Mitochondrial free [Ca2+] increases during ATP/ADP antiport and ADP phosphorylation: exploration of mechanisms

Abstract

ADP influx and ADP phosphorylation may alter mitochondrial free [Ca2+] ([Ca2+](m)) and consequently mitochondrial bioenergetics by several postulated mechanisms. We tested how [Ca2+](m) is affected by H2PO4(-) (P(i)), Mg2+, calcium uniporter activity, matrix volume changes, and the bioenergetic state. We measured [Ca2+](m), membrane potential, redox state, matrix volume, pH(m), and O2 consumption in guinea pig heart mitochondria with or without ruthenium red, carboxyatractyloside, or oligomycin, and at several levels of Mg2+ and P(i). Energized mitochondria showed a dose-dependent increase in [Ca2+](m) after adding CaCl2 equivalent to 20, 114, and 485 nM extramatrix free [Ca2+] ([Ca2+](e)); this uptake was attenuated at higher buffer Mg2+. Adding ADP transiently increased [Ca2+](m) up to twofold. The ADP effect on increasing [Ca2+](m) could be partially attributed to matrix contraction, but was little affected by ruthenium red or changes in Mg2+ or P(i). Oligomycin largely reduced the increase in [Ca2+](m) by ADP compared to control, and [Ca2+](m) did not return to baseline. Carboxyatractyloside prevented the ADP-induced [Ca2+](m) increase. Adding CaCl2 had no effect on bioenergetics, except for a small increase in state 2 and state 4 respiration at 485 nM [Ca2+](e). These data suggest that matrix ADP influx and subsequent phosphorylation increase [Ca2+](m) largely due to the interaction of matrix Ca2+ with ATP, ADP, P(i), and cation buffering proteins in the matrix.

Figure 1

Time line for adding substances to experimental buffer. PA, pyruvic acid; RRS and RRL, ruthenium red at start or later; ADP, adenosine diphosphate; CATR, carboxyatractyloside; OMN, oligomycin; CCCP, carbonylcyanide m-chlorophenylhydrazone.

Figure 2

Extramatrix free [Ca²⁺] ([Ca²⁺]_e) over time measured using indo-1 (in the presence of 40 μM EGTA). Adding CaCl₂ increased [Ca²⁺]_e in a concentration-dependent manner. Adding 25 μM CaCl₂ increased [Ca²⁺]_e up to 485 nM, which then slowly declined as Ca²⁺ was taken up into the matrix. Adding 10 μM CaCl₂ increased [Ca²⁺]_e to 114 nM with a lesser and slower subsequent Ca²⁺ uptake. RR blocked matrix uptake of Ca²⁺ through the CU so that [Ca²⁺]_e remained constant. ADP caused a small decrease in [Ca²⁺]_e only in the presence of RR.

Figure 3

Effects of ADP and RR on [Ca²⁺]_m over time, measured using indo-1 AM. Dynamic, time-dependent changes (left column) and mean changes (right column) taken at the vertical dotted lines in the left column are displayed. (A) Adding 10 and 25 μM CaCl₂ in energized mitochondria (after PA) caused abrupt, graded increases in [Ca²⁺]_m to steady-state values. Adding ADP caused an abrupt but transient rise in [Ca²⁺]_m above the values established with added CaCl₂. (B) Adding RR after 25 μM CaCl₂ (RRL) did not blunt the rise in [Ca²⁺]_m induced by adding ADP. (C) Adding RR before CaCl₂ (RRS) blocked Ca²⁺ uptake, but the ADP-induced increase in [Ca²⁺]_m remained evident, although it was much smaller (note scale) after adding 25 μM CaCl₂. In the right column, for P < 0.05, # indicates state 3 versus states 2 and 4 respiration; ^∗ indicates RRL and RRS versus the CON group at the same respiration state and CaCl₂.

Figure 4

[Ca²⁺]_m as a function of [Ca²⁺]_e. [Ca²⁺]_e and [Ca²⁺]_m were measured at t = 100 s (before added CaCl₂), at t =180 s (after added CaCl₂), and at t = 250 s (in the presence of ADP). ADP caused a more than additive increase in [Ca²⁺]_m as a function of [Ca²⁺]_e, and the increase was dependent on the existing [Ca²⁺]_m level. Data were taken from Figs. 2 and 3A. For P < 0.05, # indicates state 3 versus states 2 and 4 respiration.

Figure 5

Effects of ADP and altered buffer P_i on [Ca²⁺]_m. (A) Same as Fig. 3A, where [P_i] is 5 mM. (B) In both the 10-mM and 1-mM [P_i] groups, there were no significant differences in response to added CaCl₂ or ADP versus the corresponding 5-mM [P_i] group. For P < 0.05, # indicates state 3 versus states 2 and 4 respiration; ^∗ indicates the HP and LP versus CON groups at the same respiration state and CaCl₂. See Fig. 3 legend for additional details.

Figure 6

Effect of ADP on [Ca²⁺]_m during inhibited phosphorylation and blocked ADP/ATP transport. (A) Same as Fig. 3A. (B) Adding ADP after CaCl₂ and blocking F₁F₀-ATPase with OMN caused a significant increase in [Ca²⁺]_m after addition of both 10 and 25 μM CaCl₂. (C) Adding ADP after CaCl₂ when the ADP/ATP carrier was blocked with CATR inhibited the ADP-induced increase in [Ca²⁺]_m. For P < 0.05, # indicates state 3 versus states 2 and 4 respiration; ^∗ indicates the OMN and CATR versus CON groups at the same respiration state and CaCl₂. See Fig. 3 for additional details.

Figure 7

Effects of adding CaCl₂ and ADP on mitochondrial bioenergetics. All data correspond to [Ca²⁺]_m data displayed in Fig. 3A (CON). (A) PA increased NADH as the TCA cycle was activated. Adding CaCl₂ did not significantly alter NADH values. (B) ADP transiently oxidized mitochondria (i.e., reduced NADH (A)) as energy contained in ΔΨ_m was consumed, as shown by the transiently lowered ΔΨ_m. (C) PA induced a mild alkalinization; adding CaCl₂ did not further alter the matrix pH. ADP transiently reduced matrix pH as matrix proton influx temporarily exceeded proton pumping. (D) O₂ consumption (respiration) increased on addition of PA, and more so on addition of 25 μM CaCl₂. Adding ADP markedly enhanced O₂ consumption, but adding CaCl₂ did not produce any additional effect on respiration. See Table 1 for summary data and statistics on respiration.