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First published online 17 February 2004
doi: 10.1242/jcs.00923

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

¹ Laboratories of Pathology, Dipartimento di Scienze Mediche, Università del Piemonte Orientale `A. Avogadro', via Solaroli 17, 28100 Novara, Italy
<sup>2</sup> Molecular Pathology<sup>2</sup>, Dipartimento di Scienze Mediche, Università del Piemonte Orientale `A. Avogadro', via Solaroli 17, 28100 Novara, Italy

View larger version (56K): [in a new window]   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., 2001), 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).

View larger version (30K): [in a new window]   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., 1999). The enhancement of CD secretion was completely prevented if preconditioning treatment was performed in the presence of 3MA, Cyt D or WM.

View larger version (27K): [in a new window]   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.)

View larger version (23K): [in a new window]   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.)

View larger version (25K): [in a new window]   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.

View larger version (43K): [in a new window]   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.

View larger version (31K): [in a new window]   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.)

View larger version (30K): [in a new window]   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., 2001a) 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.