reported groups, and derived changes in intracellular pH (pH_i). This allowed calculations of the free concentrations of ADP (ADP_f) and AMP (AMP_f) from the creatine kinase and adenylate kinase equilibria, and hence the derivation of ATP/ADP_f ratios. In performing these calculations we initially assumed that the free intracellular Mg²⁺ concentration remained unchanged at 1 mM. However we also explored how a change in Mg ²⁺_i of the type described by Brooks and Bachelard (1989) influenced the calculation. The results showed that ADP_f must have risen to 150–200% of control within 15 s, and to 330–350% of control within 45 s of anoxia. The concentration of AMP_f should have increased 2–4 fold in 15 s and 10–20 fold in 45 s. Thus although tissue ATP concentration usually remains >90% of control within the first 30s of anoxia, and >80% of control within the first 45 s, the ATP/ADP_f ratios change markedly at a time when alterations in ion homeostasis are dominated by a moderate rise in K ⁺_e , and long before massive ion fluxes occur and the cells depolarise (after about 60–70 s). Such early changes in ATP/ADP_f ratio, or in phosphate potential, could well influence reactions which are coupled to ATP hydrolysis, and perhaps lead to activation of ATP-dependent K⁺ conductances.