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. 1999 Aug;104(3):253-62.
doi: 10.1172/JCI6373.

Pathogenic Escherichia Coli Increase Cl- Secretion From Intestinal Epithelia by Upregulating galanin-1 Receptor Expression

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Free PMC article

Pathogenic Escherichia Coli Increase Cl- Secretion From Intestinal Epithelia by Upregulating galanin-1 Receptor Expression

G Hecht et al. J Clin Invest. .
Free PMC article

Abstract

Galanin is widely distributed in enteric nerve terminals lining the human gastrointestinal (GI) tract. We have shown previously that galanin-1 receptors (Gal1-R) are expressed by epithelial cells lining the human GI tract, and upon activation cause Cl- secretion. Because expression of this receptor is transcriptionally regulated by nuclear factor-kappa B (NF-kappa B), which is activated by enteric pathogens as a part of the host epithelial response to infection, we investigated whether such bacterial pathogens could directly increase Gal1-R expression in the T84-cell model system. Pathogenic Escherichia coli, but not nonpathogenic E. coli, activate a p50/p65 NF-kappa B complex that binds to oligonucleotides corresponding to a recognition site located within the 5' flanking region of the human GAL1R gene. Pathogenic E. coli, but not normal commensal organisms, increase Gal1-R mRNA synthesis and [(125)I]galanin binding sites. Whereas galanin increases short-circuit current (Isc) approximately 5-fold in uninfected T84 cells, exposure to pathogenic, but not nonpathogenic, E. coli results in galanin increasing Isc approximately 20-fold. To confirm the validity of these in vitro observations, we also studied C57BL/6J mice infected with enterohemorrhagic E. coli (EHEC) by gavage. Infection caused a progressive increase in both NF-kappa B activation and Gal1-R expression, with maximal levels of both observed 3 days after gavage. Ussing chamber studies revealed that colons infected with EHEC, but not those exposed to normal colonic flora, markedly increased Isc in response to galanin. These data indicate that pathogen-induced increases in Gal1-R expression by epithelial cells lining the colon may represent a novel unifying pathway responsible for at least a portion of the excessive fluid secretion observed during infectious diarrhea.

Figures

Figure 1
Figure 1
Gel shift (a) and supershift (b) studies performed on T84-cell nuclear proteins using oligonucleotides directed to the downstream NF-κB recognition site located within the 5′ flanking region of the human GAL1R gene. (a) Nuclear proteins from control T84 cells (Control), or from T84 cells exposed to normal commensal E. coli (NC) or the indicated enteric pathogens, were mixed with 32P end-labeled oligonucleotide corresponding to the NF-κB recognition site located at –809 bp from the site of translation initiation. (b) Nuclear proteins from T84 cells exposed to EHEC for 1 hour and combined with 32P end-labeled oligonucleotide in the presence of antibody to the indicated NF-κB subunits. For both a and b, gels are representative of at least 3 separately performed experiments.
Figure 2
Figure 2
Effect of pathogenic and normal commensal E. coli on Gal1-R mRNA expression. (a) Time course of alterations in Gal1-R mRNA concentration after a 1-hour infection with EHEC (filled squares) or normal commensal E. coli (NC, open squares). Total cellular RNA was extracted from T84 cells cultured to confluence in 24-well plates after exposure to the indicated bacteria, as described in Methods. Gal1-R mRNA amount was determined by performing RT-PCR with serial dilutions of a mimic whose concentration is known (inset). (b) Peak increases (at t = 4 hours) in Gal1-R mRNA after infection with the indicated bacteria. Data represent the mean ± SEM for a minimum of 3 separate experiments.
Figure 3
Figure 3
Galanin-induced increases in Isc in T84 cells with or without prior E. coli exposure. (a) T84 cells were cultured to confluence in Transwells, exposed to EHEC (filled circles) or buffer (open circles) for 1 hour, and treated with gentamicin; the ability of 1 μM galanin to alter Isc was determined 24 hours later. (b) Maximal increases in Isc in T84 cells after stimulation with 1 μM galanin in cells exposed to buffer only (Uninfected controls), or 24 hours after a 1-hour infection with normal commensal E. coli (Normal commensals) or the indicated pathogenic E. coli. Data represent the mean ± SEM for a minimum of 3 separate experiments.
Figure 4
Figure 4
Effect of NF-κB inhibitors on Gal1-R mRNA synthesis and response to galanin after infection with pathogenic E. coli. (a) Dose-dependent effect of the NF-κB–specific inhibitor CAPE (25) on peak Gal1-R mRNA synthesis 4 hours after a 1-hour exposure to EHEC. Confluent T84 cells were exposed to the indicated concentration of CAPE for 1 hour, infected with EHEC for 1 hour in the continued presence of CAPE, and washed; RNA was extracted 4 hours later. (b) Dose-dependent effect of dexamethasone on Gal1-R mRNA synthesis 4 hours after a 1-hour exposure to EHEC. Conditions were identical to those described for a. (c) Effects of dexamethasone on galanin-induced Isc after infection with EHEC. T84 cells were infected with EHEC for 1 hour in the presence or absence of 200 nM dexamethasone, and the Isc response to 1 μM galanin was determined 24 hours later. Data represent the mean ± SEM for a minimum of 3 separate experiments.
Figure 5
Figure 5
Evaluation of Gal1-R antibody sensitivity and specificity. Immunohistochemistry was performed using Gal1-R antibody (concentration 1:500) on a 5-μm-thick section of formalin-fixed, paraffin-embedded mouse pancreas, as described in Methods. White arrowheads identify the Gal1-R immunostaining islets. Inset a: control tissue processed similarly, except not exposed to primary antibody showing islet detail (arrow). ×100. Inset b: Western blot analysis of uninfected T84 cells (Control) and T84 cells exposed to EHEC for 1 hour and then studied 24 hours later. Proteins (50 μg) were resolved by SDS-PAGE (10% acrylamide), transferred to nitrocellulose, and exposed to antibody overnight at a dilution of 1:1,000 at room temperature. Gal1-R (arrowhead) is identified by reacting with goat anti-rabbit alkaline phosphatase–conjugated IgG, and developing with an Immunoblot AP kit (BCIP-NBT; Cymed, South San Francisco, California, USA).
Figure 6
Figure 6
Immunohistochemistry performed on colonic epithelial cells isolated from C57BL/6J mice using antibodies against the Gal1-R (ac) or the activated subunit of NF-κB p65 (df). Results from control mice do not show evidence of Gal1-R expression (a) or activated NF-κB (d). In contrast, 3 days after instilling 2 × 105 log-growth EHEC by gavage, both Gal1-R expression (b) and NF-κB activation (e) are readily apparent. Concomitant parenteral administration of dexamethasone (DEX) during the same period of EHEC infection, however, markedly attenuates Gal1-R expression (c) and evidence of NF-κB activation (f). Data are representative of 5 separately treated animals per condition. ×400.
Figure 7
Figure 7
Ability of galanin to increase Isc in murine colonocytes with or without prior EHEC infection. Mouse colonic epithelium was rapidly mounted in an Ussing chamber after sacrifice; after the development of a stable baseline, epithelium was treated with 1 μM galanin as indicated. Whereas control murine colonocytes do not respond to galanin (open circles), those from mice given EHEC by gavage 3 days earlier increase Isc by approximately 5-fold (filled circles). In contrast, concomitant parenteral administration of dexamethasone (DEX) during the same period of EHEC infection completely eliminated galanin’s ability to alter Isc (filled squares). Data represent the mean ± SEM for a minimum of 5 separate experiments.

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