Mark R. Montgomery

Immunocytochemical localization of lysozyme and surfactant protein A in rat type II cells and extracellular surfactant forms.

Using immunogold labeling of fixed, cryosubstituted tissue sections, we compared the distribution of lysozyme, an oxidant-sensitive lamellar body protein, with that of surfactant protein A (SP-A) in rat Type II cells, extracellular surfactant forms, and alveolar macrophages. Morphometric analysis of gold particle distribution revealed that lysozyme and SP-A were present throughout the secretory and endosomal pathways of Type II cells, with prominent localization of lysozyme in the peripheral compartment of lamellar bodies. All extracellular surfactant forms were labeled for both proteins with preferential labeling of tubular myelin and unilamellar vesicles. Labeling of tubular myelin for SP-A was striking when compared with that of lamellar bodies and other extracellular surfactant forms. Lamellar body-like forms and multilamellar structures were uniformly labeled for lysozyme, suggesting that this protein is rapidly redistributed within these forms after secretion of lysozyme-laden lamellar bodies. By contrast, increased labeling for SP-A was observed over peripheral membranes of lamellar body-like forms and multilamellar structures, apparently reflecting progressive SP-A enrichment of these membranes during tubular myelin formation. The results indicate that lysozyme is an integral component of the lamellar body peripheral compartment and secreted surfactant membranes, and support the concept that lysozyme may participate in the structural organization of lung surfactant.

Acute ozone-induced lung injury in rats: structural-functional relationships of developing alveolar edema.

As part of a study on the effects of acute ozone stress on the lung surfactant system, we correlated morphometric, biochemical, and functional indices of lung injury using male rats exposed to 3 ppm ozone for 1, 2, 4, and 8 hr. Evaluation of lung mechanics, using the Pulmonary Evaluation and Diagnostic Laboratory System, revealed a significant decrease in dynamic lung compliance (ml/cmH2O/kg) from a control value of 0.84 +/- 0.02 (SEM) to 0.72 +/- 0.04 and 0.57 +/- 0.06 at 4 and 8 hr, respectively. At 2 hr there was a transient increase in PaO2 to 116 torr (control = 92 torr) followed by a decrease at 4 hr (65 torr) and 8 hr (55 torr). Morphometry of lung tissue, fixed by perfusion of fixative via the pulmonary artery at 12 cm H2O airway distending pressure, demonstrated an increase in the area of the intravascular compartment at 8 hr, in association with a 65 and 39% replacement of the alveolar area by fluid in ventral and dorsal lung regions, respectively. There was a positive correlation (r = 0.966) between alveolar edema and transudated proteins in lavage fluid. A stepwise multiple regression model, with edema as the dependent variable, suggested that pulmonary vasodilatation, hypoxemia, and depletion of surfactant tubular myelin in lavage fluid were indices for predicting alveolar edema. In a second model, with lavage protein concentration as the dependent variable, decreasing dynamic compliance and hypoxemia were predictors of progressive, intraalveolar transudation of plasma proteins. The above structural-functional relationships support the concept that ozone-induced high-protein alveolar edema is pathogenetically linked to pulmonary hyperemia, deficiency of surfactant tubular myelin, and associated lung dysfunctions.

Impairment in Pulmonary Bioenergetics Following Chlorphentermine Administration to Rats

Biochemical alteration in pulmonary oxidative metabolism and morphological integrity of lung mitochondria were examined in rats following administration of chlorphentermine (30 mg/kg, ip, 5 days per week) for 1 or 2 weeks. During the first week of treatment, body weight gain and food intake were decreased markedly but returned to control levels during the second week. Phospholipid content of the lung was increased 31% and 110% after 1 and 2 weeks of treatment, respectively. This was accompanied by a striking intraalveolar accumulation of hypertrophic alveolar macrophages. The metabolism of both (1-14C)- and (6-14C)-glucose was decreased 27% and 26%, respectively, after 2 weeks of drug treatment. In rat lung mitochondria, chlorphentermine significantly lowered the RCR and ADP/O ratio and stimulated state 4 respiration. State 3 respiration and uncoupled state respiration were unaffected. These data indicate that chlorphentermine functions as a true uncoupler of oxidative phosphorylation when administered in vivo. Furthermore, disruption of mitochondrial membranes was observed frequently in lung mitochondria from treated animals. These combined data indicate that the induction of pulmonary phospholipidosis by chlorphentermine is accompanied by marked alterations in subcellular bioenergetics and mitochondrial structure.

Age-related difference in pulmonary response to ozone

Acute exposure to 1.5 ppm O3 produced different responses in adult and aged rat lungs. Total triphosphonucleotides were only slightly decreased in adult animals, but were markedly decreased in aged animals. Also, adult animals maintained a greater proportion of their available triphosphonucleotides in the reduced form (NADPH) compared to aged animals. These results suggest that aged animals may not be able to maintain pulmonary reducing equivalents as efficiently as adult animals in the face of an oxidant insult.

Ascorbic acid potentiates the substrate‐specific inhibition of mixed‐function oxidation and the stimulation of NADPH oxidation caused by paraquat

Paraquat inhibits the in vitro hepatic microsomal metabolism of both ethylmorphine and aniline. Inclusion of ascorbate with paraquat in the incubations did not alter the paraquat effect on ethylmorphine N-demethylase activity but potentiated the inhibition of aniline p-hydroxylase activity. Ascorbate alone was without effect on the metabolism of either substrate. Paraquat stimulated the hepatic microsomal oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) equally in the absence of mixed-function oxidase (MFO) substrates or in the presence of ethylmorphine; in the presence of aniline the rate of NADPH oxidation was significantly greater. Also, in the presence of aniline, ascorbate potentiated the paraquat-induced NADPH oxidation, while it was ineffective with paraquat on NADPH oxidation in the presence of ethylmorphine or in the absence of substrates for the microsomal MFO system. The potentiated inhibition of aniline metabolism, concomitant with the potentiated stimulation of NADPH oxidation, was consistent whether liver microsomal fractions were prepared from control rats or from animals induced with phenobarbital. Investigation of possible influences on NADPH cytochrome c reductase activity was precluded by the rapid nonenzymatic reduction of cytochrome c by ascorbate. The paraquat-ascorbate redox couple would not reduce cytochrome P-450. These data suggest that a paraquat interaction with the active microsomal MFO enzyme system plays a role in the depletion of cellular NADPH stores that occurs after paraquat administration in vivo. This mechanism may play a significant role in the development of paraquat toxicity and in the potentiated toxicity observed with ascorbate and paraquat.

Ascorbic acid nutritional status does not affect the biochemical response to paraquat

Guinea pigs were subjected to dietary control of ascorbic acid intake to produce deficient, normal, and supplemented tissue ascorbate concentrations. Paraquat, a potent pulmonary toxin, was then administered. Tissue distribution and covalent binding of paraquat were not affected significantly by ascorbate nutritional status except for decreased 24-hr kidney burdens in deficient animals. Paraquat increased pulmonary thiobarbituric acid reactivity and mixed disulfide formation, but no evidence of either a protective or a potentiative interaction of endogenous ascorbate and paraquat was observed. This study indicates that alteration of endogenous ascorbic acid levels does not alter the biochemical response to paraquat.

Ozone Increases Atrial Natriuretic Peptides in Heart, Lung and Circulation of Aged vs. Adult Animals

Ozone can cause pulmonary edema and simultaneously decrease blood pressure. Atrial natriuretic peptides may mediate both of these affects since they increase pulmonary capillary permeability and are potent vasodilating peptides. To examine this possibility and determine if aged animals respond differently to ozone, adult (4-6 months old) and aged (24-26 months old) Fischer 344 rats were exposed to ozone (0.5 parts per million) for 8 h. Ozone increased atrial natriuretic peptides recognized by the proANF 1-30, proANF 31-67, and atrial natriuretic factor (ANF) radioimmunoassays in lung from 3.7 +/- 0.2, 3.5 +/- 0.1, and 3.0 +/- 0.2 ng/g of lung, respectively, to 9.7 +/- 1.0, 9.2 +/- 0.4, and 13.3 +/- 2.7 ng/g in adult rats (n = 6) and from 3.6 +/- 0.1, 3.5 +/- 0.1, and 3.2 +/- 0.1 ng/g of lung of aged rats (n = 6) to 10.2 +/- 0.3, 10.0 +/- 0.2, and 12.6 +/- 0.2 ng/g. Ozone increased the content of these peptides in the heart 2- to 5-fold from 266 +/- 25, 226 +/- 22, and 288 +/- 40 ng/g of heart to 716 +/- 26, 471 +/- 14, and 1,473 +/- 235 ng/g in the same adult animals and from 495 +/- 17, 483 +/- 22, and 501 +/- 18 ng/g to 903 +/- 16,879 +/- 21, and 1,489 +/- 31 ng/g of heart in the aged animals. Ozone also doubled the concentration of these atrial natriuretic peptides in the circulatory system. This study demonstrates that ozone increases atrial natriuretic peptides in the lung, heart, and circulation of equal magnitude in aged versus adult animals, indicating an equal response to ozone with aging. This study further suggests that atrial natriuretic peptides may mediate the decreased blood pressure and pulmonary edema observed with ozone exposure.

Alterations in monoamine oxidase activity after single and repeated exposure of rats to chlorphentermine

Monoamine oxidase (MAO) activity was determined in rat tissues following in vivo treatments with chlorphentermine (CP). Oxidation of seven amine substrates by liver, lung or brain mitochondrial MAO was investigated at 2 h after a single i.p. injection (60 mg/kg) or after repeated injection (once daily for 3 days, 20 mg/kg, i.p.). Deamination of the type A substrates, norepinephrine, serotonin and octopamine, was decreased significantly in liver, lung and brain after both single and repeated injections. Oxidative deamination of tyramine and dopamine (type A + B substrates) was also lowered in all organs after single and repeated exposure to CP, but to a lesser degree than the type A substrates. However, oxidation of the type B substrates, benzylamine and tryptamine, was unaffected by CP administration in comparison to control. These data indicate that CP is a specific inhibitor of mitochondrial MAO, form A.

Recovery of lung pyridine nucleotides following acute exposure of adult and aged rats to ozone

Male, pathogen-free Fischer 344 rats aged 6 and 24 mo were exposed to 1.5 or 3.0 ppm for 8 h and recovery rates of diphosphonucleotides (NAD+ and NADH) and triphosphonucleotides (NADP+ and NADPH) were measured and compared to controls. Recovery after 0.5 ppm was not examined because no significant changes occurred in either age group after this lower exposure. At zero time (immediately after exposures) both concentrations are depressed in adults and aged animals except for NADH in aged animals at 3.0 ppm; NADP+ in adults at 1.5 and 3.0 ppm was decreased, but not significantly. For NAD+ and NADH, recovery of whole lung concentrations is complete by 24 h following an 8-h exposure to 1.5 or 3.0 ppm of ozone. Only after 3.0 ppm of ozone was the ratio of the reduced to oxidized form (NADH/NAD+) still elevated after 24 h; however, it also returned to control levels by 96 h. For the triphosphonucleotides, an 8-h exposure to 1.5 ppm of ozone resulted in a sustained depression of whole lung concentrations of NADPH throughout the 96-h recovery period. Also, only after the 1.5 ppm exposure was the reduced to oxidized ratio (NADPH/NADP+) significantly depressed throughout the 96-h recovery period. Unexpectedly, recovery of whole lung levels returned to normal within 24 h after the 8-h exposure to both the 1.5 and the 3.0 ppm concentrations. With the exception of the sustained effect on NADPH levels, these data indicate that di- and triphosphonucleotide concentrations rapidly return to normal in the lung after severe, acute oxidant injury. There were no differences in recovery rates between the adult and the aged groups.

Artifacts in the determination of microsomal xenobiotic N-demethylation in the presence of ascorbic acid Tris buffer and Nash reagent

1. During investigation of microsomal xenobiotic N-demethylation in the presence of ascorbic acid, large increases in apparent enzymic activity were observed. 2. Examination of incubation components indicated that a non-enzymic interaction between ascorbic acid and Tris buffer, in the presence of acetylacetone and ammonium acetate (Nash reagent), was occurring. 3. The resulting chromophore had an absorption maximum at 412 nm that coincided with the absorption for the chromophore resulting from the interaction of the Nash reagent with the product (formaldehyde) of the enzymic reaction. 4. Strict controls of ascorbic acid potential chemical interaction with incubation components are required in enzymic studies.