Abstract
Mitochondria were prepared by a method including a Percoll purification step after the rapid homogenization of livers of fed rats which had been perfused either under unstimulated conditions or in the presence of vasopressin and/or glucagon. The two hormones separately or together increased the total calcium content of the mitochondria. This enhancement was accompanied by parallel increases in activities of the Ca2+-sensitive intramitochondrial enzymes pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. The effects of the two hormones on total mitochondrial calcium and on the activities of the oxidative enzymes were additive. The persistent enhancements of mitochondrial calcium content and enzyme activities were partially reversed by the addition of Na+ ions to the mitochondrial incubations; these effects of Na+ were blocked by diltiazem, a selective inhibitor of Na+-induced Ca2+ release. Mitochondria from control livers were incubated in vitro with CaCl2 to achieve various calcium content, and mitochondrial enzyme activities and calcium content were measured. A good correlation was obtained between the total calcium content and the activities of pyruvate dehydrogenase and oxoglutarate dehydrogenase. The results obtained are consistent with the hypothesis that vasopressin and glucagon additively cause increases in intramitochondrial [Ca2+] and so bring about the activations of these key enzymes of mitochondrial oxidative metabolism.
Highlights
Zation of livers of fed rats which had been perfused isocitrate dehydrogenase)
We present evidence that vasopressin and/or gIucagon added to the perfused liver lead to increases in 2oxoglutarate dehydrogenase activity and pyruvate dehydrogenase (PDHa) content, within subsequently prepared and incubated mitochondria
Mitochondria were incubated a t 30 "Cfor 5 min as described under "Materials and Methods." PDHa activity measured in livers was 5 -+
Summary
Ated form of the pyruvatedehydrogenase complex; EGTA; [ethyl- Livers from fed Sprague-Dawleymale rats (250-280 g) were perenebis~oxyethylenenitriIo)]tetraaceticacid. Mitochondria were prepared from control orglucagon vasopressin-treated Iivers Their calcium content and2-oxoglutarate dehydrogenase activity were measured (Table 11, MI). Total calcium measured in mitochondria incubated under the same conditiaosns mafateand "COz production wag followedafter the addition of 50pM for theoxidative enzyme activities was increased by glucagon a-[l-"C]oxoglutarate after a 5-min preincubation periotdh;e reaction and by vasopressin. When both hormones were added towas terminated by the addition of 1N perchloric acid[17].Total calcium content was measuredin mitochondria incubated itnhe same conditions as for PDHa activity except that 4-6 mg of protein/ml were used.
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