Bile acids inhibit Na⁺/H⁺ exchanger and Cl⁻/HCO3⁻ exchanger activities via cellular energy breakdown and Ca²âº overload in human colonic crypts.

Bile acids play important physiological role in the solubilisation and absorption of dietary lipids. However, under pathophysiological conditions, such as short bowel syndrome, they can reach the colon in high concentrations inducing diarrhoea. In this study, our aim was to characterise the cellular pathomechanism of bile-induced diarrhoea using human samples. Colonic crypts were isolated from biopsies of patients (controls with negative colonoscopic findings) and of cholecystectomised/ileum-resected patients with or without diarrhoea. In vitro measurement of the transporter activities revealed impaired Na⁺/H⁺ exchanger (NHE) and Cl⁻/HCO3⁻ exchanger (CBE) activities in cholecystectomised/ileum-resected patients suffering from diarrhoea, compared to control patients. Acute treatment of colonic crypts with 0.3 mM chenodeoxycholate caused dose-dependent intracellular acidosis; moreover, the activities of acid/base transporters (NHE and CBE) were strongly impaired. This concentration of chenodeoxycholate did not cause morphological changes in colonic epithelial cells, although significantly reduced the intracellular ATP level, decreased mitochondrial transmembrane potential and caused sustained intracellular Ca²âº elevation. We also showed that chenodeoxycholate induced Ca²âº release from the endoplasmic reticulum and extracellular Ca²âº influx contributing to the Ca²âº elevation. Importantly, our results suggest that the chenodeoxycholate-induced inhibition of NHE activities was ATP-dependent, whereas the inhibition of CBE activity was mediated by the sustained Ca²âº elevation. We suggest that bile acids inhibit the function of ion transporters via cellular energy breakdown and Ca²âº overload in human colonic epithelial cells, which can reduce fluid and electrolyte absorption in the colon and promote the development of diarrhea.

Ethanol and its non-oxidative metabolites profoundly inhibit CFTR function in pancreatic epithelial cells which is prevented by ATP supplementation.

Excessive alcohol consumption is a major cause of acute pancreatitis, but the mechanism involved is not well understood. Recent investigations suggest that pancreatic ductal epithelial cells (PDECs) help defend the pancreas from noxious agents such as alcohol. Because the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel plays a major role in PDEC physiology and mutated CFTR is often associated with pancreatitis, we tested the hypothesis that ethanol affects CFTR to impair ductal function. Electrophysiological studies on native PDECs showed that ethanol (10 and 100 mM) increased basal, but reversibly blocked, forskolin-stimulated CFTR currents. The inhibitory effect of ethanol was mimicked by its non-oxidative metabolites, palmitoleic acid ethyl ester (POAEE) and palmitoleic acid (POA), but not by the oxidative metabolite, acetaldehyde. Ethanol, POAEE and POA markedly reduced intracellular ATP (ATPi) which was linked to CFTR inhibition since the inhibitory effects were almost completely abolished if ATPi depletion was prevented. We propose that ethanol causes functional damage of CFTR through an ATPi-dependent mechanism, which compromises ductal fluid secretion and likely contributes to the pathogenesis of acute pancreatitis. We suggest that the maintenance of ATPi may represent a therapeutic option in the treatment of the disease.

The role of NF-kappaB activation in the pathogenesis of acute pancreatitis.

Acute pancreatitis is an inflammatory disease of the pancreas which, in its most severe form, is associated with multi-organ failure and death. Recently, signalling molecules and pathways which are responsible for the initiation and progression of this disease have been under intense scrutiny. One important signalling molecule, nuclear factor kappaB (NF-kappaB), has been shown to play a critical role in the development of acute pancreatitis. NF-kappaB is a nuclear transcription factor responsible for regulating the transcription of a wide variety of genes involved in immunity and inflammation. Many of these genes have been implicated as central players in the development and progression of acute pancreatitis. This review discusses recent advances in the investigation of pancreatic and extrapancreatic (lungs, liver, monocytes and macrophages, and endothelial cells) NF-kappaB activation as it relates to acute pancreatitis.

The role of the glucocorticoid-dependent mechanism in the progression of sodium taurocholate-induced acute pancreatitis in the rat.

The effects of glucocorticoids on acute pancreatitis (AP) have remained contradictory. The aim of this study was to investigate the time courses of the effects of the exogenous glucocorticoid agonists dexamethasone (DEX) and hydrocortisone (HYD) and a glucocorticoid antagonist (RU-38486) and to characterize the local and systemic responses in AP in rats. The glucocorticoid antagonist and agonists were administered just before AP induction. Serum amylase activity determinations, IL-6 bioassays, pancreatic weight/body weight ratio measurements, and survival analysis were performed. Liver and lung injuries were assessed via neutrophil leukocyte infiltration in myeloperoxidase (MPO) assays, tissue adenosine triphosphate (ATP) level determinations, and histology. In the glucocorticoid agonist groups, the survival rate increased, while the serum amylase level, the IL-6 activity, and the pancreatic weight/body weight ratio decreased significantly as compared with the control and RU-treated groups. AP resulted in significant decreases in tissue ATP levels in both the liver and the lung. In the DEX- or HYD-treated groups, the liver ATP levels were significantly elevated, while both the liver and the lung MPO levels were attenuated as compared with the AP and RU-treated groups. These results suggest that glucocorticoids may play important roles in mitigating the progression of the inflammatory reaction during the early phases of AP.

Induction of heat shock proteins fails to produce protection against trypsin-induced acute pancreatitis in rats.

Heat shock proteins (HSPs) are necessary in the synthesis, degradation, folding, transport, and translocation of different proteins. It is well known that the increased expression of HSPs may have a protective effect against cerulein-induced pancreatitis in rats or against choline-deficient ethionine-supplemented diet model pancreatitis in mice. The aim of this study was to investigate the potential effects of HSP preinduction by cold or hot water immersion on trypsin-induced acute pancreatitis in rats. Trypsin was injected into the interlobular tissue of the duodenal part of the pancreas at the peak level of HSP synthesis, as determined by Western blot analysis. The rats were sacrificed by exsanguination through the abdominal aorta 6 h after the trypsin injection. The serum amylase activity, the tumor necrosis factor-alpha, interleukin-1, and interleukin-6 levels, the pancreatic weight/body weight ratio, and the pancreatic contents of DNA, protein, amylase, lipase, and trypsinogen were measured. A biopsy for histology was taken. Hot water immersion significantly elevated the HSP72 expression, while cold water immersion significantly increased the HSP60 expression. Cold water immersion pretreatment ameliorated the pancreatic edema in trypsin-induced pancreatitis, however this was not due to the HSP60. Hot water immersion pretreatment did not have any effect on the measured parameters in trypsin-induced pancreatitis. The findings suggest that the induction of HSP60 or HSP72 are not enough to protect rats against the early phase of this localized necrohemorrhagic pancreatitis model.

Water immersion pretreatment decreases pro-inflammatory cytokine production in cholecystokinin-octapeptide-induced acute pancreatitis in rats: possible role of HSP72.

Heat shock proteins (HSPs) are cytoprotective proteins that are expressed constitutively and/or at elevated levels upon the exposure of cells to stress. The aim of this study was to investigate the potential effects of HSP preinduction by cold- (CWI) or hot-water immersion (HWI) on pro-inflammatory cytokine production (IL-1, IL-6, TNF-alpha) in cholecystokinin-octapeptide(CCK)-induced acute pancreatitis. Rats were injected with 3 x 75 microg/kg CCK subcutaneously at intervals of 2 h at the peak level of HSP synthesis, as determined by Western blot analysis. The animals were killed by exsanguination through the abdominal aorta 2 h after the last CCK injection. The serum IL-1, IL-6, TNF-alpha, and amylase levels, the pancreatic weight/body weight ratio, and the pancreatic contents of DNA, protein, amylase, lipase and trypsinogen were measured; biopsy for histology was taken. HWI significantly elevated the HSP72 expression, while CWI significantly increased the HSP60 expression. HWI pretreatment decreased all of the measured serum cytokine levels in this acute pancreatitis model. CWI and HWI pretreatment ameliorated most of the examined laboratory and morphological parameters of CCK-induced pancreatitis. The findings suggest the possible roles of HSP60 and HSP72 in the protection against CCK-induced pancreatitis. HSP72 might also participate in the reduction of pro-inflammatory cytokine synthesis.

The effect of pesticides on carp (Cyprinus carpio L). Acetylcholinesterase and its biochemical characterization.

The activity and molecular forms of acetylcholinesterase (AChE) were characterized in tissues of the carp (Cyprinus carpio). Tissue AChE activity was determined in response to specific inhibitors (ethopropazine, BW 284 C51) or pesticides (CuSO4, paraquat (PQ), methidathion (MD)). The highest AChE activity was found in the serum (878 +/- 100 U/liter), followed by the brain (113 +/- 12 U/liter), heart (89 +/- 6 U/liter), and trunk muscle (35 +/- 5 U/liter). Experiments with specific choline esterase inhibitors revealed a very low amount of pseudocholinesterase in all tissues studied. The ratio of the membrane-bound to the cytoplasmic-free AChE molecular forms was increased in the order of brain, trunk muscle, and heart. In sera of fish treated with MD (2 ppm) there was an 80% inhibition of AChE lasting for 2 weeks. Treatment with CuSO4 or PQ (both 5 ppm) led to a 50% decrease in the serum AChE activity followed by a transient increase over the control level. After 2 weeks of chronic treatment, AChE activity in fish exposed to CuSO4 returned to the control level, whereas in fish treated with PQ an elevated level (130% when compared to the control level) of enzyme activity was found. Our present experimental data indicate that pesticides occurring in natural waters not only inhibit AChE activity in fish but may influence the resynthesis of the enzyme as well.

Cholinotoxic effects of aluminum in rat brain.

The in vivo and in vitro effects of Al on the cholinergic system of rat brain were studied. The amount of Al accumulated after the chronic, intraperitoneal administration of aluminium gluconate (Al-G) or AlCl3, both at a dose of 1 mg/ml/100 g of body weight, increased in the frontal and parietal cortices, the hippocampus, and the striatum. Significantly decreased choline acetyltransferase activities after chronic Al treatment were measured in the parietal cortex, the hippocampus, and the striatum, but not in the frontal cortex. The acetylcholinesterase activity was not changed significantly in any brain area investigated. Both Al-G and AlCl3 administrations resulted in a general decrease (to 40-70% of the control values) in the specific l-[3H]nicotine binding, involving all brain areas studied. The specific (-)-[3H]quinuclidinyl benzilate binding was reduced (to 40-60% of the control values) only after 25 days of Al treatment. Al-G and AlCl3 were equivalent in eliciting these reductions in vitro studies revealed different alterations of the cholinergic system in response to Al treatment. No changes were observed either in choline acetyltransferase activity or in cholinergic receptor bindings. Both Al-G and Al2(SO4)3 treatments, however, exhibited a biphasic effect on the acetylcholinesterase activity. At low Al concentrations (10(-8)-10(-6) M), the activity was slightly increased, whereas at higher concentrations (10(-6)-10(-4) M), it was inhibited by a maximum of 25% as compared to the controls. Thus, these cholinotoxic effects are probably due not to a direct interaction between the metal and the cholinergic marker proteins, but rather to a manifestation and consequence of its neurodegenerative effects.

Two-site immunoassay for acetylcholinesterase in brain, nerve, and muscle.

Two-site methods were developed for immunoassay of acetylcholinesterase (AChE; EC 3.1.1.7) in crude extracts of rat and human tissues. A radiometric assay for human AChE utilized a specific monoclonal AChE antibody adsorbed to polystyrene microtiter wells at alkaline pH. AChE bound strongly to this antibody after 24 h at 4 degrees C. Bound enzyme was detected with an 125I-labeled antibody against a different AChE epitope. The assay signal was quasi-linearly related to AChE concentration in purified and crude samples, with a detection threshold near 100 pg. Tetrameric and dimeric AChE behaved equivalently in the assay. Two-site methods with a different pair of species-selective antibodies worked equally well for immunoassay of rat AChE. Assays of the rat enzyme showed that immunoreactivity was lost as rapidly as enzyme activity during heating to 54 degrees C. On the other hand, immunoreactivity was preserved despite loss of enzyme activity after exposure to anticholinesterases or trypsin. A biotinylated second antibody detected by alkaline-phosphatase-conjugated avidin was used to develop an AChE enzyme-linked immunosorbent assay (ELISA) with a sensitivity similar to that of the radiometric assay. Either the ELISA or the radiometric immunoassay may be useful whenever proteolysis or other mechanisms are suspected of dissociating enzyme activity and immunoreactivity. In denervated muscle and ligated peripheral nerve, application of the two-site method showed closely parallel variations in immunoreactivity and enzyme activity.