Báo cáo khoa học: Mapacalcine specifically blocks hypoxia-induced calcium influx in rat hepatocytes potx

Mapacalcine specifically blocks hypoxia-induced calcium influx in rat hepatocytes Dominique Crenesse 1 , Ghislaine Neuilly 2 , Jean Gugenheim 3 , Catherine Ferre 1 and Michel Hugues 2 1 Laboratoire de Physiologie, Faculte ´ de Me ´ decine, Nice, France; 2 CNRS, UMR 5017, Faculte ´ de Pharmacie, Universite ´ de Bordeaux 2, France; 3 Laboratoire de chirurgie expe ´ rimentale, Faculte ´ de Me ´ decine, Nice, France Post ischaemic cell calcium invasion has been described as one of the main causes of graft failure. Protective effects of calcium antagonists have been investigated but are not con- vincing and their mechanisms of action remain unclear. In this work we tested the protective effect of a new calcium inhibitor described to block a calcium current insensitive to all known calcium blockers. Specific mapacalcine receptors were first characterized on rat hepatocytes membranes using the 125 I-labeled mapacalcine. 45 Ca fluxes were then measured on cultured hepatocytes submitted (or not) to an hypoxic period. The action of mapacalcine was investigated on the ischaemia-induced calcium influx. We demonstrate here that: (a) there are specific receptors for mapacalcine in rat hepatocytes; (b) Mapacalcine is able to specifically block ischaemia–induced calcium influx with an IC 50 of 0.3 l M and does not significantly interact with the basal calcium flux. Our work demonstrates that the mapacalcine receptor is a cellular structure directly involved in the phenomenon of postischaemic cell invasion by calcium. Specific block of ischaemia-induced Ca 2+ influx by mapacalcine suggests that the development of a panel of pharmacological drugs acting on this receptor could lead to the discovery of therapeutic agents able to protect cells against one of the events responsible for organ failure after transplantation or simply after an ischaemic period. Moreover, identification of the cellular protein which binds mapacalcine may become an important step in the research of mechanisms involved in postischaemic cell invasion by calcium. Keywords: liver; ischaemia; reperfusion; receptor; mapacalcine. Liver resection is commonly used for trauma and tumors [1]. On the other hand, liver transplantation is nowadays a common alternative for terminal stage liver diseases [2]. Ischaemia/reperfusion injuries are a significant cause of liver dysfunction (and even nonfunction) encountered after liver surgery [3]. During transplantation or surgical procedures, a critical step is the reperfusion with warm oxygenated blood after a cold or warm hypoxic step. The most commonly used preservation solution is University of Wisconsin solution (UW) [4,5], which, although having largely contributed to improve graft success, cannot preserve liver over 24 h. Usually, livers conserved in UW solution are used within the 12 h following organ harvest [6,7]. Among the multiple biological events that occur after the ischaemia/reperfusion step, calcium over-influx has been described as one of the main causes of cell death. Several studies have approached cell calcium invasion and calcium antagonists have been described to improve liver quality [8–11]. However the concentrations demonstrating a protective effect of calcium antagonists with respect to ischaemia/reperfusion injuries are generally out of the range of effect on voltage sensitive calcium channels [11]. Several mechanisms have been considered to explain the protective effects of calcium antagonists. A direct effect on Kupffer cells preventing the release of cytokines [8]; or a non specific interaction with the membrane due to the hydrophobic properties of this family of molecules then perturbing membrane permeability to calcium [11]. Although no voltage dependent calcium channels have been described on liver till now, the presence of an a 1 subunit deleted from the voltage sensor part has been reported on rat liver cell line [12]. Mapacalcine is a small homodimeric protein (M r : 19 041) purified from the Cliona vastifica marine sponge [13]. This protein has been demonstrated to block a calcium permeability insensitive to the known calcium blockers in mouse intestinal smooth muscle [13]. Specific receptors to mapacalcine have also been described in rat brain [14]. Interestingly, no drug represen- tative of the different families of calcium channel blockers have been found to interact with mapacalcine receptors [13–15]. The aim of our work was: (i) to detect mapacalcine receptors in liver; (ii) to check the ability of mapacalcine to block calcium influx in hepatocytes exposed to hypoxia/ reperfusion conditions. Our data clearly demonstrate that mapacalcine specifically inhibits the calcium influx provoked by hypoxia but not the basal influx in normal conditions. Experimental procedures Preparation of rat liver membranes Rats were treated in compliance with French rules on animal handling. Highly enriched preparation of rat liver Correspondence to M. Hugues, CNRS/UMR 5017. Universite ´ de Bordeaux 2, Faculte ´ de Pharmacie. 146 rue Le ´ o Saignat 33076 Bordeaux Cedex, France. Fax: + 5 57 57 12 42, Tel.: + 5 57 57 12 72, E-mail: michel.hugues@umr5017.u-bordeaux2.fr Abbreviations: UW, University of Wisconsin solution. (Received 18 December 2002, revised 28 February 2003, accepted 7 March 2003) Eur. J. Biochem. 270, 1952–1957 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03558.x membranes was performed as previously described by Neville [16] with small modifications. Briefly, male Wistar rats (160–300 g) were killed by decapitation. The livers were removed, minced with scissors and homogenized in an ice- cold buffer (Tris 1 m M at pH ¼ 7.40) with a Dounce homogenizer. The homogenate was then stirred at 4 °C during 30 min in 5 L of ice-cold buffer. The cellular debris were removed by filtration on 2 layers of cheesecloth, then on 4 layers of cheesecloth. The homogenate was centri- fuged at 1500 g for 10 min at 4 °C. The supernatant was discarded and the pellets homogenized again with the Dounce homogenizer. The mixture, obtained was then vigorously mixed with stock sucrose solution at 69%, to obtain a sucrose concentration of 44%. The mixture was gently covered with stock sucrose 43.5% for the discon- tinuous gradient then centrifuged at 90 000 g for 2 h at (4 °C), in a SW28 Beckman rotor. The float present under the 43.5%, sucrose layer was collected with a spatula, homogenized, resuspended in the Tris buffer (1 m M )then centrifuged at 140 000 g for 30 min at 4 °C. The pellets obtained were homogenized in the Tris 1 m M buffer. Aliquots of membranes were frozen and kept at )80 °C until use. Proteins were measured by the Bradford’s method [17] (Bio-Rad proteins assay) using lysozyme as standard. Binding of mapacalcine on rat liver membranes Labeled mapacalcine used in binding experiments was obtained by iodination of the native peptide as described by Vidalenc et al. [15]. Binding assays were conducted in a total volume of 1 mL of physiological incubation buffer (mM): 50 Tris/HCl at pH 7.4, 130 NaCl, 5.6 KCl, 2CaCl 2 ,0.24MgCl 2 , 11 glucose and 0.2% bovine serum albumin. Duplicate aliquots of membranes (0.2 mg protÆ mL )1 ) were incubated for 120 min at 4 °Cor37°Cwith various concentrations of 125 I-labeled mapacalcine (2200 CiÆmmol )1 ). Non specific binding was determined in parallel experiments conducted in presence of 1 l M native mapacalcine. Specific binding was obtained by subtracting nonspecific binding from the total binding. After incubation, 400 lL duplicate aliquots were filtered, using a cell harvester (Millipore) over glass fiber filters (Whatman GF/C) presoaked during 60 min in 10 m M Tris/HCl at pH 7.4 at 4 °C. Filters were rapidly washed twicewith5mLofthesamebufferat4°C. The radioactivity retained by the filters was then counted in a Packard gamma counter. Competition experiments Liver membranes (0.2 mgÆmL )1 ) were incubated in the presence 0.01 n M 125 I-labeled mapacalcine and increasing concentrations of unlabeled mapacalcine either at 4 °Cor at 37 °C. The amount of labeled mapacalcine remaining bound to the membranes was estimated by the filtration technique described above. Kinetic binding studies Association kinetics were performed in standard binding buffer at 4 °Corat37 °C to assess equilibrium state of toxin binding to membranes. Association was initiated by adding 125 I-labeled mapacalcine (0.005 n M ) to a constant concen- tration of membranes (0.2 mgÆmL )1 ). Bound mapacalcine was measured at different times by the filtration technique described above. Non specific binding was determined in parallel experiments in presence of 1 l M native mapacalcine. Hepatocytes isolation and culture Hepatocytes were obtained as previously described by Seglen [18] and Berry [19]. Briefly, fed Wistar male rats were anaesthetized with ether inhalation and liver perfused through the portal vein with 100 mL of Hepes buffer at pH 7 containing 6000 U of collagenase (Boehringer Mann- heim, Meylan France), during 20 min. Dissociated hepato- cytes were then collected in William’s culture medium (Eurobio, Les Ullis France) supplemented with 5% fetal bovine serum and insulin (0.1 IUÆmL )1 ). Cell viability was estimated to be superior to 90% using trypan blue exclusion. The cell suspension was adjusted to 1 · 10 6 cellsÆmL )1 and cultured in 12 well plates (5 · 10 5 cells per well) under controlled atmosphere (5%CO 2 ). The culture medium containing dexamethasone (1 l M ), was renewed 4 h later. After 24 h of culture, hepatocytes were used for the experiments. Hypoxic treatment Culture plates were maintained at 37 °Cinanhermeticbag (Bioblock, Illkirsh, France) in which PO 2 was adjusted to 50 ± 10 mmHg and maintained constant by N 2 supply. Culture medium used (Leibovitz medium, L15, Eurobio Les Ullis, France) without NaHCO 3 ,wasusedinfreegas exchange with atmosphere and was buffered via the buffering capacity of the free base aminoacids. The medium was nitrogen saturated. A sample of each medium was kept under the same conditions and immediately analyzed on a blood gas analyzer (Corning 2504, Cergy Pontoise, France) at the end of each experiment to control. PO 2 ,PCO 2 and pH values. The pH was stable at 7.20 ± 0.02. Four groups were studied: Group 1 A (control) corresponded to hepatocytes maintained in L15 at 37 °C during two hours under ambient air. Group 1B: hepatocytes treated as described previously excepted that they were maintained under hypoxic atmosphere. Group 2 A: corresponded to Group 1 A conditions but with hepatocytes treated with 1 l M mapacalcine. Group 2B: same conditions as Group 1B but with hepatocytes treated with 1 l M mapacalcine. Calcium influx measurement Calcium influx measurements on the different groups of hepatocytes were achieved by reducing the volume of incubation to 400 lL in order to limit the amount of 45 Ca needed. The 45 Ca influx was measured as described by Combette [20] with small modifications to adapt the technique to hepatocytes in culture (i.e. attached on the plastic dishes), for Groups 1B and 2B calcium influxes were measured under atmospheric PO 2 . Briefly, calcium influxes were initiated by addition of 100 lL of incubation medium containing 1 lCi of 45 Ca in each well. Under these conditions, the total incubation volumes was 500 lLand Ó FEBS 2003 Mapacalcine blocks hypoxia-induced calcium influx (Eur. J. Biochem. 270) 1953 the total calcium concentration, 1 m M which was close to the physiological rate [20]. At different times between 0 and 120 s, the 45 Ca influx was stopped by removing the incubation medium and washing the cells twice with an ice cold washing solution containing: NaCl, 114 m M ; CaCl2, 5 m M and Tris/HCl 5 m M at pH 7.4. 1 mL of NaOH 0.1 m M was then added and cells were scrapped off the well. A total of 50 lL of the mixture was kept for protein measurement using the Bradford’s method [16] (Biorad protein assay, Biorad, Ivry/Seine, France.). The rest of the mixture was mixed with 4 mL of scintillation liquid (Ultima Gold, Packard Instruments, Rungis France) and the radioactivity counted on a Tricarb 1600 TR Packard Counter. The data were normalized as function of the protein amount contained in each well. Each experimental value (mean ± SEM) resulted from independent measure- ments on different hepatocyte preparations. Statistical comparisons were obtained using variance analysis and the Student’s t-test. Results Binding of mapacalcine to liver membranes Association of mapacalcine to liver membranes was tested to assess the time necessary to reach equilibrium at 4 and 37 °C. Mapacalcine binding reached a plateau value after 90 and 120 min incubation at 37 and 4 °C, respectively (not shown) and remained stable for 4 h (maximal time meas- ured). All subsequent binding experiments were then conducted respecting an incubation time of 2 h. Direct binding of mapacalcine to liver membranes at 4 and 37 °C Direct binding experiments were performed at 4 an 37 °C. Data obtained from saturation experiments were plotted according to Scathard (Fig. 1A,B) and demonstrated a linear plot suggesting that mapacalcine was binding to a single class of noninteracting sites. Binding parameters obtained were: K d ¼ 0.08 ± 0.02 n M and B max ¼ 210 ± 12 fmolÆmg protein )1 at 4 °C(n ¼ 12); and K d ¼ 0.07 ± 0.02 n M and B max ¼ 232 ± 40 fmolÆmg protein )1 at 37 °C (n ¼ 4). Competition experiments Figure 1C,D demonstrates curves obtained from competi- tion experiments between iodinated mapacalcine and increasing concentrations of native mapacalcine at 4 and 37 °C. As the concentration of labeled mapacalcine was 10-fold lower than the K d value of the iodinated mapacal- cine, one can assume the IC 50 obtained equal to the K d value. The K d obtained at 4 °C was 0.12 ± 0.01 n M (n ¼ 4); the K d obtained at 37 °C was 0.15 ± 0.02 n M (n ¼ 4). Effect of mapacalcine on hypoxia-induced calcium influx The time course of calcium uptake was followed at different times ranging from 2 to 120 s (Fig. 2A). In Group 1A (control) the intracellular 45 Ca level rapidly reached a plateau value corresponding to 1.8 ± 0.3 pmolÆlg )1 prot after 20 s. The plateau value corresponding to group 2A (conditions of group 1A but with treatment with mapacal- cine 1 l M ) was not significantly different. When hepatocytes were subjected to hypoxic stress (group 1B) the plateau value was found slightly higher than in group 1 A (P < 0.05). Interestingly, in the group 2B (hypoxia stress plus mapacalcine 1 l M ) the plateau value was found significantly lower (P < 0.01). Effect of mapacalcine on initial 45 Ca influx At the beginning of calcium uptake measurement, the calcium flux measured can be considered as linear during the first 8 s of the time course (Fig. 2B). During this initial period, one can assume that the signal observed is only representative for the 45 Ca influx and can be fitted as a linear phenomenon. The equation fitting the uptake corresponds to the equation of a straight line (Fig. 2B). The lines did not extrapolate to 0 as previously reported [20] and must probably be due to the presence of residual amounts of 45 Ca remaining after the washing procedure. However the slope of the curve is the parameter of interest as it reflects the 45 Ca influx rate. Interestingly when hepatocytes were submitted to an hypoxic stress (Group 1B) the rate of the initial calcium uptake (vs. Group 1A) was increased by a factor of 3.25 (P < 0.01). In group 2B (treatment with mapacalcine 1 l M ) the initial 45 Ca influx rate was decreased by a factor 11 (P < 0.01). The effect of mapacalcine on calcium uptake was concentration dependent (Fig. 2C). The IC 50 obtained from the data in Fig. 2C was 0.3 ± 0.1 l M (n ¼ 15). Discussion Among the multiple cellular events occurring after hypoxic periods in liver surgery, increase in cytosolic calcium concentration has been described as an early event contri- buting to hypoxic injury [21–23]. Hypoxia will acidify the intracellular medium increasing by the way the cytosolic calcium concentration. Mitochondrial calcium exchange system will then be affected leading to mitochondria dysfunctions, depletion of intracellular ATP concentration and cell death [24–27]. Several studies have reported a protective effect of calcium channel blockers like dihydropyridines or pheno- tiazines in hypoxic damage [11,28]. However the mecha- nisms by which these molecules exert their protection remain unclear. Diltiazem protective effects occur on hepatocytes with different IC 50 according to whether the cells have been submitted to a re-oxygenation period or not [11]. Another study reports a protective effect of nitrendi- pine occurring independently from the absence or presence of calcium [29]. Usually, protective effects of calcium channel blockers are characterized by IC 50 s that do not match with the IC 50 s described for their interactions with voltage sensitive calcium channels suggesting that these effects may come from other characteristics of these molecules (like antioxydant properties). Mapacalcine appears to act on a calcium current triggered by a non classical calcium permeability [13], we have then investigated its ability to interact with calcium 1954 D. Crenesse et al. (Eur. J. Biochem. 270) Ó FEBS 2003 influxes provoked by hypoxia/reperfusion conditions on rat hepatocytes. Receptors for mapacalcine were first charac- terized on rat liver membranes using the iodinated mapa- calcine [17]. Binding data obtained at 37° and 4 °C revealed the presence of specific high affinity receptors for mapacal- cine on these membranes. Data obtained from calcium flux experiments with hepatocytes in control conditions revealed a basal calcium influx which was not significantly affected by mapacalcine (1 l M ). Conversely, when hepatocytes were submitted to hypoxia, calcium influx dramatically increased. Application of mapacalcine 1 l M under hypoxia completely inhibited the stimulated calcium influx which was maintained at the basal level (and even lower). The dose– response curve obtained for the inhibition of the hypoxia/ stimulated calcium influx demonstrated an IC 50 of 0.3 l M . This value is in good agreement with the IC 50 obtained for the inhibition of the Ônon L -typeÕ calcium channel on mouse intestinal smooth muscle (0.1 l M ) by electrophysiological measurements [13]. The shape of the dose–response curve demonstrates a decrease within 5 orders of magnitude, this Fig. 1. Scatchard representations of 125 I-mapacalcine binding to rat hepatocytes membranes at 4 °C(A)and37°C (B), and Inhibition curves obtained from competition experiments between 125 I-labeled mapacalcine and increasing concentrations of native mapacalcine on rat hepatocytes membranes at 4 °C(C)and37°C(D).The slope of the non specific binding in the saturation experiment was 298.41 ± 8.8 fmol · mgr À1 prot · n M )1 (n ¼ 12) and 161.75 ± 11.9 fmolÆmgr À1 prot Æn M )1 (n ¼ 4) at 4 °Cand37°C, respectively. For (C) and (D), under conditions used in these experiments the non specific binding corresponded to 1.2 ± 0.1% (n ¼ 4) and 8.0 ± 0.6% (n ¼ 4) of the total binding at 4 °Cand37°C, respectively. Ó FEBS 2003 Mapacalcine blocks hypoxia-induced calcium influx (Eur. J. Biochem. 270) 1955 would suggest a complex mechanism involving several biological events to lead to the calcium influx blockade by mapacalcine. This would also explain the discrepancy observed between mapacalcine affinity determined by binding experiments and the IC 50 determined for calcium influx blockade. Our data clearly demonstrate that an application of mapacalcine to hepatocytes dramatically blocks an hypoxia induced calcium influx. An interesting property of mapacalcine is that it does not interact whit basal calcium fluxes when hepatocytes are not submitted to hypoxia. These observations suggest that mapacalcine would interacts with a cellular structure which could be directly involved in the posthypoxic cell calcium invasion. Furthermore, previous work reported that mapacalcine did not interact with the known calcium channels types [13] even used at concentrations in the lmolar range. These data, taken together, would suggest that acting on mapa- calcine receptors to protect graft against postischaemic calcium invasion has little chances to induce secondary effects resulting on a side interaction with others calcium channels. However, further experiments will have (i) to establish a direct link between calcium influx sensitive to mapacalcine and the protection of biological functions of the cell, and (ii) determine the nature of mapacalcine receptor which could represent an interesting target to address therapeutic control of postischaemic/reperfusion cellular damage. 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Mapacalcine specifically blocks hypoxia-induced calcium in ux in rat hepatocytes Dominique Crenesse 1 , Ghislaine Neuilly 2 , Jean Gugenheim 3 , Catherine Ferre 1 and Michel Hugues 2 1 Laboratoire. binding was determined in parallel experiments conducted in presence of 1 l M native mapacalcine. Specific binding was obtained by subtracting nonspecific binding from the total binding. After incubation,