A NOVEL CELL-PERMEABLE CROMOGLYCATE DERIVATIVE INHIBITS TYPE-I FC-EPSILON- RECEPTOR MEDIATED CA2+ INFLUX AND MEDIATOR SECRETION IN RAT MUCOSAL MAST-CELLS

Type I Fc-epsilon receptor (Fc-epsilon-RI) mediated Ca2+ uptake and secretion of rat serosal mast cells have been shown to be inhibited by disodium 1,3-bis[(2'-carboxylatochromon-5'-yl)oxy]-2-hydroxypropane (disodium cromoglycate, DSCG), which is widely employed in the treatment of allergic asthma [Foreman et al. (1977) Br. J. Pharmacol. 59, 473P-474P; Cox (1967) Nature (London) 216, 1328-1329]. This drug was also found to modify the protein phosphorylation pattern of these mast cells [Theoharides et al. (1980) Science 207, 80-82]. We have isolated by affinity chromatography on a water-insoluble cromoglycate-carrying matrix a cytosolic enzyme recently identified as a nucleoside 5'-diphosphate kinase. In order to examine a possible intracellular activity of the drug, a cell-permeant cromoglycate derivative, 1,3-bis[[2'-[[(acetoxymethyl)oxy]carbonyl]chromon-5'-yl]oxy]-2-hydroxypropane [bis(acetoxymethyl) cromoglycate, CG/AM], has been synthesized, and its uptake and effect on the Fc-epsilon-RI-mediated exocytosis of mast cells was investigated. A tritium-labeled CG/AM derivative, used as radioactive tracer, was found to permeate mucosal mast cells of the rat line RBL-2H3 and accumulate intracellularly up to 40-fold its extracellular concentration following hydrolysis by cytoplasmic hydrolases. A CG/AM dose dependent inhibition of the Fc-epsilon-RI-induced mediator secretion was observed in RBL-2H3 cells loaded with this compound (I50 almost-equal-to 40-mu-M extracellular CG/AM). A similar dose-dependent inhibition was observed for both the Fc-epsilon-RI-mediated transient rise in the concentration of cytosolic free Ca2+ ions ([Ca2+]i) and the net Ca2+ influx, as monitored by the fluorescent indicator Quin2 and the radioactive tracer Ca-45(2+), respectively. These results clearly show that cell-permeant cromoglycate inhibits the Fc-epsilon-RI-mediated Ca2+ influx into the cell and further underscore the dominant role of this process in the coupling of stimulus to secretion in RBL cells. Furthermore, with the identification of nucleoside 5'-diphosphate kinase as a potential intracellular target for CG activity, distinct mechanisms of action may be inferred for cell-permeant and nonpermeant forms of CG.