ATP-stimulated electrolyte and mucin secretion in the human intestinal goblet cell line HT29-C1.16E

Abstract

The response of confluent monolayers of HT29-Cl.16E cells to stimulation by extracellular ATP and ATP analogues was investigated in terms of mucin and electrolyte secretion. Mucin secretion was measured as release of glucosamine-labeled macromolecules trapped at the stacking/running gel interface of polyacrylamide gels and electrolyte secretion as short-circuit current (Isc). Luminal ATP stimulated a transient increase in the release of mucins and of Isc corresponding to a secretory Cl- current. Both secretions peaked at 3 to 5 min after addition of ATP. Maximal ATP-stimulated mucin secretion over 15 min was up to 18-fold above control with an apparent ED50 of approximately 40 microM. Maximal peak Isc after stimulation with ATP was approximately 35 microA/cm2 with an apparent ED50 of about 0.4 mM. ATP-dependent Isc was at least in part due to Cl- secretion since removal of Cl- from the medium reduced the peak Isc by 40% and the Isc integrated over 40 min by 80%. The secretory responses were not associated with cell damage as assessed by failure of ethidium bromide to enter into the cells, absence of release of lactate dehydrogenase, maintenance of monolayer conductance, viability, and responses to repeated applications of ATP. The order of efficacy of nucleotide agonists was similar for both processes with ATP > ADP > AMP > or = adenosine. Luminal ATP was much more effective than basolateral addition of this compound. These results suggest involvement of a luminal P2-type receptor which can initiate signaling pathways for granule fusion and mucin release as well as for activation of Cl- channels. P2-receptor-stimulated mucin and Isc release was strongly inhibited by a 30 min preincubation with the classical K+ channel blockers quinine (1 mM), quinidine (1 mM), and Ba2+ (3 mM). Experiments with amphotericin B to measure separately the conductance changes of either luminal or basolateral plasma membrane revealed that quinidine did not directly block the ATP-induced basolateral K+ or the luminal anion channels. The quinidine inhibition after preincubation is therefore most easily explained by interference with granule fusion and location of anion channels in granule membranes. Luminal P2 receptors may play a role in intestinal defense mechanisms with both fluid and mucin secretion aiding in the removal of noxious agents from the mucosal surface.

Heterogeneity of Jagged1 expression in human and mouse intestinal tumors: implications for targeting Notch signaling

“…This is a malignant human colon carcinoma cell line that differentiates, upon contact inhibition of cell growth, into normal-appearing polarized goblet cells that synthesize and secrete colonspecific mucin (Augeron and Laboisse, 1984;Augenlicht et al, 1987;Laburthe et al, 1989;Merlin et al, 1994). Since transition between these states seems to recapitulate the normal growth arrest and lineage-specific differentiation of colonic progenitor cells that is mediated by reduction of Notch signaling in the intestine (Yang et al, 2001;Shroyer et al, 2007), we focused on this cell line to investigate the potential role of Notch signaling in general, and Jagged1 specifically, in regulating Notch signaling in the colon carcinoma cell phenotype.…”

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].…”

“…Particularly mucin and electrolyte secretion in response to ATP has been observed in the human intestinal goblet cell line HT29-Cl.16E [10]. No work has been reported about ATP-dependent mucin secretion in normal and cystic fibrosis pancreatic epithelial cells in culture: it therefore seemed interesting to us to study the differential ATP-dependent mucin secretion and the role of Ca 2+ as a second messenger.…”

Purinergic signalling in the gastrointestinal tract and related organs in health and disease

“…A study of goblet cell-like clone derived from colonic HT-29 cells led to the conclusion that ATP-stimulated increase in Cl − current does not require an increase in [Ca 2+ ] i suggesting the involvement of either another signalling pathway or direct activation of Cl − channels via purinergic receptors [305]. ATP-stimulated electrolyte and mucin secretion by this human intestinal goblet cell line has been reported [474]. Exogenous ATP added to the medium bathing the mucosal surface of the intestine inhibits calcium transport to reduce the unidirectional flux of Ca 2+ from the mucosal to serosal side [715].…”