[24] Established intestinal cell lines as model systems for electrolyte transport studies

Dharmsathaphorn, Kiertisin; Madara, James L.

doi:10.1016/0076-6879(90)92082-o

Cited by 170 publications

(148 citation statements)

References 32 publications

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“…Hanks' balanced salt solution (HBSS, containing in g/liter 0.185 CaCl 2 , 0.098 MgSO 4 , 0.4 KCl, 0.06 KH 2 PO 4 , 8 NaCl, 0.048 Na 2 HPO 4 , 1 glucose, to which was added 10 mM HEPES, pH 7.4) was used for all assays unless otherwise stated. T84 cells obtained from ATCC were cultured and passaged as described previously (20,21). Cells from passages 69 -88 were utilized for these experiments.…”

Section: Methodsmentioning

confidence: 99%

Proteolytic Activation of Cholera Toxin and Escherichia coli Labile Toxin by Entry into Host Epithelial Cells

Lencer¹,

Constable²,

Moe³

et al. 1997

Journal of Biological Chemistry

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Cholera and Escherichia coli heat-labile toxins (CT and LT) require proteolysis of a peptide loop connecting two major domains of their enzymatic A subunits for maximal activity (termed "nicking"). To test whether host intestinal epithelial cells may supply the necessary protease, recombinant rCT and rLT and a protease-resistant mutant CTR192H were prepared. Toxin action was assessed as a Cl ؊ secretory response (Isc) elicited from monolayers of polarized human epithelial T84 cells. When applied to apical cell surfaces, wild type toxins elicited a brisk increase in Isc (80 A/cm 2 ). Isc was reduced 2-fold, however, when toxins were applied to basolateral membranes. Pretreatment of wild type toxins with trypsin in vitro restored the "basolateral" secretory responses to "apical" levels. Toxin entry into T84 cells via apical but not basolateral membranes led to nicking of the A subunit by a serine-type protease. T84 cells, however, did not nick CTR192H, and the secretory response elicited by CTR192H remained attenuated even when applied to apical membranes. Thus, T84 cells express a serine-type protease(s) fully sufficient for activating the A subunits of CT and LT. The protease, however, is only accessible for activation when the toxin enters the cell via the apical membrane.

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Section: Methodsmentioning

confidence: 99%

Proteolytic Activation of Cholera Toxin and Escherichia coli Labile Toxin by Entry into Host Epithelial Cells

Lencer¹,

Constable²,

Moe³

et al. 1997

Journal of Biological Chemistry

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“…T84 intestinal epithelial cells (passages 70-95) were grown and maintained as confluent monolayers on collagen-coated permeable supports as previously described in detail (Dharmsathaphorn and Madara, 1990). Monolayers were grown on 0.33-cm 2 ring-supported polycarbonate filters (Costar Corp., Cambridge, MA) and utilized 6-14 d after plating as described previously (Madara et al, 1993).…”

Section: Cell Culturementioning

confidence: 99%

“…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). Lastly, the regulation of ion transport events seen in T~ cells, both via direct manipulation of intracellular second messengers or indirectly through receptors for hormones or toxins (Dharmsathaphorn and Madara, 1990), closely recapitulates regulatory events seen in natural crypt epithelia. For these reasons, T~ cells are viewed as a model for haman crypt epithelium-the site at which the bulk of PMN transepithelial migration occurs in a variety of active inflammatory intestinal diseases (Yardley and Donowitz, 1977;Yardley, 1986).…”

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confidence: 99%

“…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). Lastly, the regulation of ion transport events seen in T~ cells, both via direct manipulation of intracellular second messengers or indirectly through receptors for hormones or toxins (Dharmsathaphorn and Madara, 1990), closely recapitulates regulatory events seen in natural crypt epithelia.…”

mentioning

confidence: 99%

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

Colgan

Parkos

Delp

et al. 1993

The Journal of Cell Biology

158

111

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Abstract. Neutrophil, or polymorphonuclear leukocyte (PMN), migration across intestinal epithelial barriers, such as occurs in many disease states, appears to result in modifications of epithelial barrier and ion transport functions (Nash, S., J. Stafford, and J. L. . Z Clin. Invest. 80:1104-1113; Madara, J. L., C. A. Parkos, S. P. Colgan, R. J. MacLeod, S. Nash, J. B. Matthews, C. Delp, and W. I. Lencer. 1992. J. Clin. Invest. 89:1938-1944). Here we investigate the effects of epithelial exposure to IFN-,), on PMN migration across cultured monolayers of the human intestinal epithelial cell line Tu. Transepithelial migration of PMN was initially assessed in the apicalto-basolateral direction, since previous studies indicate general qualitative similarities between PMN migration in the apical-to-basolateral and in the basolateralto-apical directions. In the apical-to-basolateral direction, epithelial exposure to IFN-.,/rnarkedly upregulated transepithelial migration of PMN in a dose-and timedependent fashion as measured by both electrical and myeloperoxidase assays. This IFN-~/-elicited effect on transmigration was specifically due to a IFN-,/effect on epithelial cells and was not secondary to IFN-,y effects on epithelial tight junction permeability. Moreover, this IFN-.y effect was dependent on epithelial protein synthesis, and involved a pathway in which CDllb/18, but not ICAM-1 or CDlla/18, appeared to play a crucial role in PMN-epithelial adhesion. IFN-~/also substantially modified PMN transepithelial migration in the natural, basolateral-to-apical direction. The IFN-'y effect on naturally directed transmigration was also specifically due to an IFN-3, effect on epithelial cells, showed comparable time and dose dependency to that of oppositely directed migration, was CDllb/18 dependent, and required epithelial protein synthesis. Additionally, however, important qualitative differences existed in how IFN-3, affected transmigration in the two directions. In contrast to apical-to-basolateral directed migration, IFN-,), markedly downregulated transepithelial migration of PMN in the natural direction. This downregulation of PMN migration in the natural direction, however, was not due to failure of PMN to move across filters and into monolayers. Indeed, IFN-3, exposure to epithelia increased the number of PMN which had moved into the basolateral space of the epithelium in naturally directed transmigration. These results represent the first detailed report of influences on PMN transepithelial migration by a cytokine, define conditions under which a qualitative difference in PMN transepithelial migration exists, and suggest that migration of PMN across epithelia in the natural direction may involve multiple steps which can be differentially regulated by cytokines. Specifically, it appears that in naturally directed migration, IFN-~/ may enhance the retention time of the recruited PMN in the paracellular space below tight junctions, and does so, at least in part, by downregulating a PMN-epithelial interactive event req...

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“…Electrophysiology-T84 cells (from passages 75-100) obtained from ATCC were grown and passaged as described (18,41). Toxins were diluted in prewarmed Hanks' balanced salt solution (HBSS) containing (per liter): 0.185 g of CaCl 2 , 0.098 g of MgSO 4 , 0.4 g of KCl, 0.06 g of KH 2 PO 4 , 8 g of NaCl, 0.048 g of Na 2 HPO 4 , 1.0 g of glucose, and 10 mM HEPES at pH 7.4, and applied to the apical surface of confluent T84 cell monolayers in Transwell inserts (Costar, Cambridge, MA), followed by incubation at 37°C.…”

Section: Toxin Assaysmentioning

confidence: 99%

Structural Basis for the Differential Toxicity of Cholera Toxin and Escherichia coli Heat-labile Enterotoxin

Rodighiero

Aman

Kenny

et al. 1999

Journal of Biological Chemistry

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Cholera toxin (Ctx) and E. coli heat-labile enterotoxin (Etx) are structurally and functionally similar AB 5 toxins with over 80% sequence identity. When their action in polarized human epithelial (T84) cells was monitored by measuring toxin-induced Cl ؊ ion secretion, Ctx was found to be the more potent of the two toxins. Here, we examine the structural basis for this difference in toxicity by engineering a set of mutant and hybrid toxins and testing their activity in T84 cells. This revealed that the differential toxicity of Ctx and Etx was (i) not due to differences in the A-subunit's C-terminal KDEL targeting motif (which is RDEL in Etx), as a KDEL to RDEL substitution had no effect on cholera toxin activity; (ii) not attributable to the enzymatically active A1-fragment, as hybrid toxins in which the A1-fragment in Ctx was substituted for that of Etx (and vice versa) did not alter relative toxicity; and (iii) not due to the B-subunit, as the replacement of the B-subunit in Ctx for that of Etx caused no alteration in toxicity, thus excluding the possibility that the broader receptor specificity of EtxB is responsible for reduced activity. Remarkably, the difference in toxicity could be mapped to a 10-amino acid segment of the A2-fragment that penetrates the central pore of the B-subunit pentamer. A comparison of the in vitro stability of two hybrid toxins, differing only in this 10-amino acid segment, revealed that the Ctx A2-segment conferred a greater stability to the interaction between the A-and B-subunits than the corresponding segment from Etx A2. This suggests that the reason for the relative potency of Ctx compared with Etx stems from the increased ability of the A2-fragment of Ctx to maintain holotoxin stability during uptake and transport into intestinal epithelia.The severe, and at times fatal, diarrheal disease caused by Vibrio cholerae is due to the potent action of cholera toxin (Ctx) 1 (for a review, see Ref. 1). A structurally and functionally similar toxin, heat-labile enterotoxin (Etx), is produced by certain strains of enterotoxigenic Escherichia coli that are responsible for causing the generally milder "traveler's" diarrhea of humans and scouring in farm animals (2, 3). Both Ctx and Etx are hetero-oligomeric proteins comprised of a single A-subunit (M r 28,000) and five B-subunits (M r 12,000 each) (4 -6). The A-subunit contains two distinct structural domains linked by a disulfide bridge: an A1-fragment (residues 1-192) that displays ADP-ribosyl transferase activity and an A2-fragment (residues 193-240) that mediates interaction with the B-subunit pentamer (4, 6). The B-subunits of Ctx and Etx (CtxB and EtxB, respectively) bind to cell surface receptors, principally G M1 -ganglioside, found ubiquitously on the plasma membranes of eukaryotic cells (7). Although Ctx and Etx exhibit a remarkable degree of structural homology, with 81.6% sequence identity between CtxA and EtxA and 82.5% sequence identity between CtxB and EtxB (8 -10), there are a number of subtle physicochemical and function...

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[24] Established intestinal cell lines as model systems for electrolyte transport studies

Cited by 170 publications

References 32 publications

Proteolytic Activation of Cholera Toxin and Escherichia coli Labile Toxin by Entry into Host Epithelial Cells

Proteolytic Activation of Cholera Toxin and Escherichia coli Labile Toxin by Entry into Host Epithelial Cells

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

Structural Basis for the Differential Toxicity of Cholera Toxin and Escherichia coli Heat-labile Enterotoxin

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