IntroductionIn different tissues a brief period of ischemia increases theresistance to necrosis or apoptosis produced by a subsequentischemic insult (Murry et al., 1986; Peralta et al., 1997). Thisphenomenon known as ‘ischemic preconditioning’ hasattracted the interest of scientists and clinicians because of thepotential implications in organ transplantation. For instance,preconditioning has been shown to protect hepatocytes againstinjury from ischemia or reperfusion and to improve the successrate of liver transplants ‘taking root’ in rats (Yoshizumi et al.,1998; Yin et al., 1998; Yamagami et al., 1998). More recently,ischemic preconditioning has proved its clinical efficacy inpatients undergoing liver resection (Clavien et al., 2000). It isobvious that clinical management of transplantable organswould benefit from a better knowledge of the chemical triggers,the signal pathways and the effector mechanisms responsiblefor the cytoprotective effect of ischemic preconditioning.Apparently both necrosis and apoptosis are prevented inpreconditioned hepatocytes exposed to hypoxia. Studiesperformed on isolated and perfused liver have shown thatresistance to hypoxia induced by preconditioning is associatedwith the release of nitric oxide and adenosine (Peralta et al.,1997; Peralta et al., 1999), downregulation of caspase 3 activity(Yadav et al., 1999) and decreased production of TNFα by

Kupffer cells (Peralta et al., 2001). We have shown that thepreconditioning-induced cytoprotection can be reproduced inisolated 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., 2001a). Thesignaling pathway was shown to involve a trimeric G-inhibitorprotein, the phospholipase C, PKC isoenzymes and the p38-MAPK (Carini et al., 2000; Carini et al., 2001a). Inpreconditioned hepatocytes the hypoxia-induced acidosis andthe consequent Na+ overload, critical alterations for theappearance of hypoxic damage (Carini et al., 1997a; Carini etal., 1999), are prevented (Carini et al., 2000; Carini et al.,1995). Both these effects do not occur in preconditionedhepatocytes treated with bafilomycin A, an inhibitor of thevacuolar H+/ATPase pump (Carini et al., 2001b). The latterobservation indicates that the H+/ATPase pump, normallylocated on endosomal-lysosomal membrane, might beresponsible for the attenuation of the hypoxic acidosis inpreconditioned hepatocytes. Several reports by our and otherlaboratories show that some of the above mentioned signaltransducers act in fact as regulators of the endocytic membranetraffic (Ogier-Denis et al., 1995; Chiarpotto et al., 1999;Baldassarre et al., 2000; Petiot et al., 2000), implying thatmovement of vacuolar acidic organelles could be linked to

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A short period of hypoxia reduces the cytotoxicityproduced by a subsequent prolonged hypoxia in isolatedhepatocytes. This phenomenon, termed hypoxicpreconditioning, is mediated by the activation of adenosineA2A-receptor and is associated with the attenuation ofcellular acidosis and Na+ overload normally occurringduring hypoxia. Bafilomycin, an inhibitor of the vacuolarH+/ATPase, reverts the latter effects and abrogates thepreconditioning-induced cytoprotection. Here we provideevidence that the acquisition of preconditioning-inducedcytoprotection requires the fusion with plasma membraneand exocytosis of endosomal-lysosomal organelles. Poisonsof the vesicular traffic, such as wortmannin and 3-methyladenine, which inhibit phosphatydilinositol 3-kinase, or cytochalasin D, which disassembles the actincytoskeleton, prevented lysosome exocytosis and also

abolished the preconditioning-associated protectionfrom acidosis and necrosis provoked by hypoxia.Preconditioning was associated with thephosphatydilinositol 3-kinase-dependent increase ofcytosolic [[Ca2+]]. Chelation of free cytosolic Ca2+ inpreconditioned cells prevented lysosome exocytosis and theacquisition of cytoprotection. We conclude that lysosome-plasma membrane fusion is the mechanism through whichhypoxic preconditioning allows hepatocytes to preserve theintracellular pH and survive hypoxic stress. This process isunder the control of phosphatydilinositol 3-kinase andrequires the integrity of the cytoskeleton and the rise ofintracellular free calcium ions.

Key words: Cell death, Cathepsin D, Ischemia, Exocytosis, Signaltransduction

Summary

Preconditioning-induced cytoprotection inhepatocytes requires Ca 2+-dependent exocytosis oflysosomes Rita Carini 1, Roberta Castino 2, Maria Grazia De Cesaris 1, Roberta Splendore 1, Marina Démoz 2,Emanuele Albano 1 and Ciro Isidoro 2,*Laboratories of Pathology1 and of Molecular Pathology2, Dipartimento di Scienze Mediche, Università del Piemonte Orientale ‘A. Avogadro’,via Solaroli 17, 28100 Novara, Italy*Author for correspondence (e-mail: isidoro@med.unipmn.it)

Accepted 6 October 2003Journal of Cell Science 117, 1065-1077 Published by The Company of Biologists 2004doi:10.1242/jcs.00923

Research Article

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some biochemical features of preconditioning. The conceptthat vesicle trafficking controls the cell surface expression ofproteins has recently received confirmation in various cellmodels. Thus, in hepatocytes the cell surface exposition ofdeath receptors (Fas and TNFR1) was shown to rely onendocytic vesicle traffic (Feng et al., 2000). Also, inlymphocytes the cell surface expression of the Fas ligand wasshown to depend upon exocytic insertion of lysosomal-likecytotoxic granules (Bossi et al., 1999). Similarly, degranulationin activated neutrophils was shown to result in the insertion ofthe vacuolar H+/ATPase on the plasma membrane (Nanda etal., 1996). Based on these observations, we have hypothesizedthat endosome and lysosome translocation to the cell peripheryand fusion with plasma membrane is the (principal) mechanismthrough which the cytoprotective effect of preconditioningis established. Indeed, a rapid movement of endosomal-lysosomal organelles would be compatible with the fact thatpreconditioning establishes within minutes (5 to 10 minutes ofhypoxia is sufficient). Here we show that in preconditionedhepatocytes such acidic organelles move in fact from theperinuclear region toward the plasma membrane and fusewith it. This was demonstrated by showing the peripherallocalization of cathepsin D-positive organelles, the surfaceexposition of Lamp-1 and the extracellular release of solublelysosomal enzymes in hypoxic-preconditioned hepatocytes.The same effects were produced by stimulating the adenosineA2A-receptor with CGS21680, a condition that also conferscytoprotection. We also provide evidence that inhibition oflysosome exocytosis by disrupting the actin cytoskeleton orblocking the activity of (phosphoinositide 3-kinase) PI3Kprecludes the acquisition of preconditioning-inducedcytoprotection from hypoxia as well as the associatedattenuation of acidosis and of Na+ overload. Finally, wedemonstrate that elevation of cytosolic free calcium ions levelsin preconditioned hepatocytes is mandatory for both exocytosisof endosomal-lysosomal organelles and acquisition ofcytoprotection. To our knowledge this is the first reportshowing the occurrence of PI3K-mediated and calcium-dependent exocytosis of lysosomes upon stimulation of theadenosine A2A-receptor and the link between this cellular eventand the protection from hypoxic cell death.

Materials and MethodsMaterialsCollagenase (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 SigmaChemical Co (St Louis, MO). EGTA-AM [ethylene glycol bis-(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid acetoxymethyl ester]was from Calbiochem (St Diego, CA). All the other chemicals wereof analytical grade and were purchased from Merck (Darmstad,Germany) if not otherwise specified.

Hepatocyte isolation, treatments and estimation of cell viabilityRat hepatocytes were freshly isolated by collagenase liver perfusionof 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 ItalianMinistry of Health and by the University Commission for AnimalCare 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 mMKCl, 1.2 mM KH2PO4, 1.3 mM CaCl2, 25 mM NaHCO3– and 20 nMHEPES at pH 7.4. Hepatocytes were preconditioned either byexposure to CGS21680 or by a hypoxic-reoxygenation cycle aspreviously described (Carini et al., 2000; Carini et al., 2001a). Theinhibitors WM, 3MA and CytD were added 15 minutes beforepreconditioning and were present throughout the followingincubation. Hepatocytes were then incubated for 60 minutes at 37°Cin sealed bottles under 95% O2/5% CO2 (control condition) or 95%N2/5% CO2 (hypoxia). Substances were used at the followingconcentrations: CGS21680, 1 µM; WM, 250 nM; 3MA, 10 mM;CytD, 20 µM. Hepatocyte viability was determined by standard LDHassay, the Trypan Blue exclusion test and by measuring thefluorescence of hepatocytes stained with propidium iodide accordingto 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 mlKHH buffer and the fluorescence was determined in aspectrofluorometer at 520 nm and 605 nm excitation and emissionwavelengths, respectively. Parallel aliquots of hepatocytes werepermeabilized with digitonin (375 µM) prior to loading withpropidium iodide in order to obtain the maximal staining. Extent ofcell death was deduced from the ratio of fluorescence intensitymeasured in non-permeabilized vs digitonin-permeabilized samples.At the beginning of the experiments hepatocyte viability rangedbetween 80-85%.

ImmunofluorescenceAt the end of the treatment hepatocytes were seeded on polylisine-coated glass coverslips, allowed to adhere for 5 minutes and thenfixed in absolute methanol. This method allowed rapid cellattachment and proved valid for morphological studies since theintegrity of subcellular structures in living cells was well maintained,despite cell polarity being lost. Endosomal-lysosomal organelleswere traced by immunodetection of cathepsin D (CD), a solublelysosomal enzyme, and of Lamp-1, a lysosomal membrane-associated glycoprotein. Cell morphology could be better appreciatedby immunostaining of actin filaments. CD immunolocalization wasperformed by using a specific rabbit antiserum (Dragonetti et al.,2000), Lamp-1 and actin were revealed by using specific mousemonoclonal antibodies, respectively purchased from BDTransduction Laboratories (Lexington, KY) and Sigma. Specificsecondary antibodies, either conjugated with Texas Red or FITC,were purchased from Sigma. As a negative control, cells wereincubated with the secondary antibody alone or with pre-immuneantiserum. The experiment was repeated three times and for eachexperimental condition three coverslips were prepared. At least fourfields with about 10-20 cells per field have been analyzed ineach coverslip with a laser confocal immunofluorescent microscope(Leica DMIREZ, Leica Microsystems, Heidelberg, Germany).Representative images have been selected by two independentinvestigators. The surface expression of Lamp-1 was evaluated innon-permeabilized hepatocytes by cytofluorometric analysis. For thispurpose isolated hepatocytes were subjected to preconditioningtreatment, stained in suspension for Lamp-1 and then analyzed witha fluorescent activated cytofluorometer (FACSCAN, BecktonDickinson, Mountain View, CA). Similarly, fluorescence associatedwith intracellular CD was evaluated in permeabilized hepatocytes (byusing the FIX & PERM kit from CALTAG Laboratories, Burlingame,CA) stained with anti-CD antibodies as above. Optimalpermeabilization and intracellular fluorescent staining was set usingactin as the reference antigen. At least 100,000 events were analyzed.The experiments were repeated twice. Based on the setting with cellslabeled only with the secondary antibody, values lower than 101

arbitrary units of fluorescence intensity (abscissa axis) wereconsidered negative. Cell positivity corresponds to the area below the

Journal of Cell Science 117 (7)

1067Lysosome exocytosis in hepatocyte preconditioning

curve starting from values of fluorescence intensity higher than 101

arbitrary units and is given as a percentage of the total area.

β-Hexosaminidase assay and CD western blottingHepatocytes (106/ml KHH buffer) were incubated for 60 minutes at37°C under control conditions after being preconditioned or not inthe absence or the presence of inhibitors. The activity of thelysosomal β-hexosaminidase was assayed in hepatocytehomogenates (106 cells sonicated in 0.36 ml phosphate buffercontaining 0.25% sodium desossicholate) and in incubation media.For the assay, 18 µl of cell homogenates and 50 µl of incubationmedia (corresponding to 50×103 hepatocytes and the respectivesecretion) were incubated for 60 minutes at 37°C in sodium-citratebuffer at pH 4.5 with the substrate p-nitrophenyl-N-acetyl β-Dglucosaminide. Fluorescence was measured at 405 nm in aspectrofluorometer (Beckman DU530). This assay reveals only themature β-hexosaminidase resident within endosomal-lysosomalorganelles and therefore it is useful to monitor the exocytosis fromthese organelles. Enzyme activity was expressed as mU/mg of cellprotein. Secreted activity is expressed as percent of total(intracellular plus extracellular) β-hexosaminidase. Enzyme assayswere run in duplicate and repeated at least three times for eachsample. Secreted CD molecular forms were revealed by standardwestern blotting techniques using specific rabbit anti-rat CDimmune serum (Dragonetti et al., 2000). Proteins secreted by thehepatocytes were TCA-precipitated from aliquots of incubationmedia normalized per number of cells, separated by SDS-polyacrylamide (12.5%) gel electrophoresis and electroblottedonto nitrocellulose filter. CD-related bands were revealed byincubation with the anti-CD antiserum followed by a peroxidase-conjugated goat-anti-rabbit antibody and subsequent peroxidase-induced chemiluminescence reaction as recommended by themanufacturer (Amersham). Intensity of the bands was estimated bydensitometry.

Determination of intracellular pHCytosolic pH was measured as previously reported in detail (Carini etal., 1999; Carini et al., 2001b) using the fluorescent indicator dye2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein-acetoxymethyl ester(BCECF-AM) (Molecular Probes, Eugene OR). For pH probe loadingthe hepatocytes were incubated for 30 minutes at 25°C in KHH buffercontaining 5 µg/ml BCECF-AM. Calibration values were obtained foreach experiment by incubating hepatocytes in media at different pHcontaining 10 µM K+/H+ ionophore nigericine and 120 mM K+.Fluorescence was measured at 450/530 nm wavelength pair using aHitachi 4500 spectrofluorometer.

Measurement of intracellular Na+ concentrationIntracellular Na+ levels were measured as detailed previously (Cariniet al., 1995; Carini et al., 2001b) using the fluorescent sodium-bindingbenzofuran isophthalate acetoxymethyl ester (SBFI-AM) (MolecularProbes, Eugene OR) as Na+ probe. Briefly, the hepatocytes wereincubated for 60 minutes at 25°C in KHH buffer containing 10 µMSBFI-AM, washed and re-suspendend in fresh KHH medium forfurther treatments. At each time-point aliquots of hepatocytes werecentrifuged and re-suspended in fresh medium for measurements.Changes in SBFI-AM fluorescence were monitored using the Hitachi4500 spectrofluorometer set at 345 and 385 nm excitation and at 510nm emission wavelengths. The ratio of fluorescence values at 345 nmand 385 nm excitation was calculated after correction for spontaneousSBFI-AM fluorescence. Calibration of SBFI-AM fluorescence wascarried 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 ionsCytosolic free calcium ions levels were determined by using thefluorescent cell permeable dye Fura2-AM (Sigma) as previouslydetailed (Carini et al., 1997b). Hepatocytes were loaded with this dyeby a 15 minute incubation in KHH medium containing 4 µM Fura2-AM. Cells were then washed to remove the excess and furtherincubated 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 excitationwavelength alternately at 340 nm or 380 nm and the emissionwavelength at 509 nm. Calibration was done by measuring thefluorescence in cells permeabilized with 10 µg/ml digitonin. Cytosolicfree Ca2+ concentration was calculated assuming a Fura-2 Kd of 225nM.

To inhibit intracellular calcium signaling the membrane-permeablecalcium chelant EGTA-AM was employed. For this purpose isolatedhepatocytes were loaded with EGTA-AM (15 minutes at roomtemperature in KHH containing 25 µM EGTA-AM) prior to thepreconditioning treatment. Hepatocytes were then processed forviability assay and fluorescence analysis in suspension (Lamp-1surface expression) or on glass coverslip (CD subcellular localization)as described above.

StatisticsAll experiments on cell viability, [Na+]i, pHi and [Ca2+]iconcentrations were done in triplicate and repeated at least threetimes. Data were expressed as mean ± s.d. Statistical analysis wasperformed with the Instat-3 statistical software (GraphPad Software,San Diego, CA) using a one-way ANOVA test with Bonferroni’scorrection for multiple comparisons when more than two groups wereanalyzed. Normality of data distribution of all groups was verified bythe Kolmogorov and Smirnov test. Significance was taken at a P valueof less than 0.005.

ResultsPreconditioning is associated with endosome andlysosome translocation to the cell periphery and fusionwith plasma membraneWe first sought to determine the effect of preconditioning onthe subcellular localization of acidic vacuolar compartment. Tothis end, hepatocytes were either maintained for 20 minutesunder oxygen fluxing (control) or preconditioned with a cycle of10 minutes of hypoxia followed by 10 minutes ofreoxygenation. Cells were then processed for confocalimmunofluorescence analysis using the soluble proteasecathepsin D (CD) and the membrane glycoprotein Lamp-1 astracers of endosomal-lysosomal organelles (Démoz et al., 1999;Sarafian et al., 1998). The image in Fig. 1A (left panels) showsthat, in control hepatocytes, endosomes and lysosomes aredistributed throughout the cytoplasm with a preferentialaccumulation in the perinuclear region, in accord with theirusual location in normal cells (Matteoni et al., 1987; Démozet al., 1999). By contrast, in non-preconditioned hepatocyteskilled by prolonged exposure to hypoxia CD fluorescence wasgreatly reduced and diffused in the cytoplasm, suggestive oflysosome rupture (not shown). The population of hypoxic-preconditioned hepatocytes appeared heterogeneous forendosome and lysosome localization. In almost 40 to 50% ofthe hepatocytes these organelles appear intact and many of themaccumulate at the periphery of the cell, close to the plasmamembrane (Fig. 1A, right panels). About 20 to 30% of thehepatocytes showed a distribution of CD-positive organelles

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similar to that observed in controls, whereas the remaininghepatocytes (about 30%) appeared partially or totally devoid oflysosomal organelles (not shown). Similar pictures wereobtained by immunostaining endosomes and lysosomes withantibodies specific for an integral membrane glycoprotein (Fig.1A). The appearance of Lamp-1 on the cell surface proves thatat least some of these organelles fused with the plasmamembrane in preconditioned hepatocytes. We extended ourinvestigation to a chemical-induced preconditioning systembased on the stimulation of the adenosine A2A-receptor with theagonist CGS21680 (Carini et al., 2001a). In this case we alsoobserved that endosomes and lysosomes localized to theperiphery of the cell in a large fraction of hepatocytes (Fig. 1B).Hepatocytes were also stained for actin, which localizes closeto the plasma membrane, to mark the cell border (Fig. 1Bc).The image in Fig. 1B (panel d), showing the overlap of corticalactin (green) and CD (red) staining, demonstrates that, in thesehepatocytes, endosomes and lysosomes move to the extremeperiphery of the cell upon preconditioning. To quantify theextent of lysosome-plasma membrane fusion we analyzed bycytofluorometry the expression of Lamp-1 on the cell surfacein non-permeabilized control and preconditioned hepatocytesand obtained the following values (percent of positivity, averageof two experiments in duplicate): control, 1%; hypoxic-preconditioned, 35%; CGS221680-preconditioned, 33% (Fig.1C).

To corroborate these data we also analyzed bycytofluorometry the intracellular content of CD inpermeabilized hepatocytes. Compared with controls, inpreconditioned hepatocytes about 29% cells (average of twoexperiments in duplicate) were judged negative for intracellularCD (arbitrary units of fluorescence). Taken together, these dataare consistent with the exocytosis of a large fraction oflysosomes that leads to the insertion of lysosomal membraneproteins in the plasma membrane and extracellular release ofthe lumenal content in preconditioned hepatocytes. This eventoccurred in about one-third of the hepatocytes subjectedto preconditioning treatments. The morphological featuresdescribed above occurred in living cells, as the preconditioningtreatments 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 withplasma membrane takes place then soluble enzymes normallyconfined within them should be found at higher levels inthe extracellular milieu. In fact, the proportion of β-hexosaminidase activity measured in the extracellular medium,compared with that found in the cell, was increased inhepatocytes preconditioned either by a brief hypoxic-reoxygenation cycle or by exposition to the adenosine A2A-receptor agonist CGS21680 (Fig. 2A). Conversely, the activitymeasured within the cells was 31.27±4.3 mU/mg and

Journal of Cell Science 117 (7)

Fig. 1.Preconditioning induces the translocation ofendosomal-lysosomal organelles to the extremeperiphery of the cell. Hepatocytes were incubated undercontrol conditions prior to (control) or after beingpreconditioned 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, fixedand permeabilized, and processed forimmunofluorescence confocal microscopy. Endosomesand lysosomes were identified by immunofluorescentdetection of CD or Lamp-1. Representative images areshown. (A) Hypoxic preconditioning (PC) caused thedislocation of endosomes and lysosomes from theperinuclear region (see controls, Co) toward the cellularperiphery. (B) CGS21680-preconditioned hepatocyteswere stained for both CD (red fluorescence, panel b)and for actin (green fluorescence, panel c). Staining ofcortical actin marked the cell border. Cell morphologycan be appreciated in the phase contrast image (panela). The translocation of endosomes and lysosomes tothe extreme periphery of the cells can be appreciated inpanel d, showing the overlap of actin and CD staining.The arrow in panel d points to the plasma membraneregion in which cortical actin appears disassembled, asexpected in the exocytosis process (Miyake et al.,2001), while lysosomal CD appears to be extruded fromthe cell (see also panels b and c). (C) Typicalcytofluorograms of cell surface expression of Lamp-1are shown. The positivity for this lysosomal membraneprotein is increased in hepatocytes preconditioned bytransient hypoxia (PC) or by CGS21680 treatment(CGS).

1069Lysosome exocytosis in hepatocyte preconditioning

23.12±3.8 mU/mg in control and preconditioned hepatocytes,respectively.

Movement of endocytic vesicles requires the integrity of thecytoskeleton and involves various signaling enzymes.Cytochalasin D (Cyt D), which affects the actin cytoskeleton,and wortmannin (WM) and 3-methyladenine (3MA), whichinhibit the lipid kinase PI3K, have been shown to interfere withthe 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 toinhibit endosome and lysosome translocation to the peripheryand fusion with plasma membrane associated withpreconditioning. As predicted, when hepatocytes were treatedin the presence of 3MA (see below for details) thepreconditioning-induced increase in β-hexosaminidase in themedium was completely prevented. In fact, the level of β-hexosaminidase was much lower than that observed underbasal conditions in control hepatocytes (Fig. 2A). Thisoutcome is consistent with the morphological data showing theclustering of endosomes and lysosomes at one pole of thenucleus in preconditioned hepatocytes treated with 3MA (notshown). We attempted to better define whether fusion of CD-

positive organelles with plasma membrane involved mainlyendosomes or lysosomes. For this purpose we took advantageof the fact that the molecular forms of CD accumulate indifferent proportions in these organelles and can therefore beexploited as markers to discriminate between endosomes andlysosomes (Chiarpotto et al., 1999; Dragonetti et al., 2000). Inrat cells CD is present as a 52 kDa precursor (proCD) withinthe endoplasmic reticulum and Golgi complex, as a 43 kDamature 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 hepatocytesthe extracellular release of the three CD molecular formswas 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 kDalarge chain of the double-chain is visible in the gel) inthe medium, but in different proportions (Fig. 2C). Inpreconditioned hepatocytes the secretion of proCD (from pre-endosomal organelles) and the mature double-chain CD (fromlysosomes) was stimulated by a factor of three, whereassecretion of the mature single-chain form of CD (typicallyresident within endosomes) was stimulated by nearly 1.5-fold

Fig. 2.Preconditioning induces the secretion of lysosomal hydrolases: inhibition by WM, 3MA and CytD. Isolated hepatocytes were subjectedto hypoxic- or CGS21680-preconditioning (PC or CGS samples, respectively) or not (Co, controls) and further incubated for 60 minutes undercontrol conditions. In some samples preconditioning and subsequent incubation were performed in the presence of 10 mM 3MA, 250 nM WMor 20 µM Cyt D as indicated (see the Materials and Methods section for details). The extracellular release of β-hexosaminidase activity (A) andCD protein (B) were determined in the incubation media by enzyme assay and western blotting, respectively. (A) There is an excess of secretedβ-hexosaminidase activity in media from preconditioned hepatocytes compared with that from controls. This secretion is largely inhibited by3MA. (B) The CD-related bands identified by western blotting were quantified by densitometry. Total CD found in media from preconditionedhepatocytes (PC and CGS lanes) was approximately double that found in media from control (Co) or preconditioned hepatocytes treated with3MA, 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 datawas statistically significant (P<0.001). 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 isshown. Control hepatocytes showed a basal level of CD secretion. Preconditioning stimulated the secretion of the three CD molecular forms ina different manner. By densitometry (D), proCD and mature double-chain CD were the most stimulated forms. Stimulation of proCD secretionin preconditioned hepatocytes is probably due to the activation of PKC (Chiarpotto et al., 1999). The enhancement of CD secretion wascompletely prevented if preconditioning treatment was performed in the presence of 3MA, Cyt D or WM.

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(Fig. 2C,D). When the experiment was performed inhepatocytes preconditioned and incubated in the presence ofWM, 3MA or Cyt D, the increase in extracellular release of thethree CD forms was completely prevented (Fig. 2C,D). Fromthese data one can conclude that preconditioning inducedfusion of mainly lysosomal organelles with the plasmamembrane and stimulated the exocytosis of pre-endosomalvesicles.

Inhibition of lysosome-plasma membrane fusion inpreconditioned hepatocytes abrogates thecytoprotection against hypoxic cell death and the abilityto counteract the hypoxia-induced overload of Na+

Preconditioning has been reported to protect hepatocytes fromsubsequent exposure to cytotoxic hypoxic conditions (Carini etal., 2000). We tested the hypothesis that fusion of endosomal-lysosomal organelles with plasma membrane is mandatory forthe acquisition of preconditioning-induced cytoprotection. Tothis end we analyzed the effects of WM, 3MA or Cyt D, whichhave been shown to inhibit lysosome exocytosis (Fig. 2), onthe viability of hepatocytes subjected to hypoxia before or afterbeing preconditioned. Hepatocytes were pre-incubated with theinhibitors under control conditions and then exposed for 60minutes to control or hypoxic conditions. The hypoxiccondition was also applied to hepatocytes that had beenpreconditioned with a short hypoxic-reoxygenation cycle in thepresence of WM, 3MA or Cyt D. These drugs were presentthroughout the entire period of the experiment (Fig. 3A). WM,3MA and Cyt D did not affect the viability of hepatocytesin control oxygenated samples nor did they alter thecytotoxicity observed in hepatocytes incubated under hypoxicconditions. Consistent with our previous report (Carini et al.,2000), hypoxic preconditioning protected the hepatocytessubsequently exposed to hypoxic conditions by increasing theirviability by more than 50% (Fig. 3B). This protective effectwas not apparent in hepatocytes that had been treated withWM, 3MA or Cyt D (Fig. 3B). In this latter case, mortalityinduced by hypoxia was identical to that observed in non-preconditioned hepatocytes.

We have shown that CGS21680, an agonist of the adenosineA2A-receptor, reproduces the hepatoprotective effect ofhypoxic preconditioning (Carini et al., 2001a). We thereforechecked whether inhibitors of the endocytic traffic couldreverse the cytoprotection induced by this drug. Consistentwith the above observation, the results shown in Fig. 4demonstrate that stimulation of the adenosine A2A-receptorconfers resistance to hypoxia killing unless the hepatocytes arenot treated with WM, 3MA or Cyt D.

Overload of Na+ in cultured hepatocytes has been shown tobe a critical event in the onset of hypoxic cell death (Cariniet al., 1995; Gasbarrini et al., 1992). In hypoxic orpharmacologically preconditioned hepatocytes exposed tohypoxic conditions the Na+ influx is limited (Carini et al.,2000; Carini et al., 2001b). To further substantiate our data onthe link between lysosomal exocytosis and cytoprotection weinvestigated the effects of WM, 3MA and Cyt D on the abilityof preconditioned hepatocytes to counteract the hypoxia-induced overload of Na+. For this purpose, the time course (0to 60 minutes) of intracellular Na+ concentration changesduring 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 ofincubation). WM, 3MA and Cyt D did not influence the [Na+]changes in hepatocytes incubated under control or hypoxicconditions. The Na+ concentration increased by a factor of sixin hepatocytes incubated under hypoxic conditions for 60minutes (Fig. 5A). As already reported (Carini et al., 2000),preconditioning limited the hypoxia-induced Na+ overload sothat at the end of the experimental period the intracellularconcentration of Na+ was about half that measured in non-preconditioned hypoxic hepatocytes (Fig. 5B,C). However,when preconditioning and subsequent incubation wereperformed in the presence of WM, 3MA or Cyt D,

Journal of Cell Science 117 (7)

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Fig. 3. Inhibitors of endocytic vesicular traffic abrogate hypoxicpreconditioning-induced cytoprotection from hypoxia.(A) Hepatocytes were incubated for 60 minutes in control or hypoxicconditions. Prior to this incubation, some samples werepreconditioned by a 10 minute exposure to hypoxia followed by a 10minute re-oxygenation (PC). In a parallel set of samples, hepatocyteswere treated with 250 nM WM, 10 mM 3MA or 20 µM Cyt D. Thesedrugs were added 15 minutes before the start of preconditioning andwere present throughout the entire experimental period.(B) Hepatocyte viability was evaluated after incubation for 60minutes under control or hypoxic conditions in non-preconditionedand preconditioned cells treated as explained above. The experimentdemonstrates that WM, 3MA and Cyt D completely nullified thecytoprotective 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 incubatedunder hypoxia in the presence of inhibitors.)

1071Lysosome exocytosis in hepatocyte preconditioning

preconditioned hepatocytes failed to prevent the influx of Na+

associated with hypoxia (Fig. 5B,C).Together these data clearly rule out the possibility that

preconditioning-associated fusion of vacuolar acidiccompartments with plasma membrane is just anepiphenomenon and rather strongly indicate that thisphenomenon is mechanistically linked to the cytoprotectiveeffect. Consistent with this interpretation, 3MA attenuatedthe secretion of the soluble lysosomal hydrolases CD andβ-hexosaminidase associated with CGS21680-inducedpreconditioning (Fig. 2A and not shown). Moreover, whenpreconditioning, either by hypoxic-reoxygenation or bytreatment with CGS21680, was performed in the presenceof 3MA, CD-positive organelles did not translocate to theperiphery, rather these organelles localized to a perinucleararea, mostly at one pole of the nucleus (not shown).

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Fig. 4. Inhibitors of endocytic vesicular traffic reversehepatoprotection induced by CGS21680. (A) Hepatocytes wereincubated as explained in the legend to Fig. 3A, except thatpreconditioning was performed by incubating the cells for 15minutes with the adenosine A2A-receptor agonist CGS21680 (1 µM).In some samples, 250 nM WM, 10 mM 3MA or 20 µM Cyt D wereadded during the treatment as indicated (see also text for details).(B) Hepatocyte viability was evaluated after incubating for 60minutes under control or hypoxic conditions in non-preconditionedand preconditioned hepatocytes. Data (means±s.d. of fourexperiments) demonstrate that WM, 3MA and Cyt D nullified thecytoprotective effect of CGS21680. (*P<0.001, statisticalsignificance vs hypoxia and vs CGS incubated under hypoxia in thepresence of inhibitors.)

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Fig. 5. Inhibitors of endocytic vesicular traffic abrogate theprotective effect of preconditioning on Na+ homeostasis. IntracellularNa+ content was assessed in isolated hepatocytes using a fluorescentsodium probe as detailed in Materials and Methods. Two parallel setof samples were prepared in which hepatocytes were preconditionedby a hypoxia-reoxygenation cycle (PC) or by treatment with 1 µMCGS21680 (CGS). In some samples, preconditioning and subsequentincubation under hypoxic conditions were performed in the presenceof 250 nM WM, 10 mM 3MA or 20 µM Cyt D, as for theexperiments described in Figs 3 and 4. Changes in cellular [Na+]were monitored during a 60 minute incubation. WM, 3MA and CytD were shown not to influence the intracellular Na+ content inhepatocytes incubated under control or hypoxic conditions (A). Incontrol (Co) hepatocytes incubated under control conditions thehomeostasis of intracellular Na+ was preserved, whereas inhepatocytes incubated under hypoxic conditions an influx of Na+ isobserved. This influx is limited in preconditioned hepatocytes but itreturns to the same values as in non-preconditioned hepatocyteswhen preconditioning and subsequent incubation are performed inthe presence of WM, 3MA or Cyt D (B,C). Data are the means±s.d.of three independent experiments.

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Lysosome-plasma membrane fusion and establishmentof cytoprotection in preconditioned hepatocytes arecalcium-dependentIn a variety of animal cell models the fusion and exocytosisof lysosomal-like organelles with plasma membrane havebeen 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 fibroblastsabout 25% of the lysosomal population is exocytosed uponelevation of the intracellular free Ca2+ concentration (Jaiswalet al., 2002). We therefore measured intracellular Ca2+ levelsin hepatocytes exposed to CGS21680-induced preconditioningand checked for any causal correlation between Ca2+

concentration and lysosome-plasma membrane fusion. Theconcentration of free cytosolic Ca2+, measured at the end ofthe preconditioning treatment, was in fact augmented bya factor of 3.65±0.28 (mean±s.d. of three independentexperiments) with respect to the value at time zero (averagevalues were 144 nM and 527 nM in control and preconditionedhepatocytes, respectively). An increase in cytosolic calciumwas not observed when preconditioning was performed in thepresence of WM, indicating that this event requires theupstream activation of PI3K. To test whether such intracellularCa2+ elevation was directly implicated in the exocytosis oflysosomes we loaded the hepatocytes with the membrane-permeable Ca2+-chelator EGTA-AM (Carini et al., 1997b).In preconditioned hepatocytes this compound caused theaccumulation of CD-positive organelles near the plasmamembrane (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 β-hexosaminidase and of CD associated with preconditioning(not shown).

We then checked whether this inhibitory effect of EGTA-AM reflected negatively on the preconditioning-inducedcytoprotection. In effect, EGTA-AM abolished the CGS21680-induced cytoprotection as much as WM did in hepatocytesincubated for 60 minutes under hypoxic conditions (Table 1).Similar data were obtained with hepatocytes preconditioned byhypoxic-reoxygenation treatment (not shown). Thus, chelationof free cytosolic Ca2+ prevented the fusion of endosomal-lysosomal organelles with the plasma membrane and abrogatedthe cytoprotective effects of preconditioning.

Inhibition of lysosome-plasma membrane fusionabolishes the ability of preconditioned hepatocytes tocounteract hypoxia-induced cellular acidosisIn hepatocytes exposed to hypoxic conditions cytosolicacidification occurs (Gores et al., 1989; Carini et al., 2001b),an effect that is not observed if hepatocytes have beenpreconditioned (Carini et al., 2001b). However, the ability ofpreconditioned hepatocytes to maintain the pH homeostasisunder hypoxic conditions is lost in the presence of bafilomycinA, an inhibitor of the lysosomal type H+/ATPase pump (Cariniet al., 2001b). We suspected a link between lysosome exocytosisand the ability to counteract cellular acidosis in preconditionedhepatocytes. Our hypothesis predicts that impairment of thefusion with plasma membrane of endosomal-lysosomalorganelles should also impact negatively on the ability ofpreconditioning to prevent hypoxia-associated acidosis.

The intracellular pH was measured in hepatocytesincubated under hypoxic conditions before or after beingpreconditioned either by a brief hypoxic-reoxygenation cycleor by preincubation with CGS21680. Before and during theinduction 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 shownin Fig. 7A, these drugs did not alter the intracellular pH inhepatocytes incubated under control conditions, nor werethey able to influence the drop in pH caused by hypoxia.However, the ability of preconditioning to prevent hypoxia-associated cellular acidosis was completely abolished byWM, 3MA and Cyt D (Fig. 6B,C). These data, together withthe observation reported in Figs 1 and 2, strongly support the

Journal of Cell Science 117 (7)

Fig. 6.Chelation of cytosolic Ca2+ prevents preconditioning-inducedlysosome exocytosis. Hepatocytes were preconditioned by treatmentwith 1 µM CGS21680 (CGS) for 15 minutes in the absence or thepresence of WM (250 nM) or the membrane-permeable cytosoliccalcium chelator EGTA-AM (25 µM). Pre-conditioned hepatocyteswere then seeded on glass coverslips for CD immunofluorescencestaining (A) or processed in suspension for cell surface Lamp-1immuofluorescence staining (B). Control untreated hepatocytes (Co)were also included in the analysis. In CGS2680-treated hepatocyteslysosomes appear located beneath the plasma membrane. Thetranslocation of endosomal-lysosomal organelles to the cellperiphery induced by preconditioning is not followed by fusion withplasma membrane when intracellular calcium ions are chelated byEGTA-AM (A). Hepatocytes stained for cell surface Lamp-1 andanalyzed by cytofluorometry reveal that EGTA-AM, much like WM,prevented insertion of this lysosomal membrane glycoprotein into theplasma membrane (B; the shift to the right of the cytofluorogram,indicative of increased fluorescence, is evident in CGS21680-treatedhepatocytes). Representative confocal microscopic images andcytofluorometric profiles are shown.

1073Lysosome exocytosis in hepatocyte preconditioning

view that the preconditioning-induced fusion of lysosomeswith the plasma membrane is instrumental to the preservationof intracellular pH in hepatocytes subjected to hypoxicconditions.

DiscussionIschemic preconditioning, i.e. a sub-lethal exposure to hypoxicconditions, renders the organ tolerant to hypoxic injury (Yellonet al., 1998). Understanding the mechanism underlying thisphenomenon is essential for a pharmacological approach toorgan preservation. Several triggers and mediators and anumber of signaling pathways activated during hepaticpreconditioning have been discovered (Yamagami et al., 1998;Peralta et al., 2001; Carini et al., 2001a). Much less is knownabout the cellular events that accompany the onset ofpreconditioning. In this study we investigated the effect ofhypoxia on endosomal and lysosomal traffic, and testedwhether these organelles were involved in the cytoprotectiveeffect against hypoxic damage seen in preconditionedhepatocytes. In oxygenated hepatocytes, endosomes andlysosomes appeared as punctuate organelles concentratedmainly in the perinuclear region whereas, in hypoxichepatocytes, the bright punctuate fluorescence was not visible,which indicates that these organelles had disintegrated andreleased their content into the cytosol and extracellular space.These features have been associated with necrotic cell death(Zahrebelski et al., 1995) whereas, in cells undergoingapoptotic cell death, endosomes and lysosomes were shown tomaintain their integrity and cluster at one pole of the cell(Démoz et al., 2002).

In the present study we found that preconditioning inducesa movement of endosomes and lysosomes toward the plasmamembrane, which is followed by the fusion of these organelleswith the plasma membrane. This was demonstrated by theappearance of Lamp-1 on the cell surface and the release oflysosomal soluble enzymes by preconditioned hepatocytes.The exocytosis process involved mainly lysosomes, based onthe molecular forms of CD released extracellularly. We can

Table 1. Chelation of cytosolic Ca2+ abolishes the preconditioning-induced cytoprotection% Cell viability (60 min)

Controls Hypoxic

Oxygen 81±4 Hypoxia 44±8Oxygen+CGS 80±5 Hypoxia+CGS 65±6Oxygen+CGS+WM 82±5 Hypoxia+CGS+WM 44±5Oxygen+CGS+EGTA-AM 80±7 Hypoxia+CGS+EGTA-AM 45±8

Control and CGS21680-preconditioned hepatocytes were incubated under control or hypoxic conditions for 60 minutes in the absence or presence of the PI3Kinhibitor WM (250 nM) or the intracellular Ca2+ chelator EGTA-AM (25 µM). Where indicated, WM and EGTA-AM were added before the start of, and werepresent throughout, the preconditioning and subsequent incubations. Data on cell viability represent the means±s.d. of three experiments carried out in duplicate.

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*Fig. 7. Inhibitors of vesicular traffic abolish the pH bufferingcapability of preconditioned hepatocytes. Isolated hepatocytes wereloaded with a pH fluorescence indicator (see Materials and Methodsfor details) and then incubated under control or hypoxic conditions.Two sets of preconditioned hepatocytes were prepared, either byhypoxic-reoxygenation cycle (PC) or by incubation for 15 minuteswith 1 µM CGS21680 (CGS). In some samples incubation wereperformed in the presence of 250 nM WM, 10 mM 3MA or 20 µMCyt D. Panel A shows that WM, 3MA and Cyt D did not affect thepHi of hepatocytes incubated under control or hypoxic conditions.These drugs, however, abolished the pH buffering effect associatedwith hypoxic- or CGS21680-induced preconditioning (panels B andC, respectively). The results are means of three experiments ± s.d.(*P<0.001, statistical significance vs hypoxia and PC or CGSincubated under hypoxia in the presence of inhibitors.)

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exclude that such translocation of endosomes and lysosomesto the cell periphery is the consequence of cytosolicacidification, as observed in fibroblasts (Heuser, 1989), sincethe preconditioning treatment does not affect the intracellularpH in hepatocytes (Carini et al., 2001b). Inhibiting thetranslocation of these acidic organelles to the periphery, byWM, 3MA or Cyt D, abrogates the cytoprotective effect ofpreconditioning as well as the preservative effects on cellularpH and Na+ homeostasis exerted by preconditioning. WM andCyt D were shown to prevent the ischemic preconditioning-induced cytoprotection in the heart (Tong et al., 2000; Baineset al., 1999). The mechanisms underlying such an effect arestill obscure, although inhibition of PI3K by WM is knownto have severe consequences for cell survival (Rameh andCantley, 1999).

Here we propose a new mechanism to explain thecytoprotective effect of preconditioning that involvesendosomal and lysosomal organelles. To demonstrate ourhypothesis we used various drugs acting on different targetsthat eventually converge in a common inhibitory effect on thetrafficking of endosomes and lysosomes. We in fact employedWM and 3MA, two known inhibitors of PI3K, and Cyt D,which is known to disrupt the organization of the actincytoskeleton. All these drugs had a similar effect on theendosomal and lysosomal traffic and on the preconditioning-associated protection of hepatocytes exposed to hypoxia. Itfollows that the mechanism by which WM reverts thepreconditioning cytoprotection in heart (Tong et al., 2000) orhepatic cells (the present study) cannot exclusively beattributed to impairment of the PI3K-dependent transcriptionof survival factors. Consistent with our hypothesis is theobservation that preconditioning, regardless of the mode itwas induced (either by sub-lethal hypoxia or stimulation ofadenosine A2A-receptor), was always associated with theperipheral redistribution and subsequent fusion with plasmamembrane of Lamp-1/CD-positive organelles. Two otherinteresting novel findings arise from the present study: first,preconditioning is associated with an increase in the freecytosolic Ca2+ concentration and, second, this increase isnecessary to allow the externalization of lysosomal membraneproteins. Chelation of cytosolic Ca2+ nullified the protectiveeffect of preconditioning against hypoxia. It should be notedthat lysosome exocytosis occurred at best in about one-third ofthe whole culture subjected to preconditioning, as assessed alsoby cytofluorometric analysis of Lamp-1 and CD positivity. Onaverage, the rate of cell survival in hepatocytes incubated undercontrol conditions was about 80 to 85%, whereas the ratesof cell survival under hypoxic conditions were about 40%in the non-preconditioned population and about 65% inthe preconditioned population. Therefore, the proportion ofhepatocytes 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 thepreconditioning-induced translocation to the periphery oflysosomes and of their fusion with the plasma membrane andconsequent release in the extracellular environment of acidhydrolases? At present it is difficult to give a definitive answerto this question, although some hypotheses can be made.Perhaps the most attractive one is the possibility that fusionof endosomal-lysosomal organelles allows the insertion on the

plasma membrane of the H+/ATPase pump. This wouldprovide an explanation for the activation of the bafilomycinA-sensitive pH buffering system occurring in preconditionedhepatocytes (Carini et al., 2001b). Consistent with thisinterpretation is the fact that exocytosis involved mainlylysosomes, i.e. the most acidic organelles in which thevacuolar H+/ATPase pump is highly concentrated (Arai et al.,1993). Further support for this interpretation comes from theobservation that, in stimulated neutrophils, cell surfaceexpression of the vacuolar type H+/ATPase pump followsexocytosis of secretory acid granules (Nanda et al., 1996),which represent a specialized lysosome subpopulation. Theplasma membrane insertion of the vacuolar type H+/ATPasepump 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 observationthat, in ileal cells, the brush border expression of a Na+/H+

exchanger was found to depend on the recycling of endosomalvesicles and it was affected by destabilizing the actincytoskeleton with Cyt D (Li et al., 2001). It is tempting tospeculate that, by preconditioning, endocytic retrieval isaccelerated and so membrane expression of such an exchangeris diminished, an event that could also account for the limitedNa+ influx occurring during hypoxia. We have previouslyshown that preservation of intracellular pH and consequentprevention of Na+ overload in preconditioned hepatocytesdepend on the activity of p38-MAPK (Carini et al., 2001a).Intriguingly, our preliminary data indicate that inhibition ofp38-MAPK by SB203580 precludes the translocation to theperiphery of acidic organelles in preconditioned hepatocytes.

Another possibility to take into consideration is thatpreconditioning induces a transient upregulation of theautophagocytic pathway shortly followed by extrusion of thelysosomal content. Sustained autophagy would lead to celldeath (Bursch, 2001), whereas temporally limited autophagymight exert a protective effect on cell death, for instance bysequestering mitochondrial death-promoting factor (Bauvy etal., 2001; Lemasters et al., 1998). Intriguingly, at least twosignaling molecules, the heterotrimeric Galfa-inhibitor proteinand PI3K, implicated in the onset of preconditioning (Carini etal., 2001a) (this study) have been shown to positively regulateautophagy (Ogier-Denis et al., 1995; Blommart et al., 1997).It has been reported that preconditioning protects from caspase3-dependent (Yadav et al., 1999) and TNFα-induced cell death(Peralta et al., 2001). Interestingly, acidic compartments areclearly involved in TNFα-induced activation of caspase 3(Monney et al., 1998) and lysosomal cathepsins have beenshown to play an important role in TNFα-mediatedcytotoxicity in various cell types (Deiss et al., 1996; Démoz etal., 2002), including hepatocytes (Guicciardi et al., 2000).Thus, the extrusion of lysosomal enzymes in preconditionedhepatocytes could contribute indirectly to the downregulationof cell death pathways activated by cytotoxic cytokinesreleased by neighboring cells during prolonged ischemia.Finally, it cannot be excluded that a controlled extracellularrelease of lysosomal hydrolases has a positive effect on cellsurvival ‘in vivo’. In fact, it has been shown that CD can digestcomponents of the extracellular matrix with consequent releaseof the basic fibroblast growth factor (Briozzo et al., 1991),which has been demonstrated to promote cell survival andproliferation of rat liver cells after partial hepatectomy (Baruch

Journal of Cell Science 117 (7)

1075Lysosome exocytosis in hepatocyte preconditioning

et al., 1995). If this latter hypothesis is proven true, theexocytosis of lysosomal proteases would provide a mechanismof protection in vivo much more efficient than is estimated invitro.

In conclusion, this study has shown that preconditioningalters the localization of endosomes and lysosomes and inducestheir calcium-dependent fusion with the plasma membrane inhepatocytes. We have shown that this event primarily involveslysosomes and is mandatory for the acquisition of resistance tohypoxic damage. The activation of endosomal-lysosomalorganelle recycling provides a rapid mechanism to redistributemolecules among cellular compartments and plasmamembrane; this mechanism is compatible with the short periodof hypoxia exposure or adenosine A2A-receptor stimulationneeded for preconditioning. Thus, in addition to the knowncascade of molecular events, the signaling pathways activatedby triggers and mediators of preconditioning seem to convergeat a common cellular event, that is the translocation to theperiphery and fusion with plasma membrane of endosomes andlysosomes. We have proposed various interpretations for suchevents, one not excluding the other, that can explain many ofthe observations reported in the literature of preconditioning.The evidence that stimulation of the adenosine A2A-receptor

induces the PI3K-dependent translocation to the periphery andthe calcium-dependent fusion with plasma membrane oflysosomes provides new insight into the signaling pathwaysthat govern the traffic in the central vacuolar system (Fig. 8).The present study predicts that drugs activating secondmessengers implicated in the stimulation of endocyticmembrane recycling could prove useful for the therapeuticimprovement of organ protection.

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.

ReferencesArai, K., Shimaya, A., Hiratani, N. and Ohkuma, S. (1993). Purification

and characterization of lysosomal H+-ATPase. An anion-sensitive v-typeH+-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. andDowley J. M. (1999). Ischemic preconditioning depends on interactionbetween mitochondrial K+/ATP channel and actin cytoscheleton. Am. J.Physiol. 276, H1361-H1368.

Baldassarre, M., Dragonetti, A., Marra, P., Luini, A., Isidoro, C. and

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08612SGC

esaPTA-V

?esaPTA-V

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1-PMAL

DCorp

RE

MA/ATGE

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Fig. 8.Dissection of the signaling pathway activated by preconditioning and leading to lysosome-plasma membrane fusion. The schemeproposed is based on our previous (Carini et al., 2001a) and present data. Stimulation of the adenosine A2 receptor activates a cascade of signalsinvolving PI3K, PKC and p38MAPK that eventually impact on the vesicular traffic. Consequently, endosomal and lysosomal organelles aretranslocated to the periphery of the cell and fuse with the plasma membrane in a calcium-dependent fashion. This process requires the integrityof 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.

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Buccione, R. (2000). Regulation of protein sorting at the TGN by plasmamembrane receptor activation. J. Cell Science 113, 741-748.

Baruch, Y., Shoshany, G., Neufeld, G. and Enat, R. (1995). Basic fibroblastgrowth factor is hepatotropic for rat liver in regeneration. J. Hepatol. 23,328-332.

Bauvy, C., Gane, P., Arico, S., Codogno, P. and Ogier-Denis, E. (2001).Autophagy delays sulindac sulfide-induced apoptosis in the human intestinalcolon cancer cell line HT-29. Exp. Cell Res. 268, 139-149.

Blommaart, E. F. C., Krause, U., Schellens, P. M., Vreeling-Sindelarova,H. and Meijer, A. J. (1997). The phosphatidylinositol 3-kinase inhibitorswortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes.Eur. J. Biochem. 243, 240-246.

Bossi, G. and Griffiths, G. M. (1999). Degranulation plays an essential partin regulating cell surface expression of Fas ligand in T cells and naturalkiller cells. Nat. Med. 5, 90-96.

Briozzo, P., Badet, J., Capony, F., Pieri, I., Montcourrier, P., Barritault, D.and Rochefort, H. (1991). MCF7 mammary cancer cells respond to bFGFand internalize it following its release from extracellular matrix: apermissive role of cathepsin D. Exp. Cell. Res. 194, 252-259.

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 transportof newly synthesized lysosomal enzymes in mammalian cells. J. Cell Biol.130, 781-796.

Bursch, W. (2001). The autophagosomal-lysosomal compartment inprogrammed cell death. Cell Death Differ. 8, 569-581.

Carini, R., Bellomo, G., Benedetti, A., Fulceri, R., Gamberucci, A., Parola,M., Dianzani, M. U. and Albano, E.(1995). Alteration of Na+ homeostasisas a critical step in the development of irreversible hepatocyte injury afteradenosine triphosphate depletion. Hepatology 26, 1089-1098.

Carini, R., Bellomo, G., De Cesaris, M. G. and Albano, E. (1997a). Glycineprotects against hepatocyte killing by KCN or hypoxia by preventing Na+

overload in the rat. Hepatology 26, 107-112.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+ chelationBiochem. Biophys. Res. Commun. 232, 107-110.

Carini, R., Autelli, R., Bellomo, G. and Albano, E. (1999). Alteration of cellvolume regulation in the development of hepatocyte necrosis. Exp. Cell Res.248, 280-293.

Carini, R., De Cesaris, M. G., Splendore, R., Bagnati, M. and Albano, E.(2000). Ischemic preconditioning reduces Na+ accumulation and cell killingin isolated rat hepatocytes exposed to hypoxia. Hepatology31, 166-172.

Carini, R., De Cesaris, M. G., Splendore, R., Vay, D., Domenicotti, C.,Nitti, M. P., Paola, D., Pronzato, M. A. and Albano, E. (2001a). Signalpathway involved in the development of hypoxic preconditioning in rathepatocytes. Hepatology33, 131-139.

Carini, R., De Cesaris, M. G., Splendore,. R. and Albano, E. (2001b).Stimulation of p38 MAP kinase reduces acidosis and Na+ overload inpreconditioned hepatocytes. FEBS Lett. 491, 180-183.

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 β isoenzymes by thelipid peroxidation product 4-hydroxy-2,3-nonenal: a signaling pathwayto modulate vesicular transport of glycoproteins. Hepatology29, 1565-1572.

Clavien, P. A., Yadav, S., Sindram, D. and Bentley, R. C. (2000). Protectiveeffects of ischemic preconditioning for liver resection performed underinflow occlusion in humans. Ann. Surg. 232, 163-165.

Cordonnier, M. N., Dauzonne, D., Louvard, D. and Coudrier, E. (2001).Actin fillaments and myosin I alpha cooperate with microtubules for themovement of lysosomes. Mol. Biol. Cell12, 4013-4029.

Deiss, L. P., Galinka, H., Berissi, H., Cohen, O. and Kimchi, A. (1996).Cathepsin D protease mediates programmed cell death induced byinterferon-gamma, Fas/APO-1 and TNF-alpha. EMBO J. 15, 3861-3870.

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 lysosomalprotease cathepsin D. J. Cell Biochem. 73, 370-378.

Démoz, M., Castino, R., Cesaro, P., Baccino, F. M., Bonelli, G. and Isidoro,C. (2002). Endosomal-lysosomal proteolysis mediates death signalling byTNFα not by VP16 in L929 fibrosarcoma cells: evidence for an active roleof 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 compartmentsin the Rat Basophilic/Mast cell Line RBL. J. Cell Sci. 113, 3289-3298.

Feng, G. and Kaplowitz, N.(2000). Colchicine protects mice from the lethaleffect 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+, andcytotoxicity 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.

Gores, G. J., Nieminen, A. L., Wray, B. E., Herman, B. and Lemasters, J.J. (1989). Intracellular pH during ‘chemical hypoxia’ in cultured rathepatocytes. Protection by intracellular acidosis against the onset of celldeath. J. Clin. Invest. 83, 386-396.

Guicciardi, M. E., Deussing, J., Miyoshi, H., Bronk, S. F., Svingen, P. A.,Peters, C., Kaufmann, S. H. and Gores, G. J. (2000). Cathepsin Bcontributes to TNF-α mediated hepatocyte apoptosis by promotingmitochondrial release of cytochrome c. J. Clin. Invest. 106, 1127-1137.

Heuser, J. (1989). Changes in lysosome shape and distribution correlated withchanges in cytoplasmic pH. J. Cell Biol. 108, 855-864.

Jaiswal, J. K., Andrews, N. W. and Simon, S. M. (2002). Membraneproximal 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 transitionin cell death: a common mechanism in necrosis, apoptosis and autophagy.Bioch. Bioph. Acta1366, 177-196.

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 intrafficking and rapid stimulation of NHE3. J. Physiol. 537, 537-552.

Matteoni, R. and Kreis, T. E. (1987). Translocation and clustering ofendosomes and lysosomes depends on microtubules. J. Cell Biol. 105, 1253-1265.

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.

Murry, C. E., Jennings, R. B. and Reimer, K. A. (1986). Preconditioningwith ischemia: a delay of lethal cell injury in ischemic myocardium.Circulation 74, 1124-1136.

Nanda, A., Brumell, J. H., Nordstrom, T., Kjeldsen, L., Segelov, H.,Borregaard, N., Rotsein, O. D. and Grinstein, S. (1996). Activation ofproton pumping in human neutrophils occurs by exocytosis of vesiclesbearing vacuolar-type H+-ATPases. J. Biol. Chem. 271, 15963-15970.

Ogier-Denis, E., Couvineau, A., Maorett, J. J., Houri, J. J., Bauvy, C., DeStefanis, D., Isidoro, C., Laburthe, M. and Codogno, P. (1995). AHeterotrimeric Gi3-protein controls autophagic sequestration in the humancolon 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 tohepatic ischemia-reperfusion in the rat: role of nitric oxide and adenosine.Hepatology 25, 934-937.

Peralta, C., Closa, D., Hotter, G., Gelpi, E., Bulbena, O. and Rosello-Catafau, J. (1999). Protective role of adenosine in inducing nitric oxidesynthesis in rat liver ischemia preconditioning is mediated by the activationof adenosine A2 receptors. Hepatology29, 126-132.

Peralta, C., Fernandez, L., Panes, J., Prats, N., Sans, M., Pique, J. M.,Gelpi, E. and Rosello-Catafau, J. (2001). Preconditioning protects againstsystemic disorders associated with hepatic ischemia-reperfusion throughblockade of tumor necrosis factor-induced P-selectin up-regulation in therat. Hepatology 33, 100-113.

Petiot, A., Ogier-Denis, E., Blommart, E. F. C., Meijer, A. J. and CodognoP. (2000). Distinct classes of phosphatidylinositol 3′-Kinases are involvedin 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 incultured 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.

Journal of Cell Science 117 (7)

1077Lysosome exocytosis in hepatocyte preconditioning

Reddy, A., Caler, E. V. and Andrews, N. W. (2001). Plasmamembrane repairis mediated by Ca2+-regulated exocytosis of lysosomes. Cell 106, 157-169.

Rodriguez, A., Webster, P., Ortengo, J. and Andrews, N. W. (1997).Lysosomes behave as Ca2+-regulated exocytic vesicles in fibroblasts andepithelial 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 inhuman tumor cells. Int. J. Cancer75, 105-111.

Tapper, H., Furuya, W. and Grinstein, S. (2002). Localized exocytosis ofprimary (lysosomal) granules during phagocytosis: role of Ca2+-dependenttyrosine phosphorylation and microtubules. J. Immunol. 168, 5287-5296.

Tong, H., Chen, W., Steenbergen, C. and Murphy, E. (2000). Ischemicpreconditioning activates phosphatidylinositol-3-kinase upstream of proteinkinase C. Circ. Res. 87, 309-315.

Yadav, S., Sindram, D., Perry, D. K. and Clavien, P. A. (1999). Ischemicpreconditioning protects the mouse liver by inhibition of apoptosis througha caspase–dependent pathway.Hepatology 30, 1223-1231.

Yamagami, K., Yamamoto, Y., Kume, M., Kimoto, S., Yamamoto, H.,Ozaki, N. and Yamamoto, M. (1998). Heat shock preconditioningameliorates liver injury following normothermic ischemia-reperfusion insteatotic rat livers. J. Surg. Res. 79, 47-53.

Yellon, D. M., Baxter, G. F., Garcia-Dorado, D., Heusch, G. and Sumeray,M. S. (1998). Ischaemic preconditioning: present position and futuredirections. Cardiovasc. Res. 37, 21-33.

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 ischemicpreconditioning on liver preservation-reperfusion injury in rats.Transplant. 66, 152-157.

Yoshizumi, T., Yanaga, K., Soejima, Y., Maeda, T., Uchiyama, H. andSugimachi, K. (1998). Amelioration of the liver injury by ischemicpreconditioning. Br. J. Surg.85, 1636-1640.

Zahrebelski, G., Nieminen, A. L., Al-Ghoul, K., Qian, T., Herman, B. andLemasters, J. J. (1995). Progression of subcellular changes during chemicalhypoxia to cultured rat hepatocytes: a laser scanning confocal microscopicstudy.Hepatology 21, 1361-1372.