An improved method for the isolation of type II and clara cells from mice

Identifying the causal events and temporal aspects of lung cancer development requires the ability to isolate target and nontarget cells for comparative analyses. Current methodology can either isolate only one pure specific cell population from a lung or multiple cell types at lower purity. Previous studies in our laboratory have identified the alveolar type II cell as the progenitor cell for tumor development in the A/J mouse. The purpose of this study was to develop new protocols for the isolation and culture of type II and Clara cells from the mouse lung. Both type II and Clara cells were obtained in high purity using a sequential centrifugal elutriation protocol. In the first elutriation, cell fractions were collected using a Standard chamber. The type II and Clara cell fractions were then elutriated separately (two different separations) using a Sanderson chamber. The final purity of the type II and Clara cell preparations was 73% and 76%, respectively. Colonies of 4 to 20 Clara cells exhibiting epithelial morphology were evident 1 wk after plating in low serum medium. The growth of type II cells required the addition of bronchioalveolar lavage fluid and acidic fibroblast growth factor to the medium. The isolation of viable mouse type II and Clara cells in high purity should facilitate the identification of cell-specific changes in gene expressions or in enzymatic pathways following in vivo or in vitro exposure to environmental carcinogens.     

Automatic cell identification and enrichment in lung cancer: V. Adenocarcinoma and large cell undifferentiated carcinoma

The aims of this study were to develop a protocol for the identification and enrichment of cancer cells from sputum obtained from patients with adenocarcinoma of the lung (n = 6) and large-cell undifferentiated carcinoma of the lung (n = 2), and to compare these findings with the results from our previous studies on other cell types from lung cancer. The hypotheses tested were: Cancer cells in sputum can be preserved following flow sorting. Enrichment for cancer cells from acridine orange (AO)-stained specimens can be achieved. Discrimination of cancer cells from noncancer cells is by AO green fluorescence and discrimination of lymphocytes from other cell types is by AO red fluorescence. Cancer cells are consistently enriched in the AO high green and red fluorescence region, although, for a given cell type, maximal enrichment is patient-dependent. Finally, cancer cell enrichment and lymphocyte exclusion can be done simultaneously. Cells from sputum were initially fixed, stained with AO, sorted on a dual parameter flow sorter, and classified into six groups corresponding to two ranges of green and three ranges of red fluorescence intensities. Cells of each region were stained by the method of Papanicolaou and differential counts were performed to determine the relative frequencies (i.e., purities) of leukocytes, macrophages, squamous cells, and cancer cells, in sorted and unsorted (i.e., control) samples. The average purity of leukocytes (81%), macrophages (6%), squamous cells (11%), and cancer cells (2%) varied markedly from sample to sample. However, the largest enrichment values (i.e., ratio of purity of a cell type in a sorted sample to its purity in the unsorted control sample) achieved for cancer cells consistently occurred for each patient sample in the region corresponding to high green and high red fluorescence intensities. Experimentally, a cancer cell average enrichment of sixteen-fold was obtained by this method. Additionally, fluorescence intensity ranges which increased the enrichment for macrophages by cell sorting typically excluded leukocytes and squamous cells, and vice versa. Finally, red fluorescence intensity was the primary discriminatory parameter for all cell types studied, although the additional use of green fluorescence intensity significantly increased cancer cell enrichment rates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation of alveolar epithelial cells from lung tissue obtained at autopsy

Summary Lung alveolar epithelial cells have been studied in a variety of laboratory animal models, and studies of human alveolar epithelial cells are important for comparison to information obtained from animal studies. Autopsy material is a source of human cells for study. Studies of human autopsy material revealed variables that negatively affected the yield of viable cells. For specimens from adults, these included death greater than 12 h before cell isolation, obvious severe lung fibrosis, longstanding metabolic disorders, and lung congestion indicated by weight of the right middle lobe greater than 150 g. Samples from children yielded significant numbers of viable cells up to 18 h after death. For 17 specimens that conformed to the above criteria, approximately 8.5×10⁶ alveolar cells were obtained per gram of tissue (tissue weights ranged from 30 to 108 g) using a procedure involving instillation of proteases into the airways. The cells could be further fractionated, and 10 to 15% of the mixed cells obtained were type II pneumocytes. Analysis of NADPH cytochrome-c-reductase distribution in subcellar fractions provided evidence that the cells obtained were intact. Phospholipid enzyme activities and synthetic activity were within the ranges previously found in laboratory studies of freshly obtained animal lungs. These results suggest that significant numbers of viable and functional human lung cells, including type II pneumocytes, can be obtained from autopsy material. This research was supported by a grant (HL 33083) from the National Heart, Lung and Blood Institute, Bethesda, MD.     

Human lung mast cells: purification and characterization

Detailed studies of the biochemistry and pharmacology of mast cell-mediated inflammatory disorders have been hampered by the inability to purify human mast cells. We now report techniques to purify human lung mast cells to apparent homogeneity. The major purification steps are: 1) dispersion of lung fragments into a single-cell suspension with enzyme combinations (pronase-chymopapain, collagenase-elastase); 2) partial purification by countercurrent centrifugation elutriation (CCE); and 3) affinity column chromatography. Enzymatic dispersion yielded suspensions with congruent to 10(6) mast cells per gram of lung parenchyma in purities of 1.2 to 9.7%. Dispersed mast cells responded comparably to those in parent lung fragments to challenge with anti-human IgG and pharmacologic agonists. Elutriation of lung cell suspensions yielded mast cell-enriched fractions with purities up to 70%. High purity mast cell fractions were combined, passively sensitized with purified human penicillin (BPO)-specific IgE, and purified by a BPO-affinity column chromatography procedure. Post elutriation mast cell purities of 29 +/- 3.5% were increased to 84 +/- 3% (range 65 to 98%) by the affinity column. Short-term (24 hr) culture of column-purified mast cells allowed adherence of non-mast cell contaminants to tissue culture plates, further increasing purity (up to 100%). Purified mast cells were intact and functional as assessed by dye exclusion, survival in short-term culture, IgE-mediated histamine release, and modulation of release by the pharmacologic agonists adenosine, IBMX, prostaglandin E2, and fenoterol.     

Separation of hepatocytes of different acinar zones by flow cytometry

Hepatocytes in the proximal (zone 1) and distal (zone 3) regions of the liver acinus are selectively stained by perfusion of the isolated rat liver with 0.2-20 microM acridine orange (AO). After 10-60 min of anterograde perfusion, AO fluorescence is visible in zone 1 cells, whereas retrograde perfusion stains cells of zone 3. In this paper, we describe a technique to isolate a mixed population of fluorescent and nonfluorescent hepatocytes (cells from all acinar zones, which do not loose the zone specific AO labeling) and to separate these cells according to their zonal origin by fluorescence activated cell sorting. The zonal populations obtained were either fluorescent or nonfluorescent (purity greater than 95%). Separated cell fractions differed in their enzyme content (5' nucleotidase, succinate-dehydrogenase, beta-glucuronidase). An unidentified AO metabolite, which is not found in bile after retrograde perfusion (not formed in zone 3 cells), is also absent after retrograde perfusion in sorted fluorescent cells (zone 3 cells), indicating zonal purity of sorted cells.     

Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells

BACKGROUND: 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. METHODS: Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. RESULTS: Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. CONCLUSIONS: Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability.     

LysoTracker is a marker of differentiated alveolar type II cells

Background

LysoTracker Green DND-26 is a fluorescent dye that stains acidic compartments in live cells and has been shown to selectively accumulate in lamellar bodies in alveolar type II (AT2) cells in the lung. The aim of this study was to determine whether the accumulation of LysoTracker in lamellar bodies can be used to isolate viable AT2 cells by flow cytometry and track their differentiation in live-cell culture by microscopy.

Methods

Mouse lung cells were sorted on the basis of CD45^negCD31^negEpCAM^posLysoTracker^pos expression and characterized by immunostaining for SP-C and cultured in a three-dimensional epithelial colony-forming unit (CFU-Epi) assay. To track AT2 cell differentiation, lung epithelial stem and progenitor cells were cultured in a CFU-Epi assay with LysoTracker-supplemented media.

Results

The purity of sorted AT2 cells as determined by SP-C staining was 97.4% and viability was 85.3%. LysoTracker^pos AT2 cells generated SP-C^pos alveolar epithelial cell colonies in culture, and when added to the CFU-Epi culture medium, LysoTracker marked the differentiation of stem/progenitor-derived AT2 cells.

Conclusions

This study describes a novel method for isolating AT2 cells from mouse lungs. The high purity and viability of cells attained by this method, makes them suitable for functional analysis in vitro. The application of LysoTracker to live cell cultures will allow better assessment of the cellular and molecular mechanisms that regulate AT2 cell differentiation.     

A quantitative study of the development of type II pneumocytes in fetal lung

Cell populations dissociated from fetal rabbit lungs were analyzed by laser flow cytometry for the presence of type II pneumocytes. These cells are distinguishable by the staining of their lamellar bodies with the fluorescent lipophilic dye, phosphine-3R and by their intensity of low-angle light scatter. Lung cells were obtained by enzymatic dissociation from fetal rabbits at gestational ages of 24 d, 27 d, and from 2-d newborn rabbits. Flow cytometric analysis was sufficiently sensitive to discriminate between fetuses. Quantitative analysis of type II pneumocytes showed that newborn rabbits had a distinct cell subpopulation in a region of low-angle light scatter and phosphine-3R fluorescence intensity similar to that previously reported on type II cells from adult rabbits. By contrast, 24-d gestation rabbits had a negligible type II cell subpopulation. Fetuses of 27 and 30 d gestation showed a slow but progressive increase in the numbers of cells in the type II region. Mathematical analyses of light scatter and fluorescence intensity distributions were used to define statistically significant (P less than .05) boundaries that characterize the development of the type II cell subpopulation in fetal rabbit lung. The methods employed offer new possibilities for quantification of developing lung cell subpopulations of particular interest to the problem of respiratory distress syndrome in human neonates.     

Identification and isolation of mouse type II cells on the basis of intrinsic expression of enhanced green fluorescent protein

The unique morphology and cell-specific expression of surfactant genes have been used to identify and isolate alveolar type II epithelial cells. Because these attributes can change during lung injury, a novel method was developed for detecting and isolating mouse type II cells on the basis of transgenic expression of enhanced green fluorescence protein (EGFP). A line of transgenic mice was created in which EGFP was targeted to type II cells under control of the human surfactant protein (SP)-C promoter. Green fluorescent cells that colocalized by immunostaining with endogenous pro-SP-C were scattered throughout the parenchyma. EGFP was not detected in Clara cell secretory protein-expressing airway epithelial cells or other nonlung tissues. Pro-SP-C immunostaining diminished in lungs exposed to hyperoxia, consistent with decreased expression and secretion of intracellular precursor protein. In contrast, type II cells could still be identified by their intrinsic green fluorescence, because EGFP is not secreted. Type II cells could also be purified from single-cell suspensions of lung homogenates using fluorescence-activated cell sorting. Less than 1% of presorted cells exhibited green fluorescence compared with >95% of the sorted population. As expected for type II cells, ultrastructural analysis revealed that the sorted cells contained numerous lamellar bodies. SP-A, SP-B, and SP-C mRNAs were detected in the sorted population, but T1alpha and CD31 (platelet endothelial cell adhesion molecule) were not, indicating enrichment of type II epithelial cells. This method will be invaluable for detecting and isolating mouse type II cells under a variety of experimental conditions.     

Human dendritic cell lysosome-associated membrane protein expressed in lung type II pneumocytes

Human dendritic cell LAMP (hDC-LAMP) is a unique member of the lysosome-associated membrane protein (LAMP) family with a tissue distribution initially described as restricted to major histocompatibility class II (MHC II) compartments of activated DC before the translocation of MHC II to the cell surface [Immunity 9 (1998) 325]. In this report, we show that hDC-LAMP is also expressed by lung type II pneumocytes, another cell type with constitutive expression of MHC II. A recombinant hDC-LAMP protein and a monospecific anti-hDC-LAMP polyclonal antibody were prepared. The antibody reacted specifically with hDC-LAMP sequences of hDC-LAMP protein expressed in transfected cells and with a 54 kDa protein of normal human lung tissue with properties corresponding to those of transgene expressed hDC-LAMP. Immunohistochemical analysis of hDC-LAMP in human lung showed its presence in alveolar type II epithelial cells (type II pneumocytes) as well as in cells in the interfollicular area of bronchus-associated lymph nodes, where interdigitating DCs are concentrated, and with lesser staining of alveolar macrophages. The native protein contained approximately 16% carbohydrates, most of which are sialyl N-linked oligosaccharides, with an acidic isoelectric point (pI 4.8). The restricted localization of this protein to lung type II pneumocytes and DCs is in contrast to hLAMP-1, which was present in many cell types of the lung and lymph node. Type II pneumocytes are known to express MHC II and the abundant expression of hDC-LAMP in these cells as well as in DCs suggests its possible relationship to specific MHC II related function(s) of DC and type II pneumocytes.     

Type II pneumocyte changes during hyperoxic lung injury and recovery

Adult rabbits exposed to 100% O2 for 64 h and then returned to room air for up to 200 h, develop a lung injury characterized by decreased levels of alveolar surfactant followed by a rebound recovery. In the present study we isolated alveolar type II cells from rabbits at various times during hyperoxic exposure and recovery and measured rates of phosphatidylcholine (PC) synthesis, cellular lipid content, and the specific activity of glycerol 3-phosphate (G-3-P) acyltransferase, an enzyme that catalyzes one of the early reactions in phosphoglyceride biosynthesis. These biochemical parameters were compared with measurements of cell size and cell cycle phase by laser flow cytometry. Results showed that alterations in alveolar phospholipid levels in vivo correlated consistently with cellular lipid metabolic changes measured in isolated type II pneumocytes. In particular, alveolar pneumocytes isolated from lungs of rabbits exposed to 100% O2 for 64 h exhibited a 60% decrease in PC synthesis, cell lipid content, and G-3-P acyltransferase activity. All variables then followed a pattern of recovery to normal and ultimately supranormal levels beginning at approximately 3 days postexposure, at which point there was also a measured increase in the number of type II cells in S phase. These findings suggest that O2-induced changes in type II cell surfactant biosynthesis may account, at least in part, for observed changes in lung phospholipid levels in vivo.     

Release of prostaglandins and thromboxanes by guinea pig isolated type II pneumocytes

Lung cells have been isolated by enzymatic digestion of guinea pig lungs and mechanical dispersion to obtain a suspension of viable cells (approximately 500 X 10(6) cells). Type II pneumocytes have been purified to approximately 92% by centrifugal elutriation (2000 rpm, 15 ml/min) followed by a plating in plastic dishes coated with guinea pig IgG (500 micrograms/ml). We have investigated the arachidonic acid metabolism through the cyclooxygenase pathway in this freshly isolated type II cells (2 x 10(6) cells/ml). Purified type II pneumocytes produced thromboxane B2 (TxB2) predominantly and to a smaller extent the 6-keto prostaglandin PGF1 alpha (6-keto-PGF1 alpha) and prostaglandin E2 (PGE2) after incubation with 10 microM arachidonic acid. The stimulation of pneumocytes with 2 microM calcium ionophore A23187 released less eicosanoids than were produced when cells were incubated with 10 microM arachidonic acid. There was no additive effect when the cells were treated with both arachidonic acid and the ionophore A23187. Guinea pig type II pneumocytes failed to release significant amounts of TxB2, 6-keto-PGF1 alpha and PGE2 after stimulation with 10 nM leukotriene B4, 10 nM leukotriene D4, 10 nM platelet-activating factor, 5 microM formyl-methionyl-leucyl-phenylalanine, 0.2 microM bradykinin and 10 nM phorbol myristate acetate. Our findings indicate that guinea pig type II pneunomocytes possess the enzymatic machinery necessary to convert arachidonic acid to specific cyclooxygenase products, which may suggest a role for these cells in lung inflammatory processes.     

Catalase immunocytochemistry allows automatic detection of lung type II alveolar cells

In mammalian lung, type II pneumocytes are especially critical in normal alveolar functioning, as they are the major source of surfactant and the progenitors of type I alveolar cells. Moreover, they undergo proliferation and transformation into type I cells in most types of cellular injury, where flattened type I pneumocytes are selectively destroyed. Hyperplasia of alveolar type II cells has also been described in some human chronic lung diseases. In lung, type II pneumocytes and non-ciliated bronchiolar cells are the unique cell types that contain a considerable amount of peroxisomes. Due to the presence of dihydroxyacetone phosphate acyltransferase and non-specific lipid-transfer protein, these organelles have been suggested to be involved in the synthesis and/or transport of the lipid moiety of surfactant. In the present research, the peroxisomal marker enzyme catalase was immunolocalised at the light microscopic level, utilising the avidin-biotin complex method, in lung specimens excised from newborn, adult and aged rats. In all the examined stages the immunoreactivity was so selective for type II pneumocytes it allowed quantitation of these cells by an automated detection system. This was accomplished on specimens from newborn rat lung, in which labelled alveolar cells were counted by a grey level-based procedure and their main morphometric parameters were determined.     

Repopulation of a human alveolar matrix by adult rat type II pneumocytes in vitro : A novel system for type II pneumocyte culture

This paper describes the preparation of lung acellular alveolar matrix fragments and culture of rat type II pneumocytes directly on the alveolar epithelial basement membrane, thereby permitting study of the effect of lung basement membrane on the morphology and function of type II cells. Collagen types I, III, IV and V, laminin and fibronectin were located by immunofluorescence in the lung matrix with the same patterns as those described for the normal human lung. Transmission electron microscopy (TEM) of the fragments revealed intact epithelial and endothelial basement membranes. The matrix maintained the normal three-dimensional alveolar architecture. Glycosaminoglycans were still present by Alcian Blue staining. Isolated adult rat type II pneumocytes cultured on 150 micron thick fragments of acellular human alveolar extracellular matrix undergo gradual cytoplasmic flattening, with loss of lamellar bodies, mitochondria, and surface microvilli. These changes are similar to the in vivo differentiation of type II pneumocytes into type I pneumocytes. The type II pneumocyte behaviour on the lung epithelial basement membrane contrasted sharply with that of the same cell type cultured on a human amnionic basement membrane. On the latter surface the cells retained their cuboidal shape, lamellar bodies and surface microvilli for up to 8 days. These observations suggest that the basement membranes from different organ systems exert differing influences on the morphology and function of type II pneumocytes and that the alveolar and amnionic basement membranes may have differing three-dimensional organizations. The technique of direct culture of type II cells on the lung basement membrane provides a useful tool for studying the modulating effect of the basement membrane on alveolar epithelial cells.     

Isolation and biochemical studies of enriched populations of spermatogonia and early primary spermatocytes from rat testes

A method to obtain several highly enriched populations of testis cell types from rats of a single age is described. Single cell suspensions from immature rat testes were prepared after enzymatic removal of interstitial cells. Cells were separated on the basis of size into four fractions (bulk preparations) or eight fractions (analytical preparations) by centrifugal elutriation. These elutriator fractions were further separated by equilibrium density centrifugation in Percoll gradients. In this manner, populations of 2 X 10(7) type A spermatogonia (51% purity), 3 X 10(7) type B spermatogonia (76% purity), 5 X 10(7) zygotene/early pachytene spermatocytes (56% purity), 3 X 10(7) midpachytene spermatocytes (70% purity), and 4 X 10(7) Sertoli cells (89% purity) could be obtained from 50 immature rats within 6 h after killing. Purities, determined by examination of cytologic smears, were verified by Coulter volume and flow cytometric DNA determinations. These separation methods were used to obtain cell populations for characterization of levels and synthesis of high mobility group proteins in the early stages of spermatogenesis.     

In vitro primary allo-CTL generation by enucleated antigen-presenting cells

It is generally thought that only viable cells can elicit a primary cytotoxic T-lymphocyte (CTL) response. We present evidence that this is not so, since enucleated tumor cells can generate a strong cytolytic response of unprimed allogeneic human T lymphocytes. Cytoplasts (enucleated cells) were obtained by incubation with cytochalasin B and subsequent isopycnic centrifugation. Their purity was assessed by electron microscopy and flow cytometry. Membrane fractions were prepared by nitrogen cavitation, and used in parallel with cytoplasts and intact cells as stimulators in primary allo-CTL generation; although all cell fractions expressed high amounts of class I and II histocompatibility antigens, as assessed by flow cytometry and ELISA technique, only the cytoplasts generated a strong cytotoxic response of naive peripheral T cells, like that induced by intact cells. The dogma that an intact and metabolically active stimulator cell is required for the primary generation of CTLs is questioned.     

Lectin Histochemistry of Normal Human Lung

This study aimed to identify and specify the glycotypes of cell populations in normal human lung including types I and II pneumocytes, alveolar macrophages and mast cells, and also in the larger tissue structures of lung, including blood vessels and bronchi/bronchioles, using lectin- and immuno-histochemistry on paraffin-embedded tissue from 11 normal cases. The alveolar macrophages were anti-CD68 positive whereas the cells lining the alveolar walls were positive for cytokeratins. The alveolar macrophages in normal lung tissues showed a broad spectrum of staining for different subsets of N-linked saccharides, N-acetylgalactosamine, N-acetylglucosamine, terminal beta-D-galactose and sialyl groups. This study showed that some lectins could be used as specific markers for some cell types i.e. Galanthus nivalis and Narcissus pseudonarcissus lectins for macrophages, Psophocarpus tetragonolobus lectin-II for capillary endothelium, Dolichos biflorus agglutinin for bronchial epithelial cells, Lycopersicon esculentum, Phytolacca americana or Triticum vulgaris (succinylated) for type I pneumocytes and Hippeastrum hybrid or Maclura pomifera lectins for type II pneumocytes. Patchy staining of type I pneumocytes by peanut agglutinin indicated the possibility of two distinct populations of these cells or a pattern of differentiation that is unapparent morphologically.     

Separation and characterization of basal and secretory cells from the rat trachea by flow cytometry

Basal and secretory cells have been separated as highly enriched viable populations from single-cell suspensions of rat tracheal epithelial cells. Isolation of the populations was achieved by preparation of a cell suspension and separation by flow cytometry using contour maps generated from 2 degrees and 90 degrees light scatter signals. Flow cytometric analysis of cells showed 10% of the whole preparation were cells in SG2M phase of the cell cycle. The secretory cells accounted for 86% of these cycling cells; the remainder were accounted for by the basal cells. Culture of sorted populations of basal and secretory cells in serum free defined medium showed that basal cells had a lower (0.6%) colony-forming efficiency than secretory cells (3.4%). Significant differences in blue auto-fluorescence, Hoechst 33342 uptake, and lectin staining were apparent between basal and secretory cells. These results suggest that the secretory cell rather than the basal cell is primarily the cell type involved in maintenance of the normal tracheal epithelium. Secretory cells are greater in number, have a higher proliferative potential, and greater metabolic capability. Because of these traits they may be a critical cell at risk from damage by environmental agents.     

Participation of carnitine palmitoyltransferase in the synthesis of dipalmitoylphosphatidylcholine in rat alveolar type II cells

We have investigated the role of carnitine palmitoyltransferase (EC 2.3.1.21) in pulmonar type II pneumocyte, a lung cell responsible for the synthesis of surface active lipids. Adult type II pneumocytes were isolated from rat lung and purified by differential adherence. When these lung cells were incubated with radioactive palmitate, the percentage of radioactivity recovered into dipalmitoylphosphatidylcholine (DPPC), a major surface active lipid, was almost 60% with respect to total phosphatidylcholine (PC) molecular species. Cellular lysates from type II pneumocytes contained detectable amount of carnitine palmitoyltransferase (CPT) activity (1 nmol/min/mg). Most of the CPT activity found in these cells could be inhibited by incubating them for 60 min with 5 M tetradecylglycidic acid (TDGA), a specific and irreversible CPT inhibitor of the malonyl-CoA sensitive CPT isoform (CPT I). TDGA treatment of adult type II pneumocytes caused a significant reduction in the incorporation of radioactive palmitate into PC, though this effect did not seem to be specific for DPPC. TDGA affected the incorporation of radioactive palmitate at the sn2 rather than the sn1 position of the glycerol backbone of PC. The incorporation of radioactive palmitate into DPPC was also observed when these lung cells were incubated with palmitate-labeled palmitoyl-L-carnitine. Our data suggest that type II pneumocyte CPT may play an important role in remodelling PC fatty acid composition and hence DPPC synthesis.     

Cytochrome P-450 monooxygenase,epoxide hydrolase and flavin monooxygenase activities in clara cells and alveolar type II cells isolated from rabbit

The activities of several enzymes which metabolize xenobiotics were measured and compared in freshly isolated rabbit Clara cells (50–70% purity) and alveolar type II cells (80–95% purity) or microsomal preparations from the isolated cell fractions. The presence of 1 mM nicotinamide in protease and cell isolation buffers increased significantly 7-ethoxycoumarin (7-EC) deethylase and epoxide hydrolase activities in the isolated Clara and type II cells. Isolated Clara cell fractions metabolized 7-EC to umbelliferone at a rate of 241 ± 27 pmoles/mg prot/min (mean ± S.E., N=5), while the 7-EC deethylation rate in type II cells was 111 ± 15 pmoles/mg prot/min. Coumarin hydroxylation activity, however, was more than ten times greater in the Clara cells than in the type II cells on a per mg cellular protein basis. N-oxidation of N,N-dimethylaniline, catalyzed by a flavin monooxygenase, was about 2 times as great in microsomes of Clara cells as in microsomes of type II cells. Epoxide hydrolase activity with benzo(a)pyrene 4,5-oxide as substrate was about 10 times higher in Clara cells than in type II cells. Because of the greater cellular, structural and functional heterogeneity in lung, differential distribution of enzymes responsible for xenobiotic metabolism in this tissue may contribute to cell selective chemical toxicity and carcinogenesis.Abbreviations 7-EC 7-ethoxycoumarin - DMA N,N-dimethylaniline