AJP - Cell Physiology, Vol 271, Issue 2 C612-C619, Copyright © 1996 by American Physiological Society

ARTICLES

Recruitment of purinergically stimulated Cl- channels from granule membrane to plasma membrane

D. Merlin, X. Guo, K. Martin, C. Laboisse, D. Landis, G. Dubyak and U. Hopfer
Department of Physiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.

HT29-Cl.16E and HT29-Cl.19A are two different subcloned cell lines derived from the human adenocarcinoma cell line HT-29. They are similar in their ability to grow and differentiate to polarized epithelial cells but differ in that HT29-Cl.16E is goblet cell-like with many mucin granules, whereas HT29-Cl.19A lacks mucin granules. Extracellular ATP stimulates Cl- secretion in both cell lines through luminal purinergic P20 receptors and, in HT29-Cl.16E, also mucin secretion release. To evaluate whether fusion of mucin granules is associated with an increase in Cl- conductance of the plasma membrane, the effects of two fusion inhibitors on luminal Cl- conductance were measured. Blockage of actin depolymerization with phalloidin (1 microM) inhibited purinergically stimulated but not adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated luminal Cl- efflux by 50% in HT29-Cl.16E. The same treatment was without effect in HT29-Cl.19A. The fungal metabolite wortmannin, which is an inhibitor of regulated exocytosis in leukocytes, at 100 nM inhibited Cl- secretion by 70% in HT29-Cl.16E. This inhibition was not a direct effect on purinergically stimulated Cl- channels because wortmannin concentrations of up to 1 microM did not affect the secretory response in HT29-Cl.19A. The wortmannin inhibition of Cl- secretion is associated with an inhibition of granule fusion as judged by electron microscopy. The differential inhibition of Cl- secretion in the related HT-29 clones that differ with respect to the presence of mucin granules indicates that 1) the granule fusion inhibitors, phalloidin and wortmannin, have no direct inhibitory effects on purinergically and cAMP-activated Cl- channels, 2) a major portion of purinergically but not cAMP-activated Cl- channels is associated with granule fusion in HT29-Cl.16E, and 3) the signaling pathways for Cl- secretion and granule fusion are not completely identical.

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