The lysosomal protease cathepsin D is efficiently sorted to and secreted from regulated secretory compartments in the rat basophilic/mast cell line RBL

Basophils and mast cells contain a peculiar class of inflammatory granules that discharge their content upon antigen-mediated crosslinking of IgE-memb rane receptors, The pathways for granule biogenesis and exocytosis in these cells are still largely obscure. In this study we employed the rat basophi lic leukemia (RBL)/mast cell nine to verify the hypothesis that inflammator y granules share common bioactive molecules and functional properties with lysosomes. We demonstrate that inflammatory granules, as identified by the monoclonal 5G10 antibody (which recognises an integral membrane protein) or by Toluidine Blue staining, have an intralumenal acidic pH, possess lysoso mal enzymes and are accessible by fluid-phase and membrane endocytosis mark ers. In addition, we studied the targeting, subcellular localisation and re gulated secretion of the lysosomal aspartic protease cathepsin D (CD) as af fected by IgE receptor stimulation in order to obtain information on the pa thways for granule biogenesis and exocytosis. Stimulation with DNP-BSA of s pecific IgE-primed RBL cells led to a prompt release of processed forms of CD, along with other mature lysosomal hydrolases. This release could be pre vented by addition of EGTA, indicating that it was dependent on extracellul ar calcium influx, Antigen stimulation also induced exocytosis of immature CD forms accumulated by ammonium chloride, suggesting the existence of an i ntermediate station in the pathway for granule biogenesis still sensitive t o regulated exocytosis. The targeting of molecules to secretory granules ma y occur via either a mannose-6-phosphate-dependent or mannose-6-phosphate-i ndependent pathway, We conclude that endosomes and lysosomes in basophils/m ast cells can act as regulated secretory granules or actually identify with them.