Vasoactive intestinal polypeptide-induced chloride secretion by a colonic epithelial cell line. Direct participation of a basolaterally localized Na+,K+,Cl- cotransport system.

Abstract

We have used a well-differentiated human colonic cell line, the T4 cell line, as a model system to study the pathways of cellular ion transport involved in vasoactive intestinal polypeptide (VIP)-induced chloride secretion. A modified Ussing chamber was used to study transepithelial Na' and Cl-fluxes across confluent monolayer cultures of the T84 cells grown on permeable supports. In a manner analogous to isolated intestine, the Addition of VIP caused an increase of net Cl-secretion which accounted for the increase in short circuit current (1). The effect of VIP on I,, was dose dependent with a threshold stimulation at 10-10 M VIP, and a maximal effect at 10-8 M.Bumetanide prevented or reversed the response to VIP. Inhibition by bumetanide occurred promptly when it was added to the serosal, but not to the mucosal bathing media. Ion replacement studies demonstrated that the response to VIP required the simultaneous presence of Na', K+, and CF-in the serosal media. Utilizing cellular ion uptake techniques, we describe an interdependence of bumetanide-sensitive 22Na', "Rb+, and MCl-uptake, which is indicative of a Na+,K+iCl-cotransport system in this cell line. This transport pathway was localized to the basolateral membrane. Extrapolated initial velocities of uptake for each of the three ions was consistent with the electroneutral cotransport of 1 Na+:1 K+ (Rb+):2 Cl-. Our findings indicate that VIP-induced Cl-secretion intimately involves a bumetanide-sensitive Na+,K+,CF cotransport system which is functionally localized to the basolateral membrane.

The Chloride Conductance of Tight Junctions of Rat Ileum Can Be Increased by cAMP But Not by Carbachol

“…This correlates with the observation in filter-grown monolayers of T 84 cells, a mammalian intestinal cell line, where it has been found that VIP, which activates adenylate cyclase, increased not only the secretory serosato-mucosa flux but also the Cl − flux from mucosa to serosa, suggesting an increase of the diffusional, paracellular pathway in addition to the transcellular Cl − secretion [9]. In HT-29cl.19A cells (another model intestinal cell line), the conductance of the transcellular pathway can reach its maximum while the transepithelial conductance continues to increase.…”

“…This is corroborated by the observed ion selectivity of the tight junctions as determined from the mucosal dilution potentials. Figure 9 shows the relation between the dilution potential (mean 7.6 ± 0.3 mV, n ‫ס‬ 55) and the initial resistance (mean 22.5 ± 0.8 ohm ⅐ cm 2 , 1.5 ± 0.4 (7) 1.5 ± 0.5 (9) Control fluxes were calculated from samples taken at t ‫ס‬ 30 and t ‫ס‬ 40 min after start of experiment. dBcAMP or dBcAMP + bumetanide were added thereafter.…”

Regulation of intestinal uroguanylin/guanylin receptor-mediated responses by mucosal acidity

“…T84 cells were raised on collagen-coated permeable filters and mounted in custom-made Ussing chambers for measurement of transepithelial chloride secretion as described (15,25). The buffer in the basolateral reservoir was a KrebsRinger solution (pH 7.4) (25 mM sodium bicarbonate), containing 10 mM glucose.…”

Gs Protein-coupled Receptor Agonists Induce Transactivation of the Epidermal Growth Factor Receptor in T84 Cells

“…Monolayers of T 84 cells were stimulated with VIP (100 nM) for various times (0.5-30 min) before lysis. At this concentration VIP is known to maximally induce epithelial secretory responses (25). Cell lysates were then immunoprecipitated with anti-EGFr antibodies, and immunoprecipitates were Western-blotted with antiphosphotyrosine.…”