PI3K signaling is required for prostaglandin-induced mucosal recovery in ischemia-injured porcine ileum

We have previously shown that PGE(2) and PGI(2) induce recovery of transepithelial resistance (TER) in ischemia-injured porcine ileal mucosa, associated with initial increases in Cl(-) secretion. We believe that the latter generates an osmotic gradient that stimulates resealing of tight junctions. Because of evidence implicating phosphatidylinositol 3-kinase (PI3K) in regulating tight junction assembly, we postulated that this signaling pathway is involved in PG-induced mucosal recovery. Porcine ileum was subjected to 45 min of ischemia, after which TER was monitored for a 180-min recovery period. Endogenous PG production was inhibited with indomethacin (5 microM). PGE(2) (1 microM) and PGI(2) (1 microM) stimulated recovery of TER, which was inhibited by serosal application of the osmotic agent urea (300 mosmol/kgH(2)O). The PI3K inhibitor wortmannin (10 nM) blocked recovery of TER in response to PGs or mucosal urea. Immunofluorescence imaging of recovering epithelium revealed that PGs restored occludin and zonula occludens-1 distribution to interepithelial junctions, and this pattern was disrupted by pretreatment with wortmannin. These experiments suggest that PGs stimulate recovery of paracellular resistance via a mechanism involving transepithelial osmotic gradients and PI3K-dependent restoration of tight junction protein distribution.     

Prostaglandin-mediated inhibition of Na+/H+ exchanger isoform 2 stimulates recovery of barrier function in ischemia-injured intestine

Prostaglandins stimulate repair of the ischemia-injured intestinal barrier in the porcine ileum through a mechanism involving cAMP-dependent Cl- secretion and inhibition of electroneutral Na+/H+ exchanger (NHE) activity. In the present study, we focused on the role of individual NHE isoforms in the recovery of barrier function. Ischemia-injured porcine ileal mucosa was mounted on Ussing chambers. Short-circuit current (I(sc)), transepithelial electrical resistance (TER), and isotopic fluxes of 22Na were measured in response to PGE2 and selective inhibitors of epithelial NHE isoforms. Immunoassays were used to assess the expression of NHE isoforms. Forty-five minutes of intestinal ischemia resulted in a 45% reduction in TER (P < 0.01). Near-complete restitution occurred within 60 min. Inhibition of NHE2 with HOE-694 (25 microM) added to the mucosal surface of the injured ileum stimulated significant elevations in TER, independent of changes in I(sc) and histological evidence of restitution. Pharmacological inhibition of NHE3 or NHE1 with mucosal S-3226 (20 microM) or serosal cariporide (25 microM), respectively, had no effect. Ischemia-injured tissues treated with mucosal S-3226 or HOE-694 exhibited equivalent reductions in mucosal-to-serosal fluxes of 22Na+ (by approximately 35%) compared with nontreated ischemia-injured control tissues (P < 0.05). Intestinal ischemia resulted in increased expression of the cytoplasmic NHE regulatory factor EBP50 in NHE2 but not in NHE3 immunoprecipitates. Selective inhibition of NHE2, and not NHE3, induces recovery of barrier function in the ischemia-injured intestine.     

Neutrophils augment recovery of porcine ischemia-injured ileal mucosa by an IL-1beta- and COX-2-dependent mechanism

Polymorphonuclear neutrophils (PMNs) play a critical role in intestinal mucosal injury and repair. To study effects of PMNs on acutely injured mucosa, we applied PMNs isolated from circulation or peritoneal fluid from animals with chemically induced peritonitis to ischemia-injured porcine ileal mucosa. In preliminary experiments, PMNs enhanced recovery of transepithelial electrical resistance (TER), and this action was inhibited by pretreatment with the nonselective cyclooxygenase (COX) inhibitor indomethacin. Because COX-2 is upregulated by inflammatory mediators such as IL-1beta, which is released by PMNs, we postulated that PMNs enhance recovery of ischemia-injured mucosa by a pathway involving IL-1beta and COX-2. Application of 5 x 10(6) PMNs to the serosal surface of ischemia-injured mucosa significantly enhanced recovery of TER (P < 0.05), an effect that was inhibited by the selective COX-2 inhibitor NS-398 (5 microM) and by an IL-1beta receptor antagonist (0.1 mg/ml). Addition of 10 ng/ml IL-1beta to the serosal surface of injured tissues caused a significant increase in TER (P < 0.05) that was inhibited by pretreatment with NS-398. Western blot analysis of mucosal homogenates revealed dramatic upregulation of COX-2 in response to IL-1beta or peritoneal PMNs, and the latter was inhibited by an IL-1beta receptor antagonist. Real-time PCR revealed that increased mRNA COX-2 expression preceded increased COX-2 protein expression in response to IL-1beta. We concluded that PMNs augment recovery of TER in ischemia-injured ileal mucosa via IL-1beta-dependent upregulation of COX-2.     

Genistein augments prostaglandin-induced recovery of barrier function in ischemia-injured porcine ileum

We have previously shown that PGE(2) enhances recovery of transmucosal resistance (R) in ischemia-injured porcine ileum via a mechanism involving chloride secretion. Because the tyrosine kinase inhibitor genistein amplifies cAMP-induced Cl(-) secretion, we postulated that genistein would augment PGE(2)-induced recovery of R. Porcine ileum subjected to 45 min of ischemia was mounted in Ussing chambers, and R and mucosal-to-serosal fluxes of [(3)H]N-formyl-methionyl-leucyl phenylalanine (FMLP) and [(3)H]mannitol were monitored as indicators of recovery of barrier function. Treatment with genistein (10(-4) M) and PGE(2) (10(-6) M) resulted in synergistic elevations in R and additive reductions in mucosal-to-serosal fluxes of [(3)H]FMLP and [(3)H]mannitol, whereas treatment with genistein alone had no effect. Treatment of injured tissues with genistein and either 8-bromo-cAMP (10(-4) M) or cGMP (10(-4) M) resulted in synergistic increases in R. However, treatment of tissues with genistein and the protein kinase C (PKC) agonist phorbol myristate acetate (10(-5)-10(-6) M) had no effect on R. Genistein augments recovery of R in the presence of cAMP or cGMP but not in the presence of PKC agonists.     

Chloride channel ClC-2 modulates tight junction barrier function via intracellular trafficking of occludin

Previously, we have demonstrated that the chloride channel ClC-2 modulates intestinal mucosal barrier function. In the present study, we investigated the role of ClC-2 in epithelial barrier development and maintenance in Caco-2 cells. During early monolayer formation, silencing of ClC-2 with small interfering (si)RNA led to a significant delay in the development of transepithelial resistance (TER) and disruption of occludin localization. Proteomic analysis employing liquid chromatography-mass spectrometry /mass spectrometry revealed association of ClC-2 with key proteins involved in intracellular trafficking, including caveolin-1 and Rab5. In ClC-2 siRNA-treated cells, occludin colocalization with caveolin-1 was diffuse and in the subapical region. Subapically distributed occludin in ClC-2 siRNA-treated cells showed marked colocalization with Rab5. To study the link between ClC-2 and trafficking of occludin in confluent epithelial monolayers, a Caco-2 cell clone expressing ClC-2 short hairpin (sh)RNA was established. Disruption of caveolae with methyl-β-cyclodextrin (MβCD) caused a marked drop in TER and profound redistribution of caveolin-1-occludin coimmunofluorescence in ClC-2 shRNA cells. In ClC-2 shRNA cells, focal aggregations of Rab5-occludin coimmunofluorescence were present within the cytoplasm. Wortmannin caused an acute fall in TER in ClC-2 shRNA cells and subapical, diffuse redistribution of Rab5-occludin coimmunofluorescence in ClC-2 shRNA cells. An endocytosis and recycling assay for occludin revealed higher basal rate of endocytosis of occludin in ClC-2 shRNA cells. Wortmannin significantly reduced the rate of recycling of occludin in ClC-2 shRNA cells. These data clearly indicate that ClC-2 plays an important role in the modulation of tight junctions by influencing caveolar trafficking of the tight junction protein occludin.     

Nitric oxide synthase stimulates prostaglandin synthesis and barrier function in C. parvum-infected porcine ileum

Cell culture models implicate increased nitric oxide (NO) synthesis as a cause of mucosal hyperpermeability in intestinal epithelial infection. NO may also mediate a multitude of subepithelial events, including activation of cyclooxygenases. We examined whether NO promotes barrier function via prostaglandin synthesis using Cryptosporidium parvum-infected ileal epithelium in residence with an intact submucosa. Expression of NO synthase (NOS) isoforms was examined by real-time RT-PCR of ileal mucosa from control and C. parvum-infected piglets. The isoforms mediating and mechanism of NO action on barrier function were assessed by measuring transepithelial resistance (TER) and eicosanoid synthesis by ileal mucosa mounted in Ussing chambers in the presence of selective and nonselective NOS inhibitors and after rescue with exogenous prostaglandins. C. parvum infection results in induction of mucosal inducible NOS (iNOS), increased synthesis of NO and PGE2, and increased mucosal permeability. Nonselective inhibition of NOS (NG-nitro-L-arginine methyl ester) inhibited prostaglandin synthesis, resulting in further increases in paracellular permeability. Baseline permeability was restored in the absence of NO by exogenous PGE2. Selective inhibition of iNOS [L-N6-(1-iminoethyl)-L-lysine] accounted for approximately 50% of NOS-dependent PGE2 synthesis and TER. Using an entire intestinal mucosa, we have demonstrated for the first time that NO serves as a proximal mediator of PGE2 synthesis and barrier function in C. parvum infection. Expression of iNOS by infected mucosa was without detriment to overall barrier function and may serve to promote clearance of infected enterocytes.     

Prostanoids stimulate K secretion and Cl secretion in guinea pig distal colon via distinct pathways

Short-circuit current (I(sc)) and transepithelial conductance (Gt) were measured in guinea pig distal colonic mucosa isolated from submucosa and underlying muscle layers. Indomethacin (2 microM) and NS-398 (2 microM) were added to suppress endogenous production of prostanoids. Serosal addition of PGE2 (10 nM) stimulated negative I(sc) consistent with K secretion, and concentrations >30 nM stimulated positive I(sc) consistent with Cl secretion. PGE2 also stimulated Gt at low and high concentrations. Dose responses to prostanoids specific for EP prostanoid receptors were consistent with stimulating K secretion through EP2 receptors, based on a rank order potency (from EC50 values) of PGE2 (1.9 nM) > 11-deoxy-PGE1 (8.3 nM) > 19(R)-hydroxy-PGE2 (13.9 nM) > butaprost (67 nM) > 17-phenyl-trinor-PGE2 (307 nM) > sulprostone (>10 microM). An isoprostane, 8-iso-PGE2, stimulated K secretion with an EC50 of 33 nM. Cl secretory response was stimulated by PGD2 and BW-245C, a DP prostanoid receptor-specific agonist: BW-245C (15 nM) > PGD2 (30 nM) > PGE2 (203 nM). Agonists specific for FP, IP, and TP prostanoid receptors were ineffective in stimulating I(sc) and Gt at concentrations <1 microM. These results indicate that PGE2 stimulated electrogenic K secretion through activation of EP2 receptors and electrogenic KCl secretion through activation of DP receptors. Thus stimulation of Cl secretion in vivo would occur either via physiological concentrations of PGD2 (<100 nM) or pathophysiological concentrations of PGE2 (>100 nM) that could occur during inflammatory conditions.     

Mice lacking the Na+/H+ exchanger 2 have impaired recovery of intestinal barrier function

Ischemic injury induces breakdown of the intestinal barrier. Recent studies in porcine postischemic tissues indicate that inhibition of NHE2 results in enhanced recovery of barrier function in vitro via a process involving interepithelial tight junctions. To further study this process, recovery of barrier function was assessed in wild-type (NHE2(+/+)) and NHE2(-/-) mice in vivo and wild-type mice in vitro. Mice were subjected to complete mesenteric ischemia in vivo, after which barrier function was measured by blood-to-lumen mannitol clearance over a 3-h recovery period or measurement of transepithelial electrical resistance (TER) in Ussing chambers immediately following ischemia. Tissues were assessed for expression of select junctional proteins. Compared with NHE2(+/+) mice, NHE2(-/-) mice had greater intestinal permeability during the postischemic recovery process. In contrast to prior porcine studies, pharmacological inhibition of NHE2 in postischemic tissues from wild-type mice also resulted in significant reductions in TER. Mucosa from NHE2(-/-) mice displayed a shift of occludin and claudin-1 expression to the Triton-X-soluble membrane fractions and showed disruption of occludin and claudin-1 localization patterns following injury. This was qualitatively and quantitatively recovered in NHE2(+/+) mice compared with NHE2(-/-) mice by the end of the 3-h recovery period. Serine phosphorylation of occludin and claudin-1 was downregulated in NHE2(-/-) postischemia compared with wild-type mice. These data indicate an important role for NHE2 in recovery of barrier function in mice via a mechanism involving tight junctions.     

Stimulation of chloride secretion by baicalein in isolated rat distal colon

The effect of baicalein on mucosal ion transport in the rat distal colon was investigated in Ussing chambers. Mucosal addition of baicalein (1-100 microM) elicited a concentration-dependent short-circuit current (I(sc)) response. The increase in I(sc) was mainly due to Cl(-) secretion. The presence of mucosal indomethacin (10 microM) significantly reduced both the basal and subsequent baicalein-evoked I(sc) responses. The baicalein-induced I(sc) were inhibited by mucosal application of diphenylamine-2-carboxylic acid (100 microM) and glibenclamide (500 microM) and basolateral application of chromanol 293B (30 microM), a blocker of K(v)LQT1 channels and Ba(2+) ions (5 mM). Treatment of the colonic mucosa with baicalein elicited a threefold increase in cAMP production. Pretreating the colonic mucosa with carbachol (100 microM, serosal) but not thapsigargin (1 microM, both sides) abolished the baicalein-induced I(sc). Addition of baicalein subsequent to forskolin induced a further increase in I(sc). These results indicate that the baicalein evoked Cl(-) secretion across rat colonic mucosa, possibly via a cAMP-dependent pathway. However, the action of baicalein cannot be solely explained by its cAMP-elevating effect. Baicalein may stimulate Cl(-) secretion via a cAMP-independent pathway or have a direct effect on cystic fibrosis transmembrane conductance regulator.     

Physiological concentrations of bile salts inhibit recovery of ischemic-injured porcine ileum

We have previously shown rapid in vitro recovery of barrier function in porcine ischemic-injured ileal mucosa, attributable principally to reductions in paracellular permeability. However, these experiments did not take into account the effects of luminal contents, such as bile salts. Therefore, the objective of this study was to evaluate the role of physiological concentrations of deoxycholic acid in recovery of mucosal barrier function. Porcine ileum was subjected to 45 min of ischemia, after which mucosa was mounted in Ussing chambers and exposed to varying concentrations of deoxycholic acid. The ischemic episode resulted in significant reductions in transepithelial electrical resistance (TER), which recovered to control levels of TER within 120 min, associated with significant reductions in mucosal-to-serosal (3)H-labeled mannitol flux. However, treatment of ischemic-injured tissues with 10(-5) M deoxycholic acid significantly inhibited recovery of TER with significant increases in mucosal-to-serosal (3)H-labeled mannitol flux, whereas 10(-6) M deoxycholic acid had no effect. Histological evaluation at 120 min revealed complete restitution regardless of treatment, indicating that the breakdown in barrier function was due to changes in paracellular permeability. Similar effects were noted with the application of 10(-5) M taurodeoxycholic acid, and the effects of deoxycholic acid were reversed with application of the Ca(2+)-mobilizing agent thapsigargin. Deoxycholic acid at physiological concentrations significantly impairs recovery of epithelial barrier function by an effect on paracellular pathways, and these effects appear to be Ca(2+) dependent.     

Lubiprostone activates non-CFTR-dependent respiratory epithelial chloride secretion in cystic fibrosis mice

Periciliary fluid balance is maintained by the coordination of sodium and chloride channels in the apical membranes of the airways. In the absence of the cystic fibrosis transmembrane regulator (CFTR), chloride secretion is diminished and sodium reabsorption exaggerated. ClC-2, a pH- and voltage-dependent chloride channel, is present on the apical membranes of airway epithelial cells. We hypothesized that ClC-2 agonists would provide a parallel pathway for chloride secretion. Using nasal potential difference (NPD) measurements, we quantified lubiprostone-mediated Cl(-) transport in sedated cystic fibrosis null (gut-corrected), C57Bl/6, and A/J mice during nasal perfusion of lubiprostone (a putative ClC-2 agonist). Baseline, amiloride-inhibited, chloride-free gluconate-substituted Ringer with amiloride and low-chloride Ringer plus lubiprostone (at increasing concentrations of lubiprostone) were perfused, and the NPD was continuously recorded. A clear dose-response relationship was detected in all murine strains. The magnitude of the NPD response to 20 muM lubiprostone was -5.8 +/- 2.1 mV (CF, n = 12), -8.1 +/- 2.6 mV (C57Bl/6 wild-type, n = 12), and -5.3 +/- 1.2 mV (AJ wild-type, n = 8). A cohort of ClC-2 knockout mice did not respond to 20 muM lubiprostone (n = 6, P = 0.27). In C57Bl/6 mice, inhibition of CFTR with topical application of CFTR inhibitor-172 did not abolish the lubiprostone response, thus confirming the response seen is independent of CFTR regulation. RT-PCR confirmed expression of ClC-2 mRNA in murine lung homogenate. The direct application of lubiprostone in the CF murine nasal airway restores nearly normal levels of chloride secretion in nasal epithelia.     

Effects of lubiprostone on human uterine smooth muscle cells

Lubiprostone, a bicyclic fatty acid derivative and member of a new class of compounds called prostones, locally activates ClC-2 Cl(-) channels without activation of prostaglandin receptors. The present study was specifically designed to test and compare lubiprostone and prostaglandin effects at the cellular level using human uterine smooth muscle cells. Effects on [Ca(2+)](i), membrane potential and [cAMP](i) in human uterine smooth muscle cells were measured. 10 nM lubiprostone significantly decreased [Ca(2+)](i) from 188 to 27 nM, which was unaffected by 100 nM SC-51322, a prostaglandin EP receptor antagonist. In contrast 10nM PGE(2) and PGE(1) both increased [Ca(2+)](i) 3-5-fold which was blocked by SC-51322. Similarly, lubiprostone and prostaglandins had opposite/different effects on membrane potential and [cAMP](i). Lubiprostone caused SC-51322-insensitive membrane hyperpolarization and no effect on [cAMP](i). PGE(2) and PGE(1) both caused SC-51322-sensitive membrane depolarization and increased [cAMP](i). Lubiprostone has fundamentally different cellular effects from prostaglandins that are not mediated by EP receptors.     

ClC-2 is required for rapid restoration of epithelial tight junctions in ischemic-injured murine jejunum

Background and aims

Involvement of the epithelial chloride channel ClC-2 has been implicated in barrier recovery following ischemic injury, possibly via a mechanism involving ClC-2 localization to the tight junction. The present study investigated mechanisms of intestinal barrier repair following ischemic injury in ClC-2^−/− mice.

Methods

Wild type, ClC-2 heterozygous and ClC-2^−/− murine jejunal mucosa was subjected to complete ischemia, after which recovery of barrier function was monitored by measuring in vivo blood-to-lumen clearance of ³H-mannitol. Tissues were examined by light and electron microscopy. The role of ClC-2 in re-assembly of the tight junction during barrier recovery was studied by immunoblotting, immunolocalization and immunoprecipitation.

Results

Following ischemic injury, ClC-2^−/− mice had impaired barrier recovery compared to wild type mice, defined by increases in epithelial paracellular permeability independent of epithelial restitution. The recovering ClC-2^−/− mucosa also had evidence of ultrastructural paracellular defects. The tight junction proteins occludin and claudin-1 shifted significantly to the detergent soluble membrane fraction during post-ischemic recovery in ClC-2^−/− mice whereas wild type mice had a greater proportion of junctional proteins in the detergent insoluble fraction. Occludin was co-immunoprecipitated with ClC-2 in uninjured wild type mucosa, and the association between occludin and ClC-2 was re-established during ischemic recovery. Based on immunofluorescence studies, re-localization of occludin from diffuse sub-apical areas to apical tight junctions was impaired in ClC-2^−/− mice.

Conclusions

These data demonstrate a pivotal role of ClC-2 in recovery of the intestinal epithelium barrier by anchoring assembly of tight junctions following ischemic injury.     

Roles of ZO-1, occludin, and actin in oxidant-induced barrier disruption

Oxidants such as monochloramine (NH(2)Cl) decrease epithelial barrier function by disrupting perijunctional actin and possibly affecting the distribution of tight junctional proteins. These effects can, in theory, disturb cell polarization and affect critical membrane proteins by compromising molecular fence function of the tight junctions. To examine these possibilities, we investigated the actions of NH(2)Cl on the distribution, function, and integrity of barrier-associated membrane, cytoskeletal, and adaptor proteins in human colonic Caco-2 epithelial monolayers. NH(2)Cl causes a time-dependent decrease in both detergent-insoluble and -soluble zonula occludens (ZO)-1 abundance, more rapidly in the former. Decreases in occludin levels in the detergent-insoluble fraction were observed soon after the fall of ZO-1 levels. The actin depolymerizer cytochalasin D resulted in a decreased transepithelial resistance (TER) more quickly than NH(2)Cl but caused a more modest and slower reduction in ZO-1 levels and in occludin redistribution. No changes in the cellular distribution of claudin-1, claudin-5, or ZO-2 were observed after NH(2)Cl. However, in subsequent studies, the immunofluorescent cellular staining pattern of all these proteins was altered by NH(2)Cl. The actin-stabilizing agent phalloidin did not prevent NH(2)Cl-induced decreases in TER or increases of apical to basolateral flux of the paracellular permeability marker mannitol. However, it partially blocked changes in ZO-1 and occludin distribution. Tight junctional fence function was also compromised by NH(2)Cl, observed as a redistribution of the alpha-subunit of basolateral Na(+)-K(+)-ATPase to the apical membrane, an effect not found with the apical membrane protein Na(+)/H(+) exchanger isoform 3. In conclusion, oxidants not only disrupt perijunctional actin but also cause redistribution of tight junctional proteins, resulting in compromised intestinal epithelial barrier and fence function. These effects are likely to contribute to the development of malabsorption and dysfunction associated with mucosal inflammation of the digestive tract.     

Mitogen-activated protein kinases regulate COX-2 and mucosal recovery in ischemic-injured porcine ileum

Mitogen-activated protein kinase (MAPK) pathways transduce signals from a diverse array of extracellular stimuli. The three primary MAPK-signaling pathways are the extracellular regulated kinases (ERK1/2), p38 MAPK, and c-Jun NH(2)-terminal kinase (JNK). Previous research in our laboratory has shown that COX-2-elaborated prostanoids participate in recovery of mucosal barrier function in ischemic-injured porcine ileum. Because COX-2 expression is regulated in part by MAPKs, we postulated that MAPK pathways would play an integral role in recovery of injured mucosa. Porcine mucosa was subjected to 45 min of ischemia, after which tissues were mounted in Ussing chambers, and transepithelial electrical resistance (TER) was monitored as an index of recovery of barrier function. Treatment of tissues with the p38 MAPK inhibitor SB-203580 (0.1 mM) or the ERK1/2 inhibitor PD-98059 (0.1 mM) abolished recovery. Western blot analysis revealed that SB-203580 inhibited upregulation of COX-2 that was observed in untreated ischemic-injured mucosa, whereas PD-98059 had no effect on COX-2 expression. Inhibition of TER recovery by SB-203580 or PD-98059 was overcome by administration of exogenous prostaglandin E(2) (1 microM). The JNK inhibitor SP-600125 (0.1 mM) significantly increased TER and resulted in COX-2 upregulation. COX-2 expression appears to be positively and negatively regulated by the p38 MAPK and the JNK pathways, respectively. Alternatively, ERK1/2 appear to be involved in COX-2-independent reparative events that remain to be defined.     

Phage display screening of epithelial cell monolayers treated with EGTA: identification of peptide FDFWITP that modulates tight junction activity

Phage display was used to screen for peptides that modulate the activity of epithelial cell tight junctions. Panning with a phage library that displays random 7-mers was performed using monolayers of human bronchial epithelial cells (16HBE14o(-)) treated with a calcium chelator, ethylene glycol-bis(2-aminoethylether)- N, N, N', N'-tetraacetic acid (EGTA), to increase accessibility to the junctional complex/paracellular space, followed by subtractive panning. A novel peptide, FDFWITP, identified as a potential tight junction modulator, was synthesized in linear and cyclic forms with lysine residues added to improve solubility. The cyclic form of the peptide reduced transepithelial electrical resistance (TER) in a concentration-dependent manner (80% reduction at 100 microM and 95% reduction at 500 microM) and was reversible within 2 h; the linear form only affected TER at the highest concentration. Interestingly, the constrained peptide did not increase permeation of the model small molecule, fluorescein. The highly selective activity of FDFWITP supports the hypothesis that ions and small molecules may be transported paracellularly across tight junctions by separate pathways.     

Restitution of the bullfrog gastric mucosa is dependent on a DIDS-inhibitable pathway not related to HCO3- ion transport

This study was conducted to determine the contribution of ion transport to restitution after injury in the gastric mucosa. For this, intact sheets of stomach from the bullfrog, Rana catesbeiana, were mounted in Ussing chambers. Restitution was evaluated in the presence or absence of ion transport inhibitors amiloride, DIDS, and bumetanide to block Na(+)/H(+) exchange, Cl(-)/HCO(3)(-) exchange and Na(+)/HCO(3)(-) co-transport, and Na(+)-K(+)-2Cl(-) cotransport, respectively. Ion substitution experiments with Na(+)-free, Cl(-)-free, and HCO(3)(-)-free solutions were also performed. Injury to the mucosa was produced with 1 M NaCl, and restitution was evaluated by recovery of transepithelial resistance (TER), mannitol flux, and morphology. Amiloride, bumetanide, Cl(-)-free, or HCO(3)(-)-free solutions did not affect restitution. In Na(+)-free solutions, recovery of TER and mannitol flux did not occur because surface cells did not attach to the underlying basement membrane. In contrast, all aspects of restitution were inhibited by DIDS, a compound that inhibits Na(+)-dependent HCO(3)(-) transport. Because HCO(3)(-)-free solutions did not inhibit restitution, it was concluded that DIDS must block a yet undefined pathway not involved in HCO(3)(-) ion transport but essential for cell migration after injury and restitution in the gastric mucosa.     

ClC-2 chloride channels contribute to HTC cell volume homeostasis

Membrane Cl(-) channels play an important role in cell volume homeostasis and regulation of volume-sensitive cell transport and metabolism. Heterologous expression of ClC-2 channel cDNA leads to the appearance of swelling-activated Cl(-) currents, consistent with a role in cell volume regulation. Since channel properties in heterologous models are potentially modified by cellular background, we evaluated whether endogenous ClC-2 proteins are functionally important in cell volume regulation. As shown by whole cell patch clamp techniques in rat HTC hepatoma cells, cell volume increases stimulated inwardly rectifying Cl(-) currents when non-ClC-2 currents were blocked by DIDS (100 microM). A cDNA closely homologous with rat brain ClC-2 was isolated from HTC cells; identical sequence was demonstrated for ClC-2 cDNAs in primary rat hepatocytes and cholangiocytes. ClC-2 mRNA and membrane protein expression was demonstrated by in situ hybridization, immunocytochemistry, and Western blot. Intracellular delivery of antibodies to an essential regulatory domain of ClC-2 decreased ClC-2-dependent currents expressed in HEK-293 cells. In HTC cells, the same antibodies prevented activation of endogenous Cl(-) currents by cell volume increases or exposure to the purinergic receptor agonist ATP and delayed HTC cell volume recovery from swelling. These studies provide further evidence that mammalian ClC-2 channel proteins are functional and suggest that in HTC cells they contribute to physiological changes in membrane Cl(-) permeability and cell volume homeostasis.     

Neutrophil-mediated activation of epithelial protease-activated receptors-1 and -2 regulates barrier function and transepithelial migration

Neutrophil (PMN) infiltration and associated release of serine proteases contribute to epithelial injury during active phases of mucosal disorders such as inflammatory bowel disease. Previous studies have demonstrated that PMN contact with basolateral surfaces of intestinal epithelial cells in the presence of a chemoattractant results in disruption of barrier function even without transmigration. Similarly, serine protease-mediated activation of epithelial protease-activated receptors (PARs) has been shown to increase permeability. In this study, we assessed whether transmigrating PMNs can regulate barrier function through epithelial PAR activation. Transepithelial resistance (TER) decreased significantly after PMN contact with basolateral surfaces of T84 monolayers or after incubation with PMN elastase and proteinase-3, but not cathepsin G. Inhibition of PMN serine proteases, but not selective inhibition of elastase or cathepsin G, prevented the fall in TER induced by PMN contact and blocked PMN transepithelial migration. Basolateral, but not apical, PAR-1 and -2 activation with selective agonists also decreased TER. PAR-1 and -2 were localized intracellularly and in close proximity to lateral surfaces beneath tight junctions, and expression was increased in colonic mucosa from individuals with Crohn's disease. Combined, but not individual, transfection with small interfering RNAs targeted against epithelial PAR-1 and -2, prevented the fall in TER induced by PMN contact. Furthermore, basolateral PAR-1 and -2 activation induced phosphorylation of myosin L chain kinase and regulatory myosin L chain. Lastly, epithelial PAR-1 and -2 knockdown decreased the rate of PMN transepithelial migration. These results suggest that protease-mediated epithelial PAR-1 and -2 activation, by migrating PMNs, induces signaling events that increase epithelial permeability thereby facilitates PMN transepithelial migration.     

A synthetic prostone activates apical chloride channels in A6 epithelial cells

The bicyclic fatty acid lubiprostone (formerly known as SPI-0211) activates two types of anion channels in A6 cells. Both channel types are rarely, if ever, observed in untreated cells. The first channel type was activated at low concentrations of lubiprostone (<100 nM) in >80% of cell-attached patches and had a unit conductance of approximately 3-4 pS. The second channel type required higher concentrations (>100 nM) of lubiprostone to activate, was observed in approximately 30% of patches, and had a unit conductance of 8-9 pS. The properties of the first type of channel were consistent with ClC-2 and the second with CFTR. ClC-2's unit current strongly inwardly rectified that could be best fit by models of the channel with multiple energy barrier and multiple anion binding sites in the conductance pore. The open probability and mean open time of ClC-2 was voltage dependent, decreasing dramatically as the patches were depolarized. The order of anion selectivity for ClC-2 was Cl > Br > NO(3) > I > SCN, where SCN is thiocyanate. ClC-2 was a "double-barreled" channel favoring even numbers of levels over odd numbers as if the channel protein had two conductance pathways that opened independently of one another. The channel could be, at least, partially blocked by glibenclamide. The properties of the channel in A6 cells were indistinguishable from ClC-2 channels stably transfected in HEK293 cells. CFTR in the patches had a selectivity of Cl > Br > NO(3) congruent with SCN congruent with I. It outwardly rectified as expected for a single-site anion channel. Because of its properties, ClC-2 is uniquely suitable to promote anion secretion with little anion reabsorption. CFTR, on the other hand, could promote either reabsorption or secretion depending on the anion driving forces.