ABSTRACT
The antigen induced stimulation of mast cells by aggregation of Fc epsilon RI receptors activates a signal transduction cascade leading to release of mediators of inflammation like histamine, arachidonic acid metabolites and cytokines. In this study we investigated a series of structurally related anti-allergic drugs, containing a common lipophilic diphenylmethyl piperazinyl tail and head groups that differ in lipophilicity. Effects of these drugs on various steps of the signal transduction cascade was investigated to gain insight into the mechanism of action of these drugs. It appeared that addition of the drugs to resting cells induced changes in the tyrosine phosphorylation of cellular proteins. The most active anti-allergics in inhibiting exocytosis, AL3264 and oxatomide, also induced the largest changes in phosphorylation. The effects of the drugs on tyrosine phosphorylation after cell activation was complex. Additionally, Ca2+ fluxes were investigated. Ca2+ efflux from the cells was negligibly influenced by the active drugs. However, the drugs inhibited influx from extracellular Ca2+, which was correlated with the effects of the drugs on inhibition of exocytosis and on membrane stabilization induced by the drugs, measured as haemolysis of erythrocytes. It is concluded that inhibition of Ca2+ influx is the major mechanism with which these drugs inhibit exocytosis and that for this effect drug-membrane interactions, possibly affecting the function of membrane embedded proteins, are of importance. Possible mechanisms including drug-membrane interactions, phosphorylation and inhibition of Ca2+ influx are discussed.
Subject(s)
Anti-Allergic Agents/pharmacology , Calcium Channels/drug effects , Calcium Channels/metabolism , Erythrocyte Membrane/drug effects , Erythrocyte Membrane/metabolism , Receptors, IgE/physiology , Signal Transduction/drug effects , Animals , Calcium/metabolism , Exocytosis/drug effects , Extracellular Space/metabolism , Intracellular Fluid/drug effects , Intracellular Fluid/metabolism , Leukemia, Basophilic, Acute , Phosphorylation , Phosphotyrosine/metabolism , Rats , Thapsigargin/pharmacology , Tumor Cells, CulturedABSTRACT
In a mast cell model, oxatomide displays inhibition of mediator release which is not related to its histamine H1 receptor antagonistic activity. From a previous study it appeared that especially early steps in the signal transduction leading to exocytosis were influenced by oxatomide. We now studied effects of oxatomide on those early steps in more detail. The antigen- and thapsigargin-mediated exocytosis in rat basophilic leukemia (RBL-2H3) cells were both inhibited by oxatomide. After aggregation of high affinity receptors for immunoglobulin E (Fc epsilon RI), protein tyrosine phosphorylation is induced. Oxatomide caused remarkable changes in the tyrosine phosphorylation pattern in resting cells. Also after antigen and thapsigargin activation, changes in the tyrosine phosphorylation of cellular proteins are observed. In addition, Ca2+ fluxes were studied by means of the net influx of 45Ca2+ and by measuring intracellular free Ca2+ concentrations ([Ca2+]) with the fluorescent probe fura-2. Oxatomide inhibited the 45Ca2+ influx and the increase in [Ca2+]i upon antigen and thapsigargin activation of the cells. Neither the release of Ca2+ from internal stores nor the efflux of Ca2+ over the plasma membrane seems to be affected. The effect of oxatomide on Ca2+ influx was further characterized by studying Ba2+ influx in the absence of extracellular free Ca2+. We conclude that inhibition of mediator release is mainly caused by inhibition of influx of extracellular Ca2+, via plasma membrane Ca2+ channels that are activated by depletion of intracellular Ca2+ stores. The molecular mechanism with which oxatomide might interfere with these channels is discussed.
