Tamiko Suzuki-Nishimura

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.

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.

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.

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.

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.

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.