Effects of the mitochondria-targeted antioxidant mitoquinone in murine acute pancreatitis.

Although oxidative stress has been strongly implicated in the development of acute pancreatitis (AP), antioxidant therapy in patients has so far been discouraging. The aim of this study was to assess potential protective effects of a mitochondria-targeted antioxidant, MitoQ, in experimental AP using in vitro and in vivo approaches. MitoQ blocked H2O2-induced intracellular ROS responses in murine pancreatic acinar cells, an action not shared by the control analogue dTPP. MitoQ did not reduce mitochondrial depolarisation induced by either cholecystokinin (CCK) or bile acid TLCS, and at 10 µM caused depolarisation per se. Both MitoQ and dTPP increased basal and CCK-induced cell death in a plate-reader assay. In a TLCS-induced AP model MitoQ treatment was not protective. In AP induced by caerulein hyperstimulation (CER-AP), MitoQ exerted mixed effects. Thus, partial amelioration of histopathology scores was observed, actions shared by dTPP, but without reduction of the biochemical markers pancreatic trypsin or serum amylase. Interestingly, lung myeloperoxidase and interleukin-6 were concurrently increased by MitoQ in CER-AP. MitoQ caused biphasic effects on ROS production in isolated polymorphonuclear leukocytes, inhibiting an acute increase but elevating later levels. Our results suggest that MitoQ would be inappropriate for AP therapy, consistent with prior antioxidant evaluations in this disease.

Bile acids induce pancreatic acinar cell injury and pancreatitis by activating calcineurin.

Biliary pancreatitis is the leading cause of acute pancreatitis in both children and adults. A proposed mechanism is the reflux of bile into the pancreatic duct. Bile acid exposure causes pancreatic acinar cell injury through a sustained rise in cytosolic Ca(2+). Thus, it would be clinically relevant to know the targets of this aberrant Ca(2+) signal. We hypothesized that the Ca(2+)-activated phosphatase calcineurin is such a Ca(2+) target. To examine calcineurin activation, we infected primary acinar cells from mice with an adenovirus expressing the promoter for a downstream calcineurin effector, nuclear factor of activated T-cells (NFAT). The bile acid taurolithocholic acid-3-sulfate (TLCS) was primarily used to examine bile acid responses. TLCS caused calcineurin activation only at concentrations that cause acinar cell injury. The activation of calcineurin by TLCS was abolished by chelating intracellular Ca(2+). Pretreatment with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl ester) (BAPTA-AM) or the three specific calcineurin inhibitors FK506, cyclosporine A, or calcineurin inhibitory peptide prevented bile acid-induced acinar cell injury as measured by lactate dehydrogenase leakage and propidium iodide uptake. The calcineurin inhibitors reduced the intra-acinar activation of chymotrypsinogen within 30 min of TLCS administration, and they also prevented NF-κB activation. In vivo, mice that received FK506 or were deficient in the calcineurin isoform Aß (CnAß) subunit had reduced pancreatitis severity after infusion of TLCS or taurocholic acid into the pancreatic duct. In summary, we demonstrate that acinar cell calcineurin is activated in response to Ca(2+) generated by bile acid exposure, bile acid-induced pancreatic injury is dependent on calcineurin activation, and calcineurin inhibitors may provide an adjunctive therapy for biliary pancreatitis.

Longitudinal zipping/unzipping of self-assembled organic tubes.

Stimuli-responsive organic tubes are an attractive supramolecular assembly which has potential applications as a controlled release vehicle. We synthesize a smart organic tube by the coassembly of lithocholic acid (LCA) and taurolithocholic acid (TLCA) in aqueous solution. The coassembled LCA/TLCA tubes can be longitudinally unzipped into flat sheets by capillary force after being dehydrated on substrates. Consequently, the encapsulated guest molecules are released from the unzipping tubes. After the release of guest molecules, the flat sheets can be zipped back into hollow tubes upon hydration with aqueous solution. The zipping/unzipping LCA/TLCA tubes provide a new type of delivery vehicles, which may have potential for surface decontaminations.

Complexation of tauro- and glyco-conjugated bile salts with three neutral beta-CDs studied by ACE.

Complexation of the bile salts (BS) taurocholate, tauro-beta-muricholate, taurodeoxycholate, taurochenodeoxycholate, glycocholate, glycodeoxycholate, and glycochenodeoxycholate common in rat, dog, and man with natural beta-CD and the chemically modified beta-CDs 2-hydroxypropyl-beta-CD and 2-O-methyl-beta-CD was studied using mobility shift ACE. The CDs were selected due to their frequent use in preformulation and drug formulation as oral excipients for the solubilization of drug substances with low aqueous solubility. ACE was demonstrated to be a feasible and efficient technique for investigation of the interactions between BS and beta-CDs. All the investigated BS possessed affinity for the three CDs with stability constants ranging from 2x10(3) to 4x10(5) M(-) (1). The requirements and assumptions related to the use of ACE for estimating high affinity stability constants were discussed. The extent and pattern of hydroxylation significantly influenced the affinity of the glyco- and tauro-conjugated BS toward the beta-CDs (chenodeoxycholates >> deoxycholates > cholates) whereas the nature of the beta-CD derivatization and BS conjugation played a minor role only. The results indicate that displacement of drug substances from beta-CD inclusion complexes is likely to occur in the small intestine where BS are present potentially influencing drug bioavailability.

Hydrophobic bile salts trigger ceramide formation through endosomal acidification.

Hydrophobic bile salts activate NADPH oxidase through a ceramide- and PKCzeta-dependent pathway as an important upstream event of bile salt-induced hepatocyte apoptosis. The mechanisms underlying bile salt-induced ceramide formation have remained unclear to date and thus were studied in rat hepatocytes. Proapoptotic bile salts, such as taurolithocholylsulfate (TLCS), lowered the apparent pHves within seconds from 6.0 to 5.6 in an FITC-dextran-accessible endosomal compartment that also contains acidic sphingomyelinase. Simultaneously, a rapid decrease in N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) fluorescence was observed, suggestive of an increase in cytosolic [Cl-], which is known to activate vacuolar-type H+-ATPase. No vesicular acidification or increase in cytosolic [Cl-] was found in response to the non-apoptotic bile salt taurocholate or the anti-apoptotic bile salt tauroursodesoxycholate. Inhibition of TLCS-induced endosomal acidification by bafilomycin or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid largely abolished the TLCS-induced ceramide-formation and downstream ceramide-dependent processes, such as p47phox-serine phosphorylation, NADPH oxidase activation, CD95 activation and apoptosis. These responses were also abolished after knockdown of acidic sphingomyelinase in rat hepatocytes. In conclusion, hydrophobic, proapoptotic bile salts stimulate ceramide formation through chloride-dependent acidification of endosomes, with subsequent activation of acidic sphingomyelinase. Our data suggest that changes in ion homeostasis underlie the stimulation of ceramide formation in response to hydrophobic bile acids as an important upstream event of bile salt-induced apoptosis.

Effect of drugs on cholesterol crystallization in an artificial bile model and relation of this effect to drug binding to albumin.

Substances that can affect the crystallization of cholesterol from human bile and consequently the gallstone formation have been given considerable attention. We improved the model system for testing cholesterol crystallization-affecting activity (promoting or inhibiting) of substances and used it for some drugs that are excreted into bile. Besides other factors natural lipid-protein complexes isolated from the native human bile have been shown to be responsible for nucleation and fast crystal growth in cholesterol supersaturated model bile. Artificial lipid-protein complex of taurolithocholate, human serum albumin and Ca2+ (TLTC-HSA-Ca2+) exhibited a lower crystallization activity than both the artificial lipid-protein complexes of taurodeoxycholate, human serum albumin and Ca2+ and the lipid-protein complex isolated from native human bile. The model bile supplemented with this artificial lipid-protein complex (TLTC-HSA-Ca2+) formed a convenient system for testing of various substances (drugs) for their crystallization-affecting activity. From the 20 tested drugs, which could occur at least in small amounts in human bile, the highest crystallization-promoting activity was found for complexes with ampicillin, butorphanol and colchicine. Complexes with tetracycline, thioridazine and doxycycline were the strongest inhibitors. The drugs, which had some effect on cholesterol crystallization, affected somehow the artificial lipid-albumin complex by displacing its components. Interactions of different drugs with HSA and its artificial complexes with the conjugated bile salt and Ca2+ ions were followed by absorption spectroscopy to observe displacement interactions. On the basis of these experiments we could classify drugs into four groups which differ by their effects on spectral characteristics of complexes.

Sensitivity and selectivity of neurons in the medial region of the olfactory bulb to skin extract from conspecifics in crucian carp, Carassius carassius.

To examine the functional subdivision of the teleost olfactory bulb, extracellular recordings were made from the posterior part of the medial region of the olfactory bulb in the crucian carp, Carassius carassius. Bulbar units classified as type I or type II were frequently and simultaneously encountered at a recording site. Type I units displayed a diphasic action potential (AP) with a relatively small amplitude, a short duration (rise time approximately 1 ms) and high spontaneous activity (2.5 per s). Type II units exhibited an AP with a rise time of approximately 1.8 ms and low spontaneous activity (1.5 per s). The AP of this latter unit was nearly always followed by a slow potential, a characteristic diphasic wave with a rise time of approximately 5 ms. Chemical stimulation of the olfactory organ with a graded series of conspecific skin extract induced an increased firing of the type I units. During the period of increased activity of the type I units, the activity of the type II units was suppressed. Stimulation with nucleotides, amino acids and taurolithocholic acid did not induce firing of the type I units of the posterior part of the medial region of the olfactory bulb. These results indicate that the posterior part of the medial region of the olfactory bulb is both sensitive to and selective for skin extract from conspecifics, which has been shown to be a potent stimulus inducing alarm behaviour. The results of the present study indicate that recording single unit activity from a particular region of the olfactory bulb is a suitable method for isolating pheromones or other chemical signals that induce specific activity in the olfactory system. The projection of the neurons categorized as type II was determined by antidromic activation of their axons by electrical stimulation applied to the medial bundle of the medial olfactory tract. The anatomical basis of the type I and type II units in the fish olfactory bulb is discussed.

Taurolithocholic acid exerts cholestatic effects via phosphatidylinositol 3-kinase-dependent mechanisms in perfused rat livers and rat hepatocyte couplets.

Taurolithocholic acid (TLCA) is a potent cholestatic agent. Our recent work suggested that TLCA impairs hepatobiliary exocytosis, insertion of transport proteins into apical hepatocyte membranes, and bile flow by protein kinase Cepsilon (PKCepsilon)-dependent mechanisms. Products of phosphatidylinositol 3-kinases (PI3K) stimulate PKCepsilon. We studied the role of PI3K for TLCA-induced cholestasis in isolated perfused rat liver (IPRL) and isolated rat hepatocyte couplets (IRHC). In IPRL, TLCA (10 micromol/liter) impaired bile flow by 51%, biliary secretion of horseradish peroxidase, a marker of vesicular exocytosis, by 46%, and the Mrp2 substrate, 2,4-dinitrophenyl-S-glutathione, by 95% and stimulated PI3K-dependent protein kinase B, a marker of PI3K activity, by 154% and PKCepsilon membrane binding by 23%. In IRHC, TLCA (2.5 micromol/liter) impaired canalicular secretion of the fluorescent bile acid, cholylglycylamido fluorescein, by 50%. The selective PI3K inhibitor, wortmannin (100 nmol/liter), and the anticholestatic bile acid tauroursodeoxycholic acid (TUDCA, 25 micromol/liter) independently and additively reversed the effects of TLCA on bile flow, exocytosis, organic anion secretion, PI3K-dependent protein kinase B activity, and PKCepsilon membrane binding in IPRL. Wortmannin also reversed impaired bile acid secretion in IRHC. These data strongly suggest that TLCA exerts cholestatic effects by PI3K- and PKCepsilon-dependent mechanisms that are reversed by tauroursodeoxycholic acid in a PI3K-independent way.