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.

A new sea urchin lectin from Toxopneustes pileolus, is D(+)galactose (Gal)-, D(+)fucose (Fuc)-specific. Incubation of rat peritoneal mast cells with the lectin in the presence of 0.3 mM CaCl2 for 10 min significantly and dose-dependently inhibited the histamine release induced by N-acetyl glucosamine (GlcNAc)-specific Datura stramonium agglutinin (DSA), an activator of the Gi-protein-dependent pathway in mast cells. This inhibition by the sea urchin lectin was sugar-specifically reversed in the presence of D(+)Gal or D(+)Fuc but not L(-)Fuc. The sea urchin lectin had no effect on the histamine release induced by compound 48/80, slightly inhibited the histamine release induced by substance P and mastoparan, and slightly enhanced the histamine release induced by melittin, but these effects were not dose-dependent. Compound 48/80, substance P, mastoparan and melittin are mast cell activators without sugar residues. It is suggested that the lectin binds to D(+)Gal residues of DSA to interfere with mast cell activation induced by DSA, a glycoprotein with arabinose and Gal residues. The effects of plant lectins with affinity to D(+)Gal, N-acetyl galactosamine and/or sialic acid and L(-)Fuc on the histamine release induced by DSA, compound 48/80 and substance P were also examined.

Characterization of redox activity in resting and activated mast cells by reduction and reoxidation of lipophilic nitroxides.

1. We measured redox systems in resting and activated rat peritoneal mast cells under anoxia by using the redox metabolism of free doxyl stearic acid (5DS) and phosphatidylcholine with two 5DS molecules esterified to the glycerol (di5DSPC). 2. In the absence of oxygen, 5DS and di5DSPC were reduced to the corresponding hydroxylamines by resting mast cells, with apparent first-order kinetics of 0.085 and 0.078/min, respectively. 3. The activation of mast cells induced by compound 48/80 and bradykinin did not affect the rates of reduction of the nitroxides, and therefore the activation appeared not to be closely coupled to the redox system of these cells; this finding implies that ischemia is unlikely to affect histamine release from mast cells. 4. The oxidation of the nitroxides by the mast cells was very fast and may be nonenzymatic. 5. We concluded that nitroxides can be useful probes of redox metabolism in the mast cells but, because the characteristics of the cellular reduction-reoxidation systems differed from that of other cells, the use of this approach in other cells will require careful characterization of the redox metabolism of nitroxides in those cells.

Mouse bone marrow-derived mast cells undergo exocytosis, prostanoid generation, and cytokine expression in response to G protein-activating polybasic compounds after coculture with fibroblasts in the presence of c-kit ligand.

Polycationic mast cell activators, such as compound 48/80 and substance P, have been reported to activate connective tissue-type mast cells specifically by interacting directly with the Gi family of trimeric GTP-binding protein. We now demonstrate that mouse bone marrow-derived mast cells (BMMC) developed in IL-3, an immature mast cell population lacking responsiveness to the Gi-coupled polycationic mast cell activators, underwent maturation toward a connective tissue-type mast cells-like phenotype that responded to polycationic compounds after only 4 to 6 days of coculture with Swiss 3T3 fibroblasts in concert with recombinant soluble c-kit ligand (KL), whereas 3T3 or KL alone was insufficient to mediate this process. Under optimal conditions, cocultured BMMC released approximately 30% beta-hexosaminidase and generated approximately 1 ng of PGD2/10(6) cells within a few minutes in response to compound 48/80 or substance P. Furthermore, these cells expressed cytokines, such as IL-1beta and IL-6, and PG endoperoxide synthase-2 1 to 4 h after stimulation with compound 48/80 or substance P. All these responses were suppressed effectively by pertussis toxin, implicating functional Gi coupling. Regardless of the remarkable change in polycationic compound sensitivity, there was only a minimal change in the constitutive expression of Gi3 alpha after coculture. These results together with the observation that before coculture BMMC responded to thrombin through its Gi-coupled receptor suggest that the alteration in a certain step(s) distinct from the level of Gi3 alpha protein expression is important for the acquisition of responsiveness to the polycationic compounds by the synergistic action of KL and 3T3 fibroblast-derived factor. Several lines of evidence have revealed that 3T3-derived factor appears to differ from the known cytokines, prostanoids, and adhesion molecules and is a labile soluble substance.

Benzalkonium chloride inhibited the histamine release from rat peritoneal mast cells induced by bradykinin and GlcNAc oligomer-specific lectin Datura stramonium agglutinin, but heparin did not.

1. Benzalkonium chloride (BAC) inhibited the histamine release from rat peritoneal mast cells induced by synthetic cationic polymers (compound 48/80 and PEI6), bradykinin, des-Arg1-bradykinin and des-Arg9-bradykinin and Datura stramonium agglutinin (DSA). 2. The anionic polymers heparin, de-N-sulfated heparin, poly-aspartic acid and poly-glutamic acid dose dependently inhibited the histamine release induced by cationic polymers, suggesting counteraction between anions and cations. 3. Inhibition by heparin was diminished but that of BAC remained after removal of extracellular heparin and BAC. 4. Mast cell activation by bradykinin and DSA was not inhibited by anionic polymers, suggesting that both bradykinin and DSA recognize membrane sites as receptors.

PEI6, a new basic secretagogue in rat peritoneal mast cells: characteristics of polyethylenimine PEI6 resemble those of compound 48/80.

1. Polyethylenimine with a molecular weight of 600 (PEI6) was the simplest and the most useful to investigate mast cell-activating mechanisms via pertussis toxin (IAP)-sensitive G protein pathway. 2. IAP, lidocaine, or dibutyryl cyclic AMP were inhibitors of the histamine release induced by PEI6, but anti-allergic drug DSCG, the calcium antagonist, D-600, kinase inhibitors, H-7 and K252a, or the calmodulin inhibitor, W-7 were not. 3. The additive effects of compound 48/80 and PEI6 suggested that the action sites for PEI6 overlapped the binding sites of compound 48/80. 4. Mast cell activation induced by PEI6 was sugar-specifically inhibited by N-acetylglucosamine(Glc-NAc)-specific lectins and/or by sialic acid (Sia)-specific lectins, suggesting that the action sites for PEI6 were glycoproteins having GlcNAc and/or Sia residues. 5. Four glycoproteins seemed to be involved in histamine release, including the IAP-sensitive G-protein pathway.

Reduction of lipid-soluble nitroxides in CHO cells and macrophage tumor cells.

There is a need to understand the metabolism of nitroxides because of their usefulness in measurements in living cells of complex phenomena, such as biophysical properties, redox metabolism, and the concentration of oxygen at specific sites. As part of a systematic study of the metabolism of nitroxides in cells, the authors studied Chinese hamster ovary (CHO) cells and mouse macrophage tumor (M5076) cells, using a set of lipophilic nitroxides based on 5 doxyl stearate: the free acid, the methyl ester of the acid, and a phosphatidylcholine with two doxyl stearates esterified to the glycerol. The rates of metabolism of these nitroxides under anoxia differed significantly as a function of both the type of cell and the type of nitroxide. The rates of reduction of the three lipophilic nitroxides depended on their localization. The rates of reduction were first order for all three lipophilic nitroxides, and the only products detected were the respective hydroxylamines. Effects of freeze-thawing and incubation temperature differed in the two cell lines. The authors conclude that the metabolism of nitroxides in different cell lines can be quite different. This may be especially important in understanding studies using nitroxides in living cells, functional organs, and in vivo.

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 N-acetylglucosamine residues.

The N-acetyl glucosamine (GlcNAc)-specific lectin Datura stramonium agglutinin (DSA) rapidly and sugar-specifically released histamine from rat peritoneal mast cells, and pertussis toxin (IAP) inhibited 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 directly suggest that they shared the same mechanism of action including involvement of the IAP-sensitive G protein. 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.

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.

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 (GlcNAc). DSA induced a transient increase in intracellular free calcium concentration ([Ca2+]i) followed by cytoskeletal disassembly and reassembly in rat peritoneal mast cells. These changes induced by DSA resulted in histamine release. The time course of fluorescence intensity in mast cells loaded with fluo-3- or DCVJ and activated by DSA resembled those activated by the basic polymer compound 48/80. Inhibition of [Ca2+]i rise by antagonist lectins was responsible for the inhibition of cytoskeletal assembly and the consequent histamine release induced by DSA. At the level of the individual cell, a mast cell stimulated by DSA responds in an all-or-none fashion. DSA possible induced intracellular calcium mobilization and cytoskeletal change by recognizing the GlcNAc-oligomer residues of specific glycoproteins of mast cells.

Inhibitory effects of sialic acid- or N-acetylglucosamine-specific lectins on histamine release induced by compound 48/80, bradykinin and a polyethylenimine in rat peritoneal mast cells.

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 micrograms/ml at 37 degrees C for 10 min), which are specific for sialic acid-alpha 2,3-N-acetyl galactosamine (Sia alpha 2,3GalNAc) and N-acetyl glucosamine (GlcNAc) 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 induced by compound 48/80, they partially shared the same signalling pathways. Glycoproteins with bisecting GlcNAc 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 pertussis 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 Sia alpha 2,3Gal), GlcNAc oligomers and/or bisecting GlcNAc.

Binding of spin-labeled fatty acids and lysophospholipids to hydrophobic region of calmodulin.

In the presence of bovine brain calmodulin activated by calcium, the sharp triplet electron spin resonance (ESR) lines of free doxyl stearic acids decreased, and the broad resonance lines increased concomitantly, suggesting that the doxyl stearic acids bound to calmodulin calcium-dependently. The bound molecules were displaced by a calmodulin inhibitor, W-7, whereas their nitroxide radicals were hardly reduced by ascorbic acid, suggesting that the spin-labeled fatty acids bind to hydrophobic regions of calmodulin, and consequently inhibit calmodulin-dependent phosphodiesterase activity. These binding characteristics to calmodulin were different from those to bovine serum albumin. Moreover, the ESR spectra of two spin-labeled derivatives of lysophospholipid having a spin-labeled acyl group or a spin-labeled polar head group showed that it is the acyl chain of lysophospholipid that interacts with the hydrophobic region of calmodulin. The interactions of fatty acids and lysophospholipids with calmodulin seem to be quite different from those of acidic phospholipids, described previously [Suzuki, T., Katoh, H., & Uchida, M.K. (1986) Biochim. Biophys. Acta, 873, 379-386]. Thus, from the results of ESR study, we can obtain information on the function of fatty acids and lysophospholipids on calmodulin. Instead of enzyme assay, ESR spectroscopy is a useful means to examine lipid-protein interaction.

Sugar-specific inhibitory effects of wheat germ agglutinin and phytohemagglutinin-E4 on histamine release induced by basic secretagogues from rat peritoneal mast cells and their possible action sites.

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, respectively. 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 substance 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 detected. We assumed that the binding sites for basic secretagogues which are coupled with histamine-releasing mechanisms exist among these glycoproteins. A 41-kDa protein (alpha-subunit of pertussis toxin-sensitive G protein) was not detected by WGA, suggesting that the binding sites for the basic secretagogues were not G proteins.

Comparison of histamine release induced by synthetic polycations with that by compound 48/80 from rat mast cells.

We compared the histamine release induced by polyethylenimines and polyallylamines with that induced by compound 48/80. Lidocaine inhibited the histamine release induced by polyethylenimine with a molecular weight of 600 (PEI6), but disodium cromoglycate did not. The histamine releases induced by all polyethylenimines and polyallylamines tested were inhibited by lidocaine, but not by disodium cromoglycate. Islet activating protein inhibited the histamine release induced by PEI6. Its effects on the release by other polyethylenimines and polyallylamines were less than that on PEI6. It is likely that the inhibition of G proteins by islet activating protein resulted in a decrease of the histamine release. This possibility was supported by the finding that guanyl-5'-(beta, gamma-imino) triphosphate enhanced the histamine release. An inhibitor of polyphosphoinositide phosphodiesterase, neomycin, did not affect the histamine releases induced by these polymers. The effect of PEI6 seemed to resemble that of compound 48/80. After pretreatment of mast cells with wheat germ agglutinin and with Limax flavus agglutinin, releases of histamine induced by PEI6 and compound 48/80 decreased, suggesting that the binding sites of PEI6 and compound 48/80 had sialic acid and/or N-acetyl glucosamine residues. The binding site for PEI6 seemed to especially overlap those of compound 48/80.

The modulatory effects of phorbol esters on histamine release from mast cells induced by a polyethylenimine and a polyallylamine.

1. Histamine release from rat peritoneal mast cells induced by polyethylenimine (mol. wt = 600) was inhibited by phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu), but not by 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD). 2. Histamine release induced by polyallylamine (mol. wt = 10,000) was enhanced by PMA and PDBu, but not by 4 alpha-PDD. 3. K-252a reduced the inhibitory effect of PMA on histamine release induced by polyethylenimine and its enhancing effect on histamine release by polyallylamine.

Characteristics of activation of rat mast cells by polyethylenimines and a polyallylamine: effects on the release of histamine and of arachidonate and its metabolites.

1. Three polyethylenimines and one polyallylamine released radioactivity from rat peritoneal mast cells labeled with [1-14C]arachidonic acid and concomitantly released histamine from the cells. 2. This enhancement of the release of radioactivity was inhibited by phospholipase A2 inhibitors, quinacrine (1 mM) and p-bromophenacyl bromide (10 microM), suggesting that polyethylenimine and polyallylamine activates phospholipase A2 to generate arachidonate and its metabolites. 3. The effects of H-7 or K-252a, general kinase inhibitors for the release of histamine and of arachidonate and its metabolites induced by the polycations, were different from those of W-7, a calmodulin inhibitor. The mechanisms to generate arachidonate and its metabolites seemed to differ from those to release histamine; activation of phospholipase A2 by the polycations was calmodulin-dependent. 4. p-Bromophenacyl bromide inhibited the histamine release induced by polyethylenimines and a polyallylamine, suggesting that arachidonate production by means of phospholipase A2 activation by polycations is an important process in the release of histamine from mast cells.

The effects of clozapine on rat mast cells are different from those of benzodiazepines.

1. Clozapine caused dose-dependent inhibition of the release of [14C]serotonin and 45Ca uptake by purified rat peritoneal mast cells activated by concanavalin A, which is similar to the effect of benzodiazepines. 2. However, it had little ability to displace [3H]diazepam binding to mast cells.

Synthetic polycations, polyethylenimines and polyallylamines release histamine from rat mast cells.

The effects of synthetic polycations, which induce liposomal membrane fusion without inducing permeability changes, on histamine release from rat mast cells were investigated. Polyethylenimines and polyallylamines with various molecular weights released histamine from mast cells. Acetylated derivatives and triethylentetramine did not release histamine or serotonin from the cells. The histamine release induced by 10 micrograms/ml polyethylenimine with a molecular weight of 600 was inhibited by 1 mM dibutyryl cyclic AMP, but not by 1 MM 8-bromo cyclic GMP; 100 microM D-600, a calcium antagonist; or 30 microM W-7, a calmodulin inhibitor. In the presence of polyethylenimines with molecular weights of 600, 1,200 and 1,800, no detectable release of cytosolic lactate dehydrogenase was observed, indicating that histamine release induced by these polycations was not due to their cytotoxicity. The potencies of these polymers in inducing histamine release depended on their charges, but not on their degrees of polymerization. On the other hand, the actions of polyethylenimine with a molecular weight of 10,000 and polyallylamines with molecular weights of 3,000-4,000 and 10,000 in releasing lactate dehydrogenase were somewhat cytotoxic. These polycations did not induce serotonin release from rat platelets, suggesting that platelets have no coupling system of signal transduction by these polycations. Thus polycations seemed to interact with the mast cell membrane to induce histamine release, and the potencies of these polycations on mast cells seemed to differ from those of their effects on liposomes, which were examined previously.

Effects of benzodiazepines on serotonin release from rat mast cells.

Ro5-4864, diazepam and chlordiazepoxide inhibited the concanavalin A-induced [14C]serotonin release from rat mast cells dose dependently with IC30 values 38, 115 and 160 microM, respectively. They also inhibited concanavalin A-induced 45Ca uptake, with IC50 values 180, 860 and 1800 microM, respectively. Clonazepam slightly inhibited serotonin release, but medazepam did not, and neither compound inhibited the calcium uptake stimulated by concanavalin A. The potencies of benzodiazepines to inhibit concanavalin A-induced serotonin release and 45Ca uptake were correlated with their binding affinities to the peripheral type of benzodiazepine binding sites. At higher concentrations, these benzodiazepines caused release of both serotonin and lactate dehydrogenase, due to their cytotoxicity. The calcium channels of mast cells are probably not voltage-dependent, as the agonists of voltage-dependent calcium channels, Bay k 8644 and YC-170, did not stimulate serotonin release. Moreover, Ro5-4864, diazepam and chlordiazepoxide inhibited A23187-induced serotonin release. Mast cells have high contents of calmodulin (602 +/- 20 ng/10(6) cells), and benzodiazepines inhibited calmodulin. The benzodiazepine inhibitory effects on the serotonin release induced by A23187 seemed to be partly due to their calmodulin-inhibiting activities. These results suggest that inhibition of serotonin release by benzodiazepines in mast cells activated by concanavalin A is mainly due to inhibition of calcium channels, which may be controlled by the peripheral type of benzodiazepine binding sites.