Ca(2+)-dependent protein kinase C isoforms are critical to estradiol 17beta-D-glucuronide-induced cholestasis in the rat

Abstract

The endogenous estradiol metabolite estradiol 17beta-D-glucuronide (E(2)17G) induces an acute cholestasis in rat liver coincident with retrieval of the canalicular transporters bile salt export pump (Bsep, Abcc11) and multidrug resistance-associated protein 2 (Mrp2, Abcc2) and their associated loss of function. We assessed the participation of Ca(2+)-dependent protein kinase C isoforms (cPKC) in the cholestatic manifestations of E(2)17G in perfused rat liver (PRL) and in isolated rat hepatocyte couplets (IRHCs). In PRL, E(2)17G (2 mumol/liver; intraportal, single injection) maximally decreased bile flow, total glutathione, and [(3)H] taurocholate excretion by 61%, 62%, and 79%, respectively; incorporation of the specific cPKC inhibitor Gö6976 (500 nM) in the perfusate almost totally prevented these decreases. In dose-response studies using IRHC, E(2)17G (3.75-800 muM) decreased the canalicular vacuolar accumulation of the Bsep substrate cholyl-lysylfluorescein with an IC50 of 54.9 +/- 7.9 muM. Gö6976 (1 muM) increased the IC50 to 178.4 +/- 23.1 muM, and similarly prevented the decrease in the canalicular vacuolar accumulation of the Mrp2 substrate, glutathione methylfluorescein. Prevention of these changes by Gö6976 coincided with complete protection against E(2)17G-induced retrieval of Bsep and Mrp2 from the canalicular membrane, as detected both in the PRL and IRHC. E(2)17G also increased paracellular permeability in IRHC, which was only partially prevented by Gö6976. The cPKC isoform PKCalpha, but not the Ca(2+)-independent PKC isoform, PKCepsilon, translocated to the plasma membrane after E(2)17G administration in primary cultured rat hepatocytes; Gö6976 completely prevented this translocation, thus indicating specific activation of cPKC. This is consistent with increased autophosphorylation of cPKC by E(2)17G, as detected via western blotting.

Conclusion: Our findings support a central role for cPKC isoforms in E(2)17G-induced cholestasis, by inducing both transporter retrieval from the canalicular membrane and opening of the paracellular route.

Fig. 1. Gö6976 protects against E₂17G-induced inhibition of bile flow and biliary secretion of GSH and taurocholate in the perfused rat liver

Rat livers were perfused as described in Methods and the effects of E₂17G on bile secretory function determined in the presence and absence of the PKCα inhibitor, Gö6976 (500 nM), or its solvent (DMSO). Panels A, B and C show the effect of the treatments on bile flow, total GSH excretion and [³H]taurocholate excretion, respectively. Gö6976 alone did not induce any changes in these parameters (data not shown). Results are expressed as mean ± S.E.M, n = 4. ^aSignificantly different from E₂17G; ^bsignificantly different from control.

Fig. 2. Gö6976 partially prevents E₂17G-induced impairment of the canalicular accumulation of CLF (A) and E₂17G-induced increase in paracellular permeability in isolated rat hepatocyte couplets (B)

A- IRHC were preincubated with Gö6976 (1 μM) for 15 min and then exposed to E₂17G (3.75–800 μM) for an additional 20-min. Canalicular accumulation of CLF was determined as the percentage of couplets displaying visible fluorescence in their canalicular vacuoles from a total of at least 200 couplets per preparation and expressed as a percentage inhibition of canalicular accumulation, where 0 correspond to controls. Data are shown as mean ± S.E.M, n = 3. B- Effects of E₂17G on paracellular permeability and the protection provided by Gö6976 were determined as described in Methods. Results are expressed as mean ± S.E.M, n = 4. ^aSignificantly different from control; ^bsignificantly different from E₂17G.

Fig. 3. Confocal images of E₂17G-induced retrieval of Mrp2 and Bsep and protection by Gö6976

Representative confocal images of immunostained liver samples displaying a co-staining of Bsep (green) and occludin (red) (left panels), and Mrp2 (red) and ZO-1 (green) (right panels). In control livers, both Bsep and Mrp2 were mainly confined to the canalicular space delineated by the tight junction-associated proteins occludin or ZO-1, respectively. Following E₂17G (2 μmol/liver), some canaliculi show intracellular fluorescence associated with Bsep or Mrp2 (arrows) at a greater distance from the canalicular membrane, consistent with their endocytic retrieval. Gö6976 prevented the internalization of canalicular transporters, as illustrated by a control-like pattern of Bsep and Mrp2 distribution. Gö6976 itself did not induce any changes in transporters localization (images not shown). White bar = 10 μm.

Fig. 4. Densitometric analysis of localization of Bsep, occludin, Mrp2 and ZO-1 in perfused livers

Profiles of Bsep (left, top) and Mrp2 (right, top) associated fluorescence in confocal images shown in Figure 3. Graphs represent the intensity of fluorescence associated with the transporters along an 8 μm line (from −4 μm to +4 μm of the canalicular center) perpendicular to the canaliculus. In control livers, transporter-associated fluorescence was concentrated in the canalicular space (from −1 μm to +1 μm). E₂17G-induced retrieval of transporters from the canalicular membrane (p < 0.0001 vs control) was detected as a decrease in the fluorescence intensity in the canalicular area together with an increased fluorescence at a greater distance from the canaliculus (from ± 1 μm to ± 2 μm). Distribution profiles of livers treated with E₂17G+Gö6976 showed a significantly decreased Mrp2 internalization (p < 0.01 vs E₂17G, p < 0.0001 vs control). Bsep distribution did not differ from controls in E₂17G+Gö6976 group (p < 0.0001 vs E₂17G). n = 20–50 canaliculi per preparation, 3 independent preparations. Statistical analysis of the distribution profiles of tight junction associated proteins, used to demarcate limits of the canaliculi, occludin (left, bottom) and ZO-1 (right, bottom) (for Bsep and Mrp2, respectively) showed no changes in the normal distribution by any of the treatments.

Fig. 5. Gö6976 prevents E₂17G-induced retrieval of Bsep and Mrp2 in isolated rat hepatocyte couplets

Isolated rat hepatocyte couplets were preincubated with Gö6976 (1 μM) and then exposed to E₂17G (50 μM), and fixed and immunostained as described in Experimental Procedures. Graphs depict quantitative analysis of transporter-associated fluorescence along a line (8 μm) perpendicular to the canalicular vacuole. Statistical analysis of the profiles revealed a significant internalization of Bsep and Mrp2 under E₂17G treatment (p < 0.0001 vs control), which was completely abolished by Gö6976 (p < 0.0001 vs E₂17G). Results are expressed as mean ± S.E.M. n = 15–25 canalicular vacuoles per preparation, 3 independent preparations.

Fig. 6. Specific PKCα activation by E₂17G in primary cultured hepatocytes

A Immunoblots of PKCα and PKCε in membrane (60 μg protein) and cytosolic (40 μg protein) fractions. Primary cultured hepatocytes were treated with E₂17G (50 μM) for 5, 10, 15 or 20 min, and PKC isoform distribution evaluated. The bar graph shows membrane bound PKCα and PKCε expressed as percentage of total cellular content. B – Immunoblots of PKCα in membrane and cytosolic fractions. Primary cultured hepatocytes were treated with E₂17G (50 or 200 μM) for 10 min, in the presence or absence of Gö6976 (1 μM). The bar graph shows membrane bound PKCα expressed as percentage of total cellular content. C – Representative immunoblot of PKCε in membrane and cytosolic fractions of primary cultured hepatocytes treated for 10 min with E₂17G (50 μM) or the PKC activator PMA (1 μM). The bar graph shows membrane bound PKCε expressed as percentage of total cellular content. D – Representative immunoblots and quantitation of the phosphorylated forms of cPKC in total cellular extracts from E₂17G- (50 μM, 10 min) and PMA- (1 μM, 10 min) treated primary cultured rat hepatocytes. Results are expressed as mean ± S.E.M., n = 3. ^aSignificantly different from control, ^bsignificantly different from E₂17G 50 μM, ^csignificantly different from E₂17G 200 μM.