Age D. Boelens

Mitochondrial Free [Ca2+] Increases during ATP/ADP Antiport and ADP Phosphorylation: Exploration of Mechanisms

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.

Increases in Extra-Matrix Mg2+ Inhibit Ca2+ Uptake via the Ca2+-Uniporter but do not Acutely Alter State 3 Respiration

Magnesium is essential for all energy-dependent transport systems, glycolysis, and oxidative energy metabolism. In addition, Mg2+ binds to ADP and ATP, regulating their availability. It also controls matrix free Ca2+ concentration (m[Ca2+]) through its inhibitory effect on the mitochondrial Ca2+-uniporter. Here we investigated the interplay between Ca2+ and Mg2+ and their roles in regulating mitochondrial bioenergetics. Mitochondria from guinea pig hearts were isolated by differential centrifugation, incubated with fluorescent dyes indo-1-AM for m[Ca2+], furaptra-AM for m[Mg2+], or DMSO. Ca2+-uptake in mitochondria, which were suspended in respiration buffer containing EGTA, 0, 0.5 or 2 mM MgCl2, and 0.5 mM pyruvic acid, was measured in response to 0, 0.25 and 0.50 mM CaCl2. In separate experiments Mg2+ uptake was measured after adding 0, 0.5 and 2 mM MgCl2. O2 consumption was measured using a Clark-2 O2-electrode during states 2 through 4 respiration, i.e. before, during and after adding 250 µM ADP. Addition of MgCl2 to the buffer inhibited Ca2+-uptake in a dose-dependent manner. 0.25 mM CaCl2 increased m[Ca2+] from 59 nM to 325, 131 and 93 nM Ca2+ in the 0, 0.5 and 2 mM MgCl2 groups, respectively, whereas 0.50 mM CaCl2 increased m[Ca2+] to 1045, 360 and 279 nM, respectively. Adding 0.5 mM MgCl2 did not increase m[Mg2+], but 2 mM MgCl2 increased m[Mg2+] slowly from 0.35 to 0.55 mM over ten minutes. State 3 respiration was not different among the three MgCl2 groups, but state 4 was faster with MgCl2 present. We conclude that the inhibitory effect of extra-matrix Mg2+ on Ca2+-uptake is close to maximum in the low range of normal cytosolic [Mg2+], and that mitochondrial Mg2+ influx by itself is not fast enough to acutely modulate state 3 respiration.

Buffer Magnesium Limits Mitochondrial Calcium Uptake but not Matrix Calcium Buffering in Response to ADP

Mg2+ is known to limit Ca2+ uptake by mitochondria through the Ca2+ uniporter. Changes in matrix Ca2+ concentration are an important signaling pathway in mitochondrial function as well as in apoptosis. In a previous study we showed an increase in matrix free Ca2+ in response to added ADP in MgCl2 free buffer. Because of the presumed role of Mg2+ in mitochondrial regulation of Ca2+ we explored the effects of buffer Mg2+ on matrix Ca2+ uptake and buffering in isolated mitochondria. Guinea pig heart mitochondria were isolated by differential centrifugation, loaded with the fluorescent dye Indo 1 AM and then suspended in respiration media, containing 1 mM of EGTA, with or without added 1 mM MgCl2. To the mitochondrial suspension was added 0.5 mM pyruvic acid, either 0.25, 0.5 or 0.75 mM CaCl2, and 250 μM ADP. Adding 0.25, 0.5 and 0.75 mM Ca2+ caused a dose-dependent increase in matrix Ca2+ of 14, 35 and 45%, respectively, in the group without Mg2+ in the buffer, and 6, 18 and 42%, respectively, in the group with Mg2+ in the buffer. The differences in uptake between Mg2+ and no Mg2+ groups were significant in the 0.25 and 0.5 mM groups, but not in the 0.75 mM group. The additional increase in matrix free Ca2+ in response to ADP without Mg2+ was 9, 11 and 9% for the 0.25, 0.5 and 0.75 mM Ca2+ groups, respectively. These additional increases in matrix free Ca2+ with ADP were not significantly altered by Mg2+. We conclude that external Mg2+ alters the uptake of Ca2+ into the mitochondrial matrix, but does not alter the increase in matrix ionized Ca2+ after addition of ADP.