1 Laboratories of Pathology, Dipartimento di Scienze Mediche, Università del Piemonte Orientale `A. Avogadro', via Solaroli 17, 28100 Novara, Italy
2 Molecular Pathology2, Dipartimento di Scienze Mediche, Università del Piemonte Orientale `A. Avogadro', via Solaroli 17, 28100 Novara, Italy
* Author for correspondence (e-mail: isidoro{at}med.unipmn.it)
Accepted 6 October 2003
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Summary |
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Key words: Cell death, Cathepsin D, Ischemia, Exocytosis, Signal transduction
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Introduction |
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Materials and Methods |
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Hepatocyte isolation, treatments and estimation of cell viability
Rat hepatocytes were freshly isolated by collagenase liver perfusion of male Wistar rats (180-250 g) (Harlan Italy, S. Pietro al Natisone, Italy), as previously reported (Carini et al., 2000; Carini et al., 2001a
). The use and the care of the animals were approved by the Italian Ministry of Health and by the University Commission for Animal Care following the criteria of the Italian National Research Council. Hepatocytes were suspended at a final cell density of 106/ml in Krebs-Henseleit-HEPES (KHH) buffer containing 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.3 mM CaCl2, 25 mM NaHCO3- and 20 nM HEPES at pH 7.4. Hepatocytes were preconditioned either by exposure to CGS21680 or by a hypoxic-reoxygenation cycle as previously described (Carini et al., 2000
; Carini et al., 2001a
). The inhibitors WM, 3MA and CytD were added 15 minutes before preconditioning and were present throughout the following incubation. Hepatocytes were then incubated for 60 minutes at 37°C in sealed bottles under 95% O2/5% CO2 (control condition) or 95% N2/5% CO2 (hypoxia). Substances were used at the following concentrations: CGS21680, 1 µM; WM, 250 nM; 3MA, 10 mM; CytD, 20 µM. Hepatocyte viability was determined by standard LDH assay, the Trypan Blue exclusion test and by measuring the fluorescence of hepatocytes stained with propidium iodide according to the method of Gores et al. (Gores et al., 1989
). For the latter, 106 hepatocytes were loaded with 10 µg/ml propidium iodide in 1 ml KHH buffer and the fluorescence was determined in a spectrofluorometer at 520 nm and 605 nm excitation and emission wavelengths, respectively. Parallel aliquots of hepatocytes were permeabilized with digitonin (375 µM) prior to loading with propidium iodide in order to obtain the maximal staining. Extent of cell death was deduced from the ratio of fluorescence intensity measured in non-permeabilized vs digitonin-permeabilized samples. At the beginning of the experiments hepatocyte viability ranged between 80-85%.
Immunofluorescence
At the end of the treatment hepatocytes were seeded on polylisine-coated glass coverslips, allowed to adhere for 5 minutes and then fixed in absolute methanol. This method allowed rapid cell attachment and proved valid for morphological studies since the integrity of subcellular structures in living cells was well maintained, despite cell polarity being lost. Endosomal-lysosomal organelles were traced by immunodetection of cathepsin D (CD), a soluble lysosomal enzyme, and of Lamp-1, a lysosomal membrane-associated glycoprotein. Cell morphology could be better appreciated by immunostaining of actin filaments. CD immunolocalization was performed by using a specific rabbit antiserum (Dragonetti et al., 2000), Lamp-1 and actin were revealed by using specific mouse monoclonal antibodies, respectively purchased from BD Transduction Laboratories (Lexington, KY) and Sigma. Specific secondary antibodies, either conjugated with Texas Red or FITC, were purchased from Sigma. As a negative control, cells were incubated with the secondary antibody alone or with pre-immune antiserum. The experiment was repeated three times and for each experimental condition three coverslips were prepared. At least four fields with about 10-20 cells per field have been analyzed in each coverslip with a laser confocal immunofluorescent microscope (Leica DMIREZ, Leica Microsystems, Heidelberg, Germany). Representative images have been selected by two independent investigators. The surface expression of Lamp-1 was evaluated in non-permeabilized hepatocytes by cytofluorometric analysis. For this purpose isolated hepatocytes were subjected to preconditioning treatment, stained in suspension for Lamp-1 and then analyzed with a fluorescent activated cytofluorometer (FACSCAN, Beckton Dickinson, Mountain View, CA). Similarly, fluorescence associated with intracellular CD was evaluated in permeabilized hepatocytes (by using the FIX & PERM kit from CALTAG Laboratories, Burlingame, CA) stained with anti-CD antibodies as above. Optimal permeabilization and intracellular fluorescent staining was set using actin as the reference antigen. At least 100,000 events were analyzed. The experiments were repeated twice. Based on the setting with cells labeled only with the secondary antibody, values lower than 101 arbitrary units of fluorescence intensity (abscissa axis) were considered negative. Cell positivity corresponds to the area below the curve starting from values of fluorescence intensity higher than 101 arbitrary units and is given as a percentage of the total area.
ß-Hexosaminidase assay and CD western blotting
Hepatocytes (106/ml KHH buffer) were incubated for 60 minutes at 37°C under control conditions after being preconditioned or not in the absence or the presence of inhibitors. The activity of the lysosomal ß-hexosaminidase was assayed in hepatocyte homogenates (106 cells sonicated in 0.36 ml phosphate buffer containing 0.25% sodium desossicholate) and in incubation media. For the assay, 18 µl of cell homogenates and 50 µl of incubation media (corresponding to 50x103 hepatocytes and the respective secretion) were incubated for 60 minutes at 37°C in sodium-citrate buffer at pH 4.5 with the substrate p-nitrophenyl-N-acetyl ß-D glucosaminide. Fluorescence was measured at 405 nm in a spectrofluorometer (Beckman DU530). This assay reveals only the mature ß-hexosaminidase resident within endosomal-lysosomal organelles and therefore it is useful to monitor the exocytosis from these organelles. Enzyme activity was expressed as mU/mg of cell protein. Secreted activity is expressed as percent of total (intracellular plus extracellular) ß-hexosaminidase. Enzyme assays were run in duplicate and repeated at least three times for each sample. Secreted CD molecular forms were revealed by standard western blotting techniques using specific rabbit anti-rat CD immune serum (Dragonetti et al., 2000). Proteins secreted by the hepatocytes were TCA-precipitated from aliquots of incubation media normalized per number of cells, separated by SDS-polyacrylamide (12.5%) gel electrophoresis and electroblotted onto nitrocellulose filter. CD-related bands were revealed by incubation with the anti-CD antiserum followed by a peroxidase-conjugated goat-anti-rabbit antibody and subsequent peroxidase-induced chemiluminescence reaction as recommended by the manufacturer (Amersham). Intensity of the bands was estimated by densitometry.
Determination of intracellular pH
Cytosolic pH was measured as previously reported in detail (Carini et al., 1999; Carini et al., 2001b
) using the fluorescent indicator dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) (Molecular Probes, Eugene OR). For pH probe loading the hepatocytes were incubated for 30 minutes at 25°C in KHH buffer containing 5 µg/ml BCECF-AM. Calibration values were obtained for each experiment by incubating hepatocytes in media at different pH containing 10 µM K+/H+ ionophore nigericine and 120 mM K+. Fluorescence was measured at 450/530 nm wavelength pair using a Hitachi 4500 spectrofluorometer.
Measurement of intracellular Na+ concentration
Intracellular Na+ levels were measured as detailed previously (Carini et al., 1995; Carini et al., 2001b
) using the fluorescent sodium-binding benzofuran isophthalate acetoxymethyl ester (SBFI-AM) (Molecular Probes, Eugene OR) as Na+ probe. Briefly, the hepatocytes were incubated for 60 minutes at 25°C in KHH buffer containing 10 µM SBFI-AM, washed and re-suspendend in fresh KHH medium for further treatments. At each time-point aliquots of hepatocytes were centrifuged and re-suspended in fresh medium for measurements. Changes in SBFI-AM fluorescence were monitored using the Hitachi 4500 spectrofluorometer set at 345 and 385 nm excitation and at 510 nm emission wavelengths. The ratio of fluorescence values at 345 nm and 385 nm excitation was calculated after correction for spontaneous SBFI-AM fluorescence. Calibration of SBFI-AM fluorescence was carried out with hepatocytes incubated in solutions of known Na+ concentrations in the presence of the Na+ ionophore gramicidin D (2 µM).
Evaluation and chelation of free cytosolic calcium ions
Cytosolic free calcium ions levels were determined by using the fluorescent cell permeable dye Fura2-AM (Sigma) as previously detailed (Carini et al., 1997b). Hepatocytes were loaded with this dye by a 15 minute incubation in KHH medium containing 4 µM Fura2-AM. Cells were then washed to remove the excess and further incubated to allow complete de-esterification of Fura2-AM. Ca2+-dependent Fura-2 fluorescence was measured with a computer-assisted fluorometer (Perkin Elmer LS-5B) positioning the excitation wavelength alternately at 340 nm or 380 nm and the emission wavelength at 509 nm. Calibration was done by measuring the fluorescence in cells permeabilized with 10 µg/ml digitonin. Cytosolic free Ca2+ concentration was calculated assuming a Fura-2 Kd of 225 nM.
To inhibit intracellular calcium signaling the membrane-permeable calcium chelant EGTA-AM was employed. For this purpose isolated hepatocytes were loaded with EGTA-AM (15 minutes at room temperature in KHH containing 25 µM EGTA-AM) prior to the preconditioning treatment. Hepatocytes were then processed for viability assay and fluorescence analysis in suspension (Lamp-1 surface expression) or on glass coverslip (CD subcellular localization) as described above.
Statistics
All experiments on cell viability, [Na+]i, pHi and [Ca2+]i concentrations were done in triplicate and repeated at least three times. Data were expressed as mean ± s.d. Statistical analysis was performed with the Instat-3 statistical software (GraphPad Software, San Diego, CA) using a one-way ANOVA test with Bonferroni's correction for multiple comparisons when more than two groups were analyzed. Normality of data distribution of all groups was verified by the Kolmogorov and Smirnov test. Significance was taken at a P value of less than 0.005.
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Results |
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To corroborate these data we also analyzed by cytofluorometry the intracellular content of CD in permeabilized hepatocytes. Compared with controls, in preconditioned hepatocytes about 29% cells (average of two experiments in duplicate) were judged negative for intracellular CD (arbitrary units of fluorescence). Taken together, these data are consistent with the exocytosis of a large fraction of lysosomes that leads to the insertion of lysosomal membrane proteins in the plasma membrane and extracellular release of the lumenal content in preconditioned hepatocytes. This event occurred in about one-third of the hepatocytes subjected to preconditioning treatments. The morphological features described above occurred in living cells, as the preconditioning treatments do not affect cell viability (see below).
Preconditioning-induced exocytosis of endosomal-lysosomal organelles was further demonstrated biochemically, based on the assumption that if fusion of these organelles with plasma membrane takes place then soluble enzymes normally confined within them should be found at higher levels in the extracellular milieu. In fact, the proportion of ß-hexosaminidase activity measured in the extracellular medium, compared with that found in the cell, was increased in hepatocytes preconditioned either by a brief hypoxic-reoxygenation cycle or by exposition to the adenosine A2A-receptor agonist CGS21680 (Fig. 2A). Conversely, the activity measured within the cells was 31.27±4.3 mU/mg and 23.12±3.8 mU/mg in control and preconditioned hepatocytes, respectively.
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Movement of endocytic vesicles requires the integrity of the cytoskeleton and involves various signaling enzymes. Cytochalasin D (Cyt D), which affects the actin cytoskeleton, and wortmannin (WM) and 3-methyladenine (3MA), which inhibit the lipid kinase PI3K, have been shown to interfere with the normal trafficking of endosomal-lysosomal organelles (Cordonnier et al., 2001; Brown et al., 1995
; Punnonen et al., 1994
). We therefore checked the efficacy of these drugs to inhibit endosome and lysosome translocation to the periphery and fusion with plasma membrane associated with preconditioning. As predicted, when hepatocytes were treated in the presence of 3MA (see below for details) the preconditioning-induced increase in ß-hexosaminidase in the medium was completely prevented. In fact, the level of ß-hexosaminidase was much lower than that observed under basal conditions in control hepatocytes (Fig. 2A). This outcome is consistent with the morphological data showing the clustering of endosomes and lysosomes at one pole of the nucleus in preconditioned hepatocytes treated with 3MA (not shown). We attempted to better define whether fusion of CD-positive organelles with plasma membrane involved mainly endosomes or lysosomes. For this purpose we took advantage of the fact that the molecular forms of CD accumulate in different proportions in these organelles and can therefore be exploited as markers to discriminate between endosomes and lysosomes (Chiarpotto et al., 1999
; Dragonetti et al., 2000
). In rat cells CD is present as a 52 kDa precursor (proCD) within the endoplasmic reticulum and Golgi complex, as a 43 kDa mature single-chain in endosomal compartments, and as a 31 + 13 kDa mature double-chain in lysosomes (Démoz et al., 1999
; Dragonetti et al., 2000
). In preconditioned hepatocytes the extracellular release of the three CD molecular forms was nearly doubled, an effect completely reversed by WM, 3MA or Cyt D (Fig. 2B). Under basal conditions (control) hepatocytes released the three forms of CD (only the 31 kDa large chain of the double-chain is visible in the gel) in the medium, but in different proportions (Fig. 2C). In preconditioned hepatocytes the secretion of proCD (from pre-endosomal organelles) and the mature double-chain CD (from lysosomes) was stimulated by a factor of three, whereas secretion of the mature single-chain form of CD (typically resident within endosomes) was stimulated by nearly 1.5-fold (Fig. 2C,D). When the experiment was performed in hepatocytes preconditioned and incubated in the presence of WM, 3MA or Cyt D, the increase in extracellular release of the three CD forms was completely prevented (Fig. 2C,D). From these data one can conclude that preconditioning induced fusion of mainly lysosomal organelles with the plasma membrane and stimulated the exocytosis of pre-endosomal vesicles.
Inhibition of lysosome-plasma membrane fusion in preconditioned hepatocytes abrogates the cytoprotection against hypoxic cell death and the ability to counteract the hypoxia-induced overload of Na+
Preconditioning has been reported to protect hepatocytes from subsequent exposure to cytotoxic hypoxic conditions (Carini et al., 2000). We tested the hypothesis that fusion of endosomal-lysosomal organelles with plasma membrane is mandatory for the acquisition of preconditioning-induced cytoprotection. To this end we analyzed the effects of WM, 3MA or Cyt D, which have been shown to inhibit lysosome exocytosis (Fig. 2), on the viability of hepatocytes subjected to hypoxia before or after being preconditioned. Hepatocytes were pre-incubated with the inhibitors under control conditions and then exposed for 60 minutes to control or hypoxic conditions. The hypoxic condition was also applied to hepatocytes that had been preconditioned with a short hypoxic-reoxygenation cycle in the presence of WM, 3MA or Cyt D. These drugs were present throughout the entire period of the experiment (Fig. 3A). WM, 3MA and Cyt D did not affect the viability of hepatocytes in control oxygenated samples nor did they alter the cytotoxicity observed in hepatocytes incubated under hypoxic conditions. Consistent with our previous report (Carini et al., 2000
), hypoxic preconditioning protected the hepatocytes subsequently exposed to hypoxic conditions by increasing their viability by more than 50% (Fig. 3B). This protective effect was not apparent in hepatocytes that had been treated with WM, 3MA or Cyt D (Fig. 3B). In this latter case, mortality induced by hypoxia was identical to that observed in non-preconditioned hepatocytes.
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We have shown that CGS21680, an agonist of the adenosine A2A-receptor, reproduces the hepatoprotective effect of hypoxic preconditioning (Carini et al., 2001a). We therefore checked whether inhibitors of the endocytic traffic could reverse the cytoprotection induced by this drug. Consistent with the above observation, the results shown in Fig. 4 demonstrate that stimulation of the adenosine A2A-receptor confers resistance to hypoxia killing unless the hepatocytes are not treated with WM, 3MA or Cyt D.
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Overload of Na+ in cultured hepatocytes has been shown to be a critical event in the onset of hypoxic cell death (Carini et al., 1995; Gasbarrini et al., 1992
). In hypoxic or pharmacologically preconditioned hepatocytes exposed to hypoxic conditions the Na+ influx is limited (Carini et al., 2000
; Carini et al., 2001b
). To further substantiate our data on the link between lysosomal exocytosis and cytoprotection we investigated the effects of WM, 3MA and Cyt D on the ability of preconditioned hepatocytes to counteract the hypoxia-induced overload of Na+. For this purpose, the time course (0 to 60 minutes) of intracellular Na+ concentration changes during the hypoxic incubation was measured in preconditioned hepatocytes incubated in the absence or the presence of WM, 3MA or Cyt D (see Fig. 3A and Fig. 4A for the scheme of incubation). WM, 3MA and Cyt D did not influence the [Na+] changes in hepatocytes incubated under control or hypoxic conditions. The Na+ concentration increased by a factor of six in hepatocytes incubated under hypoxic conditions for 60 minutes (Fig. 5A). As already reported (Carini et al., 2000
), preconditioning limited the hypoxia-induced Na+ overload so that at the end of the experimental period the intracellular concentration of Na+ was about half that measured in non-preconditioned hypoxic hepatocytes (Fig. 5B,C). However, when preconditioning and subsequent incubation were performed in the presence of WM, 3MA or Cyt D, preconditioned hepatocytes failed to prevent the influx of Na+ associated with hypoxia (Fig. 5B,C).
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Together these data clearly rule out the possibility that preconditioning-associated fusion of vacuolar acidic compartments with plasma membrane is just an epiphenomenon and rather strongly indicate that this phenomenon is mechanistically linked to the cytoprotective effect. Consistent with this interpretation, 3MA attenuated the secretion of the soluble lysosomal hydrolases CD and ß-hexosaminidase associated with CGS21680-induced preconditioning (Fig. 2A and not shown). Moreover, when preconditioning, either by hypoxic-reoxygenation or by treatment with CGS21680, was performed in the presence of 3MA, CD-positive organelles did not translocate to the periphery, rather these organelles localized to a perinuclear area, mostly at one pole of the nucleus (not shown).
Lysosome-plasma membrane fusion and establishment of cytoprotection in preconditioned hepatocytes are calcium-dependent
In a variety of animal cell models the fusion and exocytosis of lysosomal-like organelles with plasma membrane have been shown to be Ca2+-regulated (Rodriguez et al., 1997; Gerasimenko et al., 2001
; Reddy et al., 2001
; Jaiswal et al., 2002
; Tapper et al., 2002
). In embryonic primary fibroblasts about 25% of the lysosomal population is exocytosed upon elevation of the intracellular free Ca2+ concentration (Jaiswal et al., 2002
). We therefore measured intracellular Ca2+ levels in hepatocytes exposed to CGS21680-induced preconditioning and checked for any causal correlation between Ca2+ concentration and lysosome-plasma membrane fusion. The concentration of free cytosolic Ca2+, measured at the end of the preconditioning treatment, was in fact augmented by a factor of 3.65±0.28 (mean±s.d. of three independent experiments) with respect to the value at time zero (average values were 144 nM and 527 nM in control and preconditioned hepatocytes, respectively). An increase in cytosolic calcium was not observed when preconditioning was performed in the presence of WM, indicating that this event requires the upstream activation of PI3K. To test whether such intracellular Ca2+ elevation was directly implicated in the exocytosis of lysosomes we loaded the hepatocytes with the membrane-permeable Ca2+-chelator EGTA-AM (Carini et al., 1997b
). In preconditioned hepatocytes this compound caused the accumulation of CD-positive organelles near the plasma membrane (Fig. 6A) and inhibited the externalization of Lamp-1 in hepatocytes as much as WM did (Fig. 6B). In addition, EGTA-AM precluded the extracellular release of ß-hexosaminidase and of CD associated with preconditioning (not shown).
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We then checked whether this inhibitory effect of EGTA-AM reflected negatively on the preconditioning-induced cytoprotection. In effect, EGTA-AM abolished the CGS21680-induced cytoprotection as much as WM did in hepatocytes incubated for 60 minutes under hypoxic conditions (Table 1). Similar data were obtained with hepatocytes preconditioned by hypoxic-reoxygenation treatment (not shown). Thus, chelation of free cytosolic Ca2+ prevented the fusion of endosomal-lysosomal organelles with the plasma membrane and abrogated the cytoprotective effects of preconditioning.
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Inhibition of lysosome-plasma membrane fusion abolishes the ability of preconditioned hepatocytes to counteract hypoxia-induced cellular acidosis
In hepatocytes exposed to hypoxic conditions cytosolic acidification occurs (Gores et al., 1989; Carini et al., 2001b
), an effect that is not observed if hepatocytes have been preconditioned (Carini et al., 2001b
). However, the ability of preconditioned hepatocytes to maintain the pH homeostasis under hypoxic conditions is lost in the presence of bafilomycin A, an inhibitor of the lysosomal type H+/ATPase pump (Carini et al., 2001b
). We suspected a link between lysosome exocytosis and the ability to counteract cellular acidosis in preconditioned hepatocytes. Our hypothesis predicts that impairment of the fusion with plasma membrane of endosomal-lysosomal organelles should also impact negatively on the ability of preconditioning to prevent hypoxia-associated acidosis.
The intracellular pH was measured in hepatocytes incubated under hypoxic conditions before or after being preconditioned either by a brief hypoxic-reoxygenation cycle or by preincubation with CGS21680. Before and during the induction of preconditioning and the subsequent incubation, some samples were exposed to WM, 3MA or Cyt D (see Fig. 3A and Fig. 4A for the scheme of the experiment). As shown in Fig. 7A, these drugs did not alter the intracellular pH in hepatocytes incubated under control conditions, nor were they able to influence the drop in pH caused by hypoxia. However, the ability of preconditioning to prevent hypoxia-associated cellular acidosis was completely abolished by WM, 3MA and Cyt D (Fig. 6B,C). These data, together with the observation reported in Figs 1 and 2, strongly support the view that the preconditioning-induced fusion of lysosomes with the plasma membrane is instrumental to the preservation of intracellular pH in hepatocytes subjected to hypoxic conditions.
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Discussion |
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In the present study we found that preconditioning induces a movement of endosomes and lysosomes toward the plasma membrane, which is followed by the fusion of these organelles with the plasma membrane. This was demonstrated by the appearance of Lamp-1 on the cell surface and the release of lysosomal soluble enzymes by preconditioned hepatocytes. The exocytosis process involved mainly lysosomes, based on the molecular forms of CD released extracellularly. We can exclude that such translocation of endosomes and lysosomes to the cell periphery is the consequence of cytosolic acidification, as observed in fibroblasts (Heuser, 1989), since the preconditioning treatment does not affect the intracellular pH in hepatocytes (Carini et al., 2001b
). Inhibiting the translocation of these acidic organelles to the periphery, by WM, 3MA or Cyt D, abrogates the cytoprotective effect of preconditioning as well as the preservative effects on cellular pH and Na+ homeostasis exerted by preconditioning. WM and Cyt D were shown to prevent the ischemic preconditioning-induced cytoprotection in the heart (Tong et al., 2000
; Baines et al., 1999
). The mechanisms underlying such an effect are still obscure, although inhibition of PI3K by WM is known to have severe consequences for cell survival (Rameh and Cantley, 1999
).
Here we propose a new mechanism to explain the cytoprotective effect of preconditioning that involves endosomal and lysosomal organelles. To demonstrate our hypothesis we used various drugs acting on different targets that eventually converge in a common inhibitory effect on the trafficking of endosomes and lysosomes. We in fact employed WM and 3MA, two known inhibitors of PI3K, and Cyt D, which is known to disrupt the organization of the actin cytoskeleton. All these drugs had a similar effect on the endosomal and lysosomal traffic and on the preconditioning-associated protection of hepatocytes exposed to hypoxia. It follows that the mechanism by which WM reverts the preconditioning cytoprotection in heart (Tong et al., 2000) or hepatic cells (the present study) cannot exclusively be attributed to impairment of the PI3K-dependent transcription of survival factors. Consistent with our hypothesis is the observation that preconditioning, regardless of the mode it was induced (either by sub-lethal hypoxia or stimulation of adenosine A2A-receptor), was always associated with the peripheral redistribution and subsequent fusion with plasma membrane of Lamp-1/CD-positive organelles. Two other interesting novel findings arise from the present study: first, preconditioning is associated with an increase in the free cytosolic Ca2+ concentration and, second, this increase is necessary to allow the externalization of lysosomal membrane proteins. Chelation of cytosolic Ca2+ nullified the protective effect of preconditioning against hypoxia. It should be noted that lysosome exocytosis occurred at best in about one-third of the whole culture subjected to preconditioning, as assessed also by cytofluorometric analysis of Lamp-1 and CD positivity. On average, the rate of cell survival in hepatocytes incubated under control conditions was about 80 to 85%, whereas the rates of cell survival under hypoxic conditions were about 40% in the non-preconditioned population and about 65% in the preconditioned population. Therefore, the proportion of hepatocytes in which lysosome exocytosis occurred (
30%) and the proportion of hepatocytes that acquired cytoprotection (
25%) following preconditioning are well in agreement.
What is the physiological significance for the preconditioning-induced translocation to the periphery of lysosomes and of their fusion with the plasma membrane and consequent release in the extracellular environment of acid hydrolases? At present it is difficult to give a definitive answer to this question, although some hypotheses can be made. Perhaps the most attractive one is the possibility that fusion of endosomal-lysosomal organelles allows the insertion on the plasma membrane of the H+/ATPase pump. This would provide an explanation for the activation of the bafilomycin A-sensitive pH buffering system occurring in preconditioned hepatocytes (Carini et al., 2001b). Consistent with this interpretation is the fact that exocytosis involved mainly lysosomes, i.e. the most acidic organelles in which the vacuolar H+/ATPase pump is highly concentrated (Arai et al., 1993
). Further support for this interpretation comes from the observation that, in stimulated neutrophils, cell surface expression of the vacuolar type H+/ATPase pump follows exocytosis of secretory acid granules (Nanda et al., 1996
), which represent a specialized lysosome subpopulation. The plasma membrane insertion of the vacuolar type H+/ATPase pump would help to preserve pH homeostasis and limit Na+ overload, which are caused by Na+/H+ exchange (Carini et al., 1995
). Relevant to the present hypothesis is the observation that, in ileal cells, the brush border expression of a Na+/H+ exchanger was found to depend on the recycling of endosomal vesicles and it was affected by destabilizing the actin cytoskeleton with Cyt D (Li et al., 2001
). It is tempting to speculate that, by preconditioning, endocytic retrieval is accelerated and so membrane expression of such an exchanger is diminished, an event that could also account for the limited Na+ influx occurring during hypoxia. We have previously shown that preservation of intracellular pH and consequent prevention of Na+ overload in preconditioned hepatocytes depend on the activity of p38-MAPK (Carini et al., 2001a
). Intriguingly, our preliminary data indicate that inhibition of p38-MAPK by SB203580 precludes the translocation to the periphery of acidic organelles in preconditioned hepatocytes.
Another possibility to take into consideration is that preconditioning induces a transient upregulation of the autophagocytic pathway shortly followed by extrusion of the lysosomal content. Sustained autophagy would lead to cell death (Bursch, 2001), whereas temporally limited autophagy might exert a protective effect on cell death, for instance by sequestering mitochondrial death-promoting factor (Bauvy et al., 2001
; Lemasters et al., 1998
). Intriguingly, at least two signaling molecules, the heterotrimeric Galfa-inhibitor protein and PI3K, implicated in the onset of preconditioning (Carini et al., 2001a
) (this study) have been shown to positively regulate autophagy (Ogier-Denis et al., 1995
; Blommart et al., 1997). It has been reported that preconditioning protects from caspase 3-dependent (Yadav et al., 1999
) and TNF
-induced cell death (Peralta et al., 2001
). Interestingly, acidic compartments are clearly involved in TNF
-induced activation of caspase 3 (Monney et al., 1998
) and lysosomal cathepsins have been shown to play an important role in TNF
-mediated cytotoxicity in various cell types (Deiss et al., 1996
; Démoz et al., 2002
), including hepatocytes (Guicciardi et al., 2000
). Thus, the extrusion of lysosomal enzymes in preconditioned hepatocytes could contribute indirectly to the downregulation of cell death pathways activated by cytotoxic cytokines released by neighboring cells during prolonged ischemia. Finally, it cannot be excluded that a controlled extracellular release of lysosomal hydrolases has a positive effect on cell survival `in vivo'. In fact, it has been shown that CD can digest components of the extracellular matrix with consequent release of the basic fibroblast growth factor (Briozzo et al., 1991
), which has been demonstrated to promote cell survival and proliferation of rat liver cells after partial hepatectomy (Baruch et al., 1995
). If this latter hypothesis is proven true, the exocytosis of lysosomal proteases would provide a mechanism of protection in vivo much more efficient than is estimated in vitro.
In conclusion, this study has shown that preconditioning alters the localization of endosomes and lysosomes and induces their calcium-dependent fusion with the plasma membrane in hepatocytes. We have shown that this event primarily involves lysosomes and is mandatory for the acquisition of resistance to hypoxic damage. The activation of endosomal-lysosomal organelle recycling provides a rapid mechanism to redistribute molecules among cellular compartments and plasma membrane; this mechanism is compatible with the short period of hypoxia exposure or adenosine A2A-receptor stimulation needed for preconditioning. Thus, in addition to the known cascade of molecular events, the signaling pathways activated by triggers and mediators of preconditioning seem to converge at a common cellular event, that is the translocation to the periphery and fusion with plasma membrane of endosomes and lysosomes. We have proposed various interpretations for such events, one not excluding the other, that can explain many of the observations reported in the literature of preconditioning. The evidence that stimulation of the adenosine A2A-receptor induces the PI3K-dependent translocation to the periphery and the calcium-dependent fusion with plasma membrane of lysosomes provides new insight into the signaling pathways that govern the traffic in the central vacuolar system (Fig. 8). The present study predicts that drugs activating second messengers implicated in the stimulation of endocytic membrane recycling could prove useful for the therapeutic improvement of organ protection.
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Acknowledgments |
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References |
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Arai, K., Shimaya, A., Hiratani, N. and Ohkuma, S. (1993). Purification and characterization of lysosomal H+-ATPase. An anion-sensitive v-type H+-ATPase from rat liver lysosomes. J. Biol. Chem. 286, 5649-5660.
Baines, C. P., Liu, G. S., Birincioglu, M., Critz, S. D., Cohen, M. V. and Dowley J. M. (1999). Ischemic preconditioning depends on interaction between mitochondrial K+/ATP channel and actin cytoscheleton. Am. J. Physiol. 276, H1361-H1368.[Medline]
Baldassarre, M., Dragonetti, A., Marra, P., Luini, A., Isidoro, C. andBuccione, R. (2000). Regulation of protein sorting at the TGN by plasma membrane receptor activation. J. Cell Science 113, 741-748.
Baruch, Y., Shoshany, G., Neufeld, G. and Enat, R. (1995). Basic fibroblast growth factor is hepatotropic for rat liver in regeneration. J. Hepatol. 23, 328-332.[CrossRef][Medline]
Bauvy, C., Gane, P., Arico, S., Codogno, P. and Ogier-Denis, E. (2001). Autophagy delays sulindac sulfide-induced apoptosis in the human intestinal colon cancer cell line HT-29. Exp. Cell Res. 268, 139-149.[CrossRef][Medline]
Blommaart, E. F. C., Krause, U., Schellens, P. M., Vreeling-Sindelarova, H. and Meijer, A. J. (1997). The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur. J. Biochem. 243, 240-246.[Abstract]
Bossi, G. and Griffiths, G. M. (1999). Degranulation plays an essential part in regulating cell surface expression of Fas ligand in T cells and natural killer cells. Nat. Med. 5, 90-96.[CrossRef][Medline]
Briozzo, P., Badet, J., Capony, F., Pieri, I., Montcourrier, P., Barritault, D. and Rochefort, H. (1991). MCF7 mammary cancer cells respond to bFGF and internalize it following its release from extracellular matrix: a permissive role of cathepsin D. Exp. Cell. Res. 194, 252-259.[Medline]
Brown, W. J., De Wald, D. B., Emr, S. D., Plunter, H. and Balch, W. E. (1995). Role for Phosphatidylinisitol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells. J. Cell Biol. 130, 781-796.[Abstract]
Bursch, W. (2001). The autophagosomal-lysosomal compartment in programmed cell death. Cell Death Differ. 8, 569-581.[CrossRef][Medline]
Carini, R., Bellomo, G., Benedetti, A., Fulceri, R., Gamberucci, A., Parola, M., Dianzani, M. U. and Albano, E. (1995). Alteration of Na+ homeostasis as a critical step in the development of irreversible hepatocyte injury after adenosine triphosphate depletion. Hepatology 26, 1089-1098.
Carini, R., Bellomo, G., De Cesaris, M. G. and Albano, E. (1997a). Glycine protects against hepatocyte killing by KCN or hypoxia by preventing Na+ overload in the rat. Hepatology 26, 107-112.[Medline]
Carini, R., De Cesaris, M. G., Bellomo, G. and Albano, E. (1997b). Role of Na+/Ca2+ exchanger in preventing Na+ overload and hepatocyte injury: role of opposite effects of extracellular and intracellular Ca2+ chelation Biochem. Biophys. Res. Commun. 232, 107-110.[CrossRef][Medline]
Carini, R., Autelli, R., Bellomo, G. and Albano, E. (1999). Alteration of cell volume regulation in the development of hepatocyte necrosis. Exp. Cell Res. 248, 280-293.[CrossRef][Medline]
Carini, R., De Cesaris, M. G., Splendore, R., Bagnati, M. and Albano, E. (2000). Ischemic preconditioning reduces Na+ accumulation and cell killing in isolated rat hepatocytes exposed to hypoxia. Hepatology 31, 166-172.[Medline]
Carini, R., De Cesaris, M. G., Splendore, R., Vay, D., Domenicotti, C., Nitti, M. P., Paola, D., Pronzato, M. A. and Albano, E. (2001a). Signal pathway involved in the development of hypoxic preconditioning in rat hepatocytes. Hepatology 33, 131-139.[CrossRef][Medline]
Carini, R., De Cesaris, M. G., Splendore,. R. and Albano, E. (2001b). Stimulation of p38 MAP kinase reduces acidosis and Na+ overload in preconditioned hepatocytes. FEBS Lett. 491, 180-183.[CrossRef][Medline]
Chiarpotto, E., Domenicotti, C., Paola, D., Vitali, A., Nitti, M., Pronzato, M. A., Biasi, F., Cottalasso, D., Marinari, U. M., Dragonetti, A. et al. (1999). Regulation of rat hepatocyte protein kinase C ß isoenzymes by the lipid peroxidation product 4-hydroxy-2,3-nonenal: a signaling pathway to modulate vesicular transport of glycoproteins. Hepatology 29, 1565-1572.[Medline]
Clavien, P. A., Yadav, S., Sindram, D. and Bentley, R. C. (2000). Protective effects of ischemic preconditioning for liver resection performed under inflow occlusion in humans. Ann. Surg. 232, 163-165.[CrossRef][Medline]
Cordonnier, M. N., Dauzonne, D., Louvard, D. and Coudrier, E. (2001). Actin fillaments and myosin I alpha cooperate with microtubules for the movement of lysosomes. Mol. Biol. Cell 12, 4013-4029.
Deiss, L. P., Galinka, H., Berissi, H., Cohen, O. and Kimchi, A. (1996). Cathepsin D protease mediates programmed cell death induced by interferon-gamma, Fas/APO-1 and TNF-alpha. EMBO J. 15, 3861-3870.[Abstract]
Démoz, M., Castino, R., De Stefanis, D., Dragonetti, A., Raiteri, E., Baccino, F. M. and Isidoro, C. (1999). Transformation by oncogenic Ras-p21 alters the processing and subcellular localization of the lysosomal protease cathepsin D. J. Cell Biochem. 73, 370-378.[CrossRef][Medline]
Démoz, M., Castino, R., Cesaro, P., Baccino, F. M., Bonelli, G. and Isidoro, C. (2002). Endosomal-lysosomal proteolysis mediates death signalling by TNF not by VP16 in L929 fibrosarcoma cells: evidence for an active role of Cathepsin D. Biological Chem. 383, 1237-1248.
Dragonetti, A., Baldassarre, M., Castino, R., Démoz, M., Luini, A., Buccione, R. and Isidoro, C. (2000). The lysosomal protease Cathepsin D is efficiently sorted to and secreted from regulated secretory compartments in the Rat Basophilic/Mast cell Line RBL. J. Cell Sci. 113, 3289-3298.
Feng, G. and Kaplowitz, N. (2000). Colchicine protects mice from the lethal effect of an agonistic anti-Fas antibody. J. Clin. Invest. 105, 329-339.
Gasbarrini, A., Borle, A. B., Farghali, H., Francavilla, A. and Van Thiel, D. (1992). Effect of anoxia on intracellular ATP, Na+, Ca2+, Mg2+, and cytotoxicity in rat hepatocytes. J. Biol. Chem. 267, 6654-6663.
Gerasimenko, J. V., Gerasimenko, O. V. and Petersen, O. H. (2001). Membrane repair: Ca2+ elicited lysosomal exocytosis. Curr. Biol. 11, R971-R974.[CrossRef][Medline]
Gores, G. J., Nieminen, A. L., Wray, B. E., Herman, B. and Lemasters, J. J. (1989). Intracellular pH during `chemical hypoxia' in cultured rat hepatocytes. Protection by intracellular acidosis against the onset of cell death. J. Clin. Invest. 83, 386-396.[Medline]
Guicciardi, M. E., Deussing, J., Miyoshi, H., Bronk, S. F., Svingen, P. A., Peters, C., Kaufmann, S. H. and Gores, G. J. (2000). Cathepsin B contributes to TNF- mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c. J. Clin. Invest. 106, 1127-1137.
Heuser, J. (1989). Changes in lysosome shape and distribution correlated with changes in cytoplasmic pH. J. Cell Biol. 108, 855-864.[Abstract]
Jaiswal, J. K., Andrews, N. W. and Simon, S. M. (2002). Membrane proximal lysosomes are the major vesicles responsible for calcium-dependent exocytosis in nonsecretory cells. J. Cell Biol. 159, 625-635.
Lemasters, J. J., Nieminen, A. L., Qian, T., Trost, L. C., Elmore, S. P., Mishimura, Y., Crowe, R. A., Cascio, W. E., Bradham, C. A., Brenner, D. A. and Herman, B. (1998). The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Bioch. Bioph. Acta 1366, 177-196.[Medline]
Li, X., Galli, T., Leu, S., Wade, J. B., Weinman, E. J., Leung, G., Cheong, A., Louvard, D. and Donowitz, M. (2001). Na+-H+ exchanger 3 (NHE3) is present in lipid rafts in the rabbit ileal brush border: a role for rafts in trafficking and rapid stimulation of NHE3. J. Physiol. 537, 537-552.
Matteoni, R. and Kreis, T. E. (1987). Translocation and clustering of endosomes and lysosomes depends on microtubules. J. Cell Biol. 105, 1253-1265.[Abstract]
Miyake, K., McNeil, P. L., Suzuki, K., Tsunoda, R. and Sugai, N. (2001). An actin barrier to resealing. J. Cell Sci. 114, 3487-3494.
Monney, L., Olivier, R., Otter, I., Jansen, B., Poirier, G. G. and Borner, C. (1998). Role of an acidic compartment in tumor-necrosis-factor-alpha-induced production of ceramide, activation of caspase-3 and apoptosis. Eur. J. Biochem. 251, 295-303.[Abstract]
Murry, C. E., Jennings, R. B. and Reimer, K. A. (1986). Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74, 1124-1136.[Abstract]
Nanda, A., Brumell, J. H., Nordstrom, T., Kjeldsen, L., Segelov, H., Borregaard, N., Rotsein, O. D. and Grinstein, S. (1996). Activation of proton pumping in human neutrophils occurs by exocytosis of vesicles bearing vacuolar-type H+-ATPases. J. Biol. Chem. 271, 15963-15970.
Ogier-Denis, E., Couvineau, A., Maorett, J. J., Houri, J. J., Bauvy, C., De Stefanis, D., Isidoro, C., Laburthe, M. and Codogno, P. (1995). A Heterotrimeric Gi3-protein controls autophagic sequestration in the human colon cancer cell line HT-29. J. Biol. Chem. 270, 13-16.
Peralta, C., Hotter, G., Closa, D., Gelpi, E., Bulbena, O. and Catafau, J. R. (1997). Protective effect of preconditioning on the injury associated to hepatic ischemia-reperfusion in the rat: role of nitric oxide and adenosine. Hepatology 25, 934-937.[Medline]
Peralta, C., Closa, D., Hotter, G., Gelpi, E., Bulbena, O. and Rosello-Catafau, J. (1999). Protective role of adenosine in inducing nitric oxide synthesis in rat liver ischemia preconditioning is mediated by the activation of adenosine A2 receptors. Hepatology 29, 126-132.[Medline]
Peralta, C., Fernandez, L., Panes, J., Prats, N., Sans, M., Pique, J. M., Gelpi, E. and Rosello-Catafau, J. (2001). Preconditioning protects against systemic disorders associated with hepatic ischemia-reperfusion through blockade of tumor necrosis factor-induced P-selectin up-regulation in the rat. Hepatology 33, 100-113.[CrossRef][Medline]
Petiot, A., Ogier-Denis, E., Blommart, E. F. C., Meijer, A. J. and Codogno P. (2000). Distinct classes of phosphatidylinositol 3'-Kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J. Biol. Chem. 275, 992-998.
Punnonen, E. L., Marjomaki, V. S. and Reunanan, H. (1994). 3-Methyladenine inhibits transport from late endosomes to lysosomes in cultured rat and mouse fibroblasts. Eur. J. Cell Biol. 665, 14-25.
Rameh, L. E. and Cantley, L. C. (1999). The role of phosphoinositide 3-kinase lipid products in cell function. J. Biol. Chem. 274, 8347-8350.
Reddy, A., Caler, E. V. and Andrews, N. W. (2001). Plasmamembrane repair is mediated by Ca2+-regulated exocytosis of lysosomes. Cell 106, 157-169.[Medline]
Rodriguez, A., Webster, P., Ortengo, J. and Andrews, N. W. (1997). Lysosomes behave as Ca2+-regulated exocytic vesicles in fibroblasts and epithelial cells. J. Cell Biol. 137, 93-104.
Sarafian, V., Jadot, M., Foidart, J.-M., Letesson, J.-J., Van den Brule, F., Castronovo, V., Wattieaux, R., Wattieaux-De Conninck, S. (1998). Expression of Lamp-1 and Lamp-2 and their interactions with galectin-3 in human tumor cells. Int. J. Cancer 75, 105-111.[CrossRef][Medline]
Tapper, H., Furuya, W. and Grinstein, S. (2002). Localized exocytosis of primary (lysosomal) granules during phagocytosis: role of Ca2+-dependent tyrosine phosphorylation and microtubules. J. Immunol. 168, 5287-5296.
Tong, H., Chen, W., Steenbergen, C. and Murphy, E. (2000). Ischemic preconditioning activates phosphatidylinositol-3-kinase upstream of protein kinase C. Circ. Res. 87, 309-315.
Yadav, S., Sindram, D., Perry, D. K. and Clavien, P. A. (1999). Ischemic preconditioning protects the mouse liver by inhibition of apoptosis through a caspasedependent pathway. Hepatology 30, 1223-1231.[Medline]
Yamagami, K., Yamamoto, Y., Kume, M., Kimoto, S., Yamamoto, H., Ozaki, N. and Yamamoto, M. (1998). Heat shock preconditioning ameliorates liver injury following normothermic ischemia-reperfusion in steatotic rat livers. J. Surg. Res. 79, 47-53.[CrossRef][Medline]
Yellon, D. M., Baxter, G. F., Garcia-Dorado, D., Heusch, G. and Sumeray, M. S. (1998). Ischaemic preconditioning: present position and future directions. Cardiovasc. Res. 37, 21-33.[CrossRef][Medline]
Yin, D. P., Sankary, H. N., Chong, A. S. F., Ma, L. L., Shen, J., Foster, P. and Williams, J. W. (1998). Protective effect of ischemic preconditioning on liver preservation-reperfusion injury in rats. Transplant. 66, 152-157.[Medline]
Yoshizumi, T., Yanaga, K., Soejima, Y., Maeda, T., Uchiyama, H. and Sugimachi, K. (1998). Amelioration of the liver injury by ischemic preconditioning. Br. J. Surg. 85, 1636-1640.[CrossRef][Medline]
Zahrebelski, G., Nieminen, A. L., Al-Ghoul, K., Qian, T., Herman, B. and Lemasters, J. J. (1995). Progression of subcellular changes during chemical hypoxia to cultured rat hepatocytes: a laser scanning confocal microscopic study. Hepatology 21, 1361-1372.[Medline]
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