Inhibition of macrophage nitric oxide production by arachidonate-cascade inhibitors

Abstract

We examined whether inhibitors of the arachidonic acid cascade inhibited nitric oxide (NO) production, as measured by nitrite concentration, either in macrophages or by their cytosolic fractions. Nitrite production by peritoneal macrophages from mice receiving OK-432 treatment was significantly inhibited by phospholipase A2 inhibitors [dexamethasone and 4-bromophenacyl bromide (4-BPB)], lipoxygenase inhibitors [nordihydroguaiaretic acid (NDGA) and ketoconazole] and a glutathione S-transferase (leukotrienes LTA4-LTC4) inhibitor (ethacrynic acid). However, caffeic acid and esculetin, inhibitors of 5- and 12-lipoxygenase respectively, were not inhibitory. On the other hand, indomethacin, a cyclooxygenase inhibitor, slightly inhibited whereas another inhibitor, ibuprofen, did not. Inhibition of the nitrite production by dexamethasone, 4-BPB, NDGA and ethacrynic acid was also demonstrated when the macrophages were restimulated ex vivo with OK-432 or with lipopolysaccharide. The inhibitory activity of dexamethasone, NDGA and ethacrynic acid was significantly reduced by ex vivo restimulation with OK-432, whereas that of 4-BPB was hardly affected. Furthermore, the inhibitory activity of dexamethasone, NDGA and ethacrynic acid was much higher when the macrophages were continuously exposed to the agents than when they were pulsed. Meanwhile, inhibition by 4-BPB was almost the same with either treatment. In addition, the inhibitory activity of these agents was not blocked with L-arginine, a substrate of NO synthases, or with arachidonate metabolites (LTB4, LTC4 and LTE4). Ethacrynic acid and 4-BPB, but not dexamethasone and NDGA, also inhibited nitrite production by the cytosolic fractions from OK-432-restimulated peritoneal macrophages, and the inhibitory activity of 4-BPB was superior to that of ethacrynic acid. These agents, however, did not inhibit nitrite production from sodium nitroprusside, a spontaneous NO-releasing compound. These results indicate that dexamethasone, 4-BPB, NDGA and ethacrynic acid inhibited the production of NO by macrophages through at least two different mechanisms: one was inhibited by dexamethasone, NDGA and ethacrynic acid and the other by 4-BPB. Furthermore, 4-BPB and ethacrynic acid directly inhibited the activity of the NO synthase in macrophages, suggesting that the agents work by binding to the active site(s) of the enzyme.

Effect of propranolol on IL-10, visfatin, Hsp70, iNOS, TLR2, and survivin in amelioration of tumor progression and survival in Solid Ehrlich Carcinoma-bearing mice

“…iNOS activity was measured according to the method of Ryoyama et al [24], whereby L-arginine and molecular oxygen were catalyzed by NOS to generate nitric oxide (NO). The rate of NO production by NOS in 1 min was determined with the Griess reaction.…”

Dexamethasone lowers cytosolic pH in macrophages by altering alkalinizing pH-regulatory mechanisms

“…In the macrophage, dexamethasone has been shown to inhibit the production of certain cytokines [15], nitric oxide [16], the mobilization of arachidonate [17], phagocytosis [18], and lysosomal secretion [19]. The exact mechanisms by which dexamethasone inhibits these processes is not known, however.…”

Anti-nitric oxide production activity of isothiocyanates correlates with their polar surface area rather than their lipophilicity

“…Production of NO in peritoneal macrophages and J774.1 cells (measured as NO 2 concentration) was determined by Griess reagent as described previously [17] except for FBS concentration; the culture medium for J774.1 cells was RPMI-1640 containing 10% FBS and gentamicin sulfate (10 mg/mL). In brief, peritoneal macrophages were prepared as described above, and J774.1 cells (4 Â 10 4 / 0.2 mL) in each well of a 96-well flat-bottomed microplate were cultured at 37 C for 1 d. The culture medium was replaced with freshly prepared warm (37 C) 10% FCS-RPMI 1640 medium.…”