Regulation of mucus secretion by cells isolated from the rat gastric mucosa
This study was undertaken to further understanding of the mechanisms which regulate mucus secretion by rat stomach cells. Particular objectives were: (i) to develop and use a radiochcmical assay to estimate the secretion of mucin by suspension of gastric mucosal cells in vitro, (ii) to develop and use a solid-phase enzyme immunoassay (EIA) to study the regulation of the release of bulk gastric mucin from the isolated cells and (iii) to compare the results obtained with the two procedures. Cells were isolated by exposure of gastric mucosa to pronase and EDTA. Cell suspensions were preincubated with D-[6-3H]glucosamine. [3H]-labelled material of high molecular mass released into the incubation medium, was purified by Fast Protein Liquid Chromatography, and appeared to be gastric mucin. Some unidentified [3H]-labellcd material of lower molecular mass was also found in the medium. Release of [3H]-labelled high molecular mass material was essentially linearly related to time. Secretin, isoprenaline and carbachol stimulated release of [3H]-labelled high molecular mass material. The half-maximally effective concentrations of secretin and isoprenaline were 2.3nM and 34nM respectively. Histamine, gastrin and epidermal growth factor were without effect. A rabbit polyclonal antibody was raised by using purified 'native' rat gastric mucin as immunogen. The antibody preparation appeared specific for rat gastric mucin and was used to establish a quantitative solid-phase EIA. Release of bulk mucin was essentially linearly related to time. Phorbol-12-myristate-13-acetate (PMA), forskolin and A23187 dose-depcndently stimulated bulk mucin release. Syncrgistic interactions were observed between PMA and forskolin, and PMA and A23187. Secretin and isoprenaline were confirmed as mucin secretogogues. In conclusion gastric mucin release was investigated for the first time by using a suspension of gastric mucosal cells. Two different assay procedures were developed. Some pathways and agents responsible for controlling mucin secretion were identified.