Livers from well-fed female Sprague-Dawley rats (100-150 g) were perfused at flow rates of 4 or 8 ml.g liver-1.min-1 to deliver O2 to the organ at various rates. During perfusion at normal flow rates (4 ml.g-1.min-1), glucagon (10 nM) increased O2 uptake in perfused liver by approximately 40 mumol.g-1.h-1. In contrast, glucagon increased O2 uptake by nearly 100 mumol.g-1.h-1 when livers were perfused at high flow rates. Increase in O2 uptake was directly proportional to flow rate and was blocked partially by infusion of phorbol myristate acetate (100 nM) before glucagon. Increase in O2 uptake due to elevated flow was not due to enhanced glucagon delivery, since infusion of 120 nM glucagon at normal flow rates only increased O2 uptake by approximately 40 mumol.g-1.h-1. On the other hand, when O2 tension in the perfusate was manipulated at normal flow rates, the stimulation of O2 uptake by glucagon increased proportional to the average O2 tension in the liver. Infusion of 8-bromo-adenosine 3',5'-cyclic monophosphate (BrcAMP; 25 microM) also increased O2 uptake more than twice as much at high compared with normal flow rates. In the presence of angiotensin II (5 nM), a hormone that increases intracellular calcium, glucagon increased O2 uptake by nearly 100 mumol.g-1.h-1 at normal flow rates. Infusion of glucagon or BrcAMP into livers perfused at normal flow rates increased state 3 rates of O2 uptake of subsequently isolated mitochondria significantly by approximately 25%. In contrast, perfusion with glucagon or BrcAMP at high flow rates increased mitochondrial respiration by 50-60%. Glucagon addition acutely to suspensions of mitochondria, however, had no effect on O2 uptake. These data are consistent with reports that glucagon administration in vivo or treatment of intact cells with glucagon increases O2 uptake of subsequently isolated mitochondria, a phenomenon that can account for the observed increase in O2 uptake in livers perfused at high flow rates with glucagon. Furthermore, these results are consistent with the hypothesis that the effect of glucagon on mitochondria is O2 dependent in the perfused liver. This is most likely due to an effect of intracellular calcium on a mechanism mediated via cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)