Novel aspects of P-amionbenzoic acid metablism in connection with arachidonic acid oxidation in human lymphoid cells and platelets

Sammanfattning: The arachidonic acid metabolites - in particular the prostaglandins, thromboxanesand leukotrienes - have diverse and potent pharmacological effects and may also provephysiologically and pathologically significant. In long term incubation of humanperipheral blood mononuclear leukocytes (PBML) in RPMI 1640 with fetal calf serumand [1-14C] arachidonic acid (AA) besides small amounts of some of these known metabolitesa radioactive substance with chromatographical behaviour like no known eicosanoidwas identified. In the present investigation its structure has been revealed as p-acetamidobenzoicacid (PACBA), mainly by data obtained from NMR and MS studies. The supposition thatthe contribution from AA was only a two carbon fragment was experimentally confirmed. Several fatty acids can act as acetyldonors in the acetylation of p-aminobenzoicacid (PABA). However, AA is clearly preferred in comparison to palmitic acid, forboth PBML and the T cell leukaemia line Jurcat. In contrast palmitic acid is clearlypreferred in ,B-oxidation for PBML whereas the T-cell leukaemia Jurcat prefer AA.As our experiments pointed out further differences between PBML and Jurcat cellsregarding AA and palmitic acid we investigated several diverse tumour cell linesand found that several among them preferred AA over palmitic acid in B-oxidation.Measurement of the total relative fatty acid content by means of GC-MS revealed thatthe relative AA content in tumour cell lines was in general approximately 1/3 ofthe content in freshly prepared PBML, monocytes, Iymphocytes but also 1/3 of thecontent in these cells kept in culture. The connection of PABA to AA and the structural similarity between PABA/PACBAand other known pharmaceutics triggered us to investigate if they influenced theAA metabolism. PABA, but not PACBA, was found to inhibit (87% at 328 uM) the thrombininduced and ELISA measured thromboxan B2 production in human platelets whereas theHPLC evaluated metabolism of exogenous [1-l4C]AA, was only slightly inhibited byPABA and unaffected by PACBA. Furthermore, PABA but also PACBA inhibit agonist inducedplatelet aggregation. The substances where equipotent to acetylsalicylic acid regardinginhibition of ADP induced aggregation whereas they where approximately 50% as potentas acetylsalicylic acid regarding AA induced aggregation. cAMP levels were not affectedby the substances, but both collagen induced ATP secretion and AA induced intracellularaequorin indicated Ca2+ transients were significantly reduced. According to the experimentalconditions PABA and PACBA inhibit mobilisation and/or utilisation of intracellularCa2+. Our results reveal that the underlying cause to the inhibitory effect of PABAis likely connected to an interference with AA liberation, although likely not througha direct phospholipase inhibition, whereas PACBA, although decreasing Ca2+, likelyacts by a different mechanism. Bruno Barbieri ISBN 91-628-2679-4 Affärstryck i Linköping AB, Linköping 1997