Adenine nucleotide-dependent Ca²⁺ buffering by mitochondria in an inorganic phosphate-free medium

Inorganic phosphate (P_i) is essential for Ca²⁺ buffering by mitochondria. Adenine nucleotides (AN) are known to strongly increase the Ca²⁺-retention capacity (CRC) of mitochondria even in the absence of P_i in the medium. Several mechanisms can explain this phenomenon. Here we examined these mechanisms in detail in isolated rat liver mitochondria. We found that, in P_i-free medium, AN dose-dependently increased the CRC. The F_OF₁-ATP synthase (F-ATPase) inhibitor oligomycin decreased the CRC and the Ca²⁺ uptake rate to a minor extent. Nuclear magnetic resonance (NMR) analysis showed that P_i in suspensions of oligomycin-treated mitochondria was formed due to AN hydrolysis. In the absence and presence of Ca²⁺, mitochondria accumulated small and large (50 and > 1000 nmol/mg protein) amounts of Pi, respectively, without detectable accumulation of AN. The average ratio of Ca²⁺ to P_i accumulated by intact mitochondria in the presence of ADP, ATP, and ATP plus P_i was about 0.68, 1, and 1.25, respectively, or lower. These values correspond to the formation of calcium dihydrogen and hydrogen orthophosphates, and tricalcium phosphate/whitlockite in different proportions. AN increased the CRC in the presence of inhibitors of both F-ATPase and adenylate translocase, the known regulators of the permeability transition pore (PTP). The PTP inhibitor NADH did not increase the CRC in the absence of P_i. Thus, the mechanism of the AN-dependent increase in the CRC in the absence of P_i includes the F-ATPase-independent production of P_i and suppression of the PTP at the site other than F-ATPase and adenylate translocase.