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Kim E Barrett

Publications and source records attributed to Kim E Barrett.

8 recordsLinked to original sources
PubMed2003

Salmonella infection induces a hypersecretory phenotype in human intestinal xenografts by inducing cyclooxygenase 2.

Enteric Salmonella infection is accompanied by inflammation and diarrhea, and yet little is known about its effects on intestinal epithelial physiology. Since species differences limit the utility of animal tissues and cell lines lack relevant cell-cell interactions, we have used a human model of fetal intestine grown as xenografts in SCID mice. We investigated here the effects of Salmonella enterica serovar Typhimurium SL1344 on xenograft ion transport. Harvested xenografts were stripped of seromuscular layers by blunt dissection, infected with Salmonella, and mounted in Ussing chambers. Salmonella infection for 1 h increased baseline ion transport without altering tissue conductance or morphology. The increased transport was blocked by the cyclooxygenase inhibitor, indomethacin, or the specific Cox-2 inhibitor, NS-398. Further, xenografts infected for 2 h showed increased secretory responses to the calcium-dependent agonist, carbachol, and the cyclic AMP-dependent agonists prostaglandin E(2) (PGE(2)) and forskolin, which were blocked by indomethacin. Western blot experiments revealed that infection was accompanied by increased cyclooxygenase 2 (Cox-2) expression, with no change in Cox-1 levels. Immunoassay demonstrated basolateral PGE(2) release, which was inhibited by indomethacin. Histological examination of infected xenografts illustrated that upregulated Cox-2 expression was restricted to the epithelium and that little or no invasion of the tissue by Salmonella occurred for up to 2 h. In summary, Salmonella infection rapidly increases Cox-2 expression in human intestinal tissue, accounting for increased epithelial ion transport characteristic of infectious diarrhea.

Animals↗
PubMed2002

p38 mitogen-activated protein kinase inhibits calcium-dependent chloride secretion in T84 colonic epithelial cells.

We have previously shown that Ca(2+)-dependent Cl(-) secretion across intestinal epithelial cells is limited by a signaling pathway involving transactivation of the epidermal growth factor receptor (EGFR) and activation of ERK mitogen-activated protein kinase (MAPK). Here, we have investigated a possible role for p38 MAPK in regulation of Ca(2+)-dependent Cl(-) secretion. Western blot analysis of T(84) colonic epithelial cells revealed that the muscarinic agonist carbachol (CCh; 100 microM) stimulated phosphorylation and activation of p38 MAPK. The p38 inhibitor SB-203580 (10 microM) potentiated and prolonged short-circuit current (I(sc)) responses to CCh across voltage-clamped T(84) cells to 157.4 +/- 6.9% of those in control cells (n = 21; P < 0.001). CCh-induced p38 phosphorylation was attenuated by the EGFR inhibitor tyrphostin AG-1478 (0.1 nM-10 microM) and by the Src family kinase inhibitor PP2 (20 nM-2 microM). The effects of CCh on p38 phosphorylation were mimicked by thapsigargin (TG; 2 microM), which specifically elevates intracellular Ca(2+), and were abolished by the Ca(2+) chelator BAPTA-AM (20 microM), implying a role for intracellular Ca(2+) in mediating p38 activation. SB-203580 (10 microM) potentiated I(sc) responses to TG to 172.4 +/- 18.1% of those in control cells (n = 18; P < 0.001). When cells were pretreated with SB-203580 and PD-98059 to simultaneously inhibit p38 and ERK MAPKs, respectively, I(sc) responses to TG and CCh were significantly greater than those observed with either inhibitor alone. We conclude that Ca(2+)-dependent agonists stimulate p38 MAPK in T(84) cells by a mechanism involving intracellular Ca(2+), Src family kinases, and the EGFR. CCh-stimulated p38 activation constitutes a similar, but distinct and complementary, antisecretory signaling pathway to that of ERK MAPK.

Animals↗
PubMed2002

Transactivation of the epidermal growth factor receptor in colonic epithelial cells by carbachol requires extracellular release of transforming growth factor-alpha.

We have shown previously that the muscarinic agonist, carbachol (CCh), transactivates the epidermal growth factor receptor (EGFr) via calmodulin, Pyk-2, and Src kinase activation. EGFr phosphorylation causes extracellular signal-regulated kinase (ERK) activation and inhibits CCh-stimulated chloride secretion across intestinal epithelial cells. Here we investigated whether CCh-stimulated EGFr transactivation involves EGFr ligand release. Pre-incubation of T(84) cell monolayers with a neutralizing antibody to the EGFr ligand binding domain decreased CCh-induced phosphorylation of EGFr and ERK. CCh-stimulated efflux of (86)Rb+ from T(84) cell monolayers, which parallels changes in chloride secretion, was potentiated by anti-EGFr pre-incubation. Anti-EGFr did not reduce CCh-stimulated Pyk-2 phosphorylation. Co-incubation with the Src kinase inhibitor PP2 and anti-EGFr had an additive inhibitory effect on CCh-induced ERK phosphorylation greater than either inhibitor alone. CCh caused the basolateral release of transforming growth factor alpha (TGF-alpha) into T(84) cell bathing media. A metalloproteinase inhibitor, WAY171318, reduced CCh-induced phosphorylation of ERK and completely blocked EGFr phosphorylation and TGF-alpha release. We conclude that CCh-stimulated EGFr transactivation and subsequent ERK activation, a pathway that limits CCh-induced chloride secretion, is mediated by metalloproteinase-dependent extracellular release of TGF-alpha and intracellular Src activation. These findings have important implications for our understanding of the role of growth factors in regulating epithelial ion secretion.

Binding Sites↗
PubMed2002

Enteroinvasive bacteria alter barrier and transport properties of human intestinal epithelium: role of iNOS and COX-2.

BACKGROUND & AIMS: Various invasive pathogens cause diarrhea, but the mechanism(s) are poorly understood. We hypothesized that nitric oxide and prostaglandins might modulate chloride secretory and barrier properties of the infected intestinal epithelium and that diarrhea is caused, in part, by altered expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2). METHODS: Studies were conducted in human intestinal epithelial cell lines (HT29/cl.19A, Caco-2, and T84). Cells were infected with enteroinvasive Escherichia coli (EIEC 029:NM) or Salmonella dublin (SD), or nonpathogenic, noninvasive bacteria (Streptococcus thermophilus [ST] and Lactobacillus acidophilus [LA]). Infected cells and controls were tested for transepithelial resistance, chloride secretion, prostaglandin E2, guanosine 3',5'-cyclic monophosphate and adenosine 3',5'-cyclic monophosphate, and protein expression. RESULTS: Cells infected with EIEC or SD, but not uninfected controls or ST/LA-exposed monolayers, showed a progressive reduction in transepithelial resistance starting at 6-12 hours. Infected HT29/cl.19A and Caco-2 cells, but not T84 cells, also showed an increase in total nitrite. Expression of iNOS, and consequently COX-2, was also increased, followed by increased production of prostaglandins and cyclic nucleotides. Furthermore, basal and stimulated chloride secretory responses to various agonists were enhanced in HT29/cl.19A and Caco-2 cells after infection with enteroinvasive bacteria, and this effect was reversed for some agonists by iNOS or COX-2 inhibitors. Increased expression of cystic fibrosis transmembrane conductance regulator and NKCC1 was also observed in EIEC or SD-infected cells vs. controls, secondary to NO synthase activity. CONCLUSIONS: Up-regulation of iNOS and COX-2 by enteroinvasive bacteria can modulate chloride secretion and barrier function in intestinal epithelial cells. Thus, these enzymes represent possible therapeutic targets in infectious diarrhea.

Adenylyl Cyclase Inhibitors↗
PubMed2002

Interferon-gamma activates EGF receptor and increases TGF-alpha in T84 cells: implications for chloride secretion.

IFN-gamma inhibits intestinal Cl(-) secretion, in part via downregulation of CFTR and Na(+)-K(+)-ATPase activity and expression, but the proximal signaling events were unknown. We have shown that transforming growth factor-alpha (TGF-alpha) inhibits calcium-activated Cl(-) secretion, and effects of IFN-gamma in other systems are mediated via EGF family members. We tested whether IFN-gamma inhibits Cl(-) secretion via EGF receptor (EGFr) activation. IFN-gamma increased tyrosine phosphorylation in T84 cells at 24 h, including the EGFr. IFN-gamma also increased cell-associated pro-TGF-alpha, as well as free TGF-alpha in the bathing media. However, whereas IFN-gamma significantly inhibited carbachol-induced Cl(-) secretion, neither neutralizing antibodies to TGF-alpha nor an EGFr inhibitor (1 microM tyrphostin AG 1478) were able to reverse this inhibitory effect. AG 1478 also failed to reverse IFN-gamma-induced tyrosine phosphorylation of the EGFr, but receptor phosphorylation was attenuated by both the neutralizing antibody to TGF-alpha and PP2, a Src kinase inhibitor. Moreover, PP2 reversed the inhibitory effect of IFN-gamma on Cl(-) secretion. In total, our findings suggest an increase in functional TGF-alpha and activation of the EGFr in response to IFN-gamma. The release of TGF-alpha and intracellular Src activation likely combine to mediate EGFr phosphorylation, but only Src appears to contribute to the inhibition of transport. Nevertheless, because TGF-alpha plays a role in restitution and repair of the intestinal epithelium after injury, we speculate that these findings reflect a feedback loop whereby IFN-gamma modulates the extent of cytokine-induced intestinal damage.

Antibodies↗