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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.

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