PRELIMINARY CHARACTERIZATION OF DIACYLGLYCEROL GENERATION IN HUMAN BASOPHILS - TEMPORAL RELATIONSHIP TO HISTAMINE-RELEASE AND RESOLUTION OFDEGRANULATION

Purified human basophils were examined for changes in diacylglycerol l evels to determine whether the transient nature of a N-formyl-methiony l-leucyl-phenylalanine (fMLP) -stimulated elevation in membrane protei n kinase C (PKC) activity could be explained by the transient producti on of diacylglycerol (DAG), In preliminary experiments total DAG level s were measured by the DAG kinase assay, Although elevations followed stimulation with 1 mu M fMLP (basal levels of 15 pmol/10(6) basophils vs, 45 pmol/l0(6) basophils at the 3-min time point), there were no de tectable changes in the first 60 s of the reaction, Histamine release is typically complete by 30-45 s, Measurement of inositol trisphosphat e indicated a rapid increase by 5 s of 2.5 pmol/10(6) basophils, If DA G were produced at similar levels, the DAG kinase assay would not have detected the elevation, Consequently, fMLP-stimulated basophils were examined for changes in 1-stearoyl, 2-arachidonoyl, 3-sn-glycerol (SA- DAG) and l-oleoyl, 2-arachidonoyl, 3-sn-glycerol by GC-NICIMS (negativ e ion chemical ionization mass spectroscopy), A 5-s elevation in these two species averaged 2 pmol/10(6) basophils, consistent with the inos itol trisphosphate levels and occurring during the period of histamine release, However, a much more pronounced second phase to the SA-DAG r esponse also occurred, mirroring the total DAG levels, This second pha se of the DAG response, either total or SA-DAG, was transient on a tim e scale temporally coincident with the appearance and resolution of de granulation sacs as measured by fluorescence microscopy, These data su ggest that there is selective generation of DAG species in the early r eaction and the later appearance of DAG may be related to the formatio n and resolution of granule structures that follow the secretion of hi stamine.