Chloride secretory mechanism induced by prostaglandin E1 in a colonic epithelial cell line.

Abstract

Confluent T8 monolayers grown on permeable supports and mounted in a modified Ussing chamber secrete chloride (Cl-) in response to prostaglandin El. The threshold stimulation was observed at 10-' M and a maximal effect at 10' M. Unidirectional flux studies showed an increase in both serosal to mucosal and mucosal to serosal CI-fluxes with 10' M prostaglandin El; the increase in serosal to mucosal Cl-flux exceeded the increase in mucosal to serosal flux, resulting in net Cl-secretion.Na' transport was not affected in either direction and the changes in net Cla flux correlated well with the changes in short circuit current. To identify the electrolyte transport pathways involved in the Cla secretory process, the effect of prostaglandin El on ion fluxes was tested in the presence of putative inhibitors. Bumetanide was used as an inhibitor for the basolaterally localized Na',K+,Ca-cotransport system whose existence and bumetanide sensitivity have been verified in earlier studies (Dharmsathaphorn et al. 1984. J. Clin. Invest. 75:462-471). Barium was used as an inhibitor for the K+ efflux pathway on the basolateral membrane whose existence and barium sensitivity were demonstrated in this study by preloading the monolayers with "Rb+ (as a tracer for K+) and simultaneously measuring MRb+ efflux into both serosal and mucosal reservoirs. Both bumetanide and barium inhibited the net chloride secretion induced by prostaglandin El suggesting the involvement of the Na+,K+,Ca-cotransport and a K+ efflux pathways on the basolateral membrane in the Clsecretory process. The activation of another Cl-transport pathway on the apical membrane by prostaglandin El was suggested by Cl-uptake studies. Our findings indicate that the prostaglandin El-stimulated Cl-secretion, which is associated with an increase in cyclic AMP level, intimately involves (a) a bumetanide-sensitive Na+,K+,Cl-cotransport pathway that serves as a C1-uptake step across the basolateral membrane, (b) the stimulation of a barium-sensitive K+ efflux mechanism on the basolateral membrane that most likely acts to recycle K+, and (c) the activation of a Cl-transport pathway on the apical membrane that serves as a Cl-exit pathway.

Stabilization of F-actin prevents cAMP-elicited Cl- secretion in T84 cells.

“…As shown in Fig. 1, elevation of intracellular cAMP elicits an increase in Isc that has been shown by others to represent a CQsecretory current (9,15,16). After addition of 1 mM 8-bromocAMP (the standard condition of cAMP stimulation unless otherwise noted), Isc rose to peak at 22.5±1.9 MA/cm2 by 30 min.…”

Carbachol-stimulated Transactivation of Epidermal Growth Factor Receptor and Mitogen-activated Protein Kinase in T84 Cells Is Mediated by Intracellular Ca2+, PYK-2, and p60

“…Cell Culture-Methods for maintenance of T 84 cells in culture were as described previously (24 , and 10 glucose. Monolayers were voltage-clamped to zero potential difference by the application of shortcircuit current (I sc ).…”

Fibroblasts modulate intestinal secretory responses to inflammatory mediators.

“…The cultured cells provide a model system in which pure populations ofthe desired cell types can be maintained in a viable state for extended periods under completely controlled conditions. T84 cells are a human colonic carcinoma cell line which has been shown to form monolayers ofmorphologically normal epithelial cells which respond to a variety of agonists with electrogenic Cl-secretion (13,14). Two types of fibroblasts were studied: P2JF, a fibroblast line derived in our laboratory from porcine small intestinal mucosa (15); and BG9, a commercially available fibroblast line derived from human foreskin (Lineberger Cancer Research Center, University ofNorth Carolina at Chapel Hill).…”

“…Prostaglandins are known to stimulate CQ-secretion by intestinal epithelial cells (13,14). The intimate spatial relationship between the fibroblasts and the intestinal epithelial cells and the ability of fibroblasts to synthesize and secrete compounds capable of stimulating epithelial C1-secretion suggest the possibility that the subepithelial fibroblasts may contribute to the regulation of intestinal epithelial electrolyte transport through the paracrine release of mediators in response to neurohumoral or inflammatory agents.…”