The relationship between cellular metabolism and tissue O2 tension was investigated using the isolated perfused rat lung. Lungs were ventilated with gas mixtures of varying PO2 under conditions of no net gas exchange so that alveolar and lung parenchymal gas tensions were in approximate equilibrium. When alveolar PO2 was reduced to 0.7 mmHg, there were significant increases in lung lactate production and perfusate lactate/pyruvate, and decreases in lung tissue ATP content and ATP/ADP. Metabolic parameters were unchanged by alveolar hypoxia when alveolar PO2 was 7 mmHg or greater. Changes of complete anoxia required a PO2 less than 0.04 mmHg. To determine competition between O2 and CO in lung metabolism, alveolar O2 was maintained at 5% and CO was varied from 0 to 90%. Significant changes in production of lactate and pyruvate and tissue ATP content occurred with an alveolar CO of 75% (CO/O2 = 15) but not with CO concentrations of 50%, (CO/O2 = 10) or less. These results with an intact organ confirm previous data with subcellular systems showing a high affinity of the mitochondrial respiratory chain for O2, and indicate that the metabolic changes of hypoxia do not occur until intracellular PO2 approaches 1 mmHg or the CO/O2 exceeds 10.
We investigated the relationship between perfusate concentration of glucose and its utilization and lactate production derived from exogenous glucose and from metabolism of endogenous substrates. Isolated rat lungs were ventilated with 5% CO2 in air and perfused for 100 min with Krebs-Ringer bicarbonate buffer containing 3% bovine serum albumin, 10(-2) U/ml insulin, [U-14C]glucose and [5-3H]glucose. Glucose utilization, total lactate production, [14C]lactate production, and 3H2O production were measured. The apparent Km and Vmax for glucose utilization were 3.4 mM and 72.5 mumol/g dry wt per h, respectively. Lactate production from endogenous substrates, calculated as the difference between total and [14C]lactate, was 37.6 +/- 2.2 mumol/g dry wt (n = 36); it was unaffected by perfusate glucose concentration and by omission of insulin, but increased threefold with anoxia. Lactate production from 1.5 mM glucose was significantly less (P less than 0.02) with insulin omitted. Glycogen content was unchanged during perfusion without glucose. These results suggest that: 1) protein catabolism contributes to lung lactate production; 2) glucose utilization by lung is not maximal at resting physiological glucose concentrations; and 3) insulin is required at low glucose concentrations for maximal glycolytic rates.
The metabolic responsiveness of lung tissue to inhibition of oxidative metabolism was determined by measurement of the redox state of the isolated perfused and ventilated rat lung. Changes in redox state were evaluated by fluorescence from the lung surface at wavelengths suitable for reduced pyridine nucleotides and by measurement of the ratios of redox couples in rapidly frozen lung tissue. Maximal change of redox state was observed during ventilation with carbon monoxide; surface fluorescence increased 6.6%, lactate/pyruvate increased 5.8 times, glycerol 3-P/dihydroxyacetone-P increased fourfold and glutamate/alpha-ketoglutarate doubled. KCN infusion resulted in similar changes. Hypoxia produced with N2 ventilation resulted in less than maximal changes in redox couple ratios until alveolar PO2 was reduced below 0.1 mmHg. Redox changes observed during infusion of 0.5 mM aminoxyacetic acid suggested that maintenance of cytoplasmic redox state depended on functioning of a malate-aspartate "shuttle." The isolated perfused lung appears suitable to study factors controlling pulmonary parenchymal oxidative metabolism. The results emphasize the need for ventilation with CO to establish intracellular anoxia.
Glucose metabolism was studied in isolated rat lungs ventilated with 95% O2.5% CO2 (control), 95% N2: 5% CO2 (hypoxia), and 95% CO:5% CO2 (carbon monoxide) and perfused for 100-120 min with Krebs-Ringer-bicarbonate buffer, pH 7.4, containing [U-14C] and [3-3H]glucose. The production of 14C-labeled lactate plus pyruvate (L + P) and of 14CO2 represented 48% and 22% respectively, of the total [14C]glucose utilization. The lactate-to-pyruvate ratio (L/P) was 8.7. Tritium was recovered predominantly as 3H2O in the perfusate. Wth carbon monoxide ventilation, L + P production was increased by 357% with an L/P of 52.9, and 14CO2 production was markedly decreased. A 56% decrease in lung ATP content was associated with decreased incorporation of 14C into fatty acids. Compared with CO, changes with N2 ventilation were less marked, indicating that ventilation with CO is a more effective method with which to study inhibtion of oxidative metabolism. The lung exhibits a Pasteur effectbintain ATP content or its supply for synthetic activity.
The effect of exogenous lactate on glycolytic rate was studied with the isolated perfused rat lung. Glucose utilization was estimated from the rate of 3H2O production from [5-3H]glucose, and lactate and pyruvate production was measured by perfusate assay. Glucose utilization was unaffected by addition of 0.5 mM lactate to the perfusate but decreased by 27% with 1 mM lactate. With 2 mM lactate, glucose utilization was decreased by 46% and lactate production decreased 95%. With addition of 0.2 mM pyruvate plus 2 mM lactate, glucose utilization was decreased 63% compared with control. These data indicate that the effect of lactate on glucose utilization was not through change in the cellular redox state. During lung anoxia produced by ventilation with CO, glucose utilization and lactate production were again markedly decreased by addition of lactate (2 mM) to the perfusate. However, addition of pyruvate plus lactate resulted in a markedly stimulated rate of glucose utilization. This result indicates that during anoxia the effect of lactate on glycolysis resulted from alteration of the redox ratio. This study indicates that lactate influences the rate of glycolysis in the normal lung through its utilization as a substrate for mitochondrial metabolism. During anoxia, changes in the lung redox state with lactate are a major determinant of the glycolytic rate.
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