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Am J Physiol Gastrointest Liver Physiol 281: G809-G815, 2001;
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Vol. 281, Issue 3, G809-G815, September 2001

Cold preservation-warm reoxygenation increases hepatocyte steady-state Ca2+ and response to Ca2+-mobilizing agonist

Aziz Elimadi and Pierre S. Haddad

Membrane Transport Research Group and Department of Pharmacology, Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada H3C 3J7


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although the role of Ca2+ in liver transplantation injury has been the object of several studies, direct evidence for alterations in intracellular Ca2+ homeostasis after cold preservation-warm reoxygenation (CP/WR) has never been presented. We thus investigated the effects of CP/WR on steady-state Ca2+ and responses to a Ca2+-mobilizing agonist. Isolated rat hepatocytes were suspended in University of Wisconsin solution, stored at 4°C for 0, 24, and 48 h, and reoxygenated at 37°C for 1 h. Cytosolic Ca2+ was measured in single cells by digitized fluorescence videomicroscopy. CP/WR caused a significant increase in steady-state cytosolic Ca2+, which was inversely proportional to cell viability. Pretreatment of hepatocytes with an agent that protects mitochondrial function attenuated the increase in steady-state cytosolic Ca2+ and improved hepatocyte viability. Ca2+ responses to the purinergic agonist ATP also increased significantly as a function of cold storage time. This increase was related to an increase in the size of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores and subsequent capacitative Ca2+ entry. Thus CP/WR significantly perturbs steady-state hepatocellular Ca2+ and responses to Ca2+-mobilizing agonists, which may contribute to hepatocyte metabolic dysfunction observed after CP/WR.

adenosine 5'-trisphosphate; endoplasmic reticulum; inositol 1,4,5-trisphosphate; mitochondria; transplantation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

LIVER TRANSPLANTATION HAS become an accepted therapy for end-stage liver disease. During transplantation, the liver suffers injury not only from cold ischemia due to the long-term cold storage but also from reperfusion with warm oxygenated blood. This results in primary graft dysfunction, which remains a major clinical problem (1, 8, 15). Among hepatocellular perturbations, dysfunction in liver cell volume (30, 40), bile secretion (26), drug metabolism (2), and mitochondrial function (37) has been reported as a result of cold preservation/warm reoxygenation (CP/WR). The exact mechanisms underlying these functional disorders are still not understood, but the involvement of intracellular Ca2+ has been proposed.

The hypothesis that intracellular Ca2+ plays a crucial role in liver transplantation injury is based essentially on the indirect observation that inhibitors of L-type voltage-dependent Ca2+ channels, such as nisoldipine or verapamil, improve the viability of preserved hepatocytes (45) or of liver allografts in the rat (9, 44). However, no evidence has ever been found for the presence of voltage-dependent Ca2+ channels on hepatocytes, either functionally (31) or at the level of mRNA (21). The effects of such Ca2+ channel antagonists may thus result from nonspecific beneficial actions on hepatocytes (22, 29) or from effects on Kupffer cells (20, 43). Hence, direct evidence for the perturbation of hepatocellular Ca2+ homeostasis after CP/WR has never been presented.

On the other hand, recent studies from our laboratory (17) and that of Kim and Southard (25) have shown that acute hypothermia alone leads to a rapid increase in steady-state intracellular Ca2+ in isolated hepatocytes (17). Kim and Southard (25) have shown that, after this initial increase, cytosolic Ca2+ subsequently decreases as cold preservation time is prolonged to 24 and 48 h (25). Both studies (17, 25) addressed perturbation of hepatocyte Ca2+ during simple cold storage. Information is still lacking on the effect of the overall process of liver transplantation, i.e., CP/WR (conditions that the grafted organ normally encounters), on liver cell Ca2+ homeostasis.

Intracellular Ca2+ homeostasis can be modulated by many extracellular stimuli, including ATP and related nucleotides, which have significant biological effects on many tissues and cell types such as hepatocytes. For instance, ATP has been shown to stimulate glycogen phosphorylase activity in hepatocytes, which is the rate-limiting step in liver glycogenolysis (24). ATP is also involved in intercellular signal propagation between hepatocytes and between hepatocytes and bile duct cells in the rat liver (38). Such nucleotides are released from hepatocytes themselves, from sympathetic nerve endings, as well as platelets and damaged cells during injury such as ischemia-reperfusion. Released nucleotides are known to trigger Ca2+ mobilization in hepatocytes (12). This mobilization consists of two phases, namely Ca2+ release from intracellular stores and Ca2+ influx across the plasma membrane (42). Should Ca2+ homeostatic mechanisms be affected by CP/WR, alterations in Ca2+-dependent hepatocyte functions such as metabolism and bile secretion may ensue and form, at least in part, the basis of primary graft dysfunction.

We thus sought to evaluate the impact of long-term CP/WR on hepatocyte Ca2+ homeostasis, particularly at the level of steady-state intracellular Ca2+, and of Ca2+ responses to the purinergic agonist ATP. We used an in vitro model that appropriately mimics CP/WR in liver cells (13, 17, 18, 39-41). We report that CP/WR significantly increases steady-state hepatocellular Ca2+. Responses to the Ca2+-mobilizing agent ATP are also increased, and this was related to an increase in the size of intracellular inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores and in the subsequent capacitative Ca2+ entry.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hepatocyte isolation. Animals used in this study were treated in accordance with the guidelines of the Canadian Council on the Care of Animals, and all experimental protocols were approved by our university's ethics committee.

Hepatocytes were isolated from the livers of fed male Sprague-Dawley rats weighing 200-250 g (Charles River Laboratories, St. Constant, QC, Canada) by a variation of the classical collagenase perfusion as described previously (19). Hepatocytes were purified by centrifugation on a Percoll gradient. The initial viability of hepatocyte preparations averaged 90% as indicated by trypan blue exclusion. Isolated rat hepatocytes were then suspended in University of Wisconsin (UW) solution and stored undisturbed in sealed 50 ml conical tubes at 4°C for 24 or 48 h. At the end of the cold preservation period, UW solution was aspirated and hepatocytes washed in cold Williams' medium E before being plated in warm Williams' medium E onto collagen-coated round glass coverslips at a density of 35 × 104 cells/ml and placed in an incubator at 37°C with continuous reoxygenation. Control (unstored) cells were treated in an identical fashion but were used immediately after isolation.

Digitized fluorescence videomicroscopy for intracellular Ca2+ measurement at single cell level. At the end of a 1-h warm culture period, hepatocytes were loaded with 5 µM fura 2-AM (Molecular Probes, Eugene, OR) for 30 min at room temperature. Coverslips were then mounted on an inverted fluorescence microscope and continually perfused with HEPES buffer (138 mM NaCl, 3.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 5 mM glucose, 1 mM pyruvate, and 10 mM HEPES, pH 7.4, at 25°C) containing 1.8 mM Ca2+. In a subset of experiments, m-iodobenzylguanidine (MIBG) at a concentration of 10 µM was added to the UW solution, the warm culture medium, and the HEPES perfusate buffer. This concentration has been shown to completely protect mitochondrial functions of rat hepatocytes subjected to long-term cold preservation (25). For experiments with EGTA, the latter was added at a concentration of 4 mM to a Ca2+-free perfusion buffer (same composition as HEPES buffer above but without added Ca2+).

Fluorescence signals from isolated hepatocytes were collected using a fluorescence imaging system. The system consisted of the following: an inverted fluorescence microscope (Nikon, TE-300, St. Laurent, QC, Canada) with appropriate fluorescence microscopy objectives; a 75-W mercury lamp source; appropriate excitation filters, dichroic mirrors, and emission filters; a computer-controlled fast excitation wavelength switcher (DX-1000, Stanford Photonics, Palo Alto, CA); an intensified progressive line scan charge-coupled device camera (GenIII+, Stanford Photonics); and a Pentium II personal computer. Ratio imaging of cell fluorescence was performed with excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. The signal was analyzed using computer-imaging software (Axon, Foster City, CA), and pseudocolor images were generated for monitoring and data analysis. Fluorescence ratio values were converted to cytosolic Ca2+ concentrations using an in situ calibration technique with ionomycin-containing solutions and a dissociation constant value of 224 nM for fura 2, as previously described by our laboratory (4) .

IP3-induced intracellular Ca2+ mobilization in permeabilized hepatocyte suspensions. After 0, 24, and 48 h of cold preservation, isolated hepatocytes were subjected to warm reoxygenation in Williams' medium E at 37°C for 1 h. The cells were then incubated at 37°C in the stirred thermostated cuvette of a SPEX model CMT-11I spectrofluorometer (Rayonics Scientific, St. Laurent, QC, Canada) in a "cytosol-like" medium (120 mM KCl, 10 mM NaCl, 1 mM KH2PO4, 1 mM MgCl2, 30 mM HEPES, and 1 mM ATP, pH 7.4, at 37°C). This medium also contained an ATP-generating system (25 mM creatine phosphate, 25 U/ml creatine kinase, and 5 mM sodium pyrophosphate) and 10 µM FCCP, a mitochondrial decoupler, to eliminate the participation of mitochondria in intracellular Ca2+ handling during this experiment. Plasma membranes were permeabilized by adding 50 µg/ml saponin. Permeabilized cells were washed once in the absence of saponin, gently pelleted, and resuspended in 1 ml cytosol-like medium without saponin at a density of 107 cells/ml, as previously described by Missiaen and co-workers (32). IP3-induced Ca2+ mobilization was carried out in the presence of 10 µM fura 2-free acid. IP3 was added at concentrations of 1 to 50 µM. Fluorescence experiments were carried out using excitation wavelengths of 340 and 380 nm and an emission wavelength of 505 nm. Fluorescent signals were captured by photomultiplier tubes, and the data were analyzed using the software supplied by SPEX.

Reagents. All reagents were of the highest quality available and obtained from Sigma-Aldrich Chemical (Mississauga, ON, Canada). Collagenase (type D) was obtained from Roche Diagnostics (Laval, QC, Canada).

Statistical analysis. Each experiment was performed with at least four to eight separate hepatocyte preparations. Values are presented as means ± SE of the indicated number of cells. Statistical analysis was performed using one- or two-way ANOVA as appropriate. P < 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of cold preservation followed by warm reoxygenation on hepatocyte steady-state intracellular Ca2+ and cell viability. Steady-state intracellular free Ca2+ concentration of control cells under our experimental conditions was 95 ± 3 nM (n = 151 cells). As shown in Fig. 1, when hepatocytes were preserved for 24 and 48 h in UW followed by 1 h of warm reoxygenation, steady-state intracellular Ca2+ concentration increased significantly to 224 ± 11 and 286 ± 21 nM, respectively (n = 100 and 130 cells, respectively; P < 0.05 for either group compared with control unstored cells). This increase in hepatocyte intracellular Ca2+ after long-term CP/WR was accompanied by a significant decrease in cell viability (Table 1).


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  		Fig. 1.   Hepatocyte steady-state intracellular Ca2+ after cold preservation followed by warm reoxygenation and effect of m-iodobenzylguanidine (MIBG). Hepatocytes were preserved in cold University of Wisconsin (UW) solution at 4°C for 0, 24, and 48 h and then reoxygenated at 37°C for 1 h in the presence (filled bars) or absence (open bars) of 10 µM MIBG. Intracellular Ca2+ was measured by means of video fluorescence imaging microscopy as described in MATERIALS AND METHODS. Values are means ± SE of 100-150 cells from at least 4 different cell preparations. * P < 0.05, significantly different from unstored controls (0 h); Dagger  P < 0.05, significantly different from respective untreated cells (saline vehicle of MIBG); § P < 0.05, significantly different from corresponding 24-h group.


                              
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  		Table 1.   Effect of MIBG on hepatocyte viability after cold preservation followed by warm reoxygenation

Protection of mitochondrial functions attenuates increase in steady-state intracellular Ca2+ after CP/WR. Mitochondria have been widely implicated in both warm and cold ischemia-reperfusion injury (10, 11, 37) and in intracellular Ca2+ homeostasis (36). Hepatocytes were thus incubated with MIBG, a pharmacological agent shown (25) to protect mitochondrial functions altered by simple cold storage. MIBG was present throughout the CP/WR periods. As shown in Fig. 1, MIBG (10 µM) has no effect on steady-state intracellular Ca2+ in control unstored cells. However, when hepatocytes were preserved for 24 and 48 h at 4°C followed by 1 h of warm reoxygenation in the continued presence of m-IGB, the rise of steady-state intracellular Ca2+ observed in untreated cells was significantly attenuated. Two-way ANOVA uncovered a significant interaction (P < 0.05) between MIBG treatment and cold preservation time. This indicates that the protective effect of MIBG was greater in the 48-h than in the 24-h preserved group. Interestingly, treatment of isolated hepatocytes with MIBG was also associated with a modest yet statistically significant improvement in hepatocyte viability after CP/WR (Table 1). Also, similarly to results on steady-state Ca2+, MIBG did not have any effect on cell viability in unstored control cells.

Effect of CP/WR on hepatocyte Ca2+ responses to the purinergic agonist ATP. ATP is known to act on P2Y2 receptors (previously known as P2U receptors) coupled to the phosphatidylinositol-Ca2+ signaling pathway and to increase cytosolic free Ca2+ concentration in rat hepatocytes (12). ATP (100 µM) induced a typical biphasic Ca2+ response in all cells. Responses were quantified by measuring the area under the Ca2+ vs. time curve (AUC) as shown in Fig. 2. Reoxygenating the hepatocytes after preserving them at 4°C for 24 and 48 h significantly increased the total amount of Ca2+ mobilized by ATP (Fig. 2, A and C). We then used EGTA (4 mM), a known Ca2+ chelator, to eliminate the Ca2+ influx phase of the ATP-induced Ca2+ response and hence to isolate Ca2+ release from intracellular stores. CP/WR also significantly increased the quantity of Ca2+ mobilized by ATP in the presence of EGTA (Ca2+-free conditions, Fig. 2, B and C), indicating a greater release from internal stores. In physiological conditions, the rise in Ca2+ response to ATP was dependent on cold preservation time [P < 0.05, for 24 and 48 h vs. unstored controls (0 h) as well as between 24- and 48-h preservation groups]. On the other hand, in Ca2+-free conditions, the increase in ATP response (AUC) in relation to unstored controls was similar for 24- and 48-h groups. This was confirmed by two-way ANOVA, which uncovered a significant interaction between extracellular Ca2+ status and cold preservation time (P < 0.05). Our results further indicate that the capacitative Ca2+ entry that follows mobilization of internal Ca2+ stores was also increased as a function of cold preservation time as indicated by the significant interaction (P < 0.05) of the two-way ANOVA.


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  		Fig. 2.   Effect of cold preservation followed by warm reoxygenation on hepatocyte Ca2+ responses to ATP. Hepatocytes were preserved in cold UW solution at 4°C for 0, 24, and 48 h and then reoxygenated at 37°C for 1 h. The total amount of intracellular Ca2+ mobilized by 100 µM ATP was measured by video fluorescence imaging microscopy in the absence (A) or presence (B) of 4 mM EGTA, using the area under the Ca2+ vs. time curve (shaded areas in A and B) as described in MATERIALS AND METHODS. Arrows indicate start of 1-min ATP administration. C: values are means ± SE of 103-145 cells from at least 4 different cell preparations. * P < 0.05, significantly different from unstored controls (0 h); Dagger  P < 0.05, significantly different from respective EGTA group; § P < 0.05, significantly different from corresponding 24-h group.

Effect of CP/WR on IP3-sensitive Ca2+ pools. The Ca2+ mobilized from intracellular stores by Gq-coupled receptor agonists, such as ATP, comes mainly from IP3-sensitive Ca2+ pools (42). The results of Fig. 2, B and C, obtained in Ca2+-free conditions thus prompted us to investigate whether the potency (EC50) of IP3 or the size (Emax) of IP3-sensitive Ca2+ pools was affected by CP/WR. For this purpose, we used permeabilized hepatocytes stimulated with exogenous IP3. Figure 3 shows the effects of increasing concentrations of IP3 on Ca2+ mobilization from IP3-sensitive Ca2+ pools in hepatocytes permeabilized after being submitted to CP/WR. As seen from both Fig. 3 and the values presented in Table 2, the Emax of IP3-sensitive Ca2+ pools increased in a statistically significant manner in both groups of cold-preserved cells (24 or 48 h) compared with unstored controls. Interestingly, and in accordance with the results shown in Fig. 2, B and C, there was no difference between the 24- and 48-h groups at the level of the increased Emax for IP3. On the other hand, the EC50 of IP3 was not affected by our experimental conditions (Table 2).


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  		Fig. 3.   Effect of cold preservation followed by warm reoxygenation on Ca2+ mobilized from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools. Hepatocytes were preserved in cold UW solution at 4°C for 24 and 48 h and then reoxygenated at 37°C for 1 h. Hepatocytes were then permeabilized with saponin, and Ca2+ was mobilized from the IP3-sensitive calcium pools by addition of different concentration of synthetic IP3. Ca2+ was measured in cell suspensions using spectrofluorometer as described in MATERIALS AND METHODS. Values are means ± SE from at least 4 groups. Depicted curves are the best nonlinear Michaelian fit of the data means for each group determined with a commercially available software (Micropharm INSERM; 46).


                              
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  		Table 2.   Effect of cold preservation followed by warm reoxygenation on potency and efficacy of IP3


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the current study, we measured hepatocyte intracellular Ca2+ concentration directly at the single cell level in experimental conditions mimicking the CP/WR of liver transplantation. We present, for the first time, direct evidence showing that the intracellular Ca2+ homeostasis of isolated hepatocytes is altered when they are submitted to CP/WR. Indeed, steady-state intracellular Ca2+ concentration is increased after CP/WR of the hepatocytes, and this increase is accompanied by a decrease in hepatocyte viability. Consistent with our observation, an increase in basal intracellular Ca2+ associated with a decrease in cell viability has also been reported (6) during oxidative stress in rat hepatocytes. This brings forth the interesting possibility that production of reactive oxygen species may be implicated in mediating the increase in steady-state cytosolic Ca2+ in hepatocytes subjected to CP/WR.

Recent research (37) has established that liver mitochondria are seriously damaged during CP/WR. Furthermore, mitochondria have been shown (3, 36) to participate actively in intracellular Ca2+ homeostasis. We thus used MIBG as a pharmacological tool to assess the implication of mitochondria in the observed effects of CP/WR on hepatocyte cytosolic Ca2+. This agent is known to prevent mitochondrial Ca2+ efflux in isolated hepatocytes without signs of cytotoxicity (23). MIBG has recently been shown (25) to protect mitochondrial functions altered by cold preservation. In the present study, pretreatment of hepatocytes with MIBG partially but significantly attenuated the elevation in steady-state intracellular Ca2+ observed when hepatocytes were subjected to CP/WR. No effect of this agent was observed on either steady-state intracellular Ca2+ or on cell viability in unstored control cells (0 h). This confirms that MIBG has no effect on hepatocellular Ca2+ and viability under normal physiological conditions. Interestingly, pretreatment of the hepatocytes with MIBG slightly but significantly improved cell viability after CP/WR. This is in accordance with the findings of Kim and Southard (25) who showed that MIBG ameliorates both mitochondrial functions and ATP regeneration in hepatocytes exposed to long-term cold preservation. Cyclosporin A, which possesses the same molecular mechanism as MIBG at the mitochondrial level, has recently been reported (16) to exert similar protective effects against reoxygenation injury in isolated rat cardiomyocytes. Both agents have long been known to inhibit mitochondrial permeability transition (MPT) (47). Furthermore, MPT has been shown to be the hallmark of mitochondrial damage observed in cold preserved-warm perfused isolated rat liver (27) as well as in warm ischemia-reperfusion injury (11). Our results thus support the notion that alterations in mitochondrial function after CP/WR are caused by opening of the MPT pore. This leads to the release of Ca2+ from mitochondria, thus reducing their Ca2+-buffering capacity and increasing intracellular Ca2+ under these pathological conditions. Indeed, treatment of hepatocytes with the mitochondrial protector and MPT inhibitor MIBG enhances mitochondrial function after CP/WR. Consequently, mitochondrial Ca2+ storage capacity is restored, thus helping to partly prevent the rise of steady-state intracellular Ca2+ observed after CP/WR.

To gain further insight into the perturbations of hepatocellular Ca2+ homeostasis after long-term CP/WR, we investigated the effects of these conditions on the Ca2+ responses to the purinergic agonist ATP. The results of the present study clearly demonstrate that compound Ca2+ responses to ATP (as evaluated by AUC analysis) are increased as a function of cold preservation time. As mentioned previously, these compound responses are generally composed of two phases, namely Ca2+ release from intracellular stores and Ca2+ influx across the plasma membrane (42). We therefore used EGTA-induced chelation of extracellular Ca2+ and show that the increase in AUC, observed in response to ATP after CP/WR, was partly due to an increase in agonist-induced Ca2+ release from intracellular stores.

ATP is known to stimulate IP3 formation, which in turn activates Ca2+ release from the endoplasmic reticulum, generally considered as the principal IP3-sensitive Ca2+ store (12). We have therefore used permeabilized hepatocytes challenged with exogenous IP3 to examine the effect of CP/WR on the sensitivity and storage capacity of IP3-sensitive Ca2+ stores. We found that the Emax of the IP3-sensitive Ca2+ pools increased with the time of preservation, whereas the EC50 of IP3 in mobilizing Ca2+ was not altered. Our results thus suggest that responses to any Ca2+-mobilizing agonist (such as alpha 1-adrenergic agonists angiotensin II or vasopressin) will be expected to increase as a function of cold preservation time for a given amount of IP3 produced.

Recent studies (28) have shown that IP3-sensitive Ca2+ pools can be influenced by intracellular Ca2+ level. An increase in steady-state intracellular Ca2+ leads to an increase in the size of IP3-sensitive Ca2+ pools, whereas a decrease in cytosolic Ca2+ reduces the size of these pools. It is therefore possible that the increase in steady-state Ca2+ that we observed after long-term CP/WR leads to a greater capacity of IP3-sensitive Ca2+ pools to store Ca2+ and hence to a greater response to Ca2+-mobilizing agonists. In addition, Missiaen et al. (32) demonstrated that increases in cytosolic Ca2+ sensitize IP3-sensitive pools to IP3, so that the increased steady-state Ca2+ after CP/WR may also have contributed to enhance the response to Ca2+-mobilizing agonists.

Finally, our results suggest that capacitative Ca2+ entry was also increased in a preservation time-dependent manner (the part of the ATP response inhibited by EGTA). Indeed, the Ca2+ entry phase of the response to Ca2+-mobilizing agonists is known to be regulated by the Ca2+ content of the stores, a process referred to as store-operated or capacitative Ca2+ entry (33). It is thus conceivable that part of the rise in cytosolic Ca2+ observed after CP/WR (possibly that part not prevented by MIBG) was related to an increased tonic influx of Ca2+ from the extracellular milieu through conductive pathways.

It is well known that the endoplasmic reticulum plays an important role in Ca2+ storage and signaling and in the folding of newly synthesized membrane and secretory proteins, reactions that are strictly Ca2+ dependent (for review, see Ref. 34). Therefore, a perturbation of the IP3-sensitive Ca2+ stores might alter these functions and participate in the functional disorders of CP/WR. In agreement with this assumption, Pinton et al. (35) recently found that overexpression of the protooncogene Bcl-2 reduces both the loading of intracellular Ca2+ stores and the capacitative Ca2+ influx. In addition, overexpression of the Bcl-2 transgene protects mouse liver and cardiac cells from ischemia-reperfusion injury (5, 7). Thus conditions with a reduced size of IP3-sensitive Ca2+ pools are associated with less ischemia-reperfusion injury. Conversely, a weakness in the functions of the Bcl-2 family of proteins (for instance, reduced inhibition of MPT; 14) could participate in the deleterious effects of CP/WR on hepatocyte Ca2+ homeostasis presented herein.

In conclusion, we find that in hepatocytes, CP/WR causes a significant increase in steady-state cytosolic Ca2+ concentration that is associated with a decrease in cell viability. The elevation in the steady-state Ca2+ can be significantly attenuated by protecting mitochondrial functions. Moreover, responses to the purinergic agonist ATP are also increased, and this is due to an increase in the size of IP3-sensitive Ca2+ pools as well as in capacitative Ca2+ entry. This alteration in both the steady-state intracellular Ca2+ and in the response to Ca2+-mobilizing agonists might contribute to the primary graft dysfunction observed after CP/WR injury of livers undergoing transplantation.


    ACKNOWLEDGEMENTS

We thank Dr. Rémy Sauvé for critical review of the manuscript.


    FOOTNOTES

This work was supported by Canadian Institutes of Health Research Operating Grant MT-14759. A. Elimadi received postdoctoral fellowships from the Membrane Transport Research Group (Research Center of the Quebec Fonds pour la Formation des Chercheurs et l'Aide à la Recherche) and the Canadian Association for the Study of the Liver, graciously provided by AXCAN Pharma (St. Hilaire, QC, Canada). P. S. Haddad is a Senior Research Scholar of the Fonds de la Recherche en Santé du Québec.

A portion of this work was presented at the annual meeting of the American Gastroenterological Association, Digestive Disease Week 2000, San Diego, CA and has been published previously in abstract form (Gastroenterology 118: A913, 2000).

Address for reprint requests and other correspondence: A. Elimadi, Groupe de Recherche en Transport Membranaire and Départment de Pharmacologie, Université de Montréal, PO Box 6128, Downtown Station, Montréal, Québec, Canada H3C 3J7 (E-mail: elimadia{at}magellan.umontreal.ca.).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11 April 2001; accepted in final form 21 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adam, R, Bismuth H, Diamond T, Ducot B, Morino M, Astarcioglu I, Johann M, Azoulay D, Chiche L, Bao YM, and Castaing D. Effect of extended cold ischaemia with UW solution on graft function after liver transplantation. Lancet 340: 1373-1376, 1992[ISI][Medline].

2.   Azoulay, D, Lemoine A, Dennison A, Gries JM, Dolizy I, Barbazza F, Bonhomme L, Beaune P, and Bismuth H. Relationship between cytochrome P-450 3A content of orthotopically transplanted liver and postoperative morbidity. Transplant Proc 25: 2630-2631, 1993[ISI][Medline].

3.   Babcock, DF, Herrington J, Goodwin PC, Park YB, and Hille B. Mitochondrial participation in the intracellular Ca2+ network. J Cell Biol 136: 833-844, 1997[Abstract/Free Full Text].

4.   Benzeroual, K, Van de Werve G, Meloche S, Mathé L, Romanelli A, and Haddad P. Insulin induces Ca2+ influx into isolated rat hepatocytes couplets. Am J Physiol Gastrointest Liver Physiol 272: G1425-G1432, 1997[Abstract/Free Full Text].

5.   Bilbao, G, Contreras JL, Eckhoff DE, Mikheeva G, Krasnykh V, Douglas JT, Thomas JM, and Curiel DT. Reduction of ischemia-reperfusion injury of the liver by in vivo adenovirus-mediated gene transfer of the antiapoptotic Bcl-2 gene. Ann Surg 230: 185-193, 1999[ISI][Medline].

6.   Brecht, M, Brecht C, and de Groot H. Late steady increase in cytosolic Ca2+ preceding hypoxic injury in hepatocytes. Biochem J 283: 399-402, 1992.

7.   Brocheriou, V, Hagege AA, Oubenaissa A, Lambert M, Mallet VO, Duriez M, Wassef M, Kahn A, Menasche P, and Gilgenkrantz H. Cardiac functional improvement by a human Bcl-2 transgene in a mouse model of ischemia/reperfusion injury. J Gene Med 2: 326-333, 2000[ISI][Medline].

8.   Brokelman, W, Stel AL, and Ploeg RJ. Risk factors for primary dysfunction after liver transplantation in the University of Wisconsin solution era. Transplant Proc 31: 2087-2090, 1999[ISI][Medline].

9.   Cheng, S, Ragsdale JR, Sasaki AW, Lee RG, Deveney CW, and Pinson CW. Verapamil improves rat hepatic preservation with UW solution. J Surg Res 50: 560-564, 1991[ISI][Medline].

10.   DiLisa, F, and Bernardi P. Mitochondrial function as a determinant of recovery or death in cell response to injury. Mol Cell Biochem 184: 379-391, 1998[ISI][Medline].

11.   Elimadi, A, Settaf A, Morin D, Sapena R, Lamchouri F, Cherrah Y, and Tillement JP. Trimetazidine counteracts the hepatic injury associated with ischemia-reperfusion by preserving mitochondrial function. J Pharmacol Exp Ther 286: 23-28, 1998[Abstract/Free Full Text].

12.   Fitz, JG, Sostman AH, and Middleton JP. Regulation of cation channels in liver cells by intracellular calcium and protein kinase C. Am J Physiol Gastrointest Liver Physiol 266: G677-G684, 1994[Abstract/Free Full Text].

13.   Forestal, DA, Haimovici J, and Haddad P. Different effect of cold storage and rewarming on three pH regulating transporters in isolated rat hepatocytes. Am J Physiol Gastrointest Liver Physiol 272: G638-G645, 1997[Abstract/Free Full Text].

14.   Green, DR, and Reed JC. Mitochondria and apoptosis. Science 281: 1309-1312, 1998[Abstract/Free Full Text].

15.   Greig, PD, Woolf GM, and Sinclair SB. Treatment of primary graft nonfunction with prostaglandin E1. Transplantation 48: 447-453, 1989[ISI][Medline].

16.   Griffiths, EJ, Ocampo CJ, Savage JS, Stern MD, and Silverman HS. Protective effects of low and high doses of cyclosporin A against reoxygenation injury in isolated rat cardiomyocytes are associated with differential effects on mitochondrial calcium levels. Cell Calcium 27: 87-95, 2000[ISI][Medline].

17.   Haddad, P, Cabrillac J-C, Piche D, Musallam L, and Huet P-M. Changes in intracellular calcium induced by acute hypothermia in parenchymal, endothelial, and Kupffer cells of the rat liver. Cryobiology 39: 69-79, 1999[Medline].

18.   Haddad, P, Noël J, Vadnais J, Mathé L, and Vallerand D. Modulation of liver cell membrane NHE-1, Na+-K+ ATPase, and GLUT-2 protein content after cold preservation and rewarming. Can J Physiol Pharmacol 77: 852-859, 1999[ISI][Medline].

19.   Haimovici, J, Beck JS, Vallerand D, Molla-Hosseini C, and Haddad P. Different modulation of hepatocellular Na+/H+ exchange activity by insulin and EGF. Am J Physiol Gastrointest Liver Physiol 267: G364-G370, 1994[Abstract/Free Full Text].

20.   Hijioka, T, Rosenberg RL, Lemasters JJ, and Thurman RG. Kupffer cells contain voltage-dependent calcium channels. Mol Pharmacol 41: 435-440, 1992[Abstract].

21.   Hughes, BP, Both K, Harland L, Hunt J, Hurst KM, Lewis M, and Barritt GJ. Identification of an mRNA species which encodes a voltage-operated Ca2+ channel in rat liver mRNA. Biochem Mol Biol Int 31: 193-200, 1993[ISI][Medline].

22.   Ioannoni, B, Hughes BP, and Barritt GJ. Identification of a class of low affinity binding sites for verapamil on liver plasma membranes. Biochem Pharmacol 42: 2239-2241, 1991[ISI][Medline].

23.   Juedes, MJ, Kass GEN, and Orrenius S. m-Iodobenzylguanidine increases the mitochondrial Ca2+ pool in isolated hepatocytes. FEBS Lett 313: 39-42, 1992[ISI][Medline].

24.   Keppens, S, and De Wulf H. Characterization of the liver P2-purinoceptor involved in the activation of glycogen phosphorylase. Biochem J 240: 367-371, 1986[ISI][Medline].

25.   Kim, JS, and Southard JH. Alterations in cellular calcium and mitochondrial functions in the rat liver during cold preservation. Transplantation 65: 369-375, 1998[ISI][Medline].

26.   Kukan, M, and Haddad PS. Role of hepatocytes and bile duct cells in preservation-reperfusion injury of liver grafts. Liver Transpl 7: 381-400, 2001[ISI][Medline].

27.   Leducq, N, Delmas-Beauvieux MC, Bourdel-Marchasson I, Four S, Llis JL, Nioni P, and Olez P. Mitochondrial permeability transition during hypothermic to normothermic reperfusion in rat liver demonstrated by the protective effect of cyclosporin A. Biochem J 336: 501-506, 1998.

28.   Mailhot, G, Petit J-L, Demers C, and Gascon-Barré M. Influence of the in vivo calcium status on cellular calcium homeostasis and the level of the calcium binding protein calreticulin in rat hepatocytes. Endocrinology 141: 891-900, 2000[Abstract/Free Full Text].

29.   Mak, IT, and Weglicki WB. Comparative antioxidant activities of propranolol, nifedipine, verapamil, and dilthiazem against sarcolemmal membrane lipid peroxidation. Circ Res 66: 1449-1452, 1990[Abstract/Free Full Text].

30.   Marsh, DC, Lindell SL, Fox LE, Belzer FO, and Southard JH. Hypothermic preservation of hepatocytes. I. Role of cell swelling. Cryobiology 26: 524-534, 1989[Medline].

31.   Mauger, JP, and Claret M. Calcium channels in hepatocytes. J Hepatol 7: 278-282, 1988[ISI][Medline].

32.   Missiaen, L, Taylor CW, and Berridge M. Spontaneous calcium release from inositol triphosphate-sensitive calcium stores. Nature 352: 241-244, 1991[Medline].

33.   Parekh, AB, and Penner R. Store depletion and calcium influx. Physiol Rev 77: 901-930, 1997[Abstract/Free Full Text].

34.   Paschen, W. Dependence of vital cell function on endoplasmic reticulum calcium levels: implications for the mechanisms underlying neuronal cell injury in different pathological states. Cell Calcium 29: 1-11, 2001[ISI][Medline].

35.   Pinton, P, Ferrari D, Magalhaes P, Schulze-Osthoff K, Di Virgilio F, Pozzan T, and Rizzuto R. Reduced loading of intracellular Ca2+ stores and downregulation of capacitative Ca2+ influx in Bcl-2-overexpressing cells. J Cell Biol 148: 857-862, 2000[Abstract/Free Full Text].

36.   Pozzan, T, and Rizzuto R. The renaissance of mitochondrial calcium transport. Eur J Biochem 267: 5269-5273, 2000[ISI][Medline].

37.   Sammut, IA, Thorniley MS, Simpkin S, Fuller BJ, Bates TE, and Green CJ. Impairment of hepatic mitochondrial respiratory function following storage and orthotopic transplantation of rat livers. Cryobiology 36: 49-60, 1998[Medline].

38.   Schlosser, SF, Burgstahler AD, and Nathanson MH. Isolated rat hepatocytes can signal to other hepatocytes and bile duct cells by release of nucleotides. Proc Natl Acad Sci USA 93: 9948-9953, 1996[Abstract/Free Full Text].

39.   Serrar, H, El-Kadi A, Du Souich P, and Haddad P. Cytochrome P-450 content and activity after cold storage of rat hepatocytes in University of Wisconsin and sodium-lactobionate-sucrose solutions. Liver Transpl Surg 5: 119-125, 1999[ISI][Medline].

40.   Serrar, H, and Haddad P. Effects of cold preservation and rewarming on rat liver cell volume regulation and concentrative amino acid uptake. Gastroenterology 112: 1344-1353, 1997[ISI][Medline].

41.   Serrar, H, Musallam L, and Haddad P. Comparative effects of UW and SLS solutions on concentrative proline uptake in cold preserved rat hepatocytes. Therapie 54: 601-606, 1999[ISI][Medline].

42.   Shuttleworth, TJ. What drives calcium entry during [Ca2+] oscillations? Challenging the capacitative model. Cell Calcium 25: 237-246, 1999[ISI][Medline].

43.   Takei, Y, Marzi I, Kauffman FC, Cowper K, Thurman RG, and Lemasters JJ. Prevention of early graft failure by the calcium channel blocker nisoldipine: involvement of Kupffer cells. Transplant Proc 22: 2202-2203, 1990[ISI][Medline].

44.   Takei, Y, Marzi I, Kauffman FC, Currin RT, Lemasters JJ, and Thurman RG. Increase in survival time of liver transplants by protease inhibitors and a calcium channel blocker, nisoldipine. Transplantation 50: 14-20, 1990[ISI][Medline].

45.   Umeshita, K, Monden M, Ukei T, Gotoh M, Nakano Y, Endoh W, Okamura J, and Mori T. Different cytoprotective effects of calcium blockers in hypothermic liver preservation. Transplant Proc 21: 1290-1291, 1989[ISI][Medline].

46.   Urien, S. Micropharm-K, a microcomputer interactive program for the analysis and simulation of pharmacokinetics processes. Pharm Res 12: 1225-1230, 1995[ISI][Medline].

47.   Weis, M, Kass GEN, and Orrenius S. Further characterization of the events involved in mitochondrial Ca2+ release and pore formation by prooxidants. Biochem Pharmacol 47: 2147-2156, 1994[ISI][Medline].

Am J Physiol Gastrointest Liver Physiol 281(3):G809-G815
0193-1857/01 $5.00 Copyright © 2001 the American Physiological Society



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