Diacyglycerol (DAG) elevations in stimulated human basophils (HB)

DAGs have a well defined role in the activation of PKC and since stimulusinduced activation of PKC in HB is transient, we examined whether this transient was due to a transient elevation of DAGs. Using the DAG kinase assay (DKA) to measure DAG changes, we were unable to detect an early DAG signal that coincided with histamine release (HR) following stimulation with FMLP although a significant DAG elevation (net peak=30 pmoles/106 basophils) .did appear after HR was complete. We hypothesized that specific molecular species of DAG might appear early and developed a novel technology based on GC-NICIMS to detect femtomole levels of DAG species. This study focuses on l-stearoyl-2-arachidonyl-3-sn-glycerol (SA-DAG). FMLP led to 1-2 pmole/106 elevations (resting=l.l pmole/106) 5 seconds after challenge. This coincided with 1-3 pmole/106 changes in IP3. The full kinetic curve is biphasic with a major mass of SA-DAG peaking at 3 minutes (5 pmoles/10s) and returning to resting levels by 20 minutes, temporally consistent with our first studies using DKA. The DKA was also unable to detect any changes in DAG following IgE-mediated stimulation while we could detect 1 pmole/106 elevations of SA-DAG following antl-IgE antibody which were also biphasic but persisted for at least 30 minutes. To explore the metabolic routes of DAG, basophils were treated with the DAG kinase inhibitor, R59-949 and the DAG lipase inhibitor, RHC-80267. RHC-80267 (10-30 U.M) had modest variable effects on both HR and SA-DAG elevations while R59-949 (10 (iM) markedly enhanced both HR and SA-DAG and together the effects were similar to R59-949 alone. Until other species of DAG are measured, these results can not be fully interpreted but it is clear that for FMLP, the early changes in DAG, relevant to PKC activation and HR, occur with a subset of species, of which SA-DAG is one.