Cell Tissue Res (1986) 244:557-563 andTissue Research 9 Springer-Verlag1986 Ultrastructural and cytochemical studies of acid phosphatase and trimetaphosphatase in rat peritoneal mast cells developing in vivo Maria Celia Jamur 1.., Ithamar Vugman 1, and Arthur R. Hand 2 1 Departamento de Morfologia, Faculdade de Medicina de Ribeirfio Preto, S~o Paulo, Brasil; 2 Laboratory of Oral Biology and Physiology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland, USA Summary. The ultrastructural and cytochemical features of peritoneal mast cells of the rat were studied. Immature mast cells show specific cytoplasmic granules of different sizes, the smaller ones localized in the Golgi region. The rough endoplasmic reticulum and Golgi apparatus are well devel- oped, and mitochondria are numerous. Nuclei show deep indentations. Acid phosphatase is present in the Golgi sac- cules, in GERL (Golgi apparatus-endoplasmic reticulum- lysosome) and in some small granules. It is not present in mature granules. Trimetaphosphatase is present in the Golgi saccules, in GERL, in most immature granules and in some mature granules. These enzymes appear to be trans- ported and packaged into granules by the Golgi apparatus, suggesting that the specific mast cell granules may be a form of lysosome. The results of this study are consistent with the hypothesis that peritoneal mast cells may be de- rived from macrophage-like precursors. Key words: Mast cell maturation - Mast cell granules Acid hydrolases - Lysosomes - Golgi apparatus Peritoneal mast cells are frequently used to study various aspects of the secretory process, especially exoeytosis of secretory granules. Biochemical studies of isolated mast cell granules of the rat indicate that they contain several lysoso- mal hydrolases, including fl-hexosaminidase, fl-glucuroni- dase, fl-I>galactosidase, and aryl sulfatase A (see reviews by Schwartz and Austen 1980, 1984; Metcalfe et al. 1981 ; Martin and Lagunoff 1984). These enzymes are released by immunologic stimulation from intact mast cells in paral- lel with histamine, indicating their secretory nature (Schwartz and Austen 1980, 1984). Cytochemical studies have demonstrated that some mast cell granules also con- tain acid phosphatase (Komiyama and Spicer 1975; Sannes and Spicer 1979), aryl sulfatase B (Bentfeld-Barker and Bainton 1980) and dipeptidyl aminopeptidase II (Sannes et al. 1979; Sannes and Spicer 1979) activities. These latter lysosomal enzymes, however, are not released by immuno- Send offprint requests to: Dr. Arthur R. Hand, National Institute of Dental Research, National Institutes of Health, Building 30, Room 211, Bethesda, MD 20892, USA * Permanent address: Departamento de Ciencias Morfologicas, Se- tor de Ciencias Biologicas, Universidade Federal do Parana logic stimulation (Schwartz and Austen 1980, 1984), and granules reactive for acid phosphatase and dipeptidyl amin- opeptidase II also incorporate colloidal gold particles endo- cytosed by the cell (Komiyama and Spicer 1975; Sannes et al. 1979; Sannes and Spicer 1979). Thus, rat mast cell granules exhibit a biochemical and functional heterogene- ity: the majority are secretory in nature, but may be defined as modified primary lysosomes based on their enzyme con- tent (Schwartz and Austen 1980, 1984), and others appear to be non-secretory and correspond to secondary lyso- somes, as revealed by their heterophagic properties (Pa- dawer 1969, 1978; Komiyama and Spicer 1975). The morphological and cytochemical features of mast cell granules have been studied principally in mature mast cells. In an attempt to obtain additional information on the formation and relationship of these two types of gran- ules, we studied the in vivo development of rat peritoneal mast cells. In this paper we describe the ultrastructural fea- tures of very young mast cells and the cytochemical localiza- tion of acid phosphatase and trimetaphosphatase activities. Materials and methods Wistar and Sprague-Dawley rats of either sex, weighing approximately 250 g, were injected intraperitoneally with 15 ml of H20. Controls received 15 ml of normal saline (0.85% NaC1). Mast cells were collected 6 or 14 days after injection by washing the peritoneal cavity with 20 ml of normal saline. The peritoneal washing was centrifuged and the cell pellet fixed in 3% glutaraldehyde in 0.1 M phos- phate buffer, pH 7.4, for 15 min at 4 ~ C, then for 60 rain at room temperature (Lawson et al. 1977). After fixation the cell pellet was washed in buffer and post-fixed in 1% OsO, in phosphate buffer for 2 h at room temperature, dehydrated in acetone and embedded in Araldite. For elec- tron microscopic cytochemistry, cell pellets were fixed in 2% glutaraldehyde - 2% formaldehyde in 0.1 M cacodylate buffer, pH 7.4, with 0.05% CaC12, for 40 rain at room tem- perature. After fixation the cell pellet was stored overnight at 4 ~ C in 0.1 M cacodylate buffer, pH 7.4, containing 7% sucrose. The cell pellet was embedded in agar and chopped on a Sorvall TC-2 Tissue Chopper into 50 gm thick sections which were used for incubation in the cytochemical media. Before incubation the sections were rinsed in cacodylate- sucrose buffer and washed twice in the incubation buffer. The trimetaphosphatase (TMPase) reaction was carried out