An Initial Signal of Activation of Rat Peritoneal Mast Cells Stimulated by Datura stramonium Agglutinin: A Confocal Fluorescence Microscopic Analysis of Intracellular Calcium Ion and Cytoskeletal Assembly

Abstract

-A confocal fluorescence microscope using fluo-3 and 9-(dicyanovinyl)-julolidine (DCVJ) was used to study the mast cell activation by the N-acetyl glucosamine oligomer specific lectin Datura stramonium agglutinin (DSA) and inhibition by antagonist lectins having affinity to N-acetyl glucosamine G1cNAc oligomer specific lectin DSA is a useful tool for examining the pertussis toxin-sensitive mechanisms of histamine release from rat peritoneal mast cells, as previ ously described (1). The G1cNAc-specific lectins WGA and STA were antagonists of DSA and compound 48/80. They may occupy sugar residues of glycoproteins that serve as binding sites for DSA, which overlap one of the binding sites of compound 48/80, prevent access of DSA and compound 48/80 to the binding sites and inhibit the histamine release induced by DSA and compound 48/80 (1-3). Con A, however, did not interfere with the inter actions and did not inhibit the histamine release, since it recognizes high-mannose sugar-residues but not G1cNAc oligomer (1, 3).DSA releases histamine (1), but it is not clear whether the increase in [Ca"]; is one of the initial signals trigger ing histamine release or whether the histamine releasing mechanisms are coupled with changing cytoskeletal assem bly. Confocal fluorescence microscopy, a new technol ogy, revealed important information about cell activation. Real time changes in the fluorescence of indicator dyes and fluorescent molecular rotors in living cells can be measured. In this study, we used the calcium ion indicator fluo-3 and the fluorescent molecular rotor DCVJ. Fluo-3 is a useful dye, but intracellular calcium ion concentra tion can not be calculated from its fluorescence. The inten sity of DCVJ fluorescence increased with the increasing cytoskeletal assembly of actin and tubulin molecules, and cytochalasin D inhibited the increase in the DCVJ fluores cence intensity in activated mast cells (4). We also exam ined the mechanisms of action of antagonist lectins (1) using real time measurement. To clarify the mechanisms of the DSA-induced histamine release, confocal fluores cence microscopic imaging using Con A and the antago nist lectins WGA and STA was carried out.

Datura stramonium Agglutinin Released Histamine from Rat Peritoneal Mast Cells That Was Inhibited by Pertussis Toxin, Haptenic Sugar and N-Acetylglucosamine-Specific Lectins: Involvement of Glycoproteins with A-Acetylglucosamine Residues

“…Our group observed that a transient increase of the intracellular calcium concentration and the consequent cytoskeletal assembly induced by DSA were an initial sig nal of histamine release (22). It is of importance that the histamine release induced by DSA was inhibited by IAP, which inactivates IAP-sensitive G-protein.…”

Prostaglandin D2 Generation by Rat Peritoneal Mast Cells Stimulated With Datura stramonium1 Agglutinin and Its Inhibition by Haptenic Sugar and Wheat Germ Agglutinin*

“…Peritoneal cells from Sprague-Dawley rats (male, 350 -400 g) were collected and purified using a Percoll density gradient, as described previously (5 …”

“…Our group reported that a GlcNAc-oligomer-specific Datura stramonium agglutinin (DSA) activated rat peritoneal mast cells to induce calcium influx and consequently histamine release in the presence of 0.3 mM Ca 2+ and the absence of phosphatidylserine (4,5). The treatment of the cells with chitooligosaccharides (a haptenic sugar of DSA) or PT resulted in the inhibition of the histamine release induced by DSA, suggesting that the mast cell activation induced by DSA was sugar-specific, PT-sensitive, and Gprotein-dependent (4).…”

D-Galactose-Specific Sea Urchin Lectin Sugar-Specifically Inhibited Histamine Release Induced by Datura stramonium Agglutinin: Differences Between Sugar-Specific Effects of Sea Urchin Lectin and Those of D-Galactose- or L-Fucose-Specific Plant Lectins

“…Peritoneal mast cells purified from male Sprague-Dawley rats (300 -350 g) using Percoll (Pharmacia LKB Biothechnology AB, Uppsala, Sweden) were suspended in HEPESbuffered Tyrode solution containing 0.3 mM Ca 2+ (137 mM NaCl, 2.7 mM KCl, 12 mM HEPES, 1 mM MgCl2, 0.3 mM CaCl 2, 5.6 mM dextrose and 0.03% (w/v) bovine serum albumin, pH 7.40) (6). The purity of the mast cells in the final preparation was greater than 90% as assessed by toluidine blue staining, and the viability of the mast cells was greater than 90%, as assessed by trypan blue extrusion.…”

“…The histamine release was extracellular calcium-independent and pertussis toxin-sensitive and not enhanced by phosphatidylserine. Suzuki-Nishimura et al (6) observed an increase in intracellular calcium ion and cytoskeletal assembly after the stimulation of mast cells with DSA as well as compound 48 / 80. Compound 48/ 80, substance P, mastoparan and melittin are activators of the IgE-independent pathway of mast cells; compound 48/ 80 is a synthetic polyamine and substance P, mastoparan and melittin are amphiphilic peptides.…”