A human colonic epithelial cell line, T84, derived from a colonic carcinoma, has been examined both morphologically and functionally. The cells grew to confluence as a monolayer with the basolateral membrane attached to the surface of the culture dish and a microvillus-studded apical membrane facing the media. Tight junctions and desmosomes were demonstrated between adjacent cells. Confluent monolayer cultures conducted vectorial electrolyte transport that could be altered by a variety of secretagogues and antisecretagogues similar to isolated intestinal tissues. This cell line will serve as an excellent model system for the study of electrolyte transport processes and their regulation by peptide hormones and neurotransmitters.

Electrical circuit analysis was used to study the structural development of occluding junctions (OJs) in cultured monolayers composed of T84 cells. The magnitude of the increments in transepithelial resistance predicted by such analysis was compared with the magnitude of the measured increments in resistance. Confluent sheets of epithelial cells were formed after cells were plated at high density on collagen-coated filters. Using Claude's OJ strand countresistance hypothesis (1978, J. Membr. Biol. 39:219-232), electrical circuit analysis of histograms describing OJ strand count distribution at different time points after plating predicted that junctional resistance should rise in a proportion of 1:21:50 from 18 h to 2 d to 5 d. This reasonably paralleled the degree of rise in transepithelial resistance over this period, which was 1:29:59. The ability to predict the observed resistance rise was eliminated if only mean strand counts were analyzed or if electrical circuit analysis of OJ strand counts were performed using an OJ strand count-resistance relationship substantially different from that proposed by Claude. Measurements of unidirectional fluxes of inulin, mannitol, and sodium indicated that restriction of transjunctional permeability accounted for the observed resistance rise, and that T84 junctional strands have finite permeability to molecules with radii ___3.6 ,g, but are essentially impermeable to molecules with radii >15 A. The results suggest that general correlates between OJ structure and OJ ability to resist passive ion flow do exist in T84 monolayers. The study also suggests that such correlates can be obtained only if OJ structural data are analyzed as an electrical circuit composed of parallel resistors.

Serosal application of carbachol to Tg cell monolayers mounted in an Ussing chamber caused an immediate increase in short circuit current (I,)

No abstract

No 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.

Vasoactive intestinal polypeptide (VIP) and the calcium ionophore A23187 caused dose-dependent changes in the potential difference and the short circuit current (I.) across confluent T84 cell monolayers mounted in modified Ussing chambers. Both VIP and A23187 stimulated net chloride secretion without altering sodium transport. Net chloride secretion accounted for the increase in I,<. When A23187 was tested in combination with VIP, net chloride secretion was significantly greater than predicted from the calculated sum of their individual responses indicating a synergistic effect. VIP increased cellular cyclic AMP (cAMP) production in a dose-dependent manner, whereas A23187 had no effect on cellular cAMP. We then determined whether VIP and A23187 activated different transport pathways. Earlier studies suggest that VIP activates a basolaterally localized, barium-sensitive potassium channel as well as an apically localized chloride conductance pathway. In this study, stimulation of basolateral membrane potassium efflux by A23187 was documented by preloading the monolayers with MRb+. Stimulation of potassium efflux by A23187 was additive to the VIP-stimulated potassium efflux. By itself, 0.3 ,uM A23187 did not alter transepithelial chloride permeability, and its stimulation of basolateral membrane potassium efflux caused only a relatively small amount of chloride secretion. However, in the presence of an increased transepithelial chloride permeability induced by VIP, the effectiveness of A23187 on chloride secretion was greatly augmented.Our studies suggest that cAMP and calcium each activate basolateral potassium channels, but cAMP also activates an apically localized chloride channel. Synergism results from cooperative interaction of potassium channels and the chloride channel.

Escherichia coli heat stable enterotoxin (ST.) caused Cl-secretion across T84 cell monolayers in a dose-dependent manner only when applied to the apical membrane surface and not when applied to the basolateral surface. Measurement of cAMP, cGMP, and free cytosolic Ca2l in response to STa suggested that cGMP alone mediated the Cl-secretory response. Studies utilizing blockers of the Na+,K+-ATPase pump, a Na+,K+,ClI cotransport system, a K+ channel, and a Cl-channel suggest that all of them participate in the Clsecretory process induced by ST.. The results suggest that the Cl-secretory response induced by STa is mediated by cGMP after the enterotoxin binds to its receptor on the apical membrane. The enterotoxin, by increasing cGMP, opens a K+ channel on the basolateral membrane as well as a Cl-channel on the apical membrane. The activation of these ion exit mechanisms, together with activations of the Na+,K+,Cl-cotransporter and the Na+,K+-ATPase pump drives Cl-exit through the Cl-channel on the apical membrane.