Institute of Experimental Physiology, CONICET-National University of Rosario, Rosario (S2002LRL), Argentina
1 To whom correspondence should be addressed at Instituto de Fisiología Experimental (IFISE), Facultad de Ciencias Bioquímicas y Farmacéuticas, Suipacha 570, S2000LRL Rosario, Argentina. Fax: +54-341-4399473. E-mail: mroma{at}fbioyf.unr.edu.ar.
Received June 7, 2004; accepted September 9, 2004
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ABSTRACT |
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Key Words: cytosolic calcium; hepatocyte; hydrophobic bile salt; necrosis; oxidative stress; protein kinase.
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INTRODUCTION |
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Hydrophobic BSs induce either necrosis or apoptosis, depending on the severity of the injury (Benz et al., 1998). These mechanisms of cell death may initiate or aggravate the original hepatocellular damage in cholestatic liver diseases, which results in hepatocellular retention of BSs, along with other normal bile constituents.
BS-induced necrosis, as indicated by hepatocellular enzyme release and morphologic signs of membrane destruction, occurs at high BS concentrations, capable to surpass the critical micellar concentration (Benz et al., 1998); this is a prerequisite for conjugated BSs to exert their detergent effects, by actively incorporating membrane cholesterol and phospholipids into the micellar hydrophobic core (Coleman, 1987
). In contrast, apoptosis is induced by low BS concentrations (Benz et al., 1998
); this process involves vesicular trafficking of Fas death receptor from the cytosol to the cell membrane, their further oligomerization, and the initiation of the caspase-dependent death-signaling pathway (Sodeman et al., 2000
). Considering the differential conditions at which BSs induce both cell death mechanisms, it seems likely that necrosis is the main mechanism of BS-induced cell death in severe cholestasis, whereas apoptosis would be predominant in less severe cholestatic conditions (Benz et al., 1998
).
Several experimental studies where BSs were administered at high doses either into the whole rat (Drew and Priestly, 1979), the isolated perfused rat liver (Baumgartner et al., 1992
; Yousef et al., 1987
), isolated hepatocytes (Scholmerich et al., 1984
), or membrane fractions (Scholmerich et al., 1984
) have shown that these compounds induce extensive membrane damage, as assessed by the release of membrane lipids, intracellular protein and, in the first two cases, bile secretory failure. In these studies, BS efficiency to induce membrane damage was shown to depend critically on BS hydrophobicity; lipophilic BSs are highly cytotoxic, whereas hydrophilic BSs have low, if any, cytotoxic effect (Scholmerich et al., 1984
).
The necrotic damage induced by hydrophobic BSs also depends on their capability to induce oxidative stress and membrane lipid peroxidation (Sokol et al., 1993, 1998
, 2001
). BSs have toxic effects on mitochondria by inducing formation of mitochondrial permeability transition (MPT) pores; this leads to collapse of the mitochondrial inner transmembrane potential, rupture of the outer membrane, blockage of the mitochondrial respiratory chain and, eventually, leakage of electrons with formation of reactive oxygen species (ROS) (Botla et al., 1995
). Both oxidative stress and MPT formation interact positively with each other, as ROS favor MPT formation, and MPT induces further ROS formation, by impairing of mitochondrial respiration (Sokol et al., 2001
). Mitochondrial dysfunction seems to be a common event in both BS-induced necrosis and apoptosis (Lemasters et al., 2002
).
BSs are able to evocate a number of signal-transduction cascades, including the Ca2+-dependent and the PKC-dependent signaling pathways. On the other hand, changes in signaling balance modify transport events involved in the hepatic handling of BSs, thus affecting their steady-state cytosolic concentrations (for a review, see Bouscarel et al., 1999). It is therefore not surprising that some BS-deleterious effects are associated, at least in part, to signal-transduction imbalance. Indeed, glycochenodeoxycolate-induced apoptosis is associated with PKC activation (Jones et al., 1997
). In addition to promote pro-apoptotic signaling pathways, BSs activate signaling cascade of opposite nature. For example, BSs activate the antiapoptotic signaling molecule, phosphoinositide-3-kinase (PI3K) (Rust et al., 2000
). Similarly, cAMP prevents BS-induced apoptosis in a PKA- and a PI3K-dependent manner (Webster et al., 2002
).
The recognition that both apoptosis and necrosis share common mechanisms of induction and that signaling pathways are involved in BS-induced apoptosis prompted us to assess the participation of signal-transduction cascades in BS-induced necrosis. For this purpose, we studied, in isolated rat hepatocytes, whether activation and/or inhibition of PKA-, PKC-, and Ca2+-dependent signaling cascades, three pathways known to influence BS-induced apoptosis, can modulate BS-induced necrotic damage as well. Since oxidative stress is thought to be a main mechanism of BS-induced hepatotoxicity (Sokol et al., 1993, 1998
, 2001
), we also ascertained whether these signal-transduction pathways influence the capability of BSs to induce lipid peroxidation, a key event in oxidative stress-induced damage.
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MATERIALS AND METHODS |
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Animals. Wistar male rats 120130 days of age (300350 g) were used throughout. Before the experiments, the animals were maintained on a standard diet (Purina Laboratory Rodent Chow 5001, Purina Mills, Inc., St. Louis, MO) and water ad libitum, and housed in a temperature- (2123°C) and humidity- (4550%) controlled room, under a constant 12 h-light, 12 h-dark cycle. All animals received humane care according to the criteria outlined in the Guide for Care and Use of Laboratory Animals (National Institutes of Health publication 2528, revised 1996).
Isolation of hepatocytes. Hepatocytes were isolated from livers by the collagenase perfusion technique, using a modification of the method of Berry and Friend (1969). Briefly, under sodium pentobarbital anesthesia (50 mg/kg body wt, ip), heparin was administered in the inferior vena cava (1500 U/kg of body weight), and a 14G catheter (Abbocath-T, Venisystem, Abbocath Ireland Ltd., Sligo, Ireland) was introduced in the portal vein. This was followed by a non-recirculant, portal perfusion of the liver for 10 min with a Ca2+-free, oxygenated (95% O2/5% CO2) Hanks' solution, pH = 7.477.50, supplemented with HEPES (3 g/l) and EGTA (0.24 g/l). The livers were perfused for a further 5-min period with the same solution without EGTA, which was supplemented with 1 mM MgSO4, 2.5 mM CaCl2 and collagenase type IV (4300 U/l). Finally, the livers were removed, and the cells isolated by mechanical dissociation by gently stirring with a glass stick for 34 min. Hepatocytes were further purified from non-parenchymal cells by low-speed centrifugation (50 x g, 2 min), followed by three consecutive washings in oxygenated Hanks' solution containing 2.5 mM CaCl2 and 5 mM Tris. The resulting preparation yielded
400600 x 106 hepatocytes per liver of high viability (>90%), as assessed by the trypan blue exclusion test (Baur et al., 1975
).
Treatments. Hepatocytes were resuspended in Krebs-Ringer-HEPES buffer, pH = 7.4, supplemented with 0.5% D-glucose and 3% BSA, to reach a final density of 2.5 x 105 cells/ml (unless otherwise indicated). The suspension was kept on ice no longer than 30 min before use. Four ml of this suspension were incubated without or with the hydrophobic BS, TCDC (0.25, 0.50, 1.00, and 1.50 mM) for 2 h in 20 ml in plastic beakers, immersed in a Dubnoff water bath at 37°C, under an atmosphere of 95% O2/5% CO2; TCDC was used as a tool, since it was shown to induce a dose- and time-dependent necrotoxic effect to hepatocytes, as apparent from loss of cell viability and leakage of cytosolic enzymes (Ohiwa et al., 1993; Sokol et al., 1993
). The selection of the concentrations and the time of exposure of TCDC was based upon a previous study by Ohiwa et al. (1993)
, which showed that necrotoxic changes occur in hepatocytes at TCDC concentrations higher than 0.1 mM, and at exposure periods longer than 1 h.
The effect of pre-incubation of the hepatocytes with a number of signaling modulators was studied to ascertain the respective roles of PKA-, PKC-, and the Ca2+-dependent signal pathways in the necrotic effect of TCDC. The compounds tested, their biological effects, their final concentrations and the volume and kind of vehicle used for delivery are also indicated in Table 1. Hepatocytes were pre-incubated with the signaling modulators for 15 min, and then exposed to increasing TCDC concentrations for a further 2-h period. The signaling modulators were kept in the incubation medium throughout TCDC exposure.
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Analytical Methods
Assessment of hepatocellular integrity. At the end of the incubation period with TCDC, aliquots of hepatocytes were removed to assess cell viability, leakage of the cytosolic enzymes, lactate dehydrogenase (LDH) and alanine aminotransferase (ALAT), as well as the release of the plasma membrane-associated protein, alkaline phosphatase (AP).
Hepatocyte viability was assessed by the trypan blue exclusion test (Baur et al., 1975). For this purpose, 5 µl of cell suspension were added to 150 µl of trypan blue (1.3 g/l), dissolved in HEPES-supplemented Hanks' solution. Viability was calculated as the percentage of hepatocytes able to exclude the dye from their cell bodies, referred to the values recorded in control cells not exposed to TCDC.
Impairment of barrier properties of the hepatocellular plasma membrane is a chief event in cellular necrosis. To evaluate plasma membrane integrity, leakage of the cytosolic enzymes, LDH (EC 1.1.1.27) and ALAT (EC 2.6.1.2), into the incubation medium was assessed. These enzymes were determined spectrophotometrically in the incubation medium (Perkin Elmer UV/Vis Spectrometer Lambda2S, Überlingen, Germany), by measuring the rate of NADH consumption at 340 nm using commercial, kinetic kits (Wiener Lab., Rosario, Argentina).
The capability of BSs to impair hepatocellular integrity is associated with their ability to remove membrane lipids, thus releasing plasma membrane-associated proteins into the incubation medium. We evaluated this process by studying the release of the plasma membrane protein, AP (EC 3.1.3.1), assessed by measuring the rate of the AP-catalyzed conversion of p-nitrophenyl phosphate to p-nitrophenol, using a commercial, kinetic kit (Wiener Lab., Rosario, Argentina).
Correction of the inhibition of these enzyme activities by the TCDC present in the reaction medium where enzyme activities had been assessed was carried out. For this purpose, a rat serum sample previously subjected to assessment of LDH, ALAT, and AP activity was used as an internal standard, by adding it into the reaction medium after the enzyme activity in the cell incubation medium had been measured. Serum sample addition increases abruptly the rate of NADH consumption (for LDH and ALAT) or p-nitrophenol apparition (for AP), as it becomes proportional to the sum of the enzyme activities of both extracellular medium and serum. The apparent enzyme activity of the serum sample in the reaction medium subjected to TCDC-induced inhibition () can be therefore calculated as the difference between the slope of NADH consumption (or p-nitrophenol apparition) before and after the serum sample is added into the reaction medium. Inhibition of the activity of the exogenously added enzymes (I) can be then calculated as
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Assessment of lipid peroxidation. ROS production in the presence of TCDC was assessed by measuring generation of lipid peroxidation products, by a modification of the thiobarbituric acid-reactive substances (TBARS) method (Buege and Aust, 1978). Briefly, 0.2 ml of a cell suspension containing 106 cells/ml were added to 0.5 ml of trichloroacetic acid (10% w/v) and 50 µl of the antioxidant, DPPD (60 µM). The resulting supernatant was then added to 1 ml of thiobarbituric acid (0.7% w/v), and heated in a water bath to 100°C for 15 min. After cooling and centrifugation (1000 x g for 10 min), absorbance was measured at 532 nm. A standard curve using 1,1,3,3-tetramethoxypropane, which is converted mol for mol into malondialdehyde (MDA), was routinely run. Protein content in the aliquots of cell suspension used for the assay was measured by the method of Lowry et al. (1951)
. TBARS were then expressed as nmol of MDA equivalents per mg of proteins.
Measurement of intracellular Ca2+ concentration ([Ca2+]i). The effect of the pre-treatment with the intracellular Ca2+ chelator, BAPTA/AM (20 µM, 15 min), on TCDC (1 mM)-induced increase in [Ca2+]i was assessed 15 min after the administration of the BS, using Fura-2/AM as a probe. For this purpose, 2 x 106 hepatocytes were resuspended at 37°C in 3 ml of a PBS buffer solution (pH = 7.4), containing 3 mM CaCl2, and then supplemented with 10 µM Fura-2/AM. Fluorescence intensities (F) were measured by using alternating excitation of 340 and 380 nm, and a fluorescence emission wavelength of 510 nm (3 nm bandwidth), using a spectrofluorometer Shimadzu RF-5301 PC. [Ca2+]i was calculated from the 340 nm/380 nm Fura-2/AM fluorescence intensity ratio (R), according to the following equation (Grynkiewicz et al., 1985):
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Statistical analysis. The results were expressed as mean ± SE. When requirements for parametric analysis were met, a Student's unpaired t-test was used for comparison between two groups; comparisons between groups that did not meet this criterion were made by using the Mann-Whitney's rank sum test. The Kruskal-Wallis' test (one-way ANOVA by ranks) was used when more than two groups were compared, followed by the Dunn's multiple-comparison, post hoc test for pairwise comparisons, if ANOVA reached any statistical significance among groups. P values lower than 0.05 were judged to be significant.
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RESULTS |
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To confirm whether the toxic effect induced by TCDC actually involves activation of the PKC-dependent pathways, we pretreated hepatocytes with the more specific PKC inhibitor, Che. Like H7, this compound partially attenuated the hepatocellular damage, as revealed by the improvement in cell viability and a reduction in the TCDC-induced release of LDH, ALAT, and AP into the incubation medium (Fig. 2). The protective effect of Che reached a significant difference in all the parameters of cell integrity evaluated at the TCDC concentration of 1 mM, although cell viability showed an improvement in a wider range (0.251 mM), with a tendency towards protection with the remaining parameters evaluated. Lack of involvement of PKA inhibition as an artifact in the evaluation of the protective effect of H7 was further confirmed by using the specific PKA inhibitor, KT5720, which failed to affect per se TCDC-induced necrosis (data not shown).
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As depicted in Table 3, TCDC, at the dose of 1 mM, increased more than one order of magnitude lipid peroxidation levels, as assessed by MDA generation. Pretreatment with the ROS scavenger, DPPD (50 µM), completely blocked this increase. As shown in Figure 6, DPPD prevented significantly the drop of cell viability and the release induced by TCDC of the three hepatocellular enzymes studied only at the higher concentration studied (1.5 mM), although a tendency towards protection was apparent at the TCDC concentration of1 mM, which did not reach statistical significance for LDH and ALAT.
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As can be seen in Table 3, 1 mM TCDC increased more than one order of magnitude lipid peroxidation levels, as assessed by MDA generation. This increase was slightly, but significantly, counteracted by the PKC specific inhibitors, Che and staurosporine (SP); TCDC induced only a 8.1- and 8.6-fold increase in MDA generation in the presence of Che and SP treatment, respectively, as compared with the 10-fold increase for TCDC alone. On the contrary, the PKA activator, DB-cAMP, was without effect on this parameter.
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DISCUSSION |
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A likely mechanism for this additional damage is the detergent action of lipophilic BSs on plasma membranes, a contention supported by their well-recognized tensioactive properties, derived from their amphoteric structure. Our results showing a progressive release into the incubation medium by increasing concentrations of TCDC of the plasma membrane-constitutive protein, AP, support this possibility. This enzyme binds to the plasma membrane via a glycan-phosphatidylinositol anchor, which interacts strongly with plasma membrane fatty acids (Low, 1987). At TCDC concentrations higher than its critical micellar concentration (4 µM), like that employed in this study (2501500 µM), AP incorporates into TCDC micelles, which favors its stability and solubility in the extracellular aqueous medium (Coleman, 1987
). Although we cannot rule out a contribution of AP from cells other than hepatocytes present in the cell preparation (e.g., cholangiocytes), this is likely to be negligible, as our isolation procedure yields hepatocyte preparations with high (>95%) purity (Berry and Friend, 1969
).
A cross talk exists between both oxidative stress and signal-transduction pathways (Kamata and Hirata, 1999). Since BSs induce ROS generation (Sokol et al., 1993
, 2001
), it is not surprising that BS-induced hepatocellular damage is influenced by the cellular signaling status. In line with this view, previous studies carried out in primary hepatocyte cultures showed that apoptosis induced by glycochenodeoxycholate is counteracted by PKC inhibitors (Jones et al., 1997
), suggesting that PKC-dependent signaling pathways play a key role in hydrophobic BS-induced apoptosis. Taking into account the existence of common mechanisms between BS-induced apoptosis and necrosis (e.g., MPT formation, oxidative stress), it is possible to infer a similar protective effect of PKC inhibitors on TCDC-induced necrosis. Our results agree with this view. H7, a preferential PKC inhibitor (although it can inhibit in certain extent PKA as well) prevented partially the necrotoxic damage induced by TCDC (see Fig. 1). Furthermore, the specific PKA inhibitor, KT5720, was without effect, suggesting that H7 protective effect depended exclusively on its capability to block PKC activity. This was supported further using the specific PKC inhibitor, Che, which mimicked H7 protective effect. The mechanisms by which PKC inhibition protects against BS-induced necrosis can be multifactorial in nature. Our results showing here that PKC inhibitors prevented partially TCDC-induced ROS formation suggest that mitochondrial ROS production is facilitated somewhat by PKC activation. In line with this observation, lipid peroxidation induced to isolated rat hepatocytes by the oxidizing compound, tert-butyl hydroperoxide (tBOOH) (von Ruecker et al., 1989
), or by the heavy metal, cooper (Mudassar et al., 1992
), was prevented by the PKC inhibitor, H7, and exacerbated by PKC activators. Furthermore, H7 attenuates tBOOH-induced LDH leakage (Mudassar et al., 1992
).
The preventive effect of PKC inhibitors on TCDC-induced lipid peroxidation is, however, rather marginal, suggesting that other mechanisms must be involved. For example, PKC inhibition may favor TCDC efflux into the extracellular medium by stimulating the exocytic discharge of vesicles containing BSs, as PKC inhibits hepatocellular vesicular trafficking (Zegers and Hoekstra, 1998); this mechanism is thought to play a key role in BS overcharging conditions, like that occurring in our experimental setting (Erlinger, 1990
). Furthermore, we have shown that PKC inhibitors blocked, and PKC activators stimulated, vesicle-mediated trafficking of vesicle-containing BS transporters towards the apical hepatocellular pole (Roma et al., 2000
). In concordance with this, a study in isolated rat perfused liver showed that H7-induced PKC inhibition increased biliary excretion of TCDC at a concentration in the perfusate within the range used in this study (1 mM) (Nakazawa et al., 1996
).
Hydrophobic BSs induce elevation of [Ca2+]i by an inositol (1,4,5)triphosphate-independent mechanism (Combettes et al., 1988). Conceptually, Ca2+ elevations can activate different Ca2+-dependent proteases, phospholipases and endonucleases, with the consequent hepatocellular damage. Furthermore, Ca2+-elevations lead to activation of Ca2+-dependent PKC isoforms, which may be involved in TCDC-induced damage as well (see above). Therefore, we analyzed here whether the Ca2+-chelating agent, BAPTA/AM, has any beneficial effect against TCDC-induced hepatocellular necrosis. Despite this compound completely prevented TCDC-induced elevations in [Ca2+]i, the capability of TCDC to induce hepatocellular damage was not attenuated (see Fig. 5). This result, however, should not be conclusively interpreted to indicate that intracellular Ca2+ plays no role in BS-induced cytotoxicity. The predominance of other deleterious mechanisms not influenced by Ca2+ levels, e.g., the detergent properties of TCDC on cellular membranes, may have masked its contribution, particularly shortly after TCDC injury. Indeed, TCDC induced, in the perfused rat liver model, an early (4 min), transient increase in LDH release, followed by a subsequent time- and dose-dependent elevation in this parameter; only the first peak was significantly suppressed by pretreatment with the Ca2+- channel blocker, Ni2+ (Hasegawa et al., 2003
). It is therefore possible that the protective effects of intracellular Ca2+ chelation have been overlooked in our model, which evaluate events occurring later in the necrotic process. Nevertheless, Ca2+ elevations are not a prerequisite for some forms of hepatocellular necrosis to occur, like that following ATP depletion due to metabolic inhibition (Nieminen et al., 1988
).
The second messenger, cAMP, an endogenous activator of the PKA-dependent signaling pathway, was shown to have protective, dose-dependent effects in several models of hepatotoxicity (Kasai et al., 1996). Although its hepatoprotective mechanism/s have not been completely elucidated, its stabilizing effect on intracellular membrane is probably involved (Ignarro et al., 1973
). Furthermore, cAMP inhibits BS-induced apoptosis by blocking caspase activation and cytochrome c release (Webster et al., 2002
). Paradoxically, cAMP exacerbated rather than prevented TCDC-induced necrosis in a dose-dependent fashion (see Fig. 3). Although how this signaling molecule aggravates TCDC-induced damage remains elusive, changes in TCDC hepatocellular bioavailability may be involved. cAMP was shown to stimulate the Na+-dependent BS uptake by the basolateral transporter, Na+-taurocholate cotransporting polypeptide (ntcp), in the isolated rat hepatocyte model. This was attributed to the capability of cAMP to hyperpolarize the plasma membrane via PKA-mediated Na+/K+-ATPase phosphorilation (Edmondson et al., 1985
), and by stimulation of the PKA-dependent translocation of ntcp from an endosomal compartment to the sinusoidal membrane (Webster and Anwer, 1999
). Based upon these previous observations, and our own results showing the PKA dependency of DB-cAMP-induced exacerbation of TCDC-induced necrosis (see Fig. 4), we proposed that putative protective effects of cAMP could have been masked by the simultaneous increase in TCDC intracellular concentration due to enhanced uptake. Our results are in apparent contradiction to a previous study showing a protective effect of cAMP-permeant analogues on TCDC-induced necrotoxicity in hepatocytes cultured overnight (Ohiwa et al., 1993
). The explanation for these varied results may depend on the different experimental conditions employed. Whereas freshly isolated hepatocytes like those used in our study maintain an intact capability to take up BSs, this function decreases significantly under culture conditions (Follmann et al., 1990
). Indeed, uptake of the model bile salt, taurocholate, decreases to approximately half of the value recorded in freshly isolated hepatocytes during a culture period compatible to that used by Ohiwa et al. (1993)
. Therefore, our approach more clearly reflects the situation of the hepatocytes in situ, at least in terms of bile salt uptake.
In summary, the present findings clearly show that modulation of PKC- and PKA-dependent signaling pathways can modify the capability of hydrophobic BSs to induce hepatocellular necrotoxicity; whereas PKC inhibition attenuates BS-induced necrosis, PKA activation exacerbates this harmful effect. This seems to occur either or both by modulating differentially the intracellular availability of these endogenous, harmful compounds or by affecting the pathomechanisms involved in their deleterious effects. Although it is not possible at this point to establish whether these data have relevance to the situation in vivo, our results encourage future application of signaling molecules in the prevention/cure of hepatopathies occurring with elevated hepatocellular levels of endogenous BSs.
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ACKNOWLEDGMENTS |
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