Preconditioning-induced cytoprotection in hepatocytes requires Ca2+-dependent exocytosis of lysosomes

Rita Carini1, Roberta Castino2, Maria Grazia De Cesaris1, Roberta Splendore1, Marina Démoz2, Emanuele Albano1 and Ciro Isidoro2,*

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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A short period of hypoxia reduces the cytotoxicity produced by a subsequent prolonged hypoxia in isolated hepatocytes. This phenomenon, termed hypoxic preconditioning, is mediated by the activation of adenosine A2A-receptor and is associated with the attenuation of cellular acidosis and Na+ overload normally occurring during hypoxia. Bafilomycin, an inhibitor of the vacuolar H+/ATPase, reverts the latter effects and abrogates the preconditioning-induced cytoprotection. Here we provide evidence that the acquisition of preconditioning-induced cytoprotection requires the fusion with plasma membrane and exocytosis of endosomal-lysosomal organelles. Poisons of the vesicular traffic, such as wortmannin and 3-methyladenine, which inhibit phosphatydilinositol 3-kinase, or cytochalasin D, which disassembles the actin cytoskeleton, prevented lysosome exocytosis and also abolished the preconditioning-associated protection from acidosis and necrosis provoked by hypoxia. Preconditioning was associated with the phosphatydilinositol 3-kinase-dependent increase of cytosolic [Ca2+]. Chelation of free cytosolic Ca2+ in preconditioned cells prevented lysosome exocytosis and the acquisition of cytoprotection. We conclude that lysosome-plasma membrane fusion is the mechanism through which hypoxic preconditioning allows hepatocytes to preserve the intracellular pH and survive hypoxic stress. This process is under the control of phosphatydilinositol 3-kinase and requires the integrity of the cytoskeleton and the rise of intracellular free calcium ions.

Key words: Cell death, Cathepsin D, Ischemia, Exocytosis, Signal transduction


    Introduction
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 Introduction
 Materials and Methods
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In different tissues a brief period of ischemia increases the resistance to necrosis or apoptosis produced by a subsequent ischemic insult (Murry et al., 1986Go; Peralta et al., 1997Go). This phenomenon known as `ischemic preconditioning' has attracted the interest of scientists and clinicians because of the potential implications in organ transplantation. For instance, preconditioning has been shown to protect hepatocytes against injury from ischemia or reperfusion and to improve the success rate of liver transplants `taking root' in rats (Yoshizumi et al., 1998Go; Yin et al., 1998Go; Yamagami et al., 1998Go). More recently, ischemic preconditioning has proved its clinical efficacy in patients undergoing liver resection (Clavien et al., 2000Go). It is obvious that clinical management of transplantable organs would benefit from a better knowledge of the chemical triggers, the signal pathways and the effector mechanisms responsible for the cytoprotective effect of ischemic preconditioning. Apparently both necrosis and apoptosis are prevented in preconditioned hepatocytes exposed to hypoxia. Studies performed on isolated and perfused liver have shown that resistance to hypoxia induced by preconditioning is associated with the release of nitric oxide and adenosine (Peralta et al., 1997Go; Peralta et al., 1999Go), downregulation of caspase 3 activity (Yadav et al., 1999Go) and decreased production of TNF{alpha} by Kupffer cells (Peralta et al., 2001Go). We have shown that the preconditioning-induced cytoprotection can be reproduced in isolated rat hepatocytes by a short cycle of hypoxia-reoxygenation or by direct stimulation of the adenosine A2A-receptor with the agonist CGS21680 (Carini et al., 2001aGo). The signaling pathway was shown to involve a trimeric G-inhibitor protein, the phospholipase C, PKC isoenzymes and the p38-MAPK (Carini et al., 2000Go; Carini et al., 2001aGo). In preconditioned hepatocytes the hypoxia-induced acidosis and the consequent Na+ overload, critical alterations for the appearance of hypoxic damage (Carini et al., 1997aGo; Carini et al., 1999Go), are prevented (Carini et al., 2000Go; Carini et al., 1995Go). Both these effects do not occur in preconditioned hepatocytes treated with bafilomycin A, an inhibitor of the vacuolar H+/ATPase pump (Carini et al., 2001bGo). The latter observation indicates that the H+/ATPase pump, normally located on endosomal-lysosomal membrane, might be responsible for the attenuation of the hypoxic acidosis in preconditioned hepatocytes. Several reports by our and other laboratories show that some of the above mentioned signal transducers act in fact as regulators of the endocytic membrane traffic (Ogier-Denis et al., 1995Go; Chiarpotto et al., 1999Go; Baldassarre et al., 2000Go; Petiot et al., 2000Go), implying that movement of vacuolar acidic organelles could be linked to some biochemical features of preconditioning. The concept that vesicle trafficking controls the cell surface expression of proteins has recently received confirmation in various cell models. Thus, in hepatocytes the cell surface exposition of death receptors (Fas and TNFR1) was shown to rely on endocytic vesicle traffic (Feng et al., 2000). Also, in lymphocytes the cell surface expression of the Fas ligand was shown to depend upon exocytic insertion of lysosomal-like cytotoxic granules (Bossi et al., 1999). Similarly, degranulation in activated neutrophils was shown to result in the insertion of the vacuolar H+/ATPase on the plasma membrane (Nanda et al., 1996Go). Based on these observations, we have hypothesized that endosome and lysosome translocation to the cell periphery and fusion with plasma membrane is the (principal) mechanism through which the cytoprotective effect of preconditioning is established. Indeed, a rapid movement of endosomal-lysosomal organelles would be compatible with the fact that preconditioning establishes within minutes (5 to 10 minutes of hypoxia is sufficient). Here we show that in preconditioned hepatocytes such acidic organelles move in fact from the perinuclear region toward the plasma membrane and fuse with it. This was demonstrated by showing the peripheral localization of cathepsin D-positive organelles, the surface exposition of Lamp-1 and the extracellular release of soluble lysosomal enzymes in hypoxic-preconditioned hepatocytes. The same effects were produced by stimulating the adenosine A2A-receptor with CGS21680, a condition that also confers cytoprotection. We also provide evidence that inhibition of lysosome exocytosis by disrupting the actin cytoskeleton or blocking the activity of (phosphoinositide 3-kinase) PI3K precludes the acquisition of preconditioning-induced cytoprotection from hypoxia as well as the associated attenuation of acidosis and of Na+ overload. Finally, we demonstrate that elevation of cytosolic free calcium ions levels in preconditioned hepatocytes is mandatory for both exocytosis of endosomal-lysosomal organelles and acquisition of cytoprotection. To our knowledge this is the first report showing the occurrence of PI3K-mediated and calcium-dependent exocytosis of lysosomes upon stimulation of the adenosine A2A-receptor and the link between this cellular event and the protection from hypoxic cell death.


    Materials and Methods
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 Materials and Methods
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Materials
Collagenase (Type I), N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulfonic acid) (HEPES), phenylmethylsulphonyl fluoride, propidium iodide, wortmannin (WM), 3-methyladenine (3MA), cytochalasin D (Cyt D), CGS21680 were purchased from Sigma Chemical Co (St Louis, MO). EGTA-AM [ethylene glycol bis-(ß-aminoethyl ether) N,N,N',N'-tetraacetic acid acetoxymethyl ester] was from Calbiochem (St Diego, CA). All the other chemicals were of analytical grade and were purchased from Merck (Darmstad, Germany) if not otherwise specified.

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., 2000Go; Carini et al., 2001aGo). 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., 2000Go; Carini et al., 2001aGo). 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., 1989Go). 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., 2000Go), 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., 2000Go). 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., 1999Go; Carini et al., 2001bGo) 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., 1995Go; Carini et al., 2001bGo) 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., 1997bGo). 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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Preconditioning is associated with endosome and lysosome translocation to the cell periphery and fusion with plasma membrane
We first sought to determine the effect of preconditioning on the subcellular localization of acidic vacuolar compartment. To this end, hepatocytes were either maintained for 20 minutes under oxygen fluxing (control) or preconditioned with a cycle of 10 minutes of hypoxia followed by 10 minutes of reoxygenation. Cells were then processed for confocal immunofluorescence analysis using the soluble protease cathepsin D (CD) and the membrane glycoprotein Lamp-1 as tracers of endosomal-lysosomal organelles (Démoz et al., 1999Go; Sarafian et al., 1998Go). The image in Fig. 1A (left panels) shows that, in control hepatocytes, endosomes and lysosomes are distributed throughout the cytoplasm with a preferential accumulation in the perinuclear region, in accord with their usual location in normal cells (Matteoni et al., 1987; Démoz et al., 1999Go). By contrast, in non-preconditioned hepatocytes killed by prolonged exposure to hypoxia CD fluorescence was greatly reduced and diffused in the cytoplasm, suggestive of lysosome rupture (not shown). The population of hypoxic-preconditioned hepatocytes appeared heterogeneous for endosome and lysosome localization. In almost 40 to 50% of the hepatocytes these organelles appear intact and many of them accumulate at the periphery of the cell, close to the plasma membrane (Fig. 1A, right panels). About 20 to 30% of the hepatocytes showed a distribution of CD-positive organelles similar to that observed in controls, whereas the remaining hepatocytes (about 30%) appeared partially or totally devoid of lysosomal organelles (not shown). Similar pictures were obtained by immunostaining endosomes and lysosomes with antibodies specific for an integral membrane glycoprotein (Fig. 1A). The appearance of Lamp-1 on the cell surface proves that at least some of these organelles fused with the plasma membrane in preconditioned hepatocytes. We extended our investigation to a chemical-induced preconditioning system based on the stimulation of the adenosine A2A-receptor with the agonist CGS21680 (Carini et al., 2001aGo). In this case we also observed that endosomes and lysosomes localized to the periphery of the cell in a large fraction of hepatocytes (Fig. 1B). Hepatocytes were also stained for actin, which localizes close to the plasma membrane, to mark the cell border (Fig. 1Bc). The image in Fig. 1B (panel d), showing the overlap of cortical actin (green) and CD (red) staining, demonstrates that, in these hepatocytes, endosomes and lysosomes move to the extreme periphery of the cell upon preconditioning. To quantify the extent of lysosome-plasma membrane fusion we analyzed by cytofluorometry the expression of Lamp-1 on the cell surface in non-permeabilized control and preconditioned hepatocytes and obtained the following values (percent of positivity, average of two experiments in duplicate): control, 1%; hypoxic-preconditioned, 35%; CGS221680-preconditioned, 33% (Fig. 1C).



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Fig. 1. Preconditioning induces the translocation of endosomal-lysosomal organelles to the extreme periphery of the cell. Hepatocytes were incubated under control conditions prior to (control) or after being preconditioned by a cycle of hypoxia-reoxygenation (A) or by a 15 minute incubation with 1 µM CGS21680 (B). Cells were allowed to attach to glass coverslips, fixed and permeabilized, and processed for immunofluorescence confocal microscopy. Endosomes and lysosomes were identified by immunofluorescent detection of CD or Lamp-1. Representative images are shown. (A) Hypoxic preconditioning (PC) caused the dislocation of endosomes and lysosomes from the perinuclear region (see controls, Co) toward the cellular periphery. (B) CGS21680-preconditioned hepatocytes were stained for both CD (red fluorescence, panel b) and for actin (green fluorescence, panel c). Staining of cortical actin marked the cell border. Cell morphology can be appreciated in the phase contrast image (panel a). The translocation of endosomes and lysosomes to the extreme periphery of the cells can be appreciated in panel d, showing the overlap of actin and CD staining. The arrow in panel d points to the plasma membrane region in which cortical actin appears disassembled, as expected in the exocytosis process (Miyake et al., 2001Go), while lysosomal CD appears to be extruded from the cell (see also panels b and c). (C) Typical cytofluorograms of cell surface expression of Lamp-1 are shown. The positivity for this lysosomal membrane protein is increased in hepatocytes preconditioned by transient hypoxia (PC) or by CGS21680 treatment (CGS).

 

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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Fig. 2. Preconditioning induces the secretion of lysosomal hydrolases: inhibition by WM, 3MA and CytD. Isolated hepatocytes were subjected to hypoxic- or CGS21680-preconditioning (PC or CGS samples, respectively) or not (Co, controls) and further incubated for 60 minutes under control conditions. In some samples preconditioning and subsequent incubation were performed in the presence of 10 mM 3MA, 250 nM WM or 20 µM Cyt D as indicated (see the Materials and Methods section for details). The extracellular release of ß-hexosaminidase activity (A) and CD protein (B) were determined in the incubation media by enzyme assay and western blotting, respectively. (A) There is an excess of secreted ß-hexosaminidase activity in media from preconditioned hepatocytes compared with that from controls. This secretion is largely inhibited by 3MA. (B) The CD-related bands identified by western blotting were quantified by densitometry. Total CD found in media from preconditioned hepatocytes (PC and CGS lanes) was approximately double that found in media from control (Co) or preconditioned hepatocytes treated with 3MA, WM or Cyt D. Data in A and B are means±s.d. of three separate experiments. In A, the difference of PC vs Co and of CGS vs Co data was statistically significant (P0.001). In C, the typical pattern of CD molecular forms identified by western blotting in the incubation media (P, precursor; Msc, mature single-chain; LC, large chain of the mature double-chain) is shown. A representative gel from three experiments is shown. Control hepatocytes showed a basal level of CD secretion. Preconditioning stimulated the secretion of the three CD molecular forms in a different manner. By densitometry (D), proCD and mature double-chain CD were the most stimulated forms. Stimulation of proCD secretion in preconditioned hepatocytes is probably due to the activation of PKC (Chiarpotto et al., 1999Go). The enhancement of CD secretion was completely prevented if preconditioning treatment was performed in the presence of 3MA, Cyt D or WM.

 

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., 2001Go; Brown et al., 1995Go; Punnonen et al., 1994Go). 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., 1999Go; Dragonetti et al., 2000Go). 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., 1999Go; Dragonetti et al., 2000Go). 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., 2000Go). 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., 2000Go), 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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Fig. 3. Inhibitors of endocytic vesicular traffic abrogate hypoxic preconditioning-induced cytoprotection from hypoxia. (A) Hepatocytes were incubated for 60 minutes in control or hypoxic conditions. Prior to this incubation, some samples were preconditioned by a 10 minute exposure to hypoxia followed by a 10 minute re-oxygenation (PC). In a parallel set of samples, hepatocytes were treated with 250 nM WM, 10 mM 3MA or 20 µM Cyt D. These drugs were added 15 minutes before the start of preconditioning and were present throughout the entire experimental period. (B) Hepatocyte viability was evaluated after incubation for 60 minutes under control or hypoxic conditions in non-preconditioned and preconditioned cells treated as explained above. The experiment demonstrates that WM, 3MA and Cyt D completely nullified the cytoprotective effect of hypoxic preconditioning against hypoxia. Data are given as means±s.d. of four independent experiments. (*P<0.001, statistical significance vs hypoxia and vs PC incubated under hypoxia in the presence of inhibitors.)

 

We have shown that CGS21680, an agonist of the adenosine A2A-receptor, reproduces the hepatoprotective effect of hypoxic preconditioning (Carini et al., 2001aGo). 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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Fig. 4. Inhibitors of endocytic vesicular traffic reverse hepatoprotection induced by CGS21680. (A) Hepatocytes were incubated as explained in the legend to Fig. 3A, except that preconditioning was performed by incubating the cells for 15 minutes with the adenosine A2A-receptor agonist CGS21680 (1 µM). In some samples, 250 nM WM, 10 mM 3MA or 20 µM Cyt D were added during the treatment as indicated (see also text for details). (B) Hepatocyte viability was evaluated after incubating for 60 minutes under control or hypoxic conditions in non-preconditioned and preconditioned hepatocytes. Data (means±s.d. of four experiments) demonstrate that WM, 3MA and Cyt D nullified the cytoprotective effect of CGS21680. (*P<0.001, statistical significance vs hypoxia and vs CGS incubated under hypoxia in the presence of inhibitors.)

 

Overload of Na+ in cultured hepatocytes has been shown to be a critical event in the onset of hypoxic cell death (Carini et al., 1995Go; Gasbarrini et al., 1992Go). In hypoxic or pharmacologically preconditioned hepatocytes exposed to hypoxic conditions the Na+ influx is limited (Carini et al., 2000Go; Carini et al., 2001bGo). 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., 2000Go), 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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Fig. 5. Inhibitors of endocytic vesicular traffic abrogate the protective effect of preconditioning on Na+ homeostasis. Intracellular Na+ content was assessed in isolated hepatocytes using a fluorescent sodium probe as detailed in Materials and Methods. Two parallel set of samples were prepared in which hepatocytes were preconditioned by a hypoxia-reoxygenation cycle (PC) or by treatment with 1 µM CGS21680 (CGS). In some samples, preconditioning and subsequent incubation under hypoxic conditions were performed in the presence of 250 nM WM, 10 mM 3MA or 20 µM Cyt D, as for the experiments described in Figs 3 and 4. Changes in cellular [Na+] were monitored during a 60 minute incubation. WM, 3MA and Cyt D were shown not to influence the intracellular Na+ content in hepatocytes incubated under control or hypoxic conditions (A). In control (Co) hepatocytes incubated under control conditions the homeostasis of intracellular Na+ was preserved, whereas in hepatocytes incubated under hypoxic conditions an influx of Na+ is observed. This influx is limited in preconditioned hepatocytes but it returns to the same values as in non-preconditioned hepatocytes when preconditioning and subsequent incubation are performed in the presence of WM, 3MA or Cyt D (B,C). Data are the means±s.d. of three independent experiments.

 

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., 1997Go; Gerasimenko et al., 2001Go; Reddy et al., 2001Go; Jaiswal et al., 2002Go; Tapper et al., 2002Go). In embryonic primary fibroblasts about 25% of the lysosomal population is exocytosed upon elevation of the intracellular free Ca2+ concentration (Jaiswal et al., 2002Go). 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., 1997bGo). 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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Fig. 6. Chelation of cytosolic Ca2+ prevents preconditioning-induced lysosome exocytosis. Hepatocytes were preconditioned by treatment with 1 µM CGS21680 (CGS) for 15 minutes in the absence or the presence of WM (250 nM) or the membrane-permeable cytosolic calcium chelator EGTA-AM (25 µM). Pre-conditioned hepatocytes were then seeded on glass coverslips for CD immunofluorescence staining (A) or processed in suspension for cell surface Lamp-1 immuofluorescence staining (B). Control untreated hepatocytes (Co) were also included in the analysis. In CGS2680-treated hepatocytes lysosomes appear located beneath the plasma membrane. The translocation of endosomal-lysosomal organelles to the cell periphery induced by preconditioning is not followed by fusion with plasma membrane when intracellular calcium ions are chelated by EGTA-AM (A). Hepatocytes stained for cell surface Lamp-1 and analyzed by cytofluorometry reveal that EGTA-AM, much like WM, prevented insertion of this lysosomal membrane glycoprotein into the plasma membrane (B; the shift to the right of the cytofluorogram, indicative of increased fluorescence, is evident in CGS21680-treated hepatocytes). Representative confocal microscopic images and cytofluorometric profiles are shown.

 

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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Table 1. Chelation of cytosolic Ca2+ abolishes the preconditioning-induced cytoprotection

 

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., 1989Go; Carini et al., 2001bGo), an effect that is not observed if hepatocytes have been preconditioned (Carini et al., 2001bGo). 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., 2001bGo). 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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Fig. 7. Inhibitors of vesicular traffic abolish the pH buffering capability of preconditioned hepatocytes. Isolated hepatocytes were loaded with a pH fluorescence indicator (see Materials and Methods for details) and then incubated under control or hypoxic conditions. Two sets of preconditioned hepatocytes were prepared, either by hypoxic-reoxygenation cycle (PC) or by incubation for 15 minutes with 1 µM CGS21680 (CGS). In some samples incubation were performed in the presence of 250 nM WM, 10 mM 3MA or 20 µM Cyt D. Panel A shows that WM, 3MA and Cyt D did not affect the pHi of hepatocytes incubated under control or hypoxic conditions. These drugs, however, abolished the pH buffering effect associated with hypoxic- or CGS21680-induced preconditioning (panels B and C, respectively). The results are means of three experiments ± s.d. (*P<0.001, statistical significance vs hypoxia and PC or CGS incubated under hypoxia in the presence of inhibitors.)

 


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ischemic preconditioning, i.e. a sub-lethal exposure to hypoxic conditions, renders the organ tolerant to hypoxic injury (Yellon et al., 1998Go). Understanding the mechanism underlying this phenomenon is essential for a pharmacological approach to organ preservation. Several triggers and mediators and a number of signaling pathways activated during hepatic preconditioning have been discovered (Yamagami et al., 1998Go; Peralta et al., 2001Go; Carini et al., 2001aGo). Much less is known about the cellular events that accompany the onset of preconditioning. In this study we investigated the effect of hypoxia on endosomal and lysosomal traffic, and tested whether these organelles were involved in the cytoprotective effect against hypoxic damage seen in preconditioned hepatocytes. In oxygenated hepatocytes, endosomes and lysosomes appeared as punctuate organelles concentrated mainly in the perinuclear region whereas, in hypoxic hepatocytes, the bright punctuate fluorescence was not visible, which indicates that these organelles had disintegrated and released their content into the cytosol and extracellular space. These features have been associated with necrotic cell death (Zahrebelski et al., 1995Go) whereas, in cells undergoing apoptotic cell death, endosomes and lysosomes were shown to maintain their integrity and cluster at one pole of the cell (Démoz et al., 2002Go).

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, 1989Go), since the preconditioning treatment does not affect the intracellular pH in hepatocytes (Carini et al., 2001bGo). 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., 2000Go; Baines et al., 1999Go). 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, 1999Go).

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., 2000Go) 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., 2001bGo). 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., 1993Go). 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., 1996Go), 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., 1995Go). 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., 2001Go). 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., 2001aGo). 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, 2001Go), whereas temporally limited autophagy might exert a protective effect on cell death, for instance by sequestering mitochondrial death-promoting factor (Bauvy et al., 2001Go; Lemasters et al., 1998Go). Intriguingly, at least two signaling molecules, the heterotrimeric Galfa-inhibitor protein and PI3K, implicated in the onset of preconditioning (Carini et al., 2001aGo) (this study) have been shown to positively regulate autophagy (Ogier-Denis et al., 1995Go; Blommart et al., 1997). It has been reported that preconditioning protects from caspase 3-dependent (Yadav et al., 1999Go) and TNF{alpha}-induced cell death (Peralta et al., 2001Go). Interestingly, acidic compartments are clearly involved in TNF{alpha}-induced activation of caspase 3 (Monney et al., 1998Go) and lysosomal cathepsins have been shown to play an important role in TNF{alpha}-mediated cytotoxicity in various cell types (Deiss et al., 1996Go; Démoz et al., 2002Go), including hepatocytes (Guicciardi et al., 2000Go). 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., 1991Go), which has been demonstrated to promote cell survival and proliferation of rat liver cells after partial hepatectomy (Baruch et al., 1995Go). 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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Fig. 8. Dissection of the signaling pathway activated by preconditioning and leading to lysosome-plasma membrane fusion. The scheme proposed is based on our previous (Carini et al., 2001aGo) and present data. Stimulation of the adenosine A2 receptor activates a cascade of signals involving PI3K, PKC and p38MAPK that eventually impact on the vesicular traffic. Consequently, endosomal and lysosomal organelles are translocated to the periphery of the cell and fuse with the plasma membrane in a calcium-dependent fashion. This process requires the integrity of the cytoskeleton and results in the exocytic insertion of lysosomal mebrane proteins on the plasma membrane. ER, endoplasmic reticulum; GC, Golgi complex; PLC, phospholipase C; DAG, diacylglycerol; IP3, inositol (1,4,5) tris-phosphate.

 


    Acknowledgments
 
Funded by the Ministero dell'Istruzione, Università e Ricerca (MIUR, Cofin 2001), The Consiglio Nazionale delle Ricerche (CNR, Target Project Biotechnology, Contract No. 9900386pf.49 To C.I.), The Università del Piemonte Orientale and The Regione Piemonte. The authors wish to thank Denis Longhi for excellent artwork.


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 Materials and Methods
 Results
 Discussion
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