Taurocholate potentiates ethanol-induced NF-kappaB activation and inhibits caspase-3 activity in cultured rat gastric mucosal cells.

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.

[Update on current care guidelines. Safe use of non-steroidal anti-inflammatory drugs]. / Tulehduskipulääkkeiden turvallinen käyttö.

Pain medication should be based on patient's needs and risk profile. Age > 65 years, prior ulcer, co-morbidities, large daily dose, Helicobacter pylori infection, concurrent use of glucocorticoids, serotonin re-uptake inhibitors, or warfarin increase the risk of upper gastrointestinal bleeds. As a preventive strategy the use of concurrent proton pump inhibitors with non-selective NSAIDs is recommended. It is also possible to use COX-2 selective NSAIDs but they are contraindicated for persons with atherosclerotic diseases and special consideration is required for persons with risk factors of heart diseases. Paracetamol is the drug of choice for pain.

Impact of preoperative chemoradiotherapy on survival in patients with resectable pancreatic cancer.

AIM: To explore whether preoperative chemoradiation therapy improves survival of patients with pancreatic cancer undergoing resectional surgery. METHODS: Forty-seven patients with a malignant pancreatic tumor localized in the head or uncinate process of the pancreas underwent radical pancreatico-duodenectomy. Twenty-two received chemoradiation therapy (gemcitabine and radiation dose 50.4 Gy) before surgery (CRR) and 25 patients underwent surgery only (RO). The study was non-randomised. Patients were identified from a prospective database. RESULTS: The median survival time was 30.2 mo in the CRR group and 35.9 mo in the RO group. No statistically significant differences were found in subclasses according to lymph node involvement, TNM stages, tumor size, or perineural invasion. The one, three and five year survival rates were 81%, 33% and 33%, respectively, in the CRR group and 72%, 47% and 23%, respectively, in the RO group. In ductal adenocarcinoma, the median survival time was 27 mo in the CRR group and 20 mo in the RO group. No statistically significant differences were found in the above subclasses. The one, three and five year survival rates were 79%, 21% and 21%, respectively, in the CRR group and 64%, 50% and 14%, respectively, in the RO group. The overall hospital mortality rate was 2%. The morbidity rate was 45% in the CRR group and 32% (NS) in the RO group. CONCLUSION: Major multicenter randomized studies are needed to conclusively assess the impact of neoadjuvant treatment in the management of pancreatic cancer.

Effect of the ulcerogenic agents ethanol, acetylsalicylic acid and taurocholate on actin cytoskeleton and cell motility in cultured rat gastric mucosal cells.

AIM: To assess the effects of ulcerogenic agents on actin cytoskeleton and cell motility and the contribution of oxidative stress. METHODS: Rat gastric mucosal cell monolayers were cultured on coverslips. The cells were exposed, with or without allopurinol (2 mmol/L), for 15 min to ethanol (10-150 mL/L), ASA (1-20 mmol/L) or taurocholate (1-20 mmol/L), then the cells were processed for actin and vinculin staining. Cell migration after wounding was also measured. RESULTS: Exposure to 10 mL/L ethanol caused divergence of zonula adherens-associated actin bundles of adjacent cells and decreased rate of migration. These actions were opposed by xanthine oxidase inhibitor allopurinol. Exposure to 50 mL/L ethanol induced degradation and divergence of zonula adherens-associated vinculin from adjacent cells, which was, again, partially reverted by allopurinol. With 1 mmol/L ASA actin filaments became shorter and thicker. However, higher concentrations (10, 20 mmol/L) of ASA returned microfilaments thinner and longer, and decreased rate of migration. Zonula adherens-associated actin bundles were moderately distorted with 10 mmol/L ASA and with 10 mmol/L taurocholate. Exposure to taurocholate provoked changes resembling those of ASA. Taurocholate 5-20 mmol/L decreased the rate of migration dose dependently. The effects of ASA and taurocholate were not prevented by allopurinol. CONCLUSION: All ulcerogenic agents decreased the rate of migration dose dependently and induced divergence of zonula adherens-associated actin bundles of adjacent cells. In addition, ethanol and ASA caused degradation of actin cytoskeleton. Oxidative stress seems to underlie ethanol, but not ASA or taurocholate, induced cytoskeletal damage.

Calcium Signaling Is Involved in EthanolInduced Volume Decrease and Gap Junction Closure in Cultured Rat Gastric Mucosal Cells.

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.

Taurocholate-induced nitric oxide signaling and the ensuing production of reactive oxygen species lead to an increase in epithelial permeability in cultivated mouse gastric epithelium.

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.

Ethanol induced NF-{kappa}B activation protects against cell injury in cultured rat gastric mucosal epithelium.

Ethanol is a well-established irritant inducing inflammation in gastric mucosa, but the effects at the cellular level remain unclear. This study investigates NF-kappaB activation in gastric mucosal cells by ethanol and assesses the effects of heat shock pretreatment in this ulcerogenic situation. Rat gastric mucosal epithelia were exposed to ethanol for different time periods. Heat shock was induced by incubating the cells at 42 degrees C for 1 h prior to the experiments. For evaluation of NF-kappaB activation, the nuclear fraction of the cell lysates was analyzed with an EMSA or an ELISA-based assay. Caspase-3 (a promoter of apoptosis) activity was measured with a time-resolved fluorescence based assay, cell viability with a tetrazolium assay, and cell membrane integrity with a LDH assay. Ethanol (1-5%) induced NF-kappaB activation, reaching a maximum after 3 h, and also led to moderately increased COX-2 expression. Heat shock pretreatment and the intracellular calcium chelator BAPTA were able to inhibit ethanol-induced NF-kappaB activation. Heat shock pretreatment decreased ethanol-induced caspase-3 activation, decreased cell membrane damage, and retained cellular viability. Inhibition of NF-kappaB activation by NEMO-binding peptide, by decreasing RelA expression, or by inhibiting COX-2 activity by CAY-14040 promoted the effects of ethanol, such as increased caspase-3 activity and decreased cell viability. In conclusion, ethanol induces NF-kappaB activation via a calcium-dependent pathway and induces COX-2 expression. Inhibition of the NF-kappaB activation or COX-2 activity potentiates apoptosis and cell damage induced by ethanol, suggesting a protective role for NF-kappaB activation and COX-2 expression.

The effect of nitric oxide, growth factors, and estrogen on gastric cell migration.

BACKGROUND: To study gastric epithelial cell migration during nitric oxide (NO) and growth factor treatment, simulating inflammation and infection. Also, the effects of estrogen on migration of different malignant and nonmalignant gastric epithelial cell lines were explored. MATERIAL AND METHODS: Isolated primary cultured rabbit gastric epithelial cells, rat gastric mucosal cells, human gastric adenocarcinoma cells, and human colon adenocarcinoma cells (WiDr) were cultured to confluency in appropriate media (5% CO2, 37 degrees C). The cells were treated by hepatocyte growth factor (HGF), transforming growth factor-alpha (TGF-alpha) and keratinocyte growth factor (KGF), with and without sodium nitroprusside (SNP, NO donor) or 17beta-estradiol. Caspase-3 activity and cell viability and migration speed after wounding were measured. RESULTS: HGF was the most potent growth factor to stimulate migration. SNP dose-dependently decreased the speed of migration. HGF and TGF-alpha were able to overcome the SNP-induced inhibition of migration, whereas KGF was not. SNP also induced caspase-3 activity, which was inhibited by HGF and TGF-alpha. 17beta-estradiol decreased migration in all epithelial cells, but the decrease was more profound in malignant cell lines. HGF could overcome the estrogen retarded migration. CONCLUSIONS: Growth factors can overcome NO-induced retardation of cell migration and inhibit NO-induced caspase-3 activity, which altogether might also have physiological significance in in vivo inflammation and in gastric cancer. The more profound decrease in migration speed of gastric adenocarcinoma cell line may suggest that estrogen might be one of the protective factor against female gastric adenocarcinoma before menopausal age.

Epidermal growth factor enhances intracellular pH regulation via calcium signaling in acid-exposed primary cultured rabbit gastric epithelial cells.

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 during hyperosmotic shrinkage is mediated by Na+/K+-ATPase and Na+-K+-2Cl- cotransporter in Necturus gastrics surface epithelial cells.

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.

Calcium signaling is involved in ethanol-induced volume decrease and gap junction closure in cultured rat gastric mucosal cells.

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.

CD2AP contributes to cell migration and adhesion in cultured gastric epithelium.

The potential association of CD2AP with the adherens junction protein E-cadherin, co-localization with the actin cytoskeleton, and involvement in cell migration was investigated in cultured rat gastric mucosal cells. In stationary cells, CD2AP was localized perinuclearly while E-cadherin was expressed along cell-cell contacts and F-actin formed a branched network and adhesion belts. In migrating cells, CD2AP appeared as thread-like accumulations in the leading edges, colocalizing with F-actin and occasionally with E-cadherin. Intracellular injection of anti-CD2AP significantly retarded the migration speed of the cells suggesting a crucial role for CD2AP in mucosal cell migration, possibly as a scaffolding protein between cell membrane proteins and actin cytoskeleton. Co-immunoprecipitation assays revealed that CD2AP and E-cadherin are in a complex in HGF stimulated cells. It is concluded that CD2AP interacts with E-cadherin and co-localizes with F-actin in the leading edge of migrating cells, and significantly contributes to cell migration in restituting gastric epithelium.

Effect of luminal ethanol on epithelial resistances and cell volume in isolated Necturus gastric mucosa.

Ethanol is a well-established "barrier breaker" in gastric mucosa, but its effects at the cellular level remain to be elucidated. Isolated Necturus antral mucosa was exposed luminally to 5-15% (v/v) ethanol at pH 3.0. Apical, basolateral, shunt, and internal resistances in surface epithelium were measured using 2-D cable analysis. Cell volume changes were determined from tetramethylammonium-loaded surface cells. Low luminal ethanol (5%) decreased basolateral resistance, presumably by opening of K+ channels, since this decrease was partially inhibited by the K+ channel blocker, quinine. Low ethanol decreased also epithelial cell volume, which was opposed by quinine, suggesting that efflux of intracellular K+ underlies this shrinkage. High luminal ethanol (15%) markedly decreased shunt and apical cell membrane resistances, and partially closed gap junctions as judged from increased epithelial internal resistance. Opening of basolateral K+ channels with resultant epithelial cell shrinkage might be among the initial steps in ethanol induced gastric injury. The changes in intraepithelial resistances provoked by stronger ethanol probably reflect emerging structural epithelial damage.

Gastrostomy tube insertion into intestinal-cutaneous tract fistulas is a new technique to improve fistula control.

BACKGROUND: The management of gastrointestinal-cutaneous fistulas may be complicated by the difficulty in obtaining adequate control of the fistula tract. This study describes a new method to obtain better fistula control utilizing a semi-rigid stent in the form of a gastrostomy tube. METHODS: Consecutive patients with intestinal-cutaneous fistulas of at least 3 weeks duration and treated by the new technique were analyzed. The technique involved the insertion of a guide wire into the fistula tract from the luminal side using an endoscope, snaring the wire with a Dormia basket inserted into the fistula tract from the cutaneous side and then exteriorized. The gastrostomy tube was then pulled with the guide wire from the lumen along the fistula tract and out through the skin. RESULTS: Five patients had had fistulas for a median duration of 42 (range 26-140) days before insertion of the gastrostomy tube. The gastrostomy tube was replaced with a smaller diameter tube in 4 of the patients (range 1-3 changes). The patients were discharged from the hospital at a median of 14 (range 12-23) days after the tube insertion but with the tube in situ. The median time from the insertion of the tube to its removal was 42 (range 32-108) days. CONCLUSIONS: Gastrostomy tube insertion using minimally invasive techniques may improve fistula control enabling patients to be discharged home sooner than otherwise and improve the rate of healing.

Migration of primary cultured rabbit gastric epithelial cells requires intact protein kinase C and Ca2+/calmodulin activity.

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.

Eradication of Helicobacter pylori improves the healing rate and reduces the relapse rate of nonbleeding ulcers in patients with bleeding peptic ulcer.

OBJECTIVE: A causal relationship between Helicobacter pylori (H. pylori) and peptic ulcer complications remains obscure. The aim of this study was to determine the importance of H. pylori and other risk factors for healing rate, ulcer recurrence, and rebleeding in patients with bleeding peptic ulcer. METHOD: A total of 223 patients with H. pylori positive bleeding peptic ulcer were randomly allocated to three treatment groups: 1) quadruple therapy (QT) (88 patients); 2) dual therapy (DT) (88 patients); and 3) omeprazole and placebo therapy (OPl) (47 patients). Endoscopic assessment was performed initially and at 8 and 52 wk. Ulcer healing and eradication rates were assessed; endpoints were ulcer relapse and ulcer rebleeding during 52 wk. RESULTS: Results after 8 and 52 wk were available for 211 and 179 patients, respectively. Eradication rate was 100% (95% CI = 96-100%) in the QT, 84% (95% CI = 74-91%) in the DT, and 4% (95% CI = 1-15%) in the OPl group. Ulcer healing rate was 95% (95% CI = 91-98%) in H. pylori negative and 8% (95% CI = 70-91%) in H. pylori positive patients. Ulcer relapses occurred in 2% (95% CI = 0.5-6%) of H. pylori negative and in 38% (95% CI = 24-54%) of H. pylori positive patients, and rebleeding occurred in five patients (three H. pylori positive and two negative). CONCLUSIONS: Eradication of H. pylori infection enhances healing of bleeding peptic ulcers after endoscopic therapy. H. pylori infection is an important independent risk factor for relapsing of nonbleeding ulcers in patients with bleeding peptic ulcer.