Retinoic acid treatment abrogates elastase-induced pulmonary emphysema in rats.

Pulmonary emphysema is a common disease in which destruction of the lung's gas-exchange structures (alveoli) leads to inadequate oxygenation, disability and frequently death; lung transplantation provides its only remediation. Because treatment of normal rats with all-trans-retinoic acid increases the number of alveoli, we tested whether a similar effect would occur in rats with emphysema. Elastase was instilled into rat lungs, producing changes characteristic of human and experimental emphysema: increased lung volume reflecting a loss of lung elastic recoil, larger but fewer alveoli and diminished volume-corrected alveolar surface area due to destruction of alveolar walls. Treatment with all-trans-retinoic acid reversed these changes providing nonsurgical remediation of emphysema and suggesting the possibility of a similar effect in humans.

Adaption to hyperoxia. Influence on protein synthesis by lung and on granular pneumocyte ultrastructure.

We studied the influence of prolonged exposure to hyperoxia (O(2) > 98%) on protein synthesis and on the ultrastructure of the granular pneumocyte. To study protein synthesis, as indicated by l-[U-(14)C]-leucine incorporation into protein, lung slices were incubated with radioactive leucine and a surface-active fraction was obtained by ultracentrifugation of lung homogenates. We found that, following an initial depression in protein synthesis after 48 h of hyperoxia, protein synthesis in rats exposed to oxygen for 96 h rose to greater than control levels. This increase in protein synthesis was noted in whole lung protein and in protein present in the surface-active fraction. Stereologic ultrastructural analysis of granular pneumocytes revealed that the lamellar bodies occupy the same percentage of cytoplasmic volume in oxygen-exposed and control rats after 96 h; a previous study had shown lamellar bodies of oxygen-exposed rats to occupy less volume than those of control rats after 48 h of exposure at which time protein synthesis was also depressed. After 96 h of exposure there is a greater amount of rough endoplasmic reticulum in the granular pneumocytes of oxygen-exposed rats. These studies show that after 96 h of hyperoxia the lung has recovered its ability to synthesize protein including protein in the surface-active fraction and that these biochemical changes are associated with consistent ultrastructural alterations in the granular pneumocyte.

Hyperoxia: a stereologic ultrastructural examination of its influence on cytoplasmic components of the pulmonary granular pneumocyte.

We used the technique of lineal analysis to study the influence of 48 h of hyperoxia on cytoplasmic organelles of pulmonary granular pneumocytes with particular reference to their lamellar bodies. We undertook this study because lamellar bodies are considered to be storage granules for pulmonary surfactant and because we had found that hyperoxia decreased [(14)C]leucine incorporation into protein of a surface-active lung fraction. We found that for lamellar bodies the percent cytoplasmic volume was 12.8+/-1.5 (mean+/-SEM) and 8.4+/-2.2, the organelle area (mum(2)) per organelle was 0.98+/-0.13 and 0.62+/-0.10 and the organelle volume (mum(2)) was 0.35+/-0.04 and 0.18+/-0.01, for air- and oxygen-exposed rats, respectively, (P=<0.05). The surface density of the lamellar body membrane was 7.05+/-0.47 and 9.36+/-0.96 (P=<0.05) for air- and oxygen-exposed rats. There were no differences in lamellar body number per cytoplasmic area or per pneumocyte between air- and oxygen-exposed rats. There were no statistical differences in these parameters between mitochondria of air- or oxygen-exposed rats. The surface density of the rough endoplasmic reticulum was the same in both groups. This study indicates that granular pneumocytes of rats exposed to hyperoxia have the same number of lamellar bodies as control rats but the lamellar bodies are smaller. This findings in consistent with the hypothesis that the hyperoxia-induced decrease in protein synthesis by lung represents at least in part a decreased synthesis of the secretory lipoprotein-pulmonary surfactant.

In vivo regulation of secretion of bronchiolar Clara cells in rats.

We used ultrastructural morphometric methods to study the in vivo regulation of secretion in bronchiolar Clara cells of rats. The Clara cells studied were located in airways with an internal diameter of 0.21 +/- 0.06 mm (mean +/- SD) at a transpulmonary pressure of 20 cm H2O. We found that pilocarpine caused a 50% decrease in the volume density of secretory granules of Clara cells in 60 min and that atropine blocked this effect. Isoproterenol produced a similar fall in volume density and this was blocked by propranolol. Propranolol also blocked the effect of pilocarpine. The fall in volume density of the secretory granules produced by pilocarpine and by isoproterenol occurred without any change in the surface-to-volume ratio of the granules. This indicates the change in volume density reflected a decrease in number rather than in size of the secretory granules. The observation that propranolol blocks the secretory response to pilocarpine as well as the response to isoproterenol suggests a dual in series cholinergic adrenergic regulation of secretion in bronchiolar Clara cells in rats.

Studies on the regulation of secretion in Clara cells with evidence for chemical nonautonomic mediation of the secretory response to increased ventilation in rat lungs.

Using electron microscopy and morphometric methods to assess secretion, we previously found that two times tidal volume ventilation of isolated perfused rat lung stimulates secretion by bronchiolar Clara cells; this effect is not prevented by beta-adrenergic blockade (J. Clin. Invest. 1981. 67: 345-351.). In this study we used the isolated perfused rat lung and the anesthetized mechanically ventilated rat, to further study the mechanism by which large tidal volumes stimulate secretion by Clara cells. With the perfused lung we found (a) alpha-adrenergic inhibition did not block the secretory effect of ventilation at two times normal tidal volume; (b) indomethacin completely blocked the secretory action of two times tidal volume ventilation; (c) medium previously used to perfuse lungs ventilated at two times tidal volume, but not medium previously used to ventilate lungs at normal tidal volume, stimulated secretion by Clara cells when used to perfuse fresh lungs ventilated at tidal volume; (d) addition of prostacyclin to the fresh perfusate increased secretion by Clara cells of lungs ventilated at normal tidal volume. In anesthetized mechanically ventilated rats, sighs stimulated secretion by Clara cells; this increased secretion was inhibited by indomethacin but not by cholinergic blockade (bilateral vagotomy). These studies indicate that increased volume ventilation stimulates secretion by Clara cells in vivo and in vitro; they provide evidence that chemical nonadrenergic, noncholinergic mechanisms are involved in this secretion, and that prostaglandins may be the chemical messenger coupling the mechanico-secretory events.

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.

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.

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.

Pertussis toxin treatment alters manganese superoxide dismutase activity in lung. Evidence for lung oxygen toxicity in air-breathing rats.

Exposure of rats to hyperoxia or to treatment with endotoxin, increases lung manganese superoxide dismutase (MnSOD) gene expression. However, the paths by which these environmental signals are transduced into enhanced MnSOD gene expression are unknown. We now provide evidence that heterotrimeric G proteins are involved in the hyperoxia-induced increase in lung MnSOD gene expression but that pertussis toxin-sensitive G proteins are not involved in the endotoxin-induced elevation of lung MnSOD gene expression. We also show that treating rats with pertussis toxin decreased lung MnSOD activity approximately 50%. This decline in MnSOD activity occurred without a change in the lung activity of copper-zinc SOD, catalase, or glutathione peroxidase. In air-breathing rats, the pertussis toxin-induced decrease in MnSOD activity was associated with the development of lung edema, pleural effusion with a high concentration of protein, and biochemical evidence of lung oxygen toxicity. Compared to air-breathing rats, maintenance of pertussis toxin-treated rats under hypoxic or hyperoxic conditions respectively decreased or increased intrathoracic fluid. Endotoxin treatment elevated lung MnSOD activity and protected pertussis toxin-treated rats from an increase in intrathoracic fluid.

Retinoic acid receptor-beta: an endogenous inhibitor of the perinatal formation of pulmonary alveoli.

Pulmonary alveoli are formed, in part, by subdivision (septation) of the gas-exchange saccules of the immature lung. Septation is developmentally regulated, and failure to septate at the appropriate time is not followed by delayed spontaneous septation. We report retinoic acid receptor (RAR) beta knockout mice exhibit premature septation; in addition, they form alveoli twice as fast as wild-type mice during the period of septation but at the same rate as wild-type mice thereafter. Consistent with the perinatal effect of RARbeta knockout, RARbeta agonist treatment of newborn rats impairs septation. These results 1) identify RARbeta as the first recognized endogenous signaling that inhibits septation, 2) demonstrate premature onset of septation may be induced, and 3) show the molecular signaling regulating alveolus formation differs during and after the period of septation. Suppressing perinatal RARbeta signaling by RARbeta antagonists may offer a novel, nonsurgical, means of preventing, or remediating, failed septation in prematurely born children.

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.

A hypothesis relating breathing pattern to some forms of the "adult respiratory distress syndrome".

Quantitative deficiency of surfactant in neonates results in hyaline membrane disease. Although surfactant is also required for normal gas exchange in adults, no disorders have been clearly attributable to a deficient amount of surfactant. Based on studies in our laboratories as well as on information and ideas in the literature, we suggest that a physical alteration in surfactant may lead to, or contribute to, the development of some forms of "adult" respiratory distress syndrome." In particular, we suggest that an altered breathing pattern contributes to the alveolar collapse and liver-like appearance of the lung found in certain clinical entities, i.e., pulmonary embolism and oxygen toxicity. We hypothesize that in these conditions shallow breathing leads to the aggregation of surfactant into a less functional form resulting in increased alveolar surface tension and atelectasis. The increase in surface tension would also contribute to the edema found in these conditions.

Demonstration of beta-adrenergic receptors in rat bronchiolar epithelial cells employing 9-amino-acridyl propranolol fluorescent microscopy.

Prior work has provided ultrastructural evidence that beta-adrenergic agonists stimulate secretion by nonciliated bronchiolar epithelial (Clara) cells of the rat (J Clin Invest 67:345, 1981). However, since the lung is a multicellular organ it is not clear if the beta-agonists act directly on the Clara cell. The absence in Clara cells of beta-adrenergic receptors would indicate an indirect action of the beta-adrenergic agonists. In the present study, we used 9-amino-acridyl propranolol in an attempt to determine if beta-adrenergic receptors are present in rat bronchiolar Clara cells. Discrete, intense yellow fluorescent dots were identified microscopically in ciliated and in Clara cells of the rat. This anatomical localization of beta-adrenergic receptors supports the notion that beta-adrenergic agonists stimulate secretion by acting directly on Clara cells.

Pulmonary alveolus formation: critical period, retinoid regulation and plasticity.

Pulmonary alveoli, the lung's gas-exchange structures, are formed in part by subdivision (septation) of the saccules that constitute the gas-exchange region of the immature lung. Although little is known about the regulation of septation, relatively recent studies show: (1) all-trans retinoic acid (RA) treatment of newborn rats increases septation and prevents the inhibition of septation produced by treatment of newborn rats with dexamethasone, a glucocorticosteroid hormone; (2) treatment with RA of adult rats that have elastase-induced emphysema increases lung elastic recoil, induces the formation of alveoli, and increases volume-corrected alveolar surface area; and (3) in tight-skin mice, which have a genetic failure of septation, and in rats in which septation had previously been prevented by treatment with dexamethasone, treatment with RA partially rescues the failed septation. These findings raise the possibility that treatment with RA will induce the formation of alveoli in humans with pulmonary emphysema.

The Clara cell: secretory and developmental studies in the rat.

Clara cells are non-ciliated epithelial cells located mainly in small conducting airways. They give rise to ciliated cells and are also thought to secrete components of the extracellular layer lining small conducting airways. Using morphometry and electron microscopy we have shown that secretion by Clara cells in rats is stimulated by a beta-adrenergic agent, by prostacyclin and by large tidal volumes. The effect of large tidal volume ventilation breathing on secretion is blocked by indomethacin (an inhibitor of prostaglandin synthesis), but it is not blocked by cholinergic blockade or by beta- or alpha-adrenoceptor antagonists. More recently we have shown that Clara cells in rats are immature at birth, rapidly gain maturity within the first postnatal week and that maternal undernutrition delays the attainment of maturity; glucagon given intraperitoneally to the rat fetus just before term accelerates the attainment of maturity; glucagon given intraperitoneally to the rat fetus just before term accelerates the attainment of maturity.

Formation of alveoli in rats: postnatal effect of prenatal dexamethasone.

We administered a glucocorticosteroid (dexamethasone) or its diluent to pregnant rats on gestation days 17, 18, and 19. In male offspring we determined the lung's gas exchange surface area (S(a)), the average volume (v) of gas exchange saccules at age 2 days and alveoli at age 14 days, and their number (N) on these days. S(a), v, and N at 2 days and v at 14 days were not affected by the prenatal administration of dexamethasone. However, S(a) and N were lower in 14-day-old pups from dexamethasone-treated dams than in pups from diluent-treated dams. In separate experiments we found the responsiveness to prenatal dexamethasone, as a depressor of the postnatal increase in S(a), appeared earlier in female than male fetuses; it was present in female but not in male fetuses on days 16-18 and was found in male fetuses on days 17-19. We conclude 1) prenatal administration of dexamethasone diminishes the postnatal increase in S(a), 2) responsiveness to this action of dexamethasone occurs earlier in gestation in female than in male fetuses, and 3) prenatal dexamethasone does not effect the postnatal volume of an average alveolus but diminishes their number in male pups.

Postnatal lung growth: evidence that the gas-exchange region grows fastest at the periphery.

The site(s) at which the gas-exchange region enlarges after alveoli are formed from the saccules of the immature lung is unclear; however, this information might be important to studies of the regulation of lung growth. Although aware of important assumptions on which it rests, we undertook this study to test the idea that the lung's gas-exchange region enlarges more rapidly in the immediate subpleural region than more centrally. To label the interstitium of the gas-exchange region, rats were provided silver-containing water from age 23 to 135 days (112 days). Some were killed at age 135 days, others 51 days after silver exposure ended (age 186 days). We considered silver grains that formed in the interstitium as a marker of lung present or formed during silver exposure; tissue added after exposure would diminish the numerical density of grains and the fastest growing sites would have the lowest grain density. Rats killed on the 112th day of silver exposure had a silver grain density in their immediate subpleural gas-exchange matrix that was 28% lower than in their more central gas-exchange matrix. Rats killed 51 days after silver exposure ended had a grain density in the immediate subpleural region that was 65% lower than in the central matrix of the gas-exchange region. The grain density in the rats killed 51 days after silver exposure ended was 33% lower in the central matrix and 67% lower in the peripheral matrix than in the respective regions of rats killed on day 112 of exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitochondria of the pulmonary granular pneumocyte in different species.

We used stereologic methods to determine the surface density and surface-to-volume ratio of mitochondrial inner membranes and cristae in pulmonary granular pneumocytes of the mouse, rat, rabbit, and dog. We also measured mitochondrial length and width and calculated their volume. The surface density of the cristae and inner membrane and the mitochondrial volume have a direct linear correlation with the oxygen consumption of lung slices of the different species. We found an indirect linear correlation between the surface-to-volume ratio of the mitochondrial inner membrane and cristae and the lung's oxygen consumption in the different species.

Development of bronchiolar epithelium in rats.

We used ultrastructural and morphometric means to examine aspects of the regulation of the maturation of rat bronchiolar Clara cells and the development of the epithelium of small conducting airways in the perinatal period. We found the nuclear numerical density per cubed centimeter (Nvn) of Clara cells fell and the Nvn of ciliated cells increased from birth to age 60 days, the prenatal administration of a glucocorticosteroid (dexamethasone) to dams caused a 35% decrease in the Nvn of ciliated cells present at birth but did not alter the Nvn of Clara cells, the administration of dexamethasone to pups from postnatal days 1 to 6 did not alter the Nvn of bronchiolar Clara or ciliated cells present at age 7 days but did diminish the total number of lung cells as assessed by measurements of lung DNA, the administration of dexamethasone to dams from gestation days 18 to 20 or from gestation days 20 to 22 did not alter the volume density of Clara cell glycogen, secretory granules, rough endoplasmic reticulum, or mitochondria of pups at gestation day 21.5 or on the day of birth, and glucagon, epinephrine, and 8-bromoadenosine 3',5'-cyclic monophosphate resulted in a decrease of Clara cell glycogen when individually incubated in vitro for 4 h with bronchiolar tissue from rat fetuses 21.5 days of age.