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

Abstract

The arachidonic acid metabolites - in particular the prostaglandins, thromboxanes and leukotrienes - have diverse and potent pharmacological effects and may also prove physiologically and pathologically significant. In long term incubation of human peripheral blood mononuclear leukocytes (PBML) in RPMI 1640 with fetal calf serum and [1-14C] arachidonic acid (AA) besides small amounts of some of these known metabolites a radioactive substance with chromatographical behaviour like no known eicosanoid was identified. In the present investigation its structure has been revealed as p-acetamidobenzoic acid (PACBA), mainly by data obtained from NMR and MS studies. The supposition that the contribution from AA was only a two carbon fragment was experimentally confirmed.

Several fatty acids can act as acetyl donors in the acetylation of p-aminobenzoic acid (PABA). However, AA is clearly preferred in comparison to palmitic acid, for both PBML and the T cell leukaemia line Jurcat. In contrast palmitic acid is clearly preferred in, B-oxidation for PBML whereas the T-cell leukaemia Jurcat prefer AA. As our experiments pointed out further differences between PBML and Jurcat cells regarding AA and palmitic acid we investigated several diverse tumour cell lines and 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 that the relative AA content in tumour cell lines was in general approximately 1/3 of the content in freshly prepared PBML, monocytes, Iymphocytes but also 1/3 of the content in these cells kept in culture.

The connection of PABA to AA and the structural similarity between PABA/PACBA and other known pharmaceutics triggered us to investigate if they influenced the AA metabolism. PABA, but not PACBA, was found to inhibit (87% at 328 uM) the thrombin induced and ELISA measured thromboxan B2 production in human platelets whereas the HPLC evaluated metabolism of exogenous [1-l4C]AA, was only slightly inhibited by PABA and unaffected by PACBA. Furthermore, PABA but also PACBA inhibit agonist induced platelet aggregation. The substances where equipotent to acetylsalicylic acid regarding inhibition of ADP induced aggregation whereas they were approximately 50% as potent as acetylsalicylic acid regarding AA induced aggregation. cAMP levels were not affected by the substances, but both collagen induced ATP secretion and AA induced intracellular aequorin indicated Ca2+ transients were significantly reduced. According to the experimental conditions PABA and PACBA inhibit mobilisation and/or utilisation of intracellular Ca2+.

Our results reveal that the underlying cause to the inhibitory effect of PABA is likely connected to an interference with AA liberation, although likely not through a direct phospholipase inhibition, whereas PACBA, although decreasing Ca2+, likely acts by a different mechanism.