No abstract

ABSTRACT-The histamine release induced by compound 48/80, bradykinin or polyethylenimine with a molecular weight of 600 (PEI6) was inhibited by wheat germ agglutinin (WGA) and phytohemagglutinin E-subunits (PHA-E4), and the inhibition was specifically reversed by N-acetyl glucosamine and N-acetyl galactosamine, respec tively. Concanavalin A (Con A) and phytohemagglutinin L-subunits (PHA-L4) did not inhibit the histamine release induced by compound 48/80, bradykinin or PEI6. The histamine release induced by substance P was also inhibited sugar-specifically by WGA and PHA-E4. The binding sites for compound 48/80, bradykinin, PEI6 and sub stance P, therefore, seemed to especially overlap each other. These binding sites were found to be glycoproteins having affinities to WGA and PHA-E4, but not to Con A and PHA-L4. The binding of WGA and PHA-E4 to the glycoproteins resulted in inhibition of the interaction between the basic secretagogues including bradykinin and substance P and their binding sites on the mast cells. The bindings of five lectins to mast cell glycoproteins were examined by lectin-blotting. Several glycoproteins, which had specific affinities to WGA and PHA-E4, but not to Con A and PHA-L4 were de tected. We assumed that the binding sites for basic secretagogues which are coupled with histamine-releasing mechanisms exist among these glycoproteins. A 41-kDa pro tein (a-subunit of pertussis toxin-sensitive G protein) was not detected by WGA, sug gesting that the binding sites for the basic secretagogues were not G proteins.Stimulus-secretion coupling mechanisms in rat peritoneal mast cells are thought to involve G proteins, because GTP-analogues enhanced histamine release (1). Moreover, histamine re lease induced by compound 48/80 or brady kinin is inhibited by pertussis toxin, but that induced by antigen or anti-IgE is not (2-4). We found that the histamine release induced by polyethylenimine (MW = 600, PEI6) was inhibited by pertussis toxin (5). Compound 48/80, bradykinin and polyethylenimine are all basic secretagogues. There is, however, no similarity in their chemical structures. The ac tion sites for compound 48/80 on the plasma

ABSTRACT-The N-acetyl glucosamine (G1cNAc)-specific lectin Datura stramonium agglutinin (DSA) rapidly and sugar-specifically released histamine from rat peritoneal mast cells, and pertussis toxin (IAP) in hibited it, suggesting that DSA activated mast cells via an IAP-sensitive G protein pathway. The additive effects of DSA and basic secretagogues such as compound 48/80 that activate IAP-sensitive G protein direct ly suggest that they shared the same mechanism of action including involvement of the ]AP-sensitive G pro tein. Using lectin-blotting, blots of the corresponding glycoproteins detected by DSA diminished by haptenic sugar or pretreatment of the cells with N-glycosidase F, suggesting that the binding of DSA was responsible for the mast cell activation. The other GlcNAc-specific lectins such as Phytolacca americana mitogen, Solanum tuberosum agglutinin and wheat germ agglutinin (WGA) inhibited the histamine release induced by DSA, suggesting that these lectins were antagonists, but DSA was an agonist. Sialic acid-specific Macckia amurensis mitogen (MAM) inhibited the histamine release, and neuraminidase-treatment decreased mast cell activation induced by DSA. At least four mast cell glycoproteins that have affinity to DSA, WGA and MAM and are sensitive to neuraminidase-treatment were detected by lectin-blotting. Some of them may be binding sites coupled to histamine release including the IAP-sensitive G protein pathway. DSA is a useful tool for studying signal transduction of mast cells including the involvement of the IAP-sensitive G protein.

ABSTRACT-The effects of seven lectins with various sugar-specificities on histamine release from rat peritoneal mast cells induced by non-immunologic stimuli were studied. The non-immunologic stimuli used were three basic secretagogues, compound 48/80, bradykinin and PEI6 (polyethylenimine with a molecular weight of 600). In this study, we observed inhibition of the histamine release by Macckia amurensis mitogen and Solanum tuberosum agglutinin (100 tg/ml at 371C for 10 min), which are specific for sialic acid-a2,3 N-acetyl galactosamine (Siaa2,3Ga1NAc) and N-acetyl glucosamine (G1cNAc) oligomers, respectively. The effects of Phytolacca americana mitogen and Sambucus sieboldiana agglutinin were different. Three lectins specific for mucin type oligosaccharides inhibited the histamine release induced by compound 48/80 but not that induced by bradykinin or PEI6. Since bradykinin and PEI6 additively enhanced the histamine release in duced by compound 48/80, they partially shared the same signalling pathways. Glycoproteins with bisecting GIcNAc and Sia residues, as described previously (Jpn. J. Pharmacol. 57, 79-90, 1991), seemed to be one of the action sites for compound 48/80, bradykinin and PEI6. In addition to the direct activation of the per tussis toxin-sensitive G proteins, we propose another mechanism of non-immunologic stimuli via specific glycoproteins on rat peritoneal mast cells. The apparent sugar residues involved were asparagine-linked oligosaccharides with Sia (especially Siaa2,3Gal), G1cNAc oligomers and/or bisecting G1cNAc.

-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.