Modulation of 2-Oxoglutarate Dehydrogenase and Oxidative Phosphorylation by Ca2+ in Pancreas and Adrenal Cortex Mitochondria

Moreno‐Sánchez, Rafael; Rodrı́guez-Enrı́quez, Sara; Cuéllar, Adela; Corona, N.

doi:10.1006/abbi.1995.1314

Cited by 17 publications

(11 citation statements)

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“…Thus, these results indicate that 2-OGDH is one (but not the only) of the main controlling steps of oxidative phosphorylation (see also Moreno-Sá nchez et al (26)), at nonsaturing Mg 2ϩ concentrations. In this respect, control of the rate of oxidative phosphorylation by changes in the spermine/ Mg 2ϩ rates, without a concomitant increase in [Ca 2ϩ ] m , has been shown in dog pancreas mitochondria (19).…”

Section: Discussionmentioning

confidence: 91%

“…In addition to a direct interaction of Mg 2ϩ with the oxidative phosphorylation enzymes, Mg 2ϩ might also perturb matrix Ca 2ϩ homeostasis, and hence, affect the rate of ATP synthesis (16,19,26) (reviewed in Moreno-Sá nchez and Torres-Má rquez (27) Fig. 2), it can be argued that the matrix concentrations of the 2-OGDH coenzymes in intact heart mitochondria may be limiting, and that 2-OGDH activity is not the only controlling step of the pathway (25) were determined at low P i concentrations.…”

Section: Discussionmentioning

confidence: 99%

“…Then, mitochondria were centrifuged at 14,000 rpm for 1 min in a microcentrifuge. Aliquots from the pellet and supernatant were taken to measure the [ 3 H]TPP ϩ distribution; the membrane potential was determined as described previously (19).…”

Section: H]tppmentioning

confidence: 99%

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Modulation of Oxidative Phosphorylation by Mg2+ in Rat Heart Mitochondria

Rodríguez‐Zavala

Moreno‐Sánchez

1998

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 91%

Section: Discussionmentioning

confidence: 99%

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Modulation of Oxidative Phosphorylation by Mg2+ in Rat Heart Mitochondria

Rodríguez‐Zavala

Moreno‐Sánchez

1998

Journal of Biological Chemistry

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“…The supernatant was quickly removed and the mitochondrial pellet was dissolved in 0.5% sodium dodecyl sulfate; aliquots of supernatant and pellet were used for determination of radioactivity by liquid scintillation counting in a Packard liquid scintillation counter (TRI-CARB 2100TR, Meriden, CT, USA). The changes in the membrane potential were calculated using the Nernst equation and corrected for non-specific TPP + binding as described elsewhere (Rottenberg, 1984;Moreno-Sánchez et al, 1995). « m was also estimated by following the change in absorbance of safranin O at 554 minus 520 nm in a dual-wavelength spectrophotometer (2501PC Shimadzu; Japan) (Rodríguez-Enríquez et al, 2012).…”

Section: Determination Of Oxygen Consumption and Changes In Mitochondmentioning

confidence: 99%

“…Changes in the electrical membrane potential ( « m) were determined (a) quantitatively by measuring the tetraphenylphosphonium ([ 3 H]-TPP + ) distribution across the inner mitochondrial membrane (Moreno-Sánchez et al, 1995, 1999; and (b) qualitatively with the permeant cationic dye safranine O (Akerman and Wikström, 1976). For TPP + assay, mitochondria (2 mg) were incubated in 0.5 mL of KME medium under orbital shaking at 37 • C, plus 0.8 M [3H]-TPP + (specific activity 0.06-0.07 Ci/nmol), 5 mM KH 2 PO 4 and the different oxidizable substrates (as indicated in Results) without (State 4) or with 2 mM ADP (State 3).…”

Section: Determination Of Oxygen Consumption and Changes In Mitochondmentioning

confidence: 99%