Role of substrates in Sr2+-induced oscillations of ionic fluxes in rat liver mitochondria

(1) The reason for substrate specificity of Sr²⁺-induced oscillating cation fluxes in isolated rat liver mitochondria was investigated. (2) With succinate as substrate, rotenone prevented oscillation. In this case the mitochondria were only partially able to take up added Sr²⁺ and did not take up any of the released K⁺. Addition of substances decreasing the mitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio (oxaloacetate or acetoacetate) restored the ability for reuptake of K⁺ and for complete uptake of Sr²⁺ and, therefore, oscillation. (3) Inhibition of substrate-level phosphorylation by arsenite or uncoupling of substrate-level phosphorylation by arsenate in the presence of oligomycin also suppressed the reuptake of cations. This effect of inhibition of substrate-level phosphorylation on oscillation could be circumvented by addition of ATP in the presence of oligomycin. (4) Prevention of the intramitochondrial regeneration of 2-oxoglutarate from acetyl-CoA and oxaloacetate by fluorocitrate or from endogenous glutamate by aminoxyacetate shortened the time during which oscillation with succinate as substrate could be observed. (5) From the key role of substrate level phosphorylation it is concluded that for the reuptake of K⁺ and Sr²⁺ during oscillation, sufficient GTP generation by the succinyl thiokinase (EC 6.2.1.4) reaction is essential. Therefore substrate level phosphorylation seems to be a necessary energy source additional to the respiratory chain. Since the latter process drives the active cation movements, the former may be required for the restoration of a sufficiently low proton conductance of the mitochondrial inner membrane. Oscillation in the absence of exogenous ATP therefore demands 2-oxoglutarate as substrate or the intramitochondrial generation of 2-oxoglutarate for the maintenance of a sufficient GTP production for a longer time.