The T84 human colonic adenocarcinoma cell line produces mucin in culture and releases it in response to various secretagogues

Abstract

The T84 colonic adenocarcinoma cell line, which has been used extensively as a model for studies of epithelial chloride secretion, also produces mucin and secretes it in culture. Electron microscopy of fixed sections of cultured cells, along with Immunogold labelling with an antibody to human small intestine (SI) mucin, revealed the presence of goblet-like cells with mucin-containing secretory granules. The mucin was of high molecular mass, had an amino acid composition similar to that of purified human SI and colonic mucins, and competed effectively with SI mucin for binding to the anti-(SI mucin) antibody. A sensitive solid-phase immunoassay specific for intestinal mucins was developed and used to measure mucin secretion by T84 cells. Cultures were treated for 30 min at 37 degrees C with a number of agents known to cause chloride secretion by T84 cell monolayers and the amount of mucin appearing in the medium was measured. Carbachol (1 mM), A23187 (10 microM), prostaglandin E1 (PGE1) (1 microM) and vasoactive intestinal polypeptide (VIP) (0.1 microM) all stimulated mucin release, but histamine (1 mM) had no effect. Whereas VIP is reported to stimulate chloride secretion more strongly than carbachol, it was less effective than carbachol in stimulating mucin secretion. Phorbol 12-myristate 13-acetate (PMA) (0.1-10 microM) also stimulated mucin release strongly, implicating a responsive protein-kinase C-dependent pathway. Additive secretory responses were obtained with combined stimulation by VIP (10 nM-1 microM) and carbachol (1 mM). Responses to stimulation with A23187 (1-10 microM) together with PMA (10 nM-10 microM) suggest that cytosolic Ca2+ concentration is a modulator of PMA activity.

Regulated Cl transport, K and Cl permeability, and exocytosis in T84 cells.

“…Notice that very little labeled high molecular weight specific species were secreted using the 35S04-labeling protocol. Various cultured intestinal cells are able to synthesize and secrete mucins (36)(37)(38), including T84 cells (39). McCool et al (39) showed that T84 cells release 0.5-0.7% ofthe total cell mucus in 30 min, and stimulation by 1 mM carbachol increased this to 2%.…”

“…Thus, some mucin secretion is expected from the apical surface during our 2-h collection. According to McCool et al (39) these mucins have very high molecular weight and do not enter 7.5% separating gels. As shown in Fig.…”

Defective ATP‐dependent mucin secretion by cystic fibrosis pancreatic epithelial cells

“…No significant response by CFPAC-1 cells and by CFPAC-PLJ6 cells was observed with any secretagogue we used. Carbachol and VIP acting via phosphoinositide/Ca 2+ and cAMP respectively, known to be physiological secretagogues for C1 secretion [30,31], were shown to also stimulate mucin secretion in the T84 human colonic adenocarcinoma cell line [11] and in the human intestinal goblet cell line HT29-Cl.16E [10]. Forskolin, which raised intracellular cAMP, stimulated C1-secretion by increasing the magnitude of CFTR-mediated C1 conductance in the apical membrane [32], by a PKAdependent stimulus, was capable of stimulating mucin secretion in a Ca2+-independent manner [33].…”

“…Several gastrointestinal cell lines have been found to be mucin secreting and used to study the regulation of mucin secretion [10,11]. Particularly mucin and electrolyte secretion in response to ATP has been observed in the human intestinal goblet cell line HT29-Cl.16E [10].…”

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

“…There are also marked differences in Na + -dependent nucleoside transport systems in T84 cells as compared to Caco-2 cells (Ward and Tse, 1999). Furthermore, T84 cells produce mucin in culture and thus more closely mimic the intestinal surface (McCool et al, 1990); they also respond to external stimuli to exert innate immune responses similar to those initiated at the intestinal luminal surface (Ou et al, 2009).…”