Mucus glycoprotein secretion by tracheal explants: effects of pollutants.

Abstract

Tracheal slices incubated with radioactive precursors in tissue culture medium secrete labeled mucus glycoproteins into the culture medium. We have used an in vivtro approach, a combined method utilizing exposure to pneumotoxins in vivo coupled with quantitation of mucus secretion rates in vitro, to study the effects of inhaled pollutants on mucus biosynthesis by rat airways. In addition, we have purified the mucus glycoproteins secreted by rat tracheal explants in order to determine putative structural changes that might be the basis for the observed augmented secretion rates after exposure of rats to H2SO4 aerosols in combination with high ambient levels of ozone.After digestion with papain, mucus glycoproteins secreted by tracheal explants may be separated into five fractions by ion-exchange chromatography, with recovery in high yield, on columns of DEAE-cellulose.Each of these five fractions, one neutral and four acidic, migrates as a single unique spot upon cellulose acetate electrophoresis at pH values of 8.6 and 1.2. The neutral fraction, which is labeled with [3H] glucosamine, does not contain radioactivity when Na2 3"SO4 is used as the precursor. Acidic fractions I-IV are all labeled with either 3H-glucosamine or Na2 35S04 as precursor. Acidic fraction H contains sialic acid as the terminal sugar on its oligosaccharide side chains, based upon its chromatographic behavior on columns ofwheat-germ agglutinin-Agarose. Treatment of this fraction with neuraminidase shifts its elution position in the gradient to a lower salt concentration, coincident with acidic fraction I. After removal of terminal sialic acid residues with either neuraminidase or low pH treatment, the resultant terminal sugar on the oligosaccharide side chains is fucose. These results are identical with those observed with mucus glycoproteins secreted by cultured human tracheal explants and purified by these same techniques.We have been using the tracheal explant system, developed several years ago in the laboratories of Reid (1) and others (2-7) as a quantitative assay for monitoring effects of air pollutants and other pneumotoxins on the respiratory epithelium. We have used what we term an "in vivtro" approach, as indicated schematically in Figure 1, featuring exposure of animals (usually rats) in vivo to known amounts of pneumotoxin, followed by removal of their tracheae for assay of effects in vitro. With small animals such as rats, we routinely incubate the tracheae in tissue culture medium containing radioactively labelled precursors. The culture medium is then removed (Fig. 1) and an aliquot is treated with 5% trichloroacetic acid to precipitate the proteins present. With a suitable choice of labeled precursor and incubation time for the rat tracheal explants, essentially all of the acid-precipitable radioactivity in the medium is mucus glycoprotein, as we will dem-

Glycoprotein Synthesis by an Established Cell Line from Hamster Tracheal Epithelium

“…The small amount of labeling of Fraction I1 with 3sS is also consistent with our data from human tracheal explants [ 4 ] and rat tracheal explants [5]. It is not clear whether the even smaller amount of "S in Fraction I (less than 1 % of the total) is incorporated into glycoprotein or is a contaminating impurity; this problem is discussed elsewhere [4].…”

“…2(b)] is observed from the tracheal explant medium that is absent from the HTE-B cell culture medium. This additional fraction that is apparently absent from the HTE-B cell culture medium contains glycopeptides with terminal sialic acid residues, based on their susceptibility to neuraminidase digestion and their binding properties to specific lectins [ 5 ] . The concentration of LiCl at which the various The additional sialic-acid-containing peak from the tracheal medium is eluted at about 0.25 M LiC1, so is clearly separable from those fractions designated Peaks I1 and 111 in Fig.…”

Ozone-Induced Acute Pulmonary Injury in Inbred Mouse Strains

“…Other studies suggest that IL-1␤ or TNF-␣ (1), or even reactive oxygen species (26), may play a role in this process. A number of soluble by-products arising from epithelial lining fluid, and cellular reactions with O 3 , may also upregulate transcription factors and proinflammatory genes (27,28). Our data suggest that the susceptibility to acute O 3 exposure is a complex trait with strain variation in expression, combination, and distribution of several main phenotypic features (e.g., lung inflammation, increased lavageable protein levels, and airway epithelial cell injury/proliferation).…”

Mucus Glycoproteins Secreted by Respiratory Epithelial Tissue from Cystic Fibrosis Patients

“…The methodology used in the present study is identical to that currently used for animal studies in our laboratory (13,14). We have studied radionuclide-labeled mucus glycoproteins from rat and monkey tracheal explants extensively (10,II,16), and have found that papain digestion of tissue culture medium, although avoiding contamination by oropharyngeal secretions or products of airway inflammation, yields a sufficient amount of purified mucus glycopeptides to permit their relatively sharp resolution on chromatography. The primary purpose of the present study was to examine possible differences between the respiratory mucus glycoproteins of CF and non-CF subjects.…”