Modulation of Oxidative Phosphorylation by Mg2+ in Rat Heart Mitochondria

Abstract

The effect of varying the Mg 2؉ concentration on the 2-oxoglutarate dehydrogenase (2-OGDH) activity and the rate of oxidative phosphorylation of rat heart mitochondria was studied. The ionophore A23187 was used to modify the mitochondrial free Mg 2؉ concentration. Half-maximal stimulation (K 0.5 ) of ATP synthesis by Mg 2؉ was obtained with 0.13 ؎ 0.02 mM (n ‫؍‬ 7) with succinate (؉rotenone) and 0.48 ؎ 0.13 mM (n ‫؍‬ 6) with 2-oxoglutarate (2-OG) as substrates. Similar K 0.5 values were found for NAD(P)H formation, generation of membrane potential, and state 4 respiration with 2-OG. In the presence of ADP, an increase in P i concentration promoted a decrease in the K ] c has been determined, after stimulation with the muscarinic agonist carbachol, and a 10% increase was observed, after addition of forskolin in rat sublingual mucous acini (3). In acinar pancreatic cells, addition of acetylcholine or cholecystokinin-octapeptide promoted a significant diminution in [Mg 2ϩ ] c (4). Arginine-vasopressin and endothelin-1 induced an increment in [Mg 2ϩ ] c in muscle cells, probably through a Ca 2ϩ -mediated mechanism (5). Depletion of inositol 1,4,5-trisphosphate-sensitive Ca 2ϩ stores, induced by ␣-adrenergic agonists, activated the uptake of Mg 2ϩ by these organelles (6). Extracellular ATP stimulated the release of 40% of cellular Mg 2ϩ in ascites cells (7); it was proposed that cAMP promoted Mg 2ϩ release through the activation of a plasma membrane Na ϩ /Mg 2ϩ antiporter (8). However, a report indicating that addition of cAMP also induced a net release of 20 -25% of total Mg 2ϩ in rat liver mitochondria (9) was not confirmed (10). In beef heart mitochondria, the transition from basal (state 4) to active (state 3) respiration led to a small, but significant elevation in the mitochondrial matrix free ] m , can modulate the activities of the 2-OGDH and the ATP synthase and, in consequence, Mg 2ϩ may affect the rate of oxidative phosphorylation in isolated rat heart mitochondria. MATERIALS AND METHODSRat heart mitochondria were isolated from male Wistar rats of 250 -300-g weight according to a previously described method using the protease type XXVII (Nagarse) from Sigma (14).Dye Loading-Heart mitochondria were loaded with Mag-Fura-2 or BCECF (Molecular Probes) by incubating 30 -40 mg of mitochondrial protein in 2 ml of a medium composed of 250 mM sucrose, 10 mM HEPES, 1 mM EGTA (SHE medium), 1 mM MgCl 2 , 1 mM ADP, 0.2% fatty acid-free bovine serum albumin, pH 7.4, and 5 M Mag-Fura-2/AM or BCECF/AM at 25°C for 20 min. At the end of this incubation period, mitochondria were diluted 10 -15 times with ice-cold SHE medium ϩ 0.2% bovine serum albumin, centrifuged, resuspended in 1 ml of SHE medium, and kept on ice until use. Mitochondria loaded by following this procedure showed higher respiratory control values than nonloaded mitochondria, 8.6 and 4.3 (n ϭ 2), respectively, with 10 mM 2-oxoglutarate as a substrate.Determination of [Mg 2ϩ ] m -Mag-Fura-2-loaded mitochondria (0.5 mg protein/ml) were incubated in 120...

Effect of magnesium on calcium‐induced depolarisation of mitochondrial transmembrane potential

“…This is supported by rapid re-polarisation of DJ m after addition of RuR. It has been documented that ADP stimulates generation of DJ m (Panov and Scarpa, 1996;Rodriguez-Zavala and Moreno-Sanchez, 1998) and inhibits mitochondrial uniporter involved in mitochondrial Ca 2þ uptake (Litsky and Pfeiffer, 1997). Increased resistance to Ca 2þ of A23187 pre-treated mitochondria in the presence of ADP could be therefore attributable to: (1) increased rate of DJ m generation due to ADP-induced stimulation of mitochondrial respiratory chain; and (2) decreased dissipation of protons due to ADP-mediated inhibition of mitochondrial Ca 2þ uptake.…”

Glutamate‐induced calcium increase mediates magnesium release from mitochondria in rat hippocampal neurons

“…It has been reported that high concentrations of glutamate can induce increases in [Ca 21 ] i and Ca 21 accumulation in mitochondria, which in turn mediate depolarization of C mito (Stout et al, 1998;Stanika et al, 2009 (Panov and Scarpa, 1996;Rodriguez-Zavala and Moreno-Sanchez, 1998), it contributes to cellular ATP depletion. Furthermore, it has also been suggested that the increase in [Mg 21 ] i , which is attributed to the mobilization of an internal source hypothesized to be mitochondria is required for apoptosis to occur (Chien et al, 1999).…”

The Involvement of Mg2+ in Regulation of Cellular and Mitochondrial Functions

“…The earlier approach, which was focused on the description of the Mg 2+ effect on isolated mitochondrial enzymes [25, 26], has subsequently been substituted by studies focused on the effect of Mg 2+ on energy metabolism in isolated vital mitochondria [27–29] or vital cells [30, 31]. Some results obtained by the kinetic analysis of isolated enzymes have also been further analysed in more details by mathematical methods [32, 33].…”