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Stable changes to calcium fluxes in mitochondria isolated from rat livers perfused with alpha-adrenergic agonists and with glucagon.

ABSTRACT: Mitochondria isolated from rat liver after a short-term perfusion with the alpha-adrenergic agonist phenylephrine or with glucagon exhibited enhanced rates of uptake of Ca2+ and prolonged retention of Ca2+ in the presence of 4mm-P(i). The effect of Ca2+ retention was apparent after perfusion with phenylephrine for only 1min and was maximal after 7min of treatment. The changes induced by glucagon, although similar, were less rapid. Adrenaline caused similar changes to phenylephrine and its effects were blocked by the alpha-adrenergic antagonist phenoxybenzamine, but not by the beta-antagonist propranolol. The Ca2+ content of the isolated mitochondria decreased by 30% 1min after the onset of perfusion with phenylephrine; by 6min it had begun to return to the original value which was reached at 10min. A similar loss in calcium content was induced by glucagon but the changes were not as great and occurred more slowly. Mitochondria from phenylephrine-treated livers exhibited decreased rates of Ca2+ efflux induced by addition of 2mm-EGTA, a 50% increase in the contents of ADP and total adenine nucleotides, a small increase in the transmembrane pH gradient, and a reduced rate of oxaloacetate-induced NADPH oxidation. This study thus shows that stimulation of liver by alpha-adrenergic agonists, like that by glucagon, induces within minutes a stable modification of mitochondria leading to alterations in the Ca2+-translocation cycle (increased Ca2+ uptake and retention) and alterations in mitochondrial energy-linked reactions.

SUBMITTER: Taylor WM

PROVIDER: S-EPMC1161887 | biostudies-other | 1980 May

REPOSITORIES: biostudies-other

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Project description:1. Mitochondria isolated from rat liver by centrifugation of the homogenate in buffered iso-osmotic sucrose at between 4000 and 8000g-min, 1h after the administration in vivo of 30mug of glucagon/100g body wt., retain Ca(2+) for over 45min after its addition at 100nmol/mg of mitochondrial protein in the presence of 2mm-P(i). In similar experiments, but after the administration of saline (0.9% NaCl) in place of glucagon, Ca(2+) is retained for 6-8min. The ability of glucagon to enhance Ca(2+) retention is completely prevented by co-administration of 4.2mg of puromycin/100g body wt. 2. The resting rate of respiration after Ca(2+) accumulation by mitochondria from glucagon-treated rats remains low by contrast with that from saline-treated rats. Respiration in the latter mitochondria increased markedly after the Ca(2+) accumulation, reflecting the uncoupling action of the ion. 3. Concomitant with the enhanced retention of Ca(2+) and low rates of resting respiration by mitochondria from glucagon-treated rats was an increased ability to retain endogenous adenine nucleotides. 4. An investigation of properties of mitochondria known to influence Ca(2+) transport revealed a significantly higher concentration of adenine nucleotides but not of P(i) in those from glucagon-treated rats. The membrane potential remained unchanged, but the transmembrane pH gradient increased by approx. 10mV, indicating increased alkalinity of the matrix space. 5. Depletion of endogenous adenine nucleotides by P(i) treatment in mitochondria from both glucagon-treated and saline-treated rats led to a marked diminution in ability to retain Ca(2+). The activity of the adenine nucleotide translocase was unaffected by glucagon treatment of rats in vivo. 6. Although the data are consistent with the argument that the Ca(2+)-translocation cycle in rat liver mitochondria is a target for glucagon action in vivo, they do not permit conclusions to be drawn about the molecular mechanisms involved in the glucagon-induced alteration to this cycle.

Project description:Isolated liver perfusion systems have been used to characterize intrinsic metabolic changes in liver as a result of various perturbations, including systemic injury, hepatotoxin exposure, and warm ischemia. Most of these studies were done using hyperoxic conditions (95% O(2)) but without the use of oxygen carriers in the perfusate. Prior literature data do not clearly establish the impact of oxygenation, and in particular that of adding oxygen carriers to the perfusate, on the metabolic functions of the liver. Therefore, herein the effects of oxygen delivery in the perfusion system on liver metabolism were investigated by comparing three modes of oxygenation. Rat livers were perfused via the portal and hepatic veins at a constant flow rate of 3 mL/min/g liver in a recirculating perfusion system. In the first group, the perfusate was equilibrated in a membrane oxygenator with room air (21% O(2)) before entering the liver. In the second group, the perfusate was equilibrated with a 95% O(2)/5% CO(2) gas mixture. In the third group, the perfusate was supplemented with washed bovine red blood cells (RBCs) at 10% hematocrit and also equilibrated with the 95% O(2)/5% CO(2) gas mixture. Oxygen and CO(2) gradients across the liver were measured periodically with a blood gas analyzer. The rate of change in the concentration of major metabolites in the perfusate was measured over time. Net extracellular fluxes were calculated from these measurements and applied to a stoichiometric-based optimization problem to determine the intracellular fluxes and active pathways in the perfused livers. Livers perfused with RBCs consumed oxygen at twice the rate observed using hyperoxic (95% O(2)) perfusate without RBCs, and also produced more urea and ketone bodies. At the flow rate used, the oxygen supply in perfusate without RBCs was just sufficient to meet the average oxygen demand of the liver but would be insufficient if it increased above baseline, as is often the case in response to environmental perturbations. Metabolic pathway analysis suggests that significant anaerobic glycolysis occurred in the absence of RBCs even using hyperoxic perfusate. Conversely, when RBCs were used, glucose production from lactate and glutamate, as well as pathways related to energy metabolism were upregulated. RBCs also reversed an increase in PPP fluxes induced by the use of hyperoxic perfusate alone. In conclusion, the use of oxygen carriers is required to investigate the effect of various perturbations on liver metabolism.

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Stable changes to calcium fluxes in mitochondria isolated from rat livers perfused with alpha-adrenergic agonists and with glucagon.

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