Bombesin stimulates distinct time-dependent changes in the sn-1,2-diradylglycerol molecular species profile from Swiss 3T3 fibroblasts as analysed by 3,5-dinitrobenzoyl derivatization and h.p.l.c. separation

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Abstract
We have developed procedures for the analysis of endogenous diradylglycerol (DRG) molecular species using derivatization with 3,5-dinitrobenzoyl chloride. The introduction of this strong chromatophore enabled us to separate less than 1 nmol of DRG into its three classes (diacylglycerol, alkylacylglycerol and alkenylacylglycerol) using a combination of h.p.l.c. and t.l.c. followed by reversed-phase h.p.l.c. to resolve these classes into their component molecular species. When applied to Swiss 3T3 mouse fibroblasts stimulated with bombesin for 25 s, 5 min or 30 min, subtle time-dependent changes in the DRG patterns were observed, with only certain polyunsaturated 1,2-diacyglycerol species [18:0/20:3(n-9), 18:0/20:4(n-6), 18:0/20:4(n-3), 18:0/20:5(n-3), 18:1(n-9)/20:3(n-9), 18:1(n-9)/20:4(n-6), 16:0/22:6(n-3), 18:0/20:3(n-6) and 16:0/20:5(n-3)] showing significant agonist-stimulated increases. The amounts of the first six species were all raised at 25 s, whereas all except the latter two were elevated at 5 min. By 30 min these last species were also increased but 18:0/20:3(n-9) had returned to basal levels. Overall DRG levels, as measured by total molecular-species peak area, remained effectively constant. No changes in the amount or species profile of 1-alkyl-2-acylglycerol were observed. Comparison of these species with the acyl-chain structure of phospholipids supports the idea that inositol lipids could be the source of DRG at early stimulation times, but phosphatidylcholine appears to be a phospholipase substrate at all times. These results indicate sequential activation of several phospholipases with different substrate specificities and/or access to different phospholipid pools. They also suggest that only polyunsaturated DRGs act as second messengers and that changes in the relative amounts of these species may trigger activation of different proteins and/or isoforms (e.g. the different isoforms of protein kinase C).