Hepatoprotection with tauroursodeoxycholate and beta muricholate against taurolithocholate induced cholestasis: involvement of signal transduction pathways

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 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.

Figure 1

Hepatocyte couplets accumulating cholyl-lysyl-fluorescein (CLF) in the technique of canalicular vacuolar accumulation (cVA) of CLF. (A) Couplet preparation after 15 minutes of incubation with CLF. (B) Couplets exposed to 2.5 μM taurolithocholate (TLC); subsequently incubated with CLF for 15 minutes. (C) Couplets coincubated with TLC and tauroursodeoxycholate (TUDCA) (2 μM); subsequently incubated with CLF for 15 minutes.

Figure 2

Schematic representation of the mechanisms of regulation of hepatocellular signal transduction by the hepatoprotective agents (TUDC, β-MC, and DBcAMP). TUDC, and presumably β-MC, increase cytosolic Ca²⁺ levels by stimulating both Ca²⁺ release from microsomal inositol-1,4,5-trisphosphate (IP₃) sensitive stores via an IP₃ independent pathway and Ca²⁺ influx from extracellular sources via Ni²⁺ sensitive channels, leading to activation of the Ca²⁺ dependent PKC isoform, PKC-α. DBcAMP, a permeable cAMP analogue, is instrumental in both elevating cytosolic Ca²⁺ and activating PKA. Activation of Ca²⁺, PKC, and PKA dependent pathways may mediate reinsertion of canalicular transporters by stimulating their recycling from the SAC; this may counteract localisation changes of these carriers induced by TLC. To assess the role of these different signal transduction pathways, several inhibitors have been used, including the intracellular Ca²⁺ chelator BAPTA/AM, the PKC inhibitors H7 and SP, and the PKA inhibitor KT5720. BAPTA/AM, 1,2-bis-(o-aminophenoxy)-ethene-N,N,N`,N`-tetra-acetate tetra-(acetomethyl)ester; DBcAMP, dibutyryl-cAMP; β-MC, β-muricholate; PKC-α, protein kinase C α; PKA, protein kinase A; SAC, subapical compartment; SP, staurosporine; TUDC, tauroursodeoxycholate.

Figure 3

Effect of inhibitors of protein kinase C (PKC), protein kinase A (PKA), and Ca²⁺ dependent signalling pathways on hepatoprotection induced by tauroursodeoxycholate (TU) against taurolithocholate (TLC) induced decrease in canalicular vacuolar accumulation (cVA) of cholyl-lysyl-fluorescein (CLF). Couplets were incubated for 15 minutes at 37°C with dimethyl sulphoxide (DMSO, control), 1,2-bis-(o-aminophenoxy)-ethene-N,N,N`,N`-tetra-acetate tetra-(acetomethyl)ester (BAPTA/AM (B)), H7, staurosporine (S), or KT5720 (KT). Then, TLC was added to dishes containing DMSO, and TLC+TU were added to the remaining dishes, and the cells were incubated for another 30 minutes. cVA of CLF was then analysed, as described in the methods section. Values are means (SD) (n=5–6). The protective effect of TU on TLC induced cholestasis was significantly decreased by the presence of B, H7, or S, but not by KT. Significantly different from TLC, ***p<0.001; significantly different from TLC+TU, ††p<0.01, †††p<0.001; NS, not significant.

Figure 4

Effect of inhibitors of protein kinase C (PKC), protein kinase A (PKA), and Ca²⁺ dependent signalling pathways on hepatoprotection induced by β-muricholate (MA) against taurolithocholate (TLC) induced decrease in canalicular vacuolar accumulation (cVA) of cholyl-lysyl-fluorescein (CLF). Couplets were incubated for 15 minutes at 37°C with dimethyl sulphoxide (DMSO, control), 1,2-bis-(o-aminophenoxy)-ethene-N,N,N`,N`-tetra-acetate tetra-(acetomethyl)ester (BAPTA/AM (B)), H7, staurosporine (S), or KT5720 (KT). Then, TLC was added to dishes containing DMSO, and TLC+MA were added to the remaining dishes, and cells were incubated for another 30 minutes. cVA of CLF was then analysed. All values are means (SD) (n=5–6). The protective effect of MA on TLC induced cholestasis was significantly decreased by the presence of B, H7, and S, but not by KT. Significantly different from TLC, ***p<0.001; significantly different from TLC+MA, †p<0.05; NS, not significant.

Figure 5

Effect of inhibitors of protein kinase C (PKC), protein kinase A (PKA), and Ca²⁺ dependent signalling pathways on hepatoprotection induced by dibutyryl cAMP (cAMP) against taurolithocholate (TLC) induced decrease in canalicular vacuolar accumulation (cVA) of cholyl-lysyl-fluorescein (CLF). Couplets were incubated for 15 minutes at 37°C with dimethyl sulphoxide (DMSO, control), 1,2-bis-(o-aminophenoxy)-ethene-N,N,N`,N`-tetra-acetate tetra-(acetomethyl)ester (BAPTA/AM (B)), staurosporine (S), KT5720 (KT), or B+KT. Then, TLC was added to the dishes containing DMSO, and TLC+DBcAMP were added to the remaining dishes, and cells were incubated for another 30 minutes. cVA of CLF was then analysed. All values are means (SD) (n=5–6). The protective effect of DBcAMP on TLC induced cholestasis was significantly decreased by the presence of B but not by S or KT. When both KT and B were added together, their inhibiting effect was not greater than B alone. Significantly different from TLC, ***p<0.001; NS, not significant; significantly different from TLC+cAMP, †p<0.05.