Bio-synthesis of PGD2 and TXB2 by rabbit blood monocytes

A method for the preparation of a highly purified sample of rabbit blood monocytes is described. The metabolism of arachidonic acid (AA) in these cells was studied. Mononuclear cells were prepared by centrifugation on Ficoll-Paque gradients and the monocytes were obtained by further centrifugation and adherence onto plastic culture dishes. These procedures provided a preparation which contained 95% monocytes (non-specific esterase positive). Incubation of [1-14C]-AA with these cells produced four major metabolites which were separated by TLC; these corresponded to prostaglandin (PG) D2, thromboxane (TX) B2, 12-hydroxyheptadecatrienoic acid (HHT) and 12-/15-hydroxyeicosatetraenoic acid (HETE). A minor product which co-migrated with PGE2 was also detected but neither 6-keto-PGF1 alpha nor PGF2 alpha were detected. Also, there was no evidence of the formation of 5-lipoxygenase products (5-HETE and LTB4) by rabbit monocytes with or without calcium-ionophore A23187-stimulation. The production of PGD2, TXB2 and PGE2 was further confirmed by analyzing [3H]-AA metabolites using high-performance liquid chromatography (HPLC) with tritiated standards as references. The biosynthesis of these compounds from endogenous substrate in A23187-stimulated monocytes was confirmed by specific radioimmunoassays with or without prior HPLC separation. The synthesis of immunoreactive LTB4 and LTC4 by A23187-stimulated cells was also monitored and found to be relatively low. The synthesis of PGD2, TXB2 and PGE2 from both exogenous and endogenous substrate was suppressed by treatment of the monocytes with indomethacin (10(-6) M).