Effect of phenylephrine on pyruvate dehydrogenase in fasting rat livers.
Previous estimates of flux through the pyruvate-dehydrogenase complex were made by measuring 14CO2 generated from oxidation of [1-14C]pyruvate, assuming a 1:1 stoichiometry. However, this method fails to discriminate between 14CO2 produced from pyruvate dehydrogenase and 14CO2 generated from phospho-enolpyruvate carboxykinase and citric-acid-cycle dehydrogenases. While some previous reports have attempted to correct for the additional 14CO2 production by comparing 14CO2 generated by [1-14C]pyruvate with [2-14C]pyruvate or [3-14C]pyruvate, the estimates are flawed by failure to determine the radioactivity and distribution of the 14C label in the oxalacetate pool. The present method circumvents these problems by utilizing [1,4-14C]succinate to radiolabel the oxalacetate pool and by directly measuring the specific radioactivity of malate. The results demonstrate that flux through the pyruvate-dehydrogenase complex is negligible compared to the other reactions which generate 14CO2 from [1-14C]lactate in the fasted state. Phenylephrine did not significantly alter this result in the fasted state. However, 14CO2 production via the pyruvate-dehydrogenase complex is large (approximately 11.5 nmol.min-1.mg mitochondrial protein-1) compared to 14CO2 production via phosphoenolpyruvate carboxykinase and citric-acid-cycle dehydrogenases (approximately 6.4 nmol.min-1.mg-1) when the pyruvate-dehydrogenase complex is activated, in the fed state with 1 mM dichloroacetate.
Sterniczuk
A
A
Department of Cellular & Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
Hreniuk
S
S
Scaduto
R C
RC
LaNoue
K F
KF
D000818:Animals; D002245:Carbon Dioxide / Q000378:metabolism; D005215:Fasting; D005947:Glucose / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D010477:Perfusion; D010656:Phenylephrine / Q000494:pharmacology*; D011768:Pyruvate Dehydrogenase Complex / Q000032:analysis*; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D051381:Rats; D011919:Rats, Inbred Strains
D011768:Pyruvate Dehydrogenase Complex; D011773:Pyruvates; D002245:Carbon Dioxide; D010656:Phenylephrine; D019289:Pyruvic Acid; D005947:Glucose