Bile salts: natural detergents for the prevention of sexually transmitted diseases.
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The development of new, safe, topical microbicides for intravaginal use for the prevention of sexually transmitted diseases is imperative. Previous studies have suggested that bile salts may inhibit human immunodeficiency virus infection; however, their activities against other sexually transmitted pathogens have not been reported. To further explore the potential role of bile salts in preventing sexually transmitted diseases, we examined the in vitro activities and cytotoxicities of select bile salts against Chlamydia trachomatis, herpes simplex virus (types 1 and 2), Neisseria gonorrhoeae, and human immunodeficiency virus in comparison to those of nonoxynol-9 and benzalkonium chloride using both primary cells and cell lines derived from the human female genital tract. We found that taurolithocholic acid 3-sulfate and a combination of glycocholic acid and taurolithocholic acid 3-sulfate showed excellent activity against all of the pathogens assayed. Moreover, taurolithocholic acid 3-sulfate alone or in combination was less cytotoxic than nonoxynol-9 and benzalkonium chloride. Thus, taurolithocholic acid 3-sulfate alone or in combination warrants further evaluation as a candidate topical microbicidal agent. (+info)
Substrates of multidrug resistance-associated proteins block the cystic fibrosis transmembrane conductance regulator chloride channel.
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1. The effects of physiological substrates of multidrug resistance-associated proteins (MRPs) on cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel currents were examined using patch clamp recording from CFTR-transfected mammalian cell lines. 2. Two MRP substrates, taurolithocholate-3-sulphate (TLCS) and beta-estradiol 17-(beta-D-glucuronide) (E217betaG) caused a voltage-dependent block of macroscopic CFTR Cl- currents when applied to the intracellular face of excised membrane patches, with mean apparent dissociation constants (KDs) of 96+/-10 and 563+/-103 microM (at 0 mV) respectively. The unconjugated bile salts taurocholate and cholate were also effective CFTR channel blockers under these conditions, with KDs of 453+/-44 and 3760+/-710 microM (at 0 mV) respectively. 3. Reducing the extracellular Cl- concentration from 154 to 20 mM decreased the KD for block intracellular TLCS to 54+/-1 microM, and also significantly reduced the voltage dependence of block, by suggesting that TLCS blocks Cl- permeation through CFTR by binding within the channel pore. 4. Intracellular TLCS reduced the apparent amplitude of CFTR single channel currents, suggesting that the duration of block is very fast compared to the gating of the channel. 5. The apparent affinity of block by TLCs is comparable to that of other well-known CFTR channel blockers, suggesting that MRP substrates may comprise a novel class of probes of the CFTR channel pore. 6. These results also suggest that the related proteins CFTR and MRP may share a structurally similar anion binding site at the cytoplasmic face of the membrane. (+info)
Characterization of bile acid transport mediated by multidrug resistance associated protein 2 and bile salt export pump.
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Biliary excretion of certain bile acids is mediated by multidrug resistance associated protein 2 (Mrp2) and the bile salt export pump (Bsep). In the present study, the transport properties of several bile acids were characterized in canalicular membrane vesicles (CMVs) isolated from Sprague--Dawley (SD) rats and Eisai hyperbilirubinemic rats (EHBR) whose Mrp2 function is hereditarily defective and in membrane vesicles isolated from Sf9 cells infected with recombinant baculovirus containing cDNAs encoding Mrp2 and Bsep. ATP-dependent uptake of [(3)H]taurochenodeoxycholate sulfate (TCDC-S) (K(m)=8.8 microM) and [(3)H]taurolithocholate sulfate (TLC-S) (K(m)=1.5 microM) was observed in CMVs from SD rats, but not from EHBR. In addition, ATP-dependent uptake of [(3)H]TLC-S (K(m)=3.9 microM) and [(3)H]taurocholate (TC) (K(m)=7.5 microM) was also observed in Mrp2- and Bsep-expressing Sf9 membrane vesicles, respectively. TCDC-S and TLC-S inhibited the ATP-dependent TC uptake into CMVs from SD rats with IC(50) values of 4.6 microM and 1.2 microM, respectively. In contrast, the corresponding values for Sf9 cells expressing Bsep were 59 and 62 microM, respectively, which were similar to those determined in CMVs from EHBR (68 and 33 microM, respectively). By co-expressing Mrp2 with Bsep in Sf9 cells, IC(50) values for membrane vesicles from these cells shifted to values comparable with those in CMVs from SD rats (4.6 and 1.2 microM). Moreover, in membrane vesicles where both Mrp2 and Bsep are co-expressed, preincubation with the sulfated bile acids potentiated their inhibitory effect on Bsep-mediated TC transport. These results can be accounted for by assuming that the sulfated bile acids trans-inhibit the Bsep-mediated transport of TC. (+info)
Transcellular transport of organic anions across a double-transfected Madin-Darby canine kidney II cell monolayer expressing both human organic anion-transporting polypeptide (OATP2/SLC21A6) and Multidrug resistance-associated protein 2 (MRP2/ABCC2).
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Human organic anion transporting polypeptide 2 (OATP2/SLC21A6) and multidrug resistance-associated protein 2 (MRP2/ABCC2) play important roles in the vectorial transport of organic anions across hepatocytes. In the present study, we have established a double-transfected Madin-Darby canine kidney (MDCK II) cell monolayer, which expresses both OATP2 and MRP2 on basal and apical membranes, respectively. The basal-to-apical transport of 17 beta estradiol 17 beta-d-glucuronide (E(2)17 beta G), pravastatin, and leukotriene C(4) (LTC(4)), which are substrates of OATP2 and MRP2, was significantly higher than that in the opposite direction in the double-transfected cells. Such vectorial transport was also observed for taurolithocholate sulfate, which is transported by rat oatp1 and Mrp2. The K(m) values of E(2)17 beta G and pravastatin for the basal-to-apical flux were 27.9 and 24.3 microm, respectively, which were comparable with those reported for OATP2. Moreover, the MRP2-mediated export of E(2)17 beta G across the apical membrane was not saturated. In contrast, basal-to-apical transport of estrone-3-sulfate and dehydroepiandrosterone sulfate, which are significantly transported by OATP2, but not by MRP2, was not stimulated by MRP2 expression. The double-transfected MDCK II monolayer expressing both OATP2 and MRP2 may be used to analyze the hepatic vectorial transport of organic anions and to screen the transport profiles of new drug candidates. (+info)
Bile acids induce calcium signals in mouse pancreatic acinar cells: implications for bile-induced pancreatic pathology.
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The effect of the natural bile acid, taurolithocholic acid 3-sulfate (TLC-S), on calcium signalling in pancreatic acinar cells has been investigated. TLC-S induced global calcium oscillations and extended calcium transients as well as calcium signals localised to the secretory granule (apical) region of acinar cells. These calcium signals could still be triggered by TLC-S in a calcium-free external solution. TLC-S-induced calcium signals were not inhibited by atropine, but were abolished by caffeine or by depletion of calcium stores, due to prolonged application of ACh. Global calcium signals, produced by TLC-S application, displayed vectorial apical-to-basal polarity. The signals originated in the apical part and were then propagated to the basal region. Other natural bile acids, taurocholate (TC) and taurodeoxycholate (TDC), were also able to produce local and global calcium oscillations (but at higher concentrations than TLC-S). Bile, which can enter pancreas by reflux, has been implicated in the pathology of acute pancreatitis. The calcium releasing properties of bile acids suggest that calcium toxicity could be an important contributing factor in the bile acid-induced cellular damage. (+info)
Hepatoprotection with tauroursodeoxycholate and beta muricholate against taurolithocholate induced cholestasis: involvement of signal transduction pathways.
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BACKGROUND: Tauroursodeoxycholate (TUDC) provides partial protection against taurolithocholate (TLC) induced cholestasis, possibly by inducing a signalling cascade activating protein kinase C (PKC). The potential protective effects of beta muricholic acid (beta-MC), another 7-beta-hydroxylated bile salt, have not previously been studied in TLC cholestasis. AIMS: To study the effect of beta-MC on TLC induced cholestasis and also to investigate further the effects of agents affecting intracellular signalling, notably DBcAMP (a cell permeable cAMP analogue) and several protein kinase inhibitors. METHODS: Functional studies were carried out analysing the proportion of hepatocyte couplets able to accumulate the fluorescent bile acid analogue cholyl-lysyl-fluorescein (CLF) into their sealed canalicular vacuole (cVA of CLF assay). RESULTS: It was found that both beta-MC and DBcAMP were as effective as TUDC in protecting against TLC induced cholestasis. The PKC inhibitors staurosporin and H7 but not the specific protein kinase A (PKA) inhibitor KT5720 abolished the protective effects of TUDC and beta-MC. BAPTA/AM, a chelator of intracellular Ca(2+), significantly decreased the protective effect of both bile salts, and that of DBcAMP. PKC and PKA inhibitors had no effect on protection with DBcAMP. CONCLUSIONS: Beta-MC was as effective as TUDC in protecting against TLC cholestasis. Mobilisation of Ca(2+) and activation of PKC, but not of PKA, are involved in the anticholestatic effect of the two 7-beta-hydroxylated bile salts. The hepatoprotective effects of DBcAMP involved Ca(2+) mobilisation, but not PKC or PKA activation. (+info)
Lithocholylcholine, a bile acid/acetylcholine hybrid, is a muscarinic receptor antagonist.
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Previous work from our laboratory indicates that bile acids, specifically lithocholic acid conjugates, interact with muscarinic receptors on gastric chief cells. Structural similarities between acetylcholine and lithocholyltaurine suggest a potential molecular basis for their interaction with the same receptor. We synthesized a hybrid molecule consisting of the steroid nucleus of lithocholyltaurine and the choline moiety of acetylcholine. The new molecule, lithocholylcholine, is hydrolyzed by acetyl-cholinesterase. Lithocholylcholine inhibited binding of a cholinergic radioligand to Chinese hamster ovary cells expressing each of the five muscarinic receptor subtypes. The binding affinities (K(i); micromolar) of lithocholylcholine for these receptors were: M3 (1.0) > M1 (2.7) > M2 (4.1) = M4 (4.9) > M5 (6.2). Lithocholylcholine inhibited intracellular signaling pathways mediated by interaction with M1, M2, and M3 muscarinic receptors. Regarding M3 receptors, lithocholylcholine was 10-fold more potent than lithocholyltaurine in terms of binding affinity and inhibition of acetylcholine-induced increases in inositol phosphate formation and mitogen-activated protein kinase phosphorylation. In a functional assay, lithocholylcholine inhibited acetylcholine-induced relaxation of rat aortic rings. These observations indicate that lithocholylcholine is a muscarinic receptor antagonist and provide further evidence that bile acids may have gastrointestinal signaling functions that extend beyond their effects on sterol metabolism, lipid absorption, and cholesterol elimination. Hybrid molecules created from bile acids and acetylcholine may be used to develop selective muscarinic receptor ligands. (+info)
Functional interaction of lithocholic acid conjugates with M3 muscarinic receptors on a human colon cancer cell line.
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Lithocholic acid (LA) conjugates interact with M3 receptors, the muscarinic receptor subtype that modulates colon cancer cell proliferation. This observation prompted us to examine the action of bile acids on two human colon cancer cell lines: H508, which expresses M3 receptors, and SNU-C4, which does not. Cellular proliferation was determined using a colorimetric assay. Interaction with muscarinic receptors was determined by measuring inhibition of muscarinic radioligand binding and changes in cellular inositol phosphate (IP) formation. Lithocholyltaurine (LCT) caused a dose-dependent increase in H508 cell proliferation that was not observed in SNU-C4 cells. After a 6-day incubation with 300 microM LCT, H508 cell proliferation increased by 200% compared to control. Moreover, in H508 cells, LCT caused a dose-dependent inhibition of radioligand binding and an increase in IP formation. LCT did not alter the rate of apoptosis in H508 or SNU-C4 cells. These data indicate that, at concentrations achievable in the gut, LA derivatives interact with M3 muscarinic receptors on H508 human colon cancer cells, thereby causing an increase in IP formation and cell proliferation. This suggests a mechanism whereby alterations in intestinal bile acids may affect the growth of colon cancer cells. (+info)