Dexamethasone inhibits prostaglandin release from rabbit coronary microvessel endothelium.

The effects of dexamethasone on prostaglandin secretion by cultivated rabbit coronary microvascular endothelial (RCME) cells were investigated. Incubation of RCME cells with dexamethasone resulted in a time- and concentration-dependent decrease in prostaglandin accumulation in the culture media and reduced basal and A23187-stimulated prostaglandin (PG) E2 and 6-keto-PGF1 alpha release. The maximal effects of dexamethasone (50-80% inhibition) were achieved after 16-18 h of incubation with the steroid at a final concentration of 10(-7) M. The effects of dexamethasone treatment were partially reversed 24 h after removal of the steroid from the culture media. Dexamethasone treatment did not reduce arachidonic acid-stimulated prostaglandin synthesis, indicating that the level of inhibition was proximal to that of cyclooxygenase. The inhibitory effects of dexamethasone could be prevented by pretreatment of the RCME cells with actinomycin D or cycloheximide, suggesting a requirement for protein synthesis in the inhibitory action of dexamethasone. Conditioned media from dexamethasone-treated cells contained a factor that inhibited porcine pancreatic phospholipase A2 (PLA2) in vitro. Transfer of conditioned media from dexamethasone-treated cells to untreated cells did not reduce basal or stimulated prostaglandin release; in contrast, a stimulatory action was consistently observed. Adherence of rabbit peripheral polymorphonuclear leukocytes (PMN) to RCME cells was reduced when the leukocytes were pretreated with 10(-7) M dexamethasone (4 h). However, dexamethasone pretreatment of the RCME cells did not significantly effect granulocyte adhesion. Thus coronary microvascular endothelial cell prostaglandin production is regulated by glucocorticoids, and glucocorticoid-pretreated microvascular endothelial cell release an inhibitor of PLA2 activity into the culture media.(ABSTRACT TRUNCATED AT 250 WORDS)

Cultured lung cells: interplay effects of beta-mimetics, prostaglandins and corticosteroids in the biosynthesis of dipalmitoyl lecithin.

Cell lines derived from type II lung cells were used to study interplays of substances affecting incorporation of labeled precursors [1(-14)C]palmitate and [methyl-3H]choline into phosphatidyl choline. Ethanol stimulated markedly biosynthesis of dipalmitoyl phosphatidyl choline in cloned rabbit lung cells; the stimulating action of ethanol was reduced very much by cortisol and less by ritodrine. In the presence of 0.1 microM isoproterenol, two prostaglandins, E2 and F2alpha, caused marked depressions in the incorporation of both precursors by cell line A 549 derived from human lung adenocarcinoma. One concluded that among the agents studied, ethanol and cortisol are potent antagonists, and so were also the prostaglandins and isoproterenol.

The type 1 alveolar lining cells of the mammalian lung. II. In vitro identification via the cell surface and ultrastructure of isolated cells from adult rabbit lung.

Using a newly described dissociation and isolation technique, Type 1 alveolar lining cells were obtained from adult rabbit lung within a heterogeneous population. Identification of many lung cell types in this mixed population was by a) comparison of isolated cells with in situ lung cells in lung sections using identical parallel staining, b) stepwise ultrastructural examination of cells during all stages of lung dissociation so that intercellular associations were monitored throughout, and c) Type 1 cell surface changes following collagenase treatment. This phenomenon was studied with both electron and light microscopy, the latter employing tetrachrome staining of basophilic blebs as well as characteristic staining of nucleus and cytoplasm. Following their isolation, most Type 1 cells lost their surface blebs and assumed a "relaxed" state. In this condition, Type 1 cells were exposed to cytochalasin D (CD) for various times and at several concentrations. Surface knobs, having all the characteristics of zeiotic knobs produced in a number of cultured cell lines by exposure to CD, were produced in isolated Type 1 epithelial cells within 45 minutes. The reaction to CD was temperature-dependent, proceeding maximally at 37 C with inhibition at lower temperatures and was inhibited by antimetabolites such as dinitrophenol and 2-deoxyglucose in the presence of CD. As with established cell lines, formation of zeiotic knobs at the isolated Type 1 cell surface appeared closely related to microfilamentous nets located beneath the plasmalemma. The density of this net appeared to vary as isolated Type 1 cells underwent expansion and contraction in response to CD. Zeiotic knobs were formed as the result of herniation of endoplasm through the cell cortex. The significance of such a labile cortical zone is considered in relation to the deformation changes Type 1 cells undergo during inflation-deflation of alveoli and the folding-unfolding of alveolar lining cells as a result of lung volume changes.

The type 1 alveolar lining cells of the mammalian lung. I. Isolation and enrichment from dissociated adult rabbit lung.

With removal of large numbers of macrophages by airway lavage, Type 1 cells were isolated in heterogeneous cell populations following the stepwise dissociation of lung tissue. Using a carefully timed collagenase-trypsin digestive sequence at 37 C, unwanted cellular and noncellular lung components were minimized prior to selective release of Type 1 cells. Resulting heterogeneous cell suspensions containing well-preserved Type 1 cells, as determined by electron microscopy, were layered onto a shallow gradient (3 to 6% Ficoll in minimal essential medium [MEM]) and separated at unit gravity into enriched subpopulations of various cell types. These included various fractions enriched with respect to Type 1 cells (70%), Type 2 cells (82%), and macrophages (81%). Identification of Type 1 cells following their isolation and gradient enrichment was established by light microscopic staining techniques and by specific cell surface characteristics in vitro as visualized by electron microscopy.

Effects of prostaglandins E2 and F2alpha on lecithin biosynthesis by cultured lung cells.

Transformed cells from human lung carcinoma (Line A549), resembling type II pneumocytes, were cultured in monolayer at 37 degrees C and incubated for five hours with 3H-choline and 14C-palmitate in the presence of various concentrations of prostaglandins (PGS) E2 and F2alpha. In the control (no PG) the level of % palmitate incorporation was 13.5 x as high as that of choline, after taking isotope dilution into account. Between the concentrations studied, 0.1 and 10 muM, both prostaglandins stimulated markedly the incorporation of both precursors, though choline up to 3 x better than palmitate. This was indicated by a change in the palmitate/choline incorporation ratio from 13.5 to as low as 4.2. At the lowest PG concentration, 0.1 muM, PGE2 was much more effective than PGF2alpha in stimulating the incorporation of both precursors.

Ultrastructure of in vitro type 2 epithelial cell cysts derived from adult rabbit lung cells.

Fragments of adult rabbit lung, composed chiefly of terminal airway obtained by a trypsin digestion technique were maintained on collagen-coated cellulose sponges in Ham's F12 medium. Cell-sponge associations were examined with light microscopy, scanning and transmission electron microscopy over a period from 6 to 28 days. After an initial 24- to 48-hour period of cell migration from the airway fragment, sponge matrices became lined with cells suggestive of alveolar macrophages. After one week in culture, cysts appeared to be composed entirely of type 2 epithelial cells. These were characterized by a microvillous apical border and an elaborate junctional complex. The lumen of these cysts contained both myelin-like lamellar configurations and tubular myelin structures such as have been described from pulmonary washings. Consistent with the age of the sponge cultures, one or more cyst types described as young, middle and late could be found simultaneously. Middle aged cysts showed signs of active secretion into the lumen. Late cysts showed changes in the epithelium comprising the cyst wall suggestive of a cell type intermediate between type 1 and type 2 epithelial cells.

Culture characteristics of cells derived from type II pneumocyte enriched fractions from rabbit and rat.

Type II cell enriched fractions were isolated from rabbit and rat lungs using density gradient centrifugation. Cultures established from these fractions contained predominantly cells similar in most morphological respects to type II pneumocytes. These were in continuous replicating culture for 1 year and still exhibited contact inhibition. Membrane-bound structures reminiscent of, but no longer strictly identical to, type II cell lamellar cytosomes were seen in cells from these long-term cultures although their numbers were reduced in comparison to lamellar bodies in freshly isolated cells. Mitochondrial numbers and sizes, determined morphometrically, were reduced after culture in comparison to freshly isolated type II cells and those in situ. Phosphatidylcholine was synthesized by these cells and released into the extracellular medium. Application of laser activated electronic sizing data, confirmed by direct micrometry, demonstrated a significant increase in cell size as a function of culture. This sizing data, after prior confirmation by electron microscopy, was used as an aid in identifying type II cells and macrophages in dispersion, especially with those cells derived from rabbit lungs.

Sites of cytoplasmic ribonucleoprotein-filament assembly in relation to helical body formation in axenic trophozoites of Entamoeba histolytica.

Axenic trophozoites of Entamoeba histolytica showed increased logarithmic growth but absence of "chromatoid" material (stacked helical arrays of ribonucleoprotein [RNP]) when grown in an all-liquid monophasic culture. Organisms grown in a liquid overlay on a semisolid slant (biphasic medium) showed slow logarithmic growth and the presence of chromatoid material. Chromatoid material accumulated in the rapidly growing trophozoites from monophasic culture during treatment with the Vinca alkaloid, vinblastine. Many of the glycogen-free regions of vinblastine-treated trophozoites as well as, to a lesser degree, of normal cells grown in monophasic and biphasic cultures, contained free ribosomes and randomly oriented 60 A filaments. As ribonucleoprotein assumed the packed helical configuration, areas consisting of parallel, packed filaments could be detected adjacent to and continuous with the ordered RNP arrays. This arrangement could be visualized most frequently in vinblastine-treated trophozoites grown in monophasic cultures. Depending on the tilt of the section with respect to the longitudinal axis of individual helices, 60 A filamentous material could be demonstrated associated with the RNP helices. Localization of ribonucleoprotein precursors was followed by means of high resolution radioautography with uridine-(3)H and cytidine-(3)H. With a short (30-min) pulse, label could be visualized only over the glycogen-free areas containing free ribosomes and filaments. With 60-min pulses, label could also be seen over the packed helical arrays. With 30-min pulses followed by a 60-min cold chase, label was seen chiefly over RNP helices. It is postulated that the areas containing ribosomes and filaments represent sites of assembly of the RNP helices possibly on a filament protein column. The possibility that the final helical configuration may be due to a property of this protein is suggested.

Ultrastructure of bacterized and axenic trophozoites of Entamoeba histolytica with particular reference to helical bodies.

Electron microscopy of bacterized and axenic trophozoites of Entamoeba histolytica showed only slight differences in ultrastructure between the two. As with other species of Entamoeba so far studied, this species lacks typical mitochondrial structures and formed endoplasmic reticulum. Dense clusters of glycogen particles are especially characteristic in axenic amebas. Microtubular structures 360 A in diameter appear randomly oriented in both bacterized and axenic trophozoites. Ribonucleoprotein (RNP) bodies are of two typical forms-elongate, parallel arrays of helices (the classical chromatoid bodies), and short helical fragments. Both kinds of helix show a recurring pitch angle of 68-80 degrees and an over-all diameter of 480 A. RNP particles comprising the helices average 180 A in diameter. The longitudinal axes of adjacent helices are 440 A apart. Following RNase digestion of water-soluble methacrylate sections, helices show a core approximately 60 A in diameter. Short helices are also associated with digestive vacuoles. Free RNP particles per se are never seen within digestive vacuoles, but intact short helices are frequently detected closely associated with the external membrane of digestive vacuoles. In some cases, continuation of externally intact helical forms could be related to filamentous material within the vacuole. Acid phosphomonoesterase activity could be demonstrated within digestive vacuoles where deposition of reaction product is especially intense on the filamentous material.