Nitric oxide synthase and cyclo‐oxygenase pathways in the inflammatory response induced by zymosan in the rat air pouch

We have studied the participation of nitric oxide (NO) in an animal model of inflammation, the rat air pouch stimulated with zymosan. Saline or zymosan was injected into 6‐day rat air pouches at different time points and measurements were made of cell migration, levels of nitrite/nitrate (NO 2 − /NO 3 − ), prostaglandin E 2 (PGE 2 ), leukotriene B 4 (LTB 4 ) and secretory phospholipase A 2 (sPLA 2 ) in exudates. Nitric oxide synthase (NOS) activity was determined in high speed supernatants from cells present in pouch exudates. Western blot analysis was also performed on these samples. Zymosan injection induced a time‐dependent increase in leukocyte infiltration, NO 2 − /NO 3 − levels and cellular NOS activity that reached a peak by 8 h. Western blot analysis showed the same time course for induction of NOS protein. Colchicine administration to rats inhibited cellular infiltration and decreased the levels of NO metabolites and cellular NOS activity zymosan‐injected air pouch at 8 h. NOS activity was present in polymorphonuclear leukocytes (PMNs) and monocytes, but not in the lymphocytes present in exudates. This enzyme is calcium‐independent and needs NADPH for activity. PGE 2 levels in exudates showed a time course inverse to that of NOS activity and NO metabolites, with maximum levels of PGE 2 observed at 4 h after zymosan injection. Administration of N G ‐nitro‐ l ‐arginine methyl ester ( l ‐NAME) or aminoguanidine to rats significantly reduced cellular NOS activity, NO 2 − /NO 3 − levels and chemiluminescence, whereas they were without effect on cell migration and degranulation, eicosanoid levels and sPLA 2 activity. Treatment of animals with dexamethasone inhibited cellular NOS activity, NO 2 − /NO 3 − levels, chemiluminescence and the increase in the levels of PGE 2 and LTB 4 , with only a weak effect on elastase release. Administration of the selective cyclo‐oxygenase‐2 (COX‐2) inhibitor NS398 to rats strongly reduced PGE 2 levels in exudates without affecting NO metabolites or NOS activity at 4 h after zymosan injection. Our data indicate that NOS is induced in the zymosan‐stimulated rat air pouch model of inflammation. This enzyme is expressed in the cells migrating into the air pouch and caused an increased production of NO metabolites in exudates. The results also suggest the presence of an earlier phase in which eicosanoids play the main role, with participation of COX‐2 activity, and a later phase mediated by NO. The endogenous release of NO does not modify prostaglandin biosynthesis in this in vivo model.British Journal of Pharmacology (1997) 120 , 1445–1452; doi: 10.1038/sj.bjp.0701073