ATP-STIMULATED ELECTROLYTE AND MUCIN SECRETION IN THE HUMAN INTESTINAL GOBLET CELL-LINE HT29-CL.16E

The response of confluent monolayers of HT29-Cl.16E cells to stimulati on by extracellular ATP and ATP analogues was investigated in terms of mucin and electrolyte secretion. Mucin secretion was measured as rele ase of glucosamine-labeled macromolecules trapped at the stacking/runn ing gel interface of polyacrylamide gels and electrolyte secretion as short-circuit current (I-sc). Luminal ATP stimulated a transient incre ase 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 AT P. Maximal ATP-stimulated mucin secretion over 15 min was up to 18-fol d above control with an apparent ED(50) of approximately 40 mu M. Maxi mal peak I-sc after stimulation with ATP was approximately 35 mu A/cm( 2) with an apparent ED(50) of about 0.4 mM. ATP-dependent I-sc was at least in part due to Cl- secretion since removal of Cl- from the mediu m 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 a ssessed by failure of ethidium bromide to enter into the cells, absenc e of release of lactate dehydrogenase, maintenance of monolayer conduc tance, viability, and responses to repeated applications of ATP. The o rder of efficacy of nucleotide agonists was similar for both processes with ATP > ADP > AMP greater than or equal to adenosine. Luminal ATP was much more effective than basolateral addition of this compound. Th ese results suggest involvement of a luminal P-2-type receptor which c an initiate signaling pathways for granule fusion and mucin release as well as for activation of Cl- channels. P-2-receptor-stimulated mucin and I-sc release was strongly inhibited by a 30 min preincubation wit h 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 membr ane revealed that quinidine did not directly block the ATP-induced bas olateral K+ or the luminal anion channels. The quinidine inhibition af ter preincubation is therefore most easily explained by interference w ith granule fusion and location of anion channels in granule membranes . Luminal P-2 receptors may play a role in intestinal defense mechanis ms with both fluid and mucin secretion aiding in the removal of noxiou s agents from the mucosal surface.