Cytochemical analysis of sulfated glycosaminoglycans in mast cell granules of certain mammalian species.

Abstract

Cytochemicalanalysis of sulfated glycosaminoglycans was carried out in cytoplasmic granules of mast cells from the bovine aorta and rat peritoneal cavity.The cytochemical staining reactions employed for this analysis were alcian blue pH 1.0, high iron diamine and aldehyde fuchsin with or without Mast cells are known to be a type of free cell in the connective tissues of vertebrates, and to contain a number of basophilic granules in their cytoplasm (7, 19) . Although the chemical compositions of these granules vary with animal species, they are reported to contain heparin, histamine, serotonin, hyaluronic acid, various peptides and enzymes such as platelet activating factor, slow reacting substance of anaphylaxis, different kinds of proteases, decarboxylases and certain hydrolases (5, 7, 13, 19). Of these components, heparin is known to be a sulfated glycosaminoglycan which exhibits anticoagulating activity in its physiological function. Recent biochemical studies on the cytoplasmic granules of mouse mast cells in mouse mast cell-fibroblast monolayers (1) and mouse bone marrow-derived mast cells (16-18) revealed that sulfated glycosaminoglycans contained within the granules were chondroitin sulfates, in addition to heparin. More recently, a biochemical analysis of rat mucosal mast cells clarified that the major polysaccharide produced by the cells is an oversulfated galactosaminoglycan rather than heparin (4). It remains to be determined, however, whether such dual composition of sulfated glycosaminoglycans of mast cell granules can be substantiated by cytochemical means. In the present study, attempts have been made to cytochemically analyze sulfated glycosaminoglycans in the cytoplasmic granules of mast cells from bovines and rats. In keeping with the results of previous biochemical studies (1,4,(16)(17)(18), the results obtained in the present study have substantiated the dual composition of sulfated glycosaminoglycans of the mast cell granules, and this appears to be provocative of further cytophysiological studies on the precise functions of this particular cell type in the connective tissues.

Electron microscopic cytochemical studies on sulfated glycosaminoglycans in rat mast cell granules.

“…In a previous precise cytochemical analysis of sulfated glycosaminoglycans in mast cell granules of the same mammalian specimens as those used in the present study, Kida et al (17) have substantiated that a heparinase-sensitive AB pH 1.0, high iron diamine (HID), or aldehyde fuchsin (AF) reactive substance of the cytoplasmic granules is heparin, whereas chondroitinase-susceptible AB pH 1.0, HID or AF stainable ones are chondroitin sulfate D and/or E. If all these previous data are taken into consideration, heparinase-sensitive AB pH 1.0-PTA reactive ultrastructures and chondroitinase-susceptible AB pH 1.0-PTA reactive those studied here could be heparin and chondroitin sulfate D and/or E respectively.…”

“…The enzyme solution contained' proteinase inhibitors to eliminate any contaminating proteinase activities. Thus, l ml of the enzyme solution contained 100 amol of acetate buffer (pH 7.0), 10 pg of BSA,10 pmol of N-ethylmaleimide, 0.5 amol of phenylmethanesulfonyl fluoride (PMSF), 0.036 pmol of pepstatin, 10 pmol of calcium acetate and 0.6 units of heparinase (5,17).…”

“…As in the case of the heparinase solution, the enzyme solution contained proteinase inhibitors to suppress any contaminating proteinase activities. The ingredients contained in 1 ml of the enzyme solution were 100 imol of Tris-HC1 buffer (pH 8.0), 50 ag of BSA, 5 amol of N-ethylmaleimide, 2.5 umol of PMSF, 0.18 amol of pepstatin, 5 imol of sodium ethylenediamine tetraacetic acid (EDTA), 15 imol of sodium acetate and 2.5 units of chondroitinase ABC (17,30). (C) Control procedures for the enzyme digestion experiments Two types of control procedures were performed for the enzyme digestion experiments.…”

“…In a previous report by the present author's group, sulfated glycosaminoglycans contained in cytoplasmic granules of mast cells from a couple of mammalian connective tissues (bovine aorta and rat peritonaeum) were subjected to light microscopic histo-and cytochemical analyses, and as a result of such analyses, the cytoplasmic granules of mast cells were found to contain isomeric chondroitin sulfate (D and/or E) in addition to heparin (17). However, these analyses have been performed by means of light microscopic histo-and cytochemical methods, and it was impossible to elucidate the precise localizations and properties of individual sulfated glycosaminoglycans within cytoplasmic granules of mast cells.…”

The reactions of cuprolinic blue with glycoconjugate-containing tissues in light microscopy.

“…Of these ingredients, EDTA, N-ethylmaleimide, PMSF and pepstatin were employed as inhibitors of contaminating proteinase activities. 2) Digestion with heparinase (3,6).…”