Effect of fatty acid profiles on the susceptibility of cultured rabbit tracheal epithelial cells to hyperoxic injury.

To investigate the role of cellular fatty acid content on the susceptibility of airway epithelial cells to hyperoxic injury, monolayer cultures of rabbit tracheal epithelial (TE) cells were grown to confluence in serum-free media with or without a commercial mixture of cholesterol esters and phospholipid-rich lipoproteins (Excyte III, Miles-Pentex, Kankakee, IL) in conjunction with arachidonic acid complexed to BSA. Monolayer cultures were then exposed to control (5% CO2/air) or hyperoxic atmospheres (95% oxygen/5% CO2) for 2 h using an in vitro system in which cells were maintained at a gas-liquid interface analogous to in vivo conditions. Hyperoxic injury was assessed by cell viability (trypan blue exclusion) and by the generation of lipid peroxides measured as thiobarbituric acid (TBA) reactive substances. Changes in TE cell and cell culture effluent fatty acid content induced by exposure to control or hyperoxic atmospheres were analyzed by gas chromatography. TE cells grown in lipid-unsupplemented media had fatty acid profiles characteristic of essential fatty acid deficiency, whereas the fatty acid content of lipid-supplemented TE cells more closely resembled those of acutely recovered TE cells. Lipid-unsupplemented cells were more susceptible to hyperoxic injury as demonstrated by decreased viability and increased production of TBA-reactive substances compared to cells maintained in lipid-supplemented media. In both lipid-supplemented and unsupplemented cells, hyperoxic exposure was associated with a decreased relative cellular content of the monounsaturated and polyunsaturated fatty acids (PUFA) and an increased content of saturated fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiographic localization of [3H]RU 486 and [3H]progesterone in the uterus, pituitary and hypothalamus of the rat.

Twenty-one castrated oestrogen-primed Wistar rats, which were 2-months-old, were injected via the jugular vein with 100 mu Ci/100 g body weight of [3H]RU 486 or [3H]progesterone. Some of these received unlabelled compounds for competition studies. Samples of reproductive tract, pituitary and hypothalamus were excised after 15 min. The 4-microns frozen sections were processed for thaw-mounted autoradiography. The exposure time of the autoradiogram was approximately 6 months. After the injection of [3H]RU 486 and [3H]progesterone, the nuclear concentration of radioactivity was most distinct in muscular and stromal cells of the uterus, and the epithelial nuclei of lumina and glands showed weak labelling. Nuclear localization was also observed in muscle cells of the vagina, cervix and oviduct. After injection of [3H]progesterone, the radioactivity was found in the nuclei and cytoplasm of anterior pituitary cells and some cells showed a preferential nuclear concentration of radioactivity. The distribution of [3H]RU 486 in the anterior pituitary was more extensive than that of [3H]progesterone. In the hypothalamus, specific localization of [3H]RU 486 and [3H]progesterone existed in neurones accumulated in the preoptic nucleus, preoptic suprachiasmatic nucleus and the periventricular nucleus. No localization was found in the diaphragm. Pretreatment with RU 486, but not with dexamethasone, reduced the nuclear concentration of radioactivity of [3H]progesterone in the vagina, uterus, oviduct, pituitary and hypothalamus. The nuclear concentration of radioactivity after injection of [3H]RU 486 was also decreased by preinjection with progesterone. The autoradiographic results suggest that RU 486 and progesterone competed for the specific binding site (possibly a progesterone receptor) in the target cells at the levels of the uterus, pituitary and hypothalamus in vivo.

Imaging and relative quantification of 127I in human thyroid follicles by analytical ion microscope: characterization of benign thyroid epithelial tumors.

Analytical ion microscopy (AIM) can be used for imaging and relative quantification of chemical elements in tissue sections. We used this technique to assess the changes in 127I mapping within thyroid follicular cells and follicular lumina in benign thyroid epithelial abnormalities from 17 patients and in macroscopically normal perinodular tissue surrounding solitary cold nodules from 8 patients. Among the 17 patients, 9 had simple goiters, 5 had toxic nodular goiters, and 3 had hypofunctioning (cold) nodules. The tissue samples were fixed chemically and embedded in methacrylate resin to ensure preservation of organified iodine, and thin sections were analyzed by AIM. 127I was found in the follicular lumina and follicular epithelial cells of most specimens. The local concentration of 127I, which is proportional to the ratio of the two secondary ion beam currents of iodine and carbon, was evaluated in 30 follicular lumina and 30 follicular epithelial cells of each specimen. In normal tissue, the relative 127I concentration within follicular cells (mean, 0.72; range 0.01-8.30) was much lower than that in follicular lumina (mean, 4.63; range, 0.18-36.74). In simple goiter tissue, follicular lumen (mean, 0.57; range, 0.00-5.76), and cell (mean, 0.17; range, 0.002-1.82) relative 127I concentrations were below normal, but both distributions remained different. On the contrary, in toxic nodular goiter tissue the follicular cell relative 127I concentration (mean, 0.96; range, 0.003-27.3) largely overlapped that of the follicular lumina (mean, 2.1; range, 0.001-36.5). The cold nodules had the lowest relative follicular lumina 127I concentration (mean, 0.008; range, undetectable-0.07), and the relative cellular 127I concentrations were undetectable in 67%. These results demonstrate the capacity of AIM to characterize the functional activity of thyroid tissue without prior administration of radio-iodine.

X-ray microanalysis of hamster tracheal epithelium.

Studies of ion transport across respiratory epithelia are of great interest if we are to understand the pathophysiology of diseases such as cystic fibrosis in which ion transport is abnormal. Concentrations of elements were determined in various subcellular regions of normal or isoproterenol-treated hamster tracheal epithelium, using X-ray microanalysis of freeze-dried cryosections. Samples of trachea were taken from animals under anesthesia and either frozen in situ or dissected and plunge frozen. Concentrations of Mg, P, S, Cl, K and Ca were higher in cytoplasm and nuclei of control epithelial cells in dissected samples than in cryoneedle samples. Following treatment with isoproterenol, a large decrease in the concentration of Cl was observed. The results confirm that cyclic AMP-regulated chloride secretion is unaffected by anesthesia.

Epithelial bridging of the primary palate: I. Characterization of sub-cultured epithelial cells.

Primary palatogenesis involves an intricate array of events. Cell migration, proliferation, differentiation, programmed death, and fusion occur. Prior to fusion, the morphology of the epithelium undergoes marked changes. Epithelial projections form and extend across the fusion site attaching by filopodia to the opposite prominence. By appearance, the epithelium plays a critical role in facial development. In order to monitor epithelial activities, a study was done to isolate and characterize epithelial cells derived from the primary palate. The primary palate was microdissected from day 13 Sprague-Dawley rat embryos, and the epithelium and mesenchyme were separated by enzymatic digestion with a 3% trypsin-pancreatin solution (3:1). All explants were cultured in Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium (1:1) supplemented with 10% fetal calf serum (FCS), 20 ng/ml epidermal growth factor (EGF), and antibiotics. Explant cells were gathered by trypsin harvesting and sub-cultured. These sub-cultured cells were further characterized. Transmission and scanning electron microscopy showed that the cells retained many morphological features observed in vivo. In passaged cells, type IV collagen, laminin, and cytokeratins were visualized by immunocytochemistry. Gel electrophoresis analysis of the water-insoluble extracts demonstrated major bands of proteins of 50 kD and 44 kD that were synthesized by the epithelial cells but not by the mesenchymal cells. These cytokeratin types are suggestive of a simple undifferentiated embryonic epithelium. The effect of all-trans retinoic acid (RA) on cell number and [3H]-proline incorporation was assessed. At [10(-4)M] and [10(-6)M] retinoic acid resulted in significant inhibition in cell proliferation and amount of proline incorporated, with the greater inhibition occurring in the mesenchymal cells. In the concentrations studied, retinoic acid has an inhibitory effect on the two differently derived cell types. This study established that sub-cultured epithelial cells maintain their phenotype and can be used to study fusion processes. Part 2 will demonstrate how the morphology of the epithelial cells can be modified to produce the changes that are observed during fusion of the primary palate.

Essential fatty acid pattern of glycerolipids in rat hepatocytes in primary culture and in coculture with rat liver epithelial cells.

The linoleic acid content of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and triglyceride (TG) rapidly fell in rat hepatocytes in primary culture up to four days and in coculture with liver epithelial cells up to eight days. At the same time, the level of polyunsaturated fatty acids (PUFA), especially arachidonic acid, remained constant in PE, slightly decreased in PC and dropped in TG. There was no variation of the nonessential PUFA, 20:3n-9. Linoleic acid supplementation of cultures 24 hr before the harvest induced a rise in the linoleic acid level of the three lipid classes. Arachidonic acid remained constant in TG and only slightly decreased in PE and PC at day 4 of primary culture and day 8 of coculture. The level of 20:3n-9 increased in PE and PC and much more in TG. This net increase in the arachidonic acid and 20:3n-9 levels in TG could not be explained only by a transfer from the phospholipid pools of PUFA because the phospholipid content of hepatocytes and PUFA levels of phospholipids did not vary under linoleic supplementation. The low percentage of arachidonic acid in epithelial cells rules out any participation of these cells in the increase of arachidonic acid in supplemented cocultures. Triglycerides may act as a storage pool for plasma PUFA up to four days of primary culture and eight days of coculture. Besides, coculture seems more potent than primary culture to maintain the phospholipid level, to spare the essential PUFA in PE and to increase the TG synthesis in response to linoleic acid supplementation.

Isolation of an olfactory cDNA: similarity to retinol-binding protein suggests a role in olfaction.

Molecular cloning techniques were used to isolate and characterize a protein possibly involved in the signal transducing system in olfactory tissue of the frog Rana pipiens. A complementary DNA library was constructed with messenger RNA obtained from frog olfactory neuroepithelium. A 700-base pair complementary DNA clone encoding a protein with a molecular weight of 20,300 was identified by differential hybridization analysis with polyadenylated RNA from olfactory epithelium and nonsensory respiratory epithelium. The messenger RNA corresponding to this clone was abundant in the cells of Bowman's glands in olfactory tissue but not in respiratory epithelium nor in several other tissues. The predicted sequence of this protein is homologous to members of a family of proteins that bind and transport small molecules in serum, suggesting that this protein may also bind and transport odorants in the mucus secreted by Bowman's glands.

Elemental concentrations within types 2 and 3 epithelial cells of the rat thymus.

The elemental composition (Na, Mg, P, S, Cl, K, Fe and Zn) of epithelial cells of rat thymus cortex has been determined using X-ray microanalysis of freeze-dried frozen sections. The cytoplasm contained highly significantly lower levels of potassium and phosphorus than the nuclei. When the results were compared with a previous study of the elemental levels in cortical thymocytes (using the same material), only differences in the P concentration were found. This is probably attributable to the greater degree of leptochromasia usually observed in epithelial cells compared with thymocytes. Since thymocyte maturation depends very closely on the microenvironment created by the epithelial cells, the great similarity in elemental levels observed in this study could be important in the control of cell division and maturation events.

A comparison of standard lipid staining techniques used in electron microscopic studies of mammalian tissues.

Comparisons of several standard techniques for staining lipids in ultrastructural studies have been undertaken using the rat uterine epithelium as the experimental tissue. The best technique for clarity, retention of stain, and acceptability of cellular ultrastructure utilized p-phenylenediamine after primary fixation in glutaraldehyde and postfixation in osmium tetroxide. While osmium by itself stained only unsaturated lipids and p-phenylene-diamine stained no lipids in spot tests, when acting together, the staining of unsaturated lipids was enhanced and some staining of saturated lipids was seen. Further, the marked extraction of stained lipids normally found during dehydration did not then occur.

Correlation of amino acid fucoside labeling patterns with tumorigenicity of mouse mammary epithelial cells.

The amino acid fucosides of tumorigenic and nontumorigenic mouse mammary gland-derived cells were studied. The cells examined included tumorigenic cell lines derived from mammary carcinomas of the following etiologies: induction by hormone; virus; and chemical carcinogen. Also studied were cells derived from normal mammary glands and several clones of cells, which were derived from a mammary carcinoma but were not demonstrably tumorigenic at lower passage levels after cloning, while they were highly tumorigenic at higher passage levels. Cells were cultured in medium supplemented with radiolabeled fucose and extracted, and extracts were analyzed for the amino acid fucosides. Radiolabeled compounds which comigrated with the amino acid fucosides glucosylfucosylthreonine, fucosylthreonine, and fucosylserine were observed. There was a distinctive difference between the tumorigenic and nontumorigenic cells; the ratio of fucosylthreonine plus fucosylserine to glucosylfucosylthreonine was higher in all tumorigenic cells as compared to the ratio observed for the nontumorigenic cells.

Histochemical demonstration of collagen fibers in ascorbic-acid-fed cell cultures.

Nine cultures of fibroblast cell types and 13 epithelial-like cell types were maintained for 1 week in media supplemented with L-asborbic acid (50 microgram per ml). All fibroblast-like cultures produced extracellular fibers that stained positively by a silver-impregnation reticulin stain. Nine of the 13 epithelial-like cultures produced fibers that stained positively for reticulin. Nearly all cultures not supplemented with ascorbic acid showed no fiber staining. Those few lines that stained positively for reticulin in the absence of ascorbic-acid supplementation demonstrated only slight reticulin formation. Reticulin from one fibroblast culture and one epithelial culture was examined by electron microscopy, and the silver-impregnated fibrils were morphologically identical to collagen. The reticulin was digestible with collagenase, providing further evidence that the silver-impregnation reticulin stain identifies collagen in culture. The demonstartion of collagen can be performed easily in histology laboratories using Formalin-fixed cells, and provides a means of assaying a functional property of cells in culture which is characteristic of connective tissue fibroblasts in general as well as certain specialized epithelia.