Donald F. Tierney

Polyamine metabolism in rat lungs with oxygen toxicity

Ornithine decarboxylase activity increases 2-fold above control after 1 day and 25-fold after 3 days of exposure to 0.85 atm oxygen. Putrescine content nearly doubled by 72 hours which may reflect increased activity or ornithine decarboxylase. Spermidine and spermine content did not increase until after 3 days of exposure which was consistent with the delayed increase of S-adenosylmethionine decarboxylase activity. The results suggest that antimetabolites of polyamine metabolism may be useful to suppress excessive cellular proliferation in the lung after acute lung injury.

Hyperoxia and xanthine dehydrogenase/oxidase activities in rat lung and heart.

Cell injury from hyperoxia is associated with increased formation of superoxide radicals (O2-). One potential source for O2- radicals is the reduction of molecular O2 catalyzed by xanthine oxidase (XO). Physiologically, this reaction occurs at a relatively low rate, because the native form of the enzyme is xanthine dehydrogenase (XD) which produces NADH instead of O2-. Reports of accelerated conversion of XD to XO, and increased formation of O2- formation in ischemia-reperfusion injury, led us to examine whether hyperoxia, which is known to increase O2- radical formation, is associated with increased lung XO activity, and accelerated conversion of XD to XO. We exposed 3-month-old rats either to greater than 98% O2 or room air. After 48 h, we sacrificed the rats and measured XD and XO activities and uric acid contents of the lungs. We also measured the activities of the two enzymes in the heart as a control organ. We found that the activity of XD was not altered significantly by hyperoxia in rat lungs or hearts, but XO activity was markedly lower in the lung, whether expressed per whole organ or per milligram protein, and remained unchanged in the heart. Lung uric acid content was also significantly lower with hyperoxia. The decrease in lung XO activity may reflect inactivation of the enzyme by reactive O2 metabolites, possibly as a negative feedback mechanism. The concomitant decrease in uric acid content suggests either decreased production mediated by XO due to its inactivation or greater utilization of uric acid as an antioxidant. We examined these postulates in vitro using a xanthine/xanthine oxidase system and found that H2O2, but not uric acid, has an inhibitory effect on O2- formation in the system. We therefore conclude that hyperoxia is not associated with increased conversion of XD to XO, and that the exact contribution of XO to hyperoxic lung injury in vivo remains unclear.

Glucose utilization by edematous rat lungs.

Edema was produced in the isolated perfused rat lung by raising left atrial pressure. Eleven control lungs consumed 18±3.9 µmoles glucose/lung · hr−1 and released 17.1±4.2 µmoles lactate/lung · hr−1. During pulmonary edema in 13 isolated perfused lungs, glucose consumption was 35.5±8.8 µmoles/lung · hr−1 (P<.05) and lactate production was 37±5.9 µmoles/lung · hr−1 (P<.05). Separation of radiolabeled glucose and lactate indicated that all lactate was derived from glucose in control and edematous lungs. We found no important difference in14CO2 production from 1-14C, 6-14C, or14C(U)-glucose. Tissue slices of lungs made edematous in vivo had differences in glucose consumption and lactate production which were similar to those observed in the isolated lungs. Oxygen consumption by 1 mm thick lung slices was 224±9.7 µl O2/mg DNA · hr−1 in control and 218±18 µl O2/mg DNA · hr−1 in edematous lungs. When dinitrophenol was added to the medium, the QO2 was greater in the control than in the edematous lung slices (391±22 µl O2/mg DNA · hr−1 control vs. 334±33 µl O2/mg DNA · hr−1 edema,P<.05). We concluded that pulmonary edema in the isolated rat lung is accompanied by: 1) greater glucose consumption; 2) greater lactate production; 3) no important difference in14CO2 production from pentose pathway or tricarboxylic cycle activity; and 4) lower response of edematous tissue slices to dinitrophenol stimulation.

Butyrate increases catalase activity and protects rat pulmonary artery smooth muscle cells against hyperoxia

A protective effect of butyrate against hyperoxia was found with adult rat pulmonary artery smooth muscle cells. Butyrate (5mM) when added just prior to the hyperoxic exposure (95%) markedly decreased lactate dehydrogenase release from cells during 68 hours of exposure (22% release with butyrate versus 98% without). The uptake and reduction of a tetrazolium compound as another index of cell viability also showed similar improvement with butyrate. Butyrate was associated with a striking increase of catalase to three times the control in the air exposed group while GSH content and the activities of superoxide dismutase and glutathione peroxidase were not significantly changed. In the groups exposed to hyperoxia alone, both enzyme activities were decreased compared to the air exposed controls. When butyrate was present with hyperoxia, the superoxide dismutase was maintained closer to the air exposed control values and the catalase activity remained nearly twice as high as the air exposed control cells. These results suggest that butyrate protects rat pulmonary artery smooth muscle cells from hyperoxia by increasing catalase activity which may help to preserve superoxide dismutase activity. This may be a good model to determine the biological significance of catalase and its interrelationships with other antioxidant systems within the cell.

Effects of Ozone Inhalation on Polyamine Metabolism and Tritiated Thymidine Incorporation into DNA of Rat Lungs

We examined the effects of low-level ozone (O3) inhalation on polyamine metabolism and tritiated thymidine (3H-TdR) incorporation into DNA in rat lungs. We have also compared the activities of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis, and glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the pentose phosphate cycle and a typical marker of oxidant injury, to assess whether ODC can serve as a sensitive marker of O3 effects on the lung. We exposed 90-day-old male specific-pathogen-free Sprague-Dawley rats to either 0.45 +/- 0.05 ppm (882 +/- 98 micrograms/m3) O3 or filtered room air continuously for 3 days. After exposure, the rats were terminated and the lungs examined for enzyme activities, polyamine contents, DNA content, and 3H-TdR incorporation. We found that in exposed rats, the enzyme activities were significantly increased (p less than 0.05) relative to air controls. G6PD, 25%, ODC, 147%, and S-adenosylmethionine decarboxylase (AdoMet DC), 86%. Polyamine contents were also affected by O3; putrescine increased 80%, p less than 0.05, spermidine did not change, and spermine decreased 23%, p less than 0.05. 3H-TdR incorporation into DNA was significantly elevated, 155%, p less than 0.001, after O3 exposure while total lung DNA content remained unchanged. The concomitant and large increase in ODC activity (reflecting polyamine metabolism) and DNA labeling (reflecting DNA synthesis and/or repair), indicates a strong correlation between the two and suggests that polyamine metabolism may play an important role in the accelerated cell proliferation associated with O3 injury. Moreover, the greater increase in lung ODC activity compared to other enzymes offers a sensitive marker of the lung response to inhaled O3. We conclude that inhalation of O3 at levels similar to what may be encountered during some smog episodes can result in significant pulmonary biochemical alterations with a potential for long-term consequences. The possible association between ODC activity and DNA labeling may offer a new insight into the mechanism of tissue injury and repair. We also speculate that the changes in lung polyamines may reflect antioxidant and anti-inflammatory functions associated with the cellular defense against oxidant injury.

Cyclic Nucleotide Concentrations in Tissue and Perfusate of Isolated Rat Lung

Cyclic nucleotide content of lung tissue is altered by anesthesia, ventilatory pattern, and pharmacologic manipulation (e.g., isoproterenol). In addition the lung releases cyclic nucleotides into its circulation, but little is known about factors that might alter this release. We isolated and perfused rat lungs (IPL) to determine: 1) if cyclic nucleotides are released into the perfusate in the control state; and 2) if their release changes after alteration of the ventilatory pattern or the addition of isoproterenol. We demonstrated that the rat IPL releases both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) into the perfusate. Isoproterenol has no effect on cGMP release but increases cAMP release dramatically. Perfusate cAMP is not affected by ventilatory pattern, but perfusate cGMP is higher during high-pressure ventilation than it is in nonventilated or normally ventilated lungs.