Role of Ca2+ in pyruvate dehydrogenase interconversion in brain mitochondria and synaptosomes.
The steady-state content of active (dephospho) pyruvate dehydrogenase (PDHA) of suspensions of coupled rat brain mitochondria oxidizing succinate was found to be markedly increased with increasing free Ca2+ ion concentration of the medium, with a half-maximal effect at 10(-6.43) M Ca2+. Other ions were present in these studies at concentrations appropriate for the cytosol. Depolarization of the plasma membrane of synaptosomes caused an increase in the steady-state content of PDHA, with veratridine giving a larger increase than depolarization by 33 mM-KCl. Values were 68 +/- 1% (n = 13) and 81 +/- 1% (n = 19) of maximal activity, for control incubations and incubations in the presence of 30 microM-veratridine, respectively. Measurements of cytosolic free Ca2+ concentrations ([Ca2+]cyt.) in these suspensions of synaptosomes, with the use of the fluorescent Ca2+-indicator Quin-2, indicated an increase on depolarization, with the change due to 30 microM-veratridine being larger in extent than that due to 33 mM-KCl. Values were 217 +/- 21 nM (n = 15), 544 +/- 48 nM (n = 15) and 783 +/- 75 nM (n = 14) for control, KCl-depolarized and veratridine-depolarized synaptosomes respectively. Experiments in which synaptosomes were treated with Ruthenium Red, an inhibitor of mitochondrial Ca2+ uptake, gave much lower resting contents of PDHA (42 +/- 2% of maximal), but failed to prevent totally an increase on depolarization. Addition of an excess of EGTA to the synaptosomal suspension just before the addition of veratridine resulted in a partial diminution in the response of PDHA content. Parallel studies with Quin-2 indicated no increase in [Ca2+]cyt. on addition of veratridine, under these conditions. Thus an increase in [Ca2+]cyt. forms only a part of the mechanism whereby pyruvate dehydrogenase interconversion responds to depolarization. A decrease in the ATP/ADP ratio may also be important, as inferred from the results of experiments with ouabain, which inhibits the Na+ + K+-dependent ATPase.
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D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D011189:Potassium Chloride / Q000494:pharmacology; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012430:Ruthenium Red / Q000494:pharmacology; D013574:Synaptosomes / Q000187:drug effects / Q000201:enzymology; D014701:Veratridine / Q000494:pharmacology
D000634:Aminoquinolines; D005456:Fluorescent Dyes; D011768:Pyruvate Dehydrogenase Complex; D012430:Ruthenium Red; D011189:Potassium Chloride; D014701:Veratridine; C035739:Quin2; D002118:Calcium