Incorporation of N-Acetyl-[¹⁴C]<i>D</i>-Glucosamine and [³H]<i>L</i>-Leucine by Isolated Pig Gastric Mucosal Cells

Abstract

Glycoprotein and protein production of isolated pig gastric mucosal cells were determined by the incorporation of N-acetyl-[¹⁴C]D-glucosamine ([¹⁴C]G1cNAc) and [³H]L-leucine ([³H]Leu) into acid-insoluble macromolecules (AIM). In four cell fractions (F1-F4), obtained by counterflow centrifugation, specific [¹⁴C]G1cNAc incorporation was greatest in the mucous cell-enriched F2. Tracer incorporation by F2 cells, proceeded lineraly up to 20 h, was inhibited by cycloheximide or incubation at 0 ° C, and enhanced by PGE₂ 1 μmol/l. Gel chromatography of released AIM revealed that PGE₂-stimulated [¹⁴C]GlcNAc incorporation was predominantly directed into high molecular weight (2 × 10⁶ daltons) glycoproteins, whereas [³H]Leu incorporation was mainly related to proteins of albumin-like molecular weight. We conclude that incorporation of [¹⁴C]G1cNAc by enriched pig gastric mucous cells (F2), further analyzed by gel chromatography, is a suitable probe to study the production of high molecular weight gastric mucous glycoproteins in vitro.

Stimulation of glycoprotein and protein synthesis in isolated pig gastric mucosal cells by prostaglandins.

“…To avoid this problem, mucus production was examined directly at the cellular level, 3 showing that isolated and enriched pig gastric mucous cells synthesise proteins and glycoproteins in suspension culture for up to 20 hours of incubation, as measured by the incorporation of…”

Effects of histamine and activators of the cyclic AMP system on protein synthesis in and release of high molecular weight glycoproteins from isolated gastric non‐parietal cells

“…Two cell populations (F) were obtained at flow rates of 20mlmin-1 (Fl, 600ml total volume collected) and 40 ml min' 1 (F2, 1000 ml), and the remainder of cells was discarded. For this investigation we used F2 cells, consisting of more than 60% mucous cells, about 25% chief cells and less than 2% parietal cells, as determined by cell staining (Heim et al, 1989 (Heim et al, 1989 (8ml) were eluted with NaCl 0.2M, containing 0.02% sodium azide, (Pearson et al, 1980) at a pump controlled flow rate of 33.6 ml h-l. Fractions of 11.2ml volume were collected and examined for TCA/PTA precipitable radioactivity. To this end, 1 ml of each fraction was treated with 250ul TCA/PTA (50%/5%) and processed further as described for the supernatant samples above.…”

“…To confirm a function for cyclic AMP directly at the cellular level, we examined the influence of known activators of the cyclic AMP 'second messenger' system on protein and glycoprotein synthesis in and release from enriched porcine gastric mucous cells, as measured (Heim et al, 1989) by the incorporation of N-acetyl-[4C]-D-glucosamine ([`4C]-GlcNAc) and [3H]-L-leucine, respectively, into trichloroacetic acid/ phosphotungstic acid precipitable cellular and released macromolecules. As stimulants with different points of action in the cyclic AMP system we used, in addition to forskolin, histamine, shown by Gespach et al (1982Gespach et al ( , 1983 and Sewing et al (1985) to enhance cyclic AMP levels of gastric non-parietal cells via H2-receptor activation, 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cyclic AMP phosphodiesterases (Beavo, 1988), and dibutyryl cyclic AMP (db cyclic AMP), an activator of cyclic AMP-dependent protein kinases (Simon et al, 1973).…”

Effects of Histamine on Protein and Glycoprotein Production of Isolated Pig Gastric Mucosal Cells

“…To this end we used a cell mixture of isolated pig gastric chief and mucous cells practically devoid of parietal cells, prepared by pronase/collagenase treatment of the fundic mucosa fol lowed by cell elutriation. As measures of pro tein and glycoprotein production by these cells we used the incorporation of [3H]-Lleucine ([3H]Leu) and N-acetyl-[l4C]-£>-glucosamine ([l4C]GlcNAc), respectively, into acid insoluble material (AIM) as described before [6].…”