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 shortcircuit current (Isc). Luminal ATP stimulated a transient increase in the release of mucins and of I sc 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 μm. Maximal peak I sc after stimulation with ATP was approximately 35 μA/cm2 with an apparent ED50 of about 0.4 mm. ATP-dependent I sc was at least in part due to Cl− secretion since removal of Cl− from the medium reduced the peak I sc by 40% and the I sc 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≥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 I sc 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.
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Supported by grants from the National Institutes of Health (DK 39658) to U.H., the Philippe Foundation to D.M., the French Cystic Fibrosis Foundation (AFLM) and L'Association Pour La Recherche Sur Le Cancer to C.L. The authors thank Mr. J. Polack for his efforts and skill with electron microscopy and Dr. George Dubyak for helpful discussions. We also acknowledge the Cystic Fibrosis Center Core grant (DK-27651) for its support of electron and light microscopy.
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Merlin, D., Augeron, C., Tien, X.Y. et al. ATP-stimulated electrolyte and mucin secretion in the human intestinal goblet cell line HT29-C1.16E. J. Membarin Biol. 137, 137–149 (1994). https://doi.org/10.1007/BF00233483
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DOI: https://doi.org/10.1007/BF00233483