Donald Massaro

Postnatal development of alveoli. Regulation and evidence for a critical period in rats.

In many species, including humans, pulmonary alveoli are formed after birth by septal subdivision of the large gas-exchange saccules present at birth. In rats septation occurs mainly between the 4th and 14th postnatal days (Burri, P. H. 1974. Anat. Rec. 180:77-98), but little is known about the regulation of this process. We found that dexamethasone (0.1 micrograms daily) given to rats from age 4 to 13 d markedly impaired saccule septation to at least age 60 d and also diminished the extent of the increase of alveolar surface area (Sa). Underfeeding from birth to age 14 d did not diminish saccule septation but did result in diminished Sa. We conclude dexamethasone-treated rats have a critical period during which the gas-exchange saccules present at birth must be subdivided. Since Sa increased in dexamethasone-treated rats without a change in alveolar size, and, the enlargement of Sa was diminished in underfed rat pups without a deficit of saccule septation, we postulate new alveoli were formed by means other than septation of the large gas-exchange saccules present at birth. Furthermore, these various means of forming alveoli, and hence of increasing Sa, were differently regulated: dexamethasone decreased the enlargement of Sa brought about by both septation of the gas-exchange saccules present at birth and by other, as yet unidentified, means of forming alveoli; underfeeding did not diminish Sa increases produced by saccule septation but did decrease the extent of Sa enlargement due to the other means of forming alveoli.

Changes in Sedimentation of Surfactant in Ventilated Excised Rat Lungs: PHYSICAL ALTERATIONS IN SURFACTANT ASSOCIATED WITH THE DEVELOPMENT AND REVERSAL OF ATELECTASIS

We ventilated excised rat lungs at a constant tidal volume (CTV); they developed areas of atelectasis which could be reversed by a large inflation (CTV + I) or prevented by the addition of positive end-expiratory pressure to the CTV. To explore the possibility that these modes of ventilation led to changes in surfactant, we lavaged the lungs and centrifuged the returns at 500 g; we measured the amount of disaturated phosphatidylcholine (DSPC) in the resultant pellet and supernatant fluid as a marker for surfactant. We found 16.9+/-1.5 (mean+/-SE), 38.0+/-2.4, 18.3+/-1.6, and 21.7+/-2.3% of the total lavage DSPC, in the pellet from freshly excised, CTV, CTV + I, and positive end-expiratory pressure to the CTV lungs, respectively. The total amount of lavage DSPC was the same in all groups. The ultrastructure of acellular material pelleted by sequential centrifugation of lavage returns at 500, 1,000, and 60,000 g was examined. We found mostly tubular myelin in the 500-g and 1,000-g pellets, but no tubular myelin in the 60,000-g pellet. Air inflation pressure-volume measurements from the degassed state revealed that the opening pressure and recoil pressures up to 75% of total lung capacity were significantly higher in the CTV than in the CTV + I lungs. There were no differences between these groups in air deflation or in saline inflation and deflation pressure-volume measurements. Our findings suggest that CTV leads to increases in the tubular myelin form of surfactant and that this leads to increased surface tension in alveoli which results in alveolar collapse.

Rat lung Cu,Zn superoxide dismutase. Isolation and sequence of a full-length cDNA and studies of enzyme induction.

The synthesis of Cu,Zn SOD by rat lung increases spontaneously in the fetus in late gestation and during exposure of neonatal and adult rats to greater than 95% O2. To explore the regulation of these increases, we measured rat lung Cu,Zn SOD synthesis and activity. We also cloned and sequenced a rat lung Cu,Zn SOD cDNA that was used to measure Cu,Zn SOD mRNA concentration. We found that (a) under normal gestational and postgestational conditions the synthesis of this enzyme was regulated pretranslationally; (b) the increased synthesis that occurs under hyperoxia (greater than 95% O2), was pretranslationally mediated in otherwise unmanipulated neonatal rats but translationally controlled in hyperoxic adult rats; and (c) in lungs of rats made tolerant to greater than 95% O2 by allowing 24 h rest in air after an initial 48 h in greater than 95% O2, the increased Cu,Zn SOD synthesis that occurred during the second period of hyperoxia was regulated pretranslationally. We conclude Cu,Zn SOD gene expression in the lung is developmentally regulated under normal conditions and in response to an oxidant challenge. Tolerance, whether endogenous or induced, appears to require the accumulation of increased amounts of Cu,Zn SOD mRNA.

Regulation of secretion in Clara cells: studies using the isolated perfused rat lung.

Previous studies from our laboratory indicated that both beta-adrenergic and cholinergic agents stimulate in vivo secretion by rat bronchiolar Clara cells. Those studies also provided support for an in-series beta-adrenergic-cholinergic stimulation of secretion. To further explore the regulation of secretion in Clara cells, and to do it in the absence of systemic influences, we have used the isolated ventilated perfused rat lung. We have again used morphometry and electron microscopy to assess secretion by measuring the volume density (fraction of cell volume) of the secretory granules of bronchiolar Clara cells. We found that in the isolated perfused lung, as in the intact animal, isoproterenol stimulated secretion in Clara cells and that this effect was blocked by the beta-adrenergic antagonist propranolol. Pilocarpine, unlike its action in the intact animal, did not stimulate secretion in the isolated lung; rather it inhibited the secretory effect of isoproterenol. Increased tidal-volume ventilation stimulated secretion; propranolol did not block this effect. Analogs of cyclic (c)AMP and of cGMP also stimulated secretion by Clara cells. These findings indicate that there are at least two mechanisms by which Clara cells can be stimulated to secrete. One seems to be beta-adrenergic-cAMP mediated but the triggering event is unknown. The other is initiated by increased tidal volume and cGMP may be involved in the intracellular mediation of this stimulatory event. Finally, we found evidence of beta-adrenergic (stimulatory) -cholinergic (inhibitory antagonism in the regulation of secretion in Clara cells.

Exposure of rats to ozone: Evidence of damage to heart and brain

Ozone is a strong oxidizing agent, and in many locations it is a major atmospheric pollutant. It is phytotoxic and an important cause of lung dysfunction in humans. Recently, a significant association has been established between total atmospheric oxidants, of which ozone is one, and daily cardiovascular mortality rates. In this article, we show that exposure of rats to ozone for 5 days, in a concentration found in major urban centers, results in an increased concentration of thiobarbituric acid-reactive material (an indicator of lipid peroxidation) in heart and brain tissue as well as elevated activity of catalase and glutathione peroxidase (enzymic scavengers of peroxides) in these tissues. We examined the heart anatomically and found evidence of extracellular and intracellular edema. These findings indicate that the heart and brain are damaged by a concentration of ozone present in major urban centers; they may have important implications for chronic illness and degenerative processes in humans.

Gene Expression of Cellular Retinoid-Binding Proteins: Modulation by Retinoic Acid and Dexamethasone in Postnatal Rat Lung

In rats, septation of gas-exchange saccules occurs during the first 2 postnatal weeks; dexamethasone (DEX) treatment irreversibly impairs septation, and treatment with all-trans retinoic acid (RA) prevents the DEX-induced inhibition of septation. Cellular retinoic acid-binding protein I (CRABP I) and cellular retinol-binding protein I (CRBP I) are important modulators of the cellular metabolism of retinoids. In the present study, therefore, we measured the mRNA concentration of CRABP I and CRBP I in lungs of neonatal rats. In untreated rats, CRABP I and CRBP I mRNA peaked at postnatal d 8, indicating that CRABP I and CRBP I are developmentally regulated at least in part at a pretranslational level during lung septation. Daily treatment of 3-to 8-d-old rats with RA (500 µg/kg/d) had no effect on the level of CRABP I mRNA; treatment with DEX (0.25 µg/d) from d 4 to 8 caused a decrease in CRABP I mRNA that was not prevented by concomitant treatment with RA. These findings suggest that a decrease in CRABP I expression may be important in the DEX-induced block of septation but not in the prevention by RA of DEX-induced inhibition of septation. RA treatment caused an increase of CRBP I mRNA; conversely, treatment with DEX caused a decrease in CRBP I mRNA that was prevented by concomitant treatment with RA. These data suggest CRBP I may play a role in RA-induced septation, in the inhibition of septation caused by DEX, and in the ability of RA to prevent DEX-blocked septation.

Molecular mechanisms of antioxidant enzyme expression in lung during exposure to and recovery from hyperoxia

Manganese superoxide dismutase (MnSOD) activity falls approximately 50% in lung during 48 h of exposure of adult rats to > 95% O2 (L. B. Clerch and D. Massaro. J. Clin. Invest. 91: 499-508, 1993). We now show that hyperoxia also decreased MnSOD activity in lungs of adult baboons, making the phenomenon potentially more important to humans. In rats, a decrease in lung MnSOD activity during an initial 48 h of exposure to > 95% O2 and its increase during an immediately subsequent 24 h in air were due to decreases and increases, respectively, in MnSOD specific activity and synthesis rate; the latter was due to altered translational efficiency. The concentration in the lung of copper-zinc superoxide dismutase mRNA, catalase mRNA, and glutathione peroxidase mRNA, unchanged during the initial 48 h of exposure to O2, rose approximately twofold during reexposure to O2 after 24 h in air. The demonstration that the fall in MnSOD activity is translationally and posttranslationally regulated during the initial exposure to hyperoxia suggests that gene transfer to increase MnSOD activity in hyperoxic lungs may also require therapy that maintains translational efficiency and MnSOD specific activity.Manganese superoxide dismutase (MnSOD) activity falls ∼50% in lung during 48 h of exposure of adult rats to >95% O2 (L. B. Clerch and D. Massaro. J. Clin. Invest. 91: 499-508, 1993). We now show that hyperoxia also decreased MnSOD activity in lungs of adult baboons, making the phenomenon potentially more important to humans. In rats, a decrease in lung MnSOD activity during an initial 48 h of exposure to >95% O2 and its increase during an immediately subsequent 24 h in air were due to decreases and increases, respectively, in MnSOD specific activity and synthesis rate; the latter was due to altered translational efficiency. The concentration in the lung of copper-zinc superoxide dismutase mRNA, catalase mRNA, and glutathione peroxidase mRNA, unchanged during the initial 48 h of exposure to O2, rose approximately twofold during reexposure to O2after 24 h in air. The demonstration that the fall in MnSOD activity is translationally and posttranslationally regulated during the initial exposure to hyperoxia suggests that gene transfer to increase MnSOD activity in hyperoxic lungs may also require therapy that maintains translational efficiency and MnSOD specific activity.

Lung development, lung function, and retinoids.

In this issue of the Journal, Checkley et al. report that, in a region with endemic vitamin A (retinol) deficiency, children whose mothers had received vitamin A supplementation before, during, and...

Apoetm1Unc mice have impaired alveologenesis, low lung function, and rapid loss of lung function

Diminished lung function, indicated by a low forced expiratory volume in one second (FEV1), and short physical stature, predict early mortality from all causes, including cardiovascular, among smokers and never smokers. The basis for these associations is unclear, and, it is not known if there is a pulmonary morphological component to the relationship between low FEV1 and early death in a general population. Some apolipoprotein E genotypes also predict atherosclerosis and early mortality. These considerations led us to examine the Apoe(tm1Unc) (Apoe) mouse, in which the apolipoprotein E gene is deleted, and that develops dyslipidemia, atherosclerosis at an early age, and has a shorter life span than the founder wild-type (wt) strain. We asked if Apoe mice have a morphological or functional pulmonary phenotype. We measured the size, number, and surface area of pulmonary gas-exchange units (alveoli) and mechanical properties of the lung. Compared with wt mice, Apoe mice had: 1) diminished developmental alveologenesis, 2) increased airway resistance in early adulthood, 3) high lung volume and high dynamic and static compliance in later adulthood, 4) more rapid loss of lung recoil with age, and 5) were less long than wt mice. These findings in mice indicate the association of a low FEV1 with early death in humans may have developmental, and accelerated ageing, related pulmonary components, and that dietary, genetic, or dietary and genetic influences, on lipid metabolism may be an upstream cause of inflammation and oxidative stress, currently considered to be major risk factors for COPD.

Expression of lung uncoupling protein-2 mRNA is modulated developmentally and by caloric intake.

Lung expresses a high concentration of uncoupling protein-2 (UCP-2) mRNA, but neither its pulmonary regulation nor function is known. We measured lung UCP-2 mRNA expression in two animal models: in neonatal rats when both the metabolic rate, as measured by oxygen consumption, and levels of serum free fatty acids (FFAs) increase and in adult mice during decreased food intake, when levels of serum FFAs increase but the metabolic rate decreases. In rat lung, the concentration of UCP-2 mRNA was low and unchanged during late gestation, increased approximately twofold within 6 hrs after birth, and, compared with late gestation, remained approximately threefold higher from day 1 to adulthood. The early postnatal rise in the lung UCP-2 mRNA concentration was partially blocked by an antithyroid drug and was increased by treatment with triiodothyronine. Unlike lung, heart UCP-2 mRNA levels were lower during adulthood than at day 15. In adult mice, lung UCP-2 mRNA concentrations increased approximately fivefold within 12 hrs of 67% calorie restriction (CR), remained elevated during 2 weeks of CR, fell to control levels within 24 hrs of refeeding (CR-RF), and positively correlated with serum FFA concentrations. Heart UCP-2 expression during CR and CR-RF was similar to that of lung; liver UCP-2 mRNA levels were slightly lower during CR and returned to control levels during CR-RF. These data suggest that the regulation of UCP-2 is at least partly tissue-specific and that, in the adult mouse, lung UCP-2 is regulated not by oxygen consumption but by FFAs. Moreover, lung UCP-2 mRNA levels in mice fed ad libitum was increased by the intraperitoneal administration of Intralipid, a 20% fat emulsion. On the basis of these data in adult mice, together with the findings of others that levels of FFAs increase by 2 hrs after birth, we propose lung UCP-2 is regulated by FFA.