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We have previously shown that ethanol (EtOH) induces protective NF-kappaB activation in gastric surface epithelial cells. This study investigates the defense systems in rat gastric mucosal cells (RGM-1) exposed simultaneously to EtOH and taurocholate (TC) or acetylsalicylic acid (ASA). Simultaneous exposure to ASA and EtOH increased EtOH-induced caspase-3 activity and decreased cell viability, indicating synergetic damaging action. Simultaneous exposure to TC (5 mM) with EtOH (5%) increased EtOH-induced NF-kappaB activation, opposing EtOH-induced decrease in cell membrane integrity and in cell viability as shown by decreasing RelA expression with siRNA technique. Low doses of TC decreased the EtOH (5%) induced caspase-3 activity independently from NF-kappaB pathway and inhibited EtOH-induced decrease in caspase-3 precursor protein levels, also indicating the inhibition of caspase-3 pathway. The TC (5 mM)-induced protection in EtOH exposed tissues seems to have two distinct pathways, inhibition of apoptosis and enhancement of NF-kappaB signaling.

We have here elucidated whether ulcerogenic agents affect the production of NO and reactive oxygen species (ROS). The ulcerogenic agents dose dependently induced NO and ROS production in mouse gastric epithelial cells. Taurocholate (TC, 5 mM) exposure did not affect cell viability, but it increased inducible nitric oxide synthase (iNOS) expression, NO production, ROS production, and epithelial permeability. Epithelial permeability was inhibited with NOS inhibitors or antioxidants. Oxidative stress induced by acetylsalicylic acid (ASA) and ethanol was not inhibited with NOS inhibitors. ASA induced ROS production even at low concentrations (1 mM), which did not affect cell viability. Ethanol-induced ROS production was linked to cell viability, suggesting direct oxidative stress caused by ethanol. Taurocholate-induced NO signaling and the ensuing production of ROS might contribute to initiation of defensive or adaptive cellular mechanisms. ASA-induced ROS signaling might have similar effects, whereas ethanol induced direct oxidative stress, having an influence on cell viability.

We have elucidated the role of different ion transporters and epidermal growth factor(EGF) during luminal acid exposure in primary cultured rabbit surface epithelial cells by measuring intracellular calcium and pH. Amiloride, DIDS, or sodium or bicarbonate substitutions were used to inhibit ion transport. During luminal acid exposure the dominant intracellular pH regulator is the Na+/H+ antiport, and bicarbonate transport has only a secondary role, which is uncovered as the Na+/H+ function fails. The decrease in intracellular pH caused by luminal acid was significantly smaller in serosal EGF-treated epithelia than in controls. This defensive function of EGF was abolished by verapamil, BAPTA, and calmidazolium but not by TMB-8. EGF increased intracellular calcium, which was prevented by verapamil but not by TMB-8. EGF enhances gastric epithelial defense against luminal acid by inducing intracellular calcium signaling via plasma membrane verapamil-sensitive calcium channels and thereby enhancing the function of the Na+/H+ antiport.

Cell volume regulation was investigated in gastric surface epithelial cells during hypertonic conditions. Isolated Necturus antral mucosa was perfused on the serosal side with Ringer's solution (pH 7.25, 95%O2/5%CO2) and on the mucosal side successively with 150-500 mM NaCl. Amiloride, ouabain, and bumetanide were used to experimentally inhibit Na+/H+, Na+/K+ ATPase or Na+-K+-2Cl- ion transporters. Intracellular sodium activity and cell volume changes were measured with liquid sensor microelectrodes. The increase in intracellular sodium activity caused by luminal hyperosmolar exposure was mainly due to cell shrinkage. Inhibition of Na+/K+ ATPase or Na+-K+-2Cl- cotransporter increased hyperosmotic cell shrinkage (-52 +/- 5%, -85 +/- 19%, and -77 +/- 9% for control, ouabain, and bumetanide, respectively). Inhibition of Na+/K+ ATPase increased intracellular sodium activity (from 18 +/- 4 to 52 +/- 12 mM). Cell volume regulation in gastric epithelial surface cells during mucosal hyperosmolar exposure is maintained by the basolateral Na+-K+-2Cl- cotransporter, while Na+/K+ ATPase maintains sodium balance, but Na+/H+ antiport seems to have a less important role.

Ethanol is a well-established "barrier breaker" in gastric mucosa, but its detailed effects at the cellular level remain unclear. We have previously shown that the intracellular free calcium concentration is increased, gap junctions are closed, and cell volume is decreased after exposure to 5% (v/v) ethanol in primarily cultured rabbit gastric epithelial cells. Rat gastric mucosal (RGM) cells were grown to confluence on a coverslip or on a filter membrane. Gap junctional diffusion was measured in 5-carboxyfluorescein-loaded cells by bleaching a small area with a laser and measuring the recovery with confocal microscope. Intracellular calcium was measured spectrofluorometrically in fura-2-loaded cells. For cell volume measurements the cell monolayer was loaded with calcein and imaged along the Z-axis with a confocal microscope. The changes in fluorescence intensity were intercepted as a measure of cell volume change. TMB-8 was used to inhibit intracellular calcium release and lanthanum to block plasma membrane calcium selective ion channels, while BABTA served as an intracellular calcium chelating agent. Results showed that ethanol (7.5%, v/v) exposure increased intracellular calcium from 69 +/- 7 to 142 +/- 11 nM (N = 5; P < 0.05), decreased cell volume by -23 +/- 5% (N = 8; P < 0.05), and induced gap junction closure (fluorescence recovery from 37 +/- 9 to 15 +/- 3%; N = 6; P < 0.05). A serosal potassium channel blocker, quinine, almost completely prevented the ethanol-induced cell volume decrease (from -23 +/- 5 to -3 +/- 3%), suggesting that opening of basolateral potassium channels underlies cell shrinkage. BABTA inhibited completely (from 35 +/- 3 to 39 +/- 4 nM; N = 6; P < 0.05), and TMB-8 + lanthanum partially (from 60 +/- 6 to 92 +/- 12 nM; N = 6; P < 0.05), the ethanol-induced intracellular calcium increase. BABTA also abolished the ethanol-induced volume decrease (from -23 +/- 5 to 1 +/- 4%; N = 6; P < 0.05), while TMB-8 + lanthanum had a lesser effect on it (from -23 +/- 5 to -11 +/- 3%; N = 9; P < 0.05). They also abolished the closure of gap junctions induced by ethanol (fluorescence recovery, 38 +/- 5% for BABTA and 30 +/- 4% for TMB-8 + lanthanum). We conclude that luminal ethanol opens basolateral calcium-dependent potassium selective channels with resultant shrinkage of the cells and blocks the intercellular gap junctions. These actions are mediated by intracellular calcium signaling.

Ethanol is a well-established "barrier breaker" in gastric mucosa, but its detailed effects at the cellular level remain unclear. We have previously shown that the intracellular free calcium concentration is increased, gap junctions are closed, and cell volume is decreased after exposure to 5% (v/v) ethanol in primarily cultured rabbit gastric epithelial cells. Rat gastric mucosal (RGM) cells were grown to confluence on a coverslip or on a filter membrane. Gap junctional diffusion was measured in 5-carboxyfluorescein-loaded cells by bleaching a small area with a laser and measuring the recovery with confocal microscope. Intracellular calcium was measured spectrofluorometrically in fura-2-loaded cells. For cell volume measurements the cell monolayer was loaded with calcein and imaged along the Z-axis with a confocal microscope. The changes in fluorescence intensity were intercepted as a measure of cell volume change. TMB-8 was used to inhibit intracellular calcium release and lanthanum to block plasma membrane calcium selective ion channels, while BABTA served as an intracellular calcium chelating agent. Results showed that ethanol (7.5%, v/v) exposure increased intracellular calcium from 69± 7 to 142± 11 nM (N = 5; P < 0.05), decreased cell volume by -23± 5% (N = 8; P < 0.05), and induced gap junction closure (fluorescence recovery from 37± 9 to 15± 3%; N = 6; P < 0.05). A serosal potassium channel blocker, quinine, almost completely prevented the ethanol-induced cell volume decrease (from -23± 5 to -3± 3%), suggesting that opening of basolateral potassium channels underlies cell shrinkage. BABTA inhibited completely (from 35± 3 to 39± 4 nM; N = 6; P < 0.05), and TMB-8 + lanthanum partially (from 60± 6 to 92± 12 nM; N = 6; P < 0.05), the ethanol-induced intracellular calcium increase. BABTA also abolished the ethanol-induced volume decrease (from -23± 5 to 1± 4%; N = 6; P < 0.05), while TMB-8 + lanthanum had a lesser effect on it (from -23± 5 to -11± 3%; N = 9; P < 0.05). They also abolished the closure of gap junctions induced by ethanol (fluorescence recovery, 38± 5% for BABTA and 30± 4% for TMB-8 + lanthanum). We conclude that luminal ethanol opens basolateral calcium-dependent potassium selective channels with resultant shrinkage of the cells and blocks the intercellular gap junctions. These actions are mediated by intracellular calcium signaling.

Superficial gastric mucosal injury is rapidly repaired by epithelial cell migration. This study aims to characterize the intracellular signal transduction pathways underlying the repair process. Primary monolayer cultures of rabbit gastric epithelial cells were wounded. The measured spontaneous cell migration speed at the edge of the wound was 457+/-89 microm/24 hr. Epidermal growth factor stimulated and genistein (receptor tyrosine protein kinase inhibitor) inhibited cell migration significantly. Down-regulation of protein Kinase C (PKC) with long-term phorbol 12-myristate 13-acsetate or inhibition with calphostin-C significantly inhibited cell migration. Blocking of Ca2+ channels with verapamil and endogenous Ca2+ release with TMB-8 or inhibition of the Ca2+/calmodulin complex with calmidazolium likewise significantly inhibited migration speed and also abolished the rise of [Ca2+]i, which was measured in migrating cells. Modulation of the cAMP-PKA pathway or prostaglandin synthesis had no influence on cell migration. Gastric epithelial cell migration implies activation of receptor tyrosine kinase. It is associated with increased [Ca2+]i and requires an intact Ca2+/calmodulin complex. Intact PKC activity also is needed.

Due to the proposed functions in soft tissue repair, we evaluated the spatial and temporal distribution of SPARC, a counteradhesive, matricellular glycoprotein in healing intestinal anastomoses and short bowel syndrome (SBS) in rats. Intestinal anastomoses were performed in the jejunum of male Wistar rats. SBS was induced by resecting 70% of the small bowel. In situ hybridization was performed to localize SPARC mRNA and immunohistochemical studies for locating the SPARC protein. The granulation tissue in the anastomotic area exhibited immunoreactivity for SPARC at all time points. The level of expression was maximal at seven to nine days. Endothelial cells of capillaries, smooth muscle cells, fibroblastic cells, and macrophages, as well as mesothelial cells on the serosal surface, were stained. The immunoreactivity was mostly intracellular. SPARC mRNA transcripts were localized to the edges of the anastomotic area at days 1 and 4 and on the newly formed granulation tissue later. The expression of SPARC mRNA was maximal at seven days and decreased thereafter. Both in normal controls and in SBS, SPARC was expressed in endothelial cells of submucosal capillaries and in smooth muscle cells but not in epithelium. Based on the restricted temporal and spatial distribution during the healing of intestinal anastomoses and in SBS we propose that SPARC plays a significant role in intestinal repair and adaptation.

The aim of this study was to evaluate possible changes in the neuropeptide innervation pattern of the remaining porcine ileum following 75% proximal resection of the small intestine. Three-month-old piglets were operated on and two months postoperatively full-thickness specimens of the proximal part of the distal ileum wall were taken. Age-matched 3- and 5-month-old unoperated piglets were used as controls. The number and intensity of VIP-, galanin-, enkephalin-, substance P-, and somatostatin-containing nerve fibers were estimated in sections processed for immunofluorescence microscopy and subjected to quantitative scoring. The VIP-, galanin-, and enkephalin-immunoreactive fibers of the circular muscle layer and villi were also quantitated by computer-assisted morphometry. The number and intensity of VIP-immunoreactive fibers in the mucosa and circular muscle layer markedly decreased after resection as compared to 3-month-old and 5-month-old controls (P < 0.05). The galanin immunoreactivity index decreased significantly after resection in the circular muscle layer as compared to both control groups (P < 0.05). The increase in the number of enkephalin-immunoreactive nerve fibers that normally occurred from 3 to 5 months of age was inhibited by the resection. We were not able to see any differences in somatostatin or substance P immunoreactivity between the groups. The results suggest that massive resection induces significant changes in the neuropeptide-containing innervation of the remaining small intestine. These findings are compatible with altered motor activity and mucosa function in the remain intestine.

In addition to its pHi regulatory function Na(+)-H(+)-antiport is also involved in volume regulation of epithelial cells, particularly in neutral conditions. It is also known that the antiport is activated after ligand binding following growth factor receptor activation. The aim of the present study was to evaluate the role of the antiport in restitution of gastric mucosa and whether its activity is dependent on the type of superficial injury. Therefore the fundic epithelium of guinea pig stomach was perfused in an Ussing chamber in neutral conditions. Na(+)-H(+)- and Cl(-)-HCO3(-)-antiports were inhibited with 1.0 mM amiloride, 1.0 mM SITS, or with HCO3- removal and Na(+)-K(+)-2Cl(2-)-cotransporter with 0.3 M furosemide during 4 hr of restitution after superficial injury induced either by 1.25 M NaCl or by 1.0% Triton. Luminal exposure of the epithelium to amiloride had no effect on restitution but serosal application abolished the process completely. The inhibitory effect of amiloride was similar after both NaCl and Triton injury. The inhibition of Cl(-)-HCO3(-)-antiport with SITS interfered with the process as well, while HCO3- removal had no significant inhibitory effect, nor did the inhibition of Na(+)-K(+)-2Cl(-)-cotransporter. The morphologic findings were in accordance with the electrophysiologic measurements in each pair of tissues. It is concluded that the Na(+)-H(+)-antiport is essential for the epithelial cells during restitution even in neutral conditions, but a functional Cl(-)-HCO3(-)-antiport is also required. The activity of Na(+)-H(+)-antiport is sensitive to basolateral amiloride and is necessary regardless of the type of chemical injury.

Recent studies suggest that substance P (SP), vasoactive intestinal polypeptide (VIP), and mast cells play a role in inflammatory processes of the bowel. The aim of this study was to evaluate the distribution of SP and VIP immunoreactivities and to count mast cells in the ileal pouch of patients, who had pouchitis after restorative proctocolectomy performed for treatment of ulcerative colitis (UC), and to compare the findings in the same patients after a follow-up period. Nine patients with pouchitis underwent clinical evaluation, endoscopy of the pouch, and histological examination, which were repeated after the follow-up period of 14 months on average. The number and intensity of SP- and VIP-immunoreactive nerve fibers were visualized by immunofluorescence microscopy and subjected to quantitative scoring, and the number of mast cells per unit area was counted. The results were compared to the histological findings and the clinical status. Lamina propria contained fibers showing bright immunofluorescence for SP and VIP. The mean fluorescence intensity score of SP-immunoreactive nerve fibers in the lamina propria remained similar after the follow-up period (2.99 +/- 0.79 and 2.06 +/- 0.82, NS). SP-immunoreactive innervation correlated with the grade of acute (R2 = 0.5396, P = 0.0242) and chronic inflammation (R2 = 0.4561, P = 0.0459), while SP and VIP immunoreactivity, mast cell count, and histological changes did not correlate with the clinical status. The present study demonstrates an increase in the density of SP-immunoreactive nerve fibers in inflamed ileal pouch mucosa of clinically asymptomatic pouchitis patients. These results raise the possibility of therapeutic interference of SP-related processes in treatment of pouchitis.

Recent studies suggest that the intestinal polypeptides substance P (SP) and vasoactive intestinal polypeptide (VIP) play a role in the bowel inflammatory processes. The aim of this study was to evaluate the distribution of SP and VIP immunoreactivities in the ileal pouch of the patients with ulcerative colitis (UC). Thirty-six patients underwent clinical evaluation, endoscopy, and histological examinations. Samples were taken from normal ileum (N = 9), ileum of UC patients (N = 9), normal ileal pouch (N = 9) and pouchitis (N = 9). SP- and VIP-containing nerve fibers were visualized in sections processed for immunofluorescence microscopy. The number and intensity of SP and VIP immunoreactivities were subjected to quantitative scoring. On samples from all groups lamina propria contained fibers showing bright immunofluorescence for SP and VIP. The number and intensity of SP immunoreactive nerve fibers were markedly increased in pouchitis as compared to normal pouch (P < 0.005), to ileum of UC patients (P < 0.001), and to normal ileum (P < 0.05). The number and intensity of VIP-immunoreactive nerve fibers in the lamina propria were markedly increased in pouchitis patients and in those having a normal pouch as compared to pooled values of ileum of UC patients and normal ileum (P < 0.05). The results suggest that SP, which may play a role in mediating inflammatory processes, is increased in pouchitis and that VIP, which may contribute to the regulation of intestinal motility, is increased in the pouch.

The amount of colonic substance P and substance P-receptors is increased in ulcerative colitis, which may denote that substance-P is involved as a neurogenic mediator in the inflammatory process of ulcerative colitis. We studied the anatomical distribution of elevated colonic substance P in ulcerative colitis and assessed morphometrically whether the changes in substance P correlate with alterations in colonic innervation. Full-thickness specimens of colonic wall were obtained from normal human colons (N = 9) and the most and least affected regions of ulcerative colitis colons (N = 10) and immunostained for substance P. Substance P immunoreactivity index was calculated by multiplying each intensity value by the number of pixels exhibiting this intensity value. The numbers of substance P-immunoreactive nerve fibers in the lamina propria were markedly increased, and their fluorescence intensity was enhanced in ulcerative colitis. The longitudinal muscle layer contained substance P-immunoreactive nerve fibers in ulcerative colitis, but not in the controls. The substance P-immunoreactive index (= number x intensity of nerve fibers) was 3.42 +/- 1.49 in controls, 21.19 +/- 7.79 in mild ulcerative colitis regions (P < 0.05), and 29.68 +/- 9.81 in severe ulcerative colitis regions (P < 0.01). Increase in the number of substance P nerve fibers is in accordance with the hypothesis that substance P contributes to neurogenic mediation of inflammation in ulcerative colitis.

The effect of vagotomy and pyloroplasty on the density of nerve fibers containing bombesin/gastrin-releasing peptide (GRP), calcitonin gene-related peptide (CGRP), and galanin as well as histamine-, 5-hydroxytryptamine (5-HT)-, and somatostatin-containing cells in the oxyntic mucosa of the rat stomach was studied. Ten days after vagotomy and pyloroplasty the density of histamine-containing cells in the oxyntic mucosa was increased by 70% (P < 0.05), and these cells were larger and showed more extensive cell processes than in control animals. The density of 5-HT-immunoreactive (IR) cells and somatostatin-IR cells were not affected. A marked decrease in the density of CGRP-IR nerve fibers and a slighter decrease in the density of GRP-IR nerve fibers was observed in the mucosal layer, while only a minor reduction of CGRP-IR fibers, and no reduction of GRP-IR fibers was seen in the muscular layer. The density of galanin-IR nerve fibers was not affected. The height of the oxyntic mucosa was reduced by about 25% (P < 0.05). Thus, a striking effect on the histamine-IR cells was seen, supporting the view that these cells are regulated by the vagus nerve. The study also indicates that a major portion of the CGRP-IR nerve fibers, and part of the GRP-IR nerve fibers, in the mucosal layer of the fundic region are of vagal origin or regulated by normal vagus nerve activity.