Article Text
Abstract
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 β muricholic acid (β-MC), another 7-β-hydroxylated bile salt, have not previously been studied in TLC cholestasis.
Aims: To study the effect of β-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 β-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 β-MC. BAPTA/AM, a chelator of intracellular Ca2+, 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: β-MC was as effective as TUDC in protecting against TLC cholestasis. Mobilisation of Ca2+ and activation of PKC, but not of PKA, are involved in the anticholestatic effect of the two 7-β-hydroxylated bile salts. The hepatoprotective effects of DBcAMP involved Ca2+ mobilisation, but not PKC or PKA activation.
- cholestasis
- ursodeoxycholate
- muricholate
- DBcAMP cell signalling
- hepatocyte couplets
- cAMP, adenosine 3`:5`-cyclic monophosphate
- CLF, cholyl-lysyl-fluorescein
- DBcAMP (dibutyryl cAMP), N62`-o-dibutyryladenosine 3`:5`-cyclic monophosphate
- BAPTA/AM, 1,2-bis-(o-aminophenoxy)-ethene-N,N,N`,N,-tetra-acetate tetra-(acetomethyl)ester
- DMSO, dimethyl sulphoxide
- cVA, canalicular vacuolar accumulation
- HRP, horseradish peroxidase
- mrp, multidrug resistance protein
- PKA, protein kinase A
- IP3, inositol-1,4,5-trisphosphate
- L-15, Leibovitz-15
- MAPKs, mitogen activated protein kinases
- β-MC, β muricholate
- PDB, phorbol 12,13-dibutyrate
- PKC, protein kinase C
- SP, staurosporine
- SAC, subapical compartment
- TLC, taurolithocholate
- TUDC, tauroursodeoxycholate
- UDC, ursodeoxycholate
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- cAMP, adenosine 3`:5`-cyclic monophosphate
- CLF, cholyl-lysyl-fluorescein
- DBcAMP (dibutyryl cAMP), N62`-o-dibutyryladenosine 3`:5`-cyclic monophosphate
- BAPTA/AM, 1,2-bis-(o-aminophenoxy)-ethene-N,N,N`,N,-tetra-acetate tetra-(acetomethyl)ester
- DMSO, dimethyl sulphoxide
- cVA, canalicular vacuolar accumulation
- HRP, horseradish peroxidase
- mrp, multidrug resistance protein
- PKA, protein kinase A
- IP3, inositol-1,4,5-trisphosphate
- L-15, Leibovitz-15
- MAPKs, mitogen activated protein kinases
- β-MC, β muricholate
- PDB, phorbol 12,13-dibutyrate
- PKC, protein kinase C
- SP, staurosporine
- SAC, subapical compartment
- TLC, taurolithocholate
- TUDC, tauroursodeoxycholate
- UDC, ursodeoxycholate
Footnotes
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Presented in part at the annual 51st AASLD Meetings, Dallas, Texas, USA, November 2000; published in abstract form (Hepatology 2000;32(pt 2):429A).