Non-arginine based nitric oxide synthase inhibitors are potent inhibitors of cyclooxygenase activity in J774 macrophages

It has been proposed that in inflammatory conditions, in which both the mducible isolorms of nitric oxide synthase (iNOS) and cyclooxygenase (COX) are induced, inhibition of NOS also results in inhibition of prostaglandin formation. In the present study we have tested the potential direct effect of NOS inhibitors on COX activity. J774 murine macrophages were stimulated with arachidonic acid (AA, 16 μM) for 30 minutes in the presence or absence of non selective inhibitors of NOS, such as the L-arginine analogues NG-methyl-L-arginine (L-NMA) and N^G-nitro-L-arginine methyl ester (L-NAME), or the selective iNOS inhibitors, such as the non-arginine based NOS inhibitors aminoethylisothiourea (AETU) and aminoguanidine (AG). 6-keto-prostaglandin F1a (6-keto-PGF1α) and thromboxane B2 (TxB2) levels were measured in the supernatant by radioimmunoassay. AA induced a significant stimulation of 6-keto-PGF1α and TxB2 after 30 min incubation in J774 cells. L-NAME and L-NMA partially inhibited AA-induced 6-keto-PGF1α and TxB2 formation (25% and 37% inhibition of maximum production, respectively) at the highest concentration tested (3mM). Incubation of the cells with the non L-arginine based iNOS inhibitors resulted in a more pronounced, dose-related inhibition of the AA metabolites with EC₅₀ values of 86 μM and 1.2 mM for AETU and AG, respectively. It is unlikely that the inhibitory effect of these drugs is due to inhibition of nitric oxide formation and secondarily on COX since (1) nitrite/nitrate levels were undetectable in all experiments and (2) the order of inhibitory potency of the various NOS inhibitors on 6-keto-PGF1α and TxB2 formation did not parallel with the order potency of these Inhibitors on iNOS activity or on the activity of the constitutive isoforms of NOS. The results suggest that the NOS inhibitors, especially the non-arginine based inhibitors, such as AETU and AG, may directly inhibit COX activity.