FEBS Letters 582 (2008) 4131–4136
Intraluminal hydrogen peroxide induces a permeability change of the endoplasmic reticulum membrane ´ va Margittaia, Pe´ter Lo¨wb, Andra´s Szarkac, Miklo´s Csalaa, E Angelo Benedettid, Ga´bor Ba´nhegyia,d,* a
Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University and MTA-SE Pathobiochemistry Research Group, P.O. Box 260, 1444 Budapest, Hungary b Department of Anatomy, Cell and Developmental Biology, Lora´nd Eo¨tvo¨s University, Budapest, Hungary c Department of Applied Biotechnology and Food Science, Laboratory of Biochemistry and Molecular Biology, Budapest University of Technology and Economics, Budapest, Hungary d Dipartimento di Fisiopatologia, Medicina Sperimentale e Sanita` Pubblica, Universita` di Siena, Siena, Italy Received 16 September 2008; revised 1 November 2008; accepted 12 November 2008 Available online 25 November 2008 Edited by Vladimir Skulachev
Abstract Gulonolactone treatment of mice resulted in the elevation of hepatic ascorbate and hydrogen peroxide levels accompanied by transient liver swelling and reversible dilatation of endoplasmic reticulum cisternae. Although a decrease in glutathione (reduced form)/total glutathione ratio was observed in microsomes, the redox state of luminal foldases remained unchanged and the signs of endoplasmic reticulum stress were absent. Increased permeability of the microsomal membrane to various compounds of low molecular weight was substantiated. It is assumed that Gulonolactone-dependent luminal hydrogen peroxide formation in the endoplasmic reticulum provokes a temporary increase in non-selective membrane permeability, which results in the dilation of the organelle and in enhanced transmembrane fluxes of small molecules. 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Oxidative protein folding; Hydrogen peroxide; Glutathione; Membrane permeability; ER stress; Gulonolactone oxidase
1. Introduction Oxidative folding of secretory and membrane proteins is a major pathway in the endoplasmic reticulum (ER) of professional secretory cells [1]. The process, executed by an electron relay system composed by oxidoreductases, allows oxidation of cysteine residues in nascent polypeptide chains to form intramolecular disulfide bonds in the lumen of the ER [2,3]. Oxygen appears to be the final oxidant in aerobic living organisms, although the existence of alternative electron acceptors
* Corresponding author. Address: Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University and MTA-SE Pathobiochemistry Research Group, P.O. Box 260, 1444 Budapest, Hungary. Fax: +36 12662615. E-mail address:
[email protected] (G. Ba´nhegyi).
Abbreviations: ER, endoplasmic reticulum; ERO1, endoplasmic reticulum oxidoreductin 1; GLO, gulonolactone oxidase; GSH, glutathione (reduced form); IP3R, inositol 1,4,5-trisphosphate receptor; ROS, reactive oxygen species
cannot be excluded. Endoplasmic reticulum oxidoreductin 1 (ERO1), the ultimate component of the electron transfer chain catalyzes the incomplete reduction of oxygen; therefore protein oxidation in the ER is connected to generation of reactive oxygen species (ROS), presumably hydrogen peroxide [4]. In fact, reduction of molecular oxygen by recombinant yeast Ero1p yields stoichiometric hydrogen peroxide production under aerobic conditions [5]. Since ROS generated during oxidative protein folding can compromise the redox and antioxidant homeostasis of ER lumen, it has been postulated as a causative factor of ER stress in conditions characterized by the overproduction of secretory proteins [6]. However, elevation of luminal hydrogen peroxide (or ROS) levels has not been proved under these conditions. Furthermore, the antioxidant defense of the ER lumen seems more vulnerable than that of the other subcellular compartments, e.g. there are no reports of catalase or superoxide dismutase isoforms being present (for a review, see [7]). The question remains how secretory cells with heavy loads of protein thiols in the luminal compartment of the ER can accommodate to or eliminate the byproducts of disulfide bond formation. To model the situation in excessive oxidative folding, we took advantage of the presence of another hydrogen peroxide generating enzyme in the ER lumen. Gulonolactone oxidase (GLO) catalyses the last step of ascorbate synthesis with generation of hydrogen peroxide as a byproduct [8]. Gulonolactone easily crosses both the plasma and ER membranes and reaches the active site of the enzyme. Addition of gulonolactone results in an increase of ascorbate production in isolated murine hepatocytes. At the same time, a decrease in reduced glutathione (GSH) level was observed [9]. In hepatic microsomal vesicles, ascorbate synthesis stimulated by gulonolactone causes an almost equimolar consumption of added GSH [9]. In glutathione-loaded microsomes, gulonolactone addition results in the preferential oxidation of intraluminal glutathione demonstrating the intraluminal formation of hydrogen peroxide [10]. In this study, the effect of gulonolactone was investigated in vivo in mice and in vitro in hepatic microsomal vesicles. The results show that gulonolactone-dependent intraluminal hydrogen peroxide formation in the ER provokes a temporary increase in the non-selective membrane permeability, which results in ER dilation and in enhanced transmembrane fluxes of small molecules including glutathione. The mechanism can be
0014-5793/$34.00 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.11.012
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regarded as the opening of a safety valve that prevents the accumulation of ROS in the ER and the exhaustion of the antioxidant capacity of the lumen.
2. Materials and methods 2.1. Animal experiments Male CD-1 mice (20–25 g body weight) and male Hartley guinea pigs (250–300 g body weight) were purchased from Charles River Hungary (Isaszeg). Animal treatments were approved by the Committee on Animal Experiments of Semmelweis University, Budapest. Animals were kept with ad libitum access to food and water. Gulonolactone was dissolved in physiological saline and was injected intraperitoneally in a volume of 1 ml. Control animals received an equivalent volume of the vehicle. At various time points after treatment (0.5 or 24 h) animals were sacrificed. Liver samples were taken and processed for electron microscopy. The remnant of the liver was homogenized in sucrose– HEPES buffer (0.3 M sucrose, 0.02 M HEPES, pH 7.2) with a glassTeflon homogenizer. The microsomal fraction was then isolated using fractional centrifugation as detailed elsewhere [11]. Microsomes were immediately frozen in liquid nitrogen, and kept in liquid nitrogen until use. The integrity of the microsomal membranes was assessed by using the mannose-6-phosphatase assay [12] and by measuring p-nitrophenol glucuronidation [13], which showed latency greater than 95%. 2.2. Electron microscopy For transmission electron microscopy investigation, upon dissection after an in vivo treatment, the liver samples were immediately fixed in 0.5% glutaraldehyde + 2% paraformaldehyde (PolySciences Europe GmbH., Eppelheim, Germany) in 0.1 M cacodylate buffer (pH 7.2) containing 0.25 M sucrose and 2 mM CaCl2 for 2 h at room temperature, postfixed in 1% osmium tetroxide (Serva Feinbiochemica GmbH., Heidelberg, Germany) in 0.1 M cacodylate buffer, dehydrated in ascending series of ethanols, infiltrated in propylene oxide, and embedded in Durcupan ACM resin (Fluka AG, Buchs, Switzerland). Ultrathin sections were contrasted with uranyl acetate and lead citrate and electron micrographs were taken by a Jeol JEM100 CX II electron microscope operating at 60 kV. 2.3. Immunoblotting The protein concentration of samples was measured using the BioRad ‘‘microprotein assay kit’’. Equal amounts of microsomal proteins (25 lg) were separated in 9% SDS–PAGE and transferred to PVDF filter membranes by electroblotting. The filter membranes were incubated overnight with the primary antibodies (dilution 1:1000; anti-procaspase-12 and anti-eIF2a from Cell Signaling Technology, anti-ERp72 from Calbiochem, all other from Santa Cruz Biotechnology), and for 1 h with the species-specific peroxidase-conjugated secondary antibodies (dilution 1:2000; anti-goat, anti-mouse and anti-rabbit IgGs from Santa Cruz Biotechnology, anti-rat IgG from Sigma). The antibodies were detected using a chemiluminescence reagent (ECL) kit (Amersham) and blue-sensitive X-ray film. Equal protein loading was also verified by Ponceau-staining of the membrane and by b-actin detection. Band intensities were quantified by densitometry using ImageQuant software. The thiol redox state of ER foldases was investigated by alkylation with 4-acetamido-4 0 -maleimidylstilbene-2,2 0 -disulfonic acid (AMS; Molecular Probes) as reported in [14]. For the reduced and oxidized controls, microsomal samples corresponding to 0.04 g protein were incubated at 37 C for 15 min with 0.05 M dithiothreitol or with 0.025 M diamide, respectively. 2.4. Transport measurements by rapid filtration Microsomal suspensions were diluted to 15 mg of protein/ml, in a cytosol-like solution containing 100 mM KCl, 20 mM NaCl, 1 mM MgCl2, 20 mM Mops, pH 7.2 (KCl–Mops buffer). Microsomal vesicles were pre-equilibrated with 1 mM sucrose plus [U14C]sucrose (15– 16 lCi/ml). The microsomal suspensions were then 100-fold diluted in the KCl–Mops buffer at 22 C to activate the efflux. At the indicated time points, 0.4 ml samples were taken and rapidly filtered on a cellulose acetate/nitrate membrane (pore size 0.22 lm) under gentle suction. Filters were washed with 3 ml of ice-cold 0.25 M sucrose buffered with
KCl–Mops, containing 1 mM 4,4 0 -diisothiocyanatostilbene-2,2 0 -disulfonic acid. Just before diluting, 4 ll of the microsomal suspensions were withdrawn, rapidly mixed with 0.4 ml of the washing medium (see above), and filtered to determine the ÔÔzero’’ time amounts of the investigated compound associated to microsomes. The radioactivity associated with microsomes retained by filters was measured by liquid scintillation counting. For the measurement of GSH uptake, microsomal vesicles (1 mg of protein/ml) were incubated at room temperature (22 C) in KCl–Mops buffer containing 1 mM GSH and its radiolabelled analogue [3H]GSH (10 lCi/ml). At the indicated times, aliquots (0.1 ml) were withdrawn, filtered and washed quickly on the filter with the same ice-cold buffer. The radioactivity retained on the filter was measured by liquid scintillation. Alamethicin (50 lg/mg of protein) was included in parallel incubates to distinguish the intravesicular and the bound radioactivities. The alamethicin-treated vesicles were recovered on filters and washed as above. The alamethicin-releasable portion of radioactivity (regarded as intravesicular) was calculated by subtraction. 2.5. Transport measurements by light scattering technique The permeability of the microsomal membranes to various compounds was measured by the continuous detection of the osmotically induced changes in size and shape of microsomal vesicles [15]. 2.6. Measurement of enzyme activities UDP-glucuronosyltransferase and mannose-6-phosphatase activities were measured in KCl–Mops buffer containing 1 mg/ml microsomal protein and 2 mM UDP-glucuronic acid plus 0.5 mM p-nitrophenol or 5 mM mannose-6-phosphate at 37 C. p-Nitrophenol disappearance [13] or phosphate production [15] was measured. Alamethicin (50 lg/mg of protein) was included in parallel incubates to permeabilize the vesicles for the determination of total activities. 2.7. Metabolite measurements Determination of glutathione content of liver or microsomes were performed by HPLC analysis as described in [16]. Glutathione measurement from freezed–thawed microsomes gives a good approximation of the original ER glutathione levels because permeation of both GSH and GSSG through microsomal membranes is relatively slow [17,18]. Hydrogen peroxide content of liver homogenates was measured by Amplex Red Assay Kit (Invitrogen, Carlsbad, CA) according to the manufacturerÕs instruction. The protein concentration in homogenates and microsomal samples was determined using the Bio-Rad microprotein assay kit (Hercules, CA, USA) according to the manufacturerÕs instructions with bovine serum albumin as a standard.
3. Results 3.1. Gulonolactone treatment causes a transient ER dilation in mice Mice were treated with a non-lethal single intraperitoneal dose of gulonolactone (0.04, 0.4, 4 or 40 mmol/kg body weight) and sacrificed 0.5 or 24 h after the treatment. To assess the efficacy of the gulonolactone treatment, ascorbate and hydrogen peroxide levels were measured in liver homogenates. Only moderate increase in ascorbate and hydrogen peroxide concentrations was observed up to 4 mmol/kg body weight gulonolactone doses at 0.5 h (data not shown). Gulonolactone (40 mmol/kg body weight) caused a statistically significant increase in both ascorbate and hydrogen peroxide concentrations (Fig. 1A and B); therefore, this dosage was applied in the further experiments. The elevated hepatic concentrations returned to (Fig. 1A) or below (Fig. 1B) the control level after 24 h. Gulonolactone treatment at high concentration resulted in the swelling of the liver at 30 min with the significant increase in liver weight, which completely disappeared within
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*
2
1
0
150
100
50
30' GL
24h ctrl 24h GL
2
1
0
0 30' ctrl
*
3
Liver weight (g)
Hydrogen peroxide (μM)
Liver ascorbate (mM)
*
30' ctrl 30' GL 24h ctrl 24h GL
30' ctrl
30' GL
24h ctrl 24h GL
Fig. 1. Gulonolactone treatment stimulates gulonolactone oxidase activity and provokes hepatic swelling in mice. Liver homogenates were prepared from control mice and mice treated with a single intraperitoneal dose of gulonolactone (40 mmol/kg body weight) 30 min or 24 h before sacrificing. The two products of gulonolactone oxidase, ascorbate (panel A) and hydrogen peroxide (panel B) were measured in the homogenates. Liver weights (panel C) were also determined. Results are expressed as means ± S.E.M., n = 4–8, *P < 0.05.
24 h (Fig. 1C). Electron microscopy revealed a dramatic dilation of the ER cisternae in the liver, while mitochondrial and nuclear structures looked intact in the 30-min samples. The alteration was completely reversible; the 24-h samples were indistinguishable from the controls (Fig. 2). To clarify the role of hydrogen peroxide produced by gulonolactone oxidase in the observed alterations, the experiment was also done in guinea pigs. This species lacks gulonolactone oxidase activity due to multiple mutations in its gene [19]. Gulonolactone treatment resulted in a slight hepatic swelling (presumably due to osmotic effects), but morphological changes were completely absent (data not shown). 3.2. Gulonolactone treatment causes a minor change in the glutathione redox buffer of the ER Previous data obtained in microsomal experiments and in isolated murine hepatocytes indicated that gulonolactonedependent ascorbate (and hydrogen peroxide) formation was accompanied with a preferential intraluminal oxidation of glutathione [9,10]. Therefore, reduced GSH and total glutathione contents were measured in the liver homogenates and microsomal fractions. Minor (non-significant) decrease in GSH was observed together with a moderate elevation of total glutathione in microsomal fractions from gulonolactone-treated
mice (Table 1). In agreement with the previous results, the microsomal ratio of reduced/total glutathione significantly decreased upon the treatment. In liver homogenates, both GSH and total glutathione levels were slightly higher in gulonolactone-treated mice. All differences were present only in the 0.5-h samples (Table 1). The elevation of intravesicular glutathione content in the microsomes prepared from gulonolactone-treated mice could be explained by an increased uptake through a specific transporter. Therefore, GSH transport across the microsomal membrane was studied directly by the rapid filtration method. These measurements did not show any increase in the GSH uptake in microsomes from gulonolactone-treated mice (data not shown) suggesting that the stimulation of GSH transport is not responsible for the phenomenon. 3.3. Gulonolactone treatment does not provoke ER stress Redox imbalance in the ER is often associated with the appearance of the signs of ER stress and the consecutive apoptosis [7]. Therefore, the expression of ER chaperones (GRP78, GRP94), foldases (PDI, ERp72) and proapoptotic transcription factor (GADD153) as well as the phosphorylation of eIF2a and the proteolytic activation of procaspase-12 were investigated by Western blot analysis. In addition, the redox
Fig. 2. Electron microscopy of liver reveals dramatic dilatation of ER cisternae in gulonolactone-treated mice. In control, untreated samples cytoplasm of liver cells appears to be normal (panel A). After gulonolactone treatment for 30-min the ER cisterns become dilated and they form numerous small vesicles covered with ribosomes while mitochondria and nuclear structures remain intact (panel B). These drastic morphological changes in ER are completely recovered 24 h after treatment (panel C) (ER, endoplasmic reticulum; M, mitochondria. Bar = 1 lm).
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Table 1 Effect of gulonolactone treatment on hepatic glutathione levels. Glutathione (reduced and total) levels were measured in homogenates and microsomal fractions prepared from the livers of control mice and mice treated with a single intraperitoneal dose of gulonolactone (40 mmol/kg body weight) 30 min before sacrificing. GSH/total glutathione ratios were calculated separately for each sample. Results are expressed as means ± S.E.M., n = 4–8. GSH
Total glutathione
GSH/total ratio
1.48 ± 0.33 1.76 ± 0.24 47 ± 8 55 ± 13
0.62 ± 0.05 0.49 ± 0.05* 0.91 ± 0.11 0.87 ± 0.15
(nmol/mg protein) Microsomal fraction Crude liver homogenate *
Control GL-treated Control GL-treated
0.92 ± 0.15 0.86 ± 0.21 43 ± 9 48 ± 14
P < 0.05.
Table 2 Effect of gulonolactone treatment on the activity and latency of intraluminal enzymes of the ER. UDP-glucuronosyltransferase and mannose-6phosphatase activities were measured in native and permeabilized microsomal vesicles prepared from the livers of control and gulonolactone-treated mice. Results are expressed as means ± S.E.M., n = 4–8. Native
Permeabilized
100 · native/permeabilized
Activity (nmol/min/mg protein) UDP-glucuronosyltransferase Control GL-treated
2.58 ± 0.48 3.75 ± 0.67
28.11 ± 2.07 26.93 ± 1.88
9.17 ± 0.77 13.92 ± 2.86
Mannose-6-phosphatase Control GL-treated
0.78 ± 0.03 0.91 ± 0.06*
12.28 ± 0.88 12.03 ± 0.67
6.32 ± 0.27 7.60 ± 0.51*
*
P < 0.05 vs control.
state of the major intraluminal ER foldases (PDI, ERp72) was also tested by AMS staining. No differences were observed between the livers from control and gulonolactone treated animals with respect to the above mentioned proteins (data not shown).
Sucrose (nmol/mg protein)
3.4. Gulonolactone treatment increases the permeability of the ER membrane The morphological changes and the absence of the luminal GSH depletion upon gulonolactone treatment suggested that the membrane barrier of the ER was compromised. To verify this assumption, permeability of microsomal membrane was checked by measuring the activity of enzymes with intraluminal active site and by detecting the membrane permeation of various osmolytes. Gulonolactone treatment caused a significant increase in the activity of mannose-6-phosphatase and a non-significant elevation of UDP-glucuronosyltransferase activity measured in native microsomal vesicles (Table 2). The activity of these enzymes is highly latent in native ER-derived microsomal vesicles and their latency can be diminished by membrane disrupting agents. Osmotically induced vesicle shrinkage visualized by light scattering technique was greatly reduced in microsomes from gulonolactone-treated mice, while the concomitant swelling phase was more rapid. The addition of the poorly permeant sucrose or citrate resulted in less expressed vesicular shrinking and in a faster recovery of the original volume (Table 2). Similarly increased permeability was detected when the control microsomes were pretreated with gulonolactone (10 mM, 5 min) in vitro (data not shown). The increased permeation of sucrose in gulonolactone-pretreated microsomes was further validated by direct transport measurements using the rapid filtration method. The addition
5
4
3
ctrl GL-treated 2
1
0 0
5
10
15
20
Time (min) Fig. 3. Effect of gulonolactone treatment on sucrose efflux from murine liver microsomal vesicles. Microsomes (15 mg of protein/ml) were pre-equilibrated with 1 mM sucrose plus [U14C]sucrose at 22 C for 2 h, then 100-fold diluted to initiate the efflux. At the indicated time points, samples were rapidly filtered and washed on a cellulose acetate/ nitrate filter membrane. The radioactivity associated with microsomes retained by filters was measured by liquid scintillation counting. Gulonolactone (10 mM) was added to the pretreated microsomes 5 min before zero time. A representative trace out of three is shown.
E´. Margittai et al. / FEBS Letters 582 (2008) 4131–4136 Table 3 Effect of gulonolactone treatment on the permeability of microsomal membrane. Microsomal vesicles were prepared from control mice and mice treated with a single intraperitoneal dose of gulonolactone (40 mmol/kg body weight) 30 min before sacrificing. Membrane permeability was assessed by light scattering technique using citrate or sucrose (40 mM final concentration) as osmolytes. The initial peak upon the addition of osmolytes and the slope of the consecutive gradual decrease in light scattering were measured and used as indicators of the vesicular membrane permeability. Results are expressed as means ± S.E.M., n = 4–8. Osmolyte
Treatment
Peak height (AU)
Half-time of recovery (min)
Citrate
Control GL-treated Control GL-treated
41.50 ± 1.32 31.75 ± 3.64* 22.00 ± 2.34 18.20 ± 3.43
3.19 ± 0.47 2.35 ± 0.26 1.57 ± 0.48 0.88 ± 0.13*
Sucrose *
P < 0.05.
of gulonolactone (10 mM) accelerated sucrose efflux from the sucrose-preloaded microsomal vesicles (Fig. 3 and Table 3).
4. Discussion The present findings demonstrate that intraluminal hydrogen peroxide generation in the ER – provoked by a stimulated gulonolactone oxidase activity – results in a transitory permeability change in the ER membrane. Previous in vitro studies showed that the conversion of gulonolactone to ascorbate causes GSH consumption in isolated murine hepatocytes and this GSH consumption was shown to be preferentially intraluminal in glutathione-loaded hepatic microsomal vesicles. The role of hydrogen peroxide generated during ascorbate synthesis in the depletion of GSH was also verified. The major finding of the in vitro studies is confirmed by the present in vivo data; gulonolactone-dependent hydrogen peroxide formation is accompanied with a decreased GSH/total glutathione ratio preferentially in the ER lumen. In addition, this new experimental model revealed also the elevation of the hepatic total glutathione level after gulonolactone treatment. This effect is presumably due to an increased synthesis; c-glutamylcysteinyl synthetase (the key enzyme of glutathione synthesis) is known to be stimulated by increasing GSSG concentration [20]. More importantly, luminal GSH oxidation resulted in the increase of total microsomal glutathione content rather than in the depletion of the luminal GSH pool. This observation indicates a fast replenishment of luminal GSH from the cytosol, either by the activation of a specific GSH uptake transport mechanism or by an increase in the non-specific permeability of the ER membrane. Our results favor the latter option: microsomal vesicles prepared from gulonolactone-treated mice show an increased permeability to a variety of low molecular weight compounds. This permeability transition can also be modeled by gulonolactone addition to microsomal incubations. ROS (presumably hydrogen peroxide) is an undesired sideproduct of disulfide bond formation in the ER lumen. Its generation can lead to redox imbalance and hence can contribute to the ER stress caused by the overproduction of secretory proteins [6]. Although hydrogen peroxide is able to cross biological membranes, e.g. by the mediation of aquaporins [21,22], membranes still represent an obstacle for it [22,23]. Consequently, elevated hydrogen peroxide concentrations
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can be expected in the ER during extensive disulfide bond synthesis. However, direct demonstration of luminal hydrogen peroxide accumulation is still missing. It is particularly interesting, since powerful hydrogen peroxide-metabolizing system in the lumen has not been demonstrated. The findings reported here might represent an explanation for the virtual inconsistency; the increased permeability of the ER membrane caused by luminally accumulating hydrogen peroxide would allow the escape of oxidizing agents from the lumen and the influx of reducing equivalents (e.g. GSH) from the cytosol. This phenomenon can be regarded as a novel protective response to the luminal ROS overproduction. The ER lumen characterized by a limited antioxidant capacity needs to seek help from the cytosol, which is abundant in antioxidants. The transient alterations observed in our experiments are indicative of the effectiveness of this mechanism to recover the normal redox conditions and functionality of the challenged ER. It is possible, however, that a sustained increase in the permeability of the ER membrane would trigger additional responses, e.g. unfolded protein response, permeability changes in the plasma membrane or even cell death. Nevertheless, an important question emerges: what is the structural basis of the altered membrane permeability? Several ER channels – e.g. IP3R [24–26], and ryanodine receptor calcium channels [27,28] – are sensitive to redox effects, and their open probability can be governed by oxidoreductions. Moreover, some channels (e.g. translocon peptide channel and ryanodine receptor calcium channel) can mediate the transmembrane flux of small molecules. The ‘‘general’’ permeability of the ER membrane (i.e. permeability that cannot be attributed to identified pores/channels) can also be influenced by redox alterations. Further investigation is needed to explore the mechanisms underlying the observed permeability transition in the ER membrane. Acknowledgements: We would like to thank Mrs. Vale´ria Mile for skillful technical assistance. This work was supported by the Hungarian Scientific Research Fund (T48939 and IN70798), Szenta´gothai Ja´nos Knowledge Center, the Ja´nos Bolyai Research Scholarship of the Hungarian Academy of Sciences (to Miklo´s Csala) and by the Ministry of Health Hungary (ETT 182/2006 and 183/2006).
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