Occluding junction structure-function relationships in a cultured epithelial monolayer.

Abstract

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.

Neutrophil migration across cultured intestinal epithelial monolayers is modulated by epithelial exposure to IFN-gamma in a highly polarized fashion.

“…Monolayers exposed to even 1,000 U/ml IFN-'y for 72 h maintain resistances generally exceeding 250-300 ohm.cm 2 (Madara and Stafford, 1989), and we have previously shown that modulation of epithelial resistance in the range of 200-2,000 ohm.cm 2 has little effect on the efficiency of PMN transmigration (Nash et al, 1987). This is likely due to the fact that the relationship between resistance and the flux of solutes, such as those in the size-range of FMLP is asymptotic (Madara and Dharmsathaphorn, 1985;Hecht et al, 1988); that is, at higher resistance values (>200-250 ohm.cm:), large changes in resistance are accompanied by only modest changes in solute flux. The reasons for this are clear when one considers the behavior of a circuit composed of parallel resistances as we have discussed in the past (Madara and Dharmsathaphorn, 1985).…”

“…This is likely due to the fact that the relationship between resistance and the flux of solutes, such as those in the size-range of FMLP is asymptotic (Madara and Dharmsathaphorn, 1985;Hecht et al, 1988); that is, at higher resistance values (>200-250 ohm.cm:), large changes in resistance are accompanied by only modest changes in solute flux. The reasons for this are clear when one considers the behavior of a circuit composed of parallel resistances as we have discussed in the past (Madara and Dharmsathaphorn, 1985). Such sieving characteristics have other implications for the movement of PMN across epithelia (vida infra).…”

“…In contrast, cable analysis studies of the mesenteric microvasculature suggests that even physiologically confluent endothelium (except for that of the brain which has resistance >1,000 ohm.cm 2) has an average resistance of only a few ohm.cm 2 (on average, <2 ohm.cm2; Crone and Christensen, 1981). As a consequence of the capacity of epithelia to behave as very restrictive barriers to the passive transepithelial flux of hydrophilic solutes, one finds that even for solutes with hydrodynamic radii in the range of 10 /~, diffusional flux across epithelial monolayers, such as those used here, is <10 nM/h/cm 2 (Madara and Dharmsathaphorn, 1985). Indeed, even in the low resistance state induced by prolonged IFN-3, exposure, inulin fluxes, while increased sixfold from baseline, are <10 nM/h/cm 2 (Madara and Stafford, 1989).…”

“…Such monolayers are composed of columnar epithelial cells with features similar to those of natural crypt epithelia (Madara and Dharmsathaphorn, 1985;Madara et al, 1987). These cells join neighbors through apical circumferential tight junctions which maintain structure-function characteristics found in fight junctions of natural epithelia (Madara and Dharmsathaphorn, 1985). Furthermore, these ceils have a polarized surface distribution of the pumps, channels, and cotransporters which allow manifestation of electrogenic CI-secretion (Dharmsathaphorn and Madara, 1990).…”

Epithelial secretion of vinblastine by human intestinal adenocarcinoma cell (HCT-8 and T84) layers expressing P-glycoprotein

“…Inulin is a high molecular weight (-5,000) inert macromolecule which is normally excluded from cells, and is therefore used as a tracer of extracellular permeability pathways through the intercellular tight junctions, and an indicator of changes in the permeability of this paracellular pathway (Madara & Dharmsathaphorn, 1985). Bidirectional ['4C]-inulin fluxes were determined exactly as for vinblastine sulphate fluxes.…”