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Biomedical subjects

Kim E Barrett

Publications and source records attributed to Kim E Barrett.

32 records · Page 2Linked to original sources
PubMed2003

Prolonged interferon-gamma exposure decreases ion transport, NKCC1, and Na+-K+-ATPase expression in human intestinal xenografts in vivo.

IFN-gamma is elevated in intestinal inflammation and alters barrier and transport functions in human colonic epithelial cell lines, but its effects on normal human small intestinal epithelium in vivo are poorly defined. We investigated effects of prolonged IFN-gamma exposure on ion transport and expression of transporters by using human fetal small intestinal xenografts. Xenograft-bearing mice were injected with IFN-gamma, and 24 h later xenografts were harvested and mounted in Ussing chambers. Baseline potential difference (PD) was not affected by IFN-gamma treatment. However, conductance was enhanced and agonist-stimulated ion transport was decreased. IFN-gamma also decreased expression of the Na+-K+-2Cl- cotransporter and the alpha-subunit of Na+-K+-ATPase compared with controls, whereas levels of the calcium-activated Cl- channel and CFTR were unaltered. Thus prolonged exposure to IFN-gamma leads to decreased ion secretion due, in part, to decreased ion transporter levels. These findings demonstrate the implications of elevated IFN-gamma levels in human small intestine and validate the human intestinal xenograft as a model to study chronic effects of physiologically relevant stimuli.

Animals↗
PubMed2003

Growth hormone reduces chloride secretion in human colonic epithelial cells via EGF receptor and extracellular regulated kinase.

BACKGROUND AND AIMS: Growth hormone (GH) has been shown to alleviate symptoms in patients with Crohn's disease. Chloride secretion is important in driving intestinal fluid secretion. We examined whether GH inhibits chloride secretion induced by carbachol (CCh, a calcium-dependent pathway), and the downstream effectors responsible. METHODS: T(84) cells were pretreated with GH at various concentrations followed by CCh (100 micromol/L). Chloride secretion was assessed as changes in short circuit current ( triangle up I(sc)) in Ussing chambers. Tyrphostins AG1478 (an epidermal growth factor receptor [EGFr] inhibitor) and AG490 (a Janus kinase 2 [JAK2] inhibitor), SB203580 (a p38 inhibitor), and PD98059 (a MEK1 inhibitor) were used. RESULTS: GH inhibited CCh-induced chloride secretion at up to 10 nmol/L, but higher concentrations were less effective. GH caused tyrosine phosphorylation of JAK2 and EGFr. AG490 suppressed activation of JAK2 and EGFr in response to GH. AG1478 prevented GH activation of EGFr and reversed its inhibitory effect on chloride secretion. GH also induced activation of both p38 and ERK1/2. AG490 reversed GH-induced tyrosine phosphorylation of both ERK1/2 and p38, but AG1478 reversed that of ERK1/2 only. PD98059, but not SB203580, reversed the inhibitory effect of GH on chloride secretion. CONCLUSIONS: GH inhibits CCh-induced chloride secretion via a JAK2-dependent mechanism involving transactivation of EGFr and consequent recruitment of ERK1/2. Although activated, p38 does not contribute to the inhibitory effect of GH on secretion. These data elucidate mechanisms of GH inhibition of chloride secretion in intestinal epithelia, which may be relevant to therapeutic benefits of GH in Crohn's disease or other diarrheal diseases.

Biological Transport↗
PubMed2003

Epithelial transport and gut barrier function in colitis.

PURPOSE OF REVIEW: Colitis is an inflammatory bowel disease that is confined to the colon and is characterized by a watery diarrhea that can also be accompanied by blood in the stool. The inflammation associated with colitis is generally confined to the mucosal and submucosal layers, although Crohn's colitis may be transmural. The principal functions of the colonic mucosa are to act as a barrier to the luminal contents of the intestinal tract and to facilitate the bidirectional transport of water and electrolytes. It is well established that barrier and transport defects occur in colitis and may be involved in pathogenesis. Consequently, this review discusses recent evidence of potential mechanisms that may be involved in the perturbation of mucosal transport and barrier functions in colitis and therapeutic advances to counteract these defects. RECENT FINDINGS: Mechanisms responsible for transport dysfunction and barrier defects in colitis are discussed, including decreased activity of transport proteins such as CFTR, bacterial interactions with the epithelium, including understanding of the regulation and function of NOD-2, and altered expression of components of the intestinal barrier, such as mucins and multidrug resistance proteins. SUMMARY: Recent advances in our understanding of how changes in barrier and transport function occur in colitis may illuminate the pathophysiology of this condition. The work discussed may also identify novel targets that are functionally altered in colitis, which potentially can be modulated therapeutically either with existing medications or with newer agents that are in development.

Journal Article↗
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↗
PubMed2003

How can we battle the scourge of diarrhea? 2003 McKenna Memorial Lecture.

Diarrheal diseases exact a considerable toll of morbidity and mortality worldwide, including in developed countries where the incidence of foodborne illness, in particular, may be increasing. This article summarizes the current understanding of the basis of diarrheal illness, focusing particularly on intracellular signaling mechanisms that limit the extent of intestinal epithelial chloride secretion, which may offer new targets for antidiarrheal therapies. Recent information regarding the mechanisms whereby invasive bacteria cause diarrhea is also reviewed along with effects of beneficial bacteria (so-called probiotics) in limiting dysfunction associated with enteric infections. Finally, the author provides some speculations as to the possible benefits to the host of mounting a diarrheal response to an offending pathogen, and possible consequences of the failure of this primitive host defense mechanism.

Diarrhea↗
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↗
PubMedDate not supplied

A role for CagA/VacA in Helicobacter pylori inhibition of murine duodenal mucosal bicarbonate secretion.

Duodenal mucosal bicarbonate secretion is diminished in patients with Helicobacter pylori (HP)-associated duodenal ulcer disease. We examined whether HP water extracts inhibit murine duodenal mucosal bicarbonate secretion in vitro, and the mechanisms involved. Murine duodenal mucosae were mounted in Ussing chambers. Short-circuit current and bicarbonate secretion was measured. CagA/VacA-positive HP water extract (HPWE+/+) markedly inhibited PGE2-, carbachol-, or the calcium ionophore A23187-stimulated bicarbonate secretion in a dose-dependent manner. While 3-isobutyl-1-methylxanthine-stimulated bicarbonate secretion was not affected by HPWE+/+, HPWE+/+ did diminish forskolin-stimulated bicarbonate secretion. HPWE+/+ markedly diminished PGE2-induced increases in duodenal mucosal cAMP. CagA/VacA of HP decreases Ca2+-mediated bicarbonate secretion downstream of increases in intracellular Ca2+. Dimunition of PGE2-stimulated bicarbonate secretion occurs, in part, by inhibition of adenylate cyclase, which leads to decreased cAMP levels. The ability of virulent HP strains to inhibit duodenal bicarbonate secretion through multiple intracellular pathways likely contributes to the pathogenesis of HP-associated duodenal ulcer disease.

Adenylyl Cyclases↗