Cyclo-oxygenase and nitric oxide synthase isoforms in rat carrageenin-induced pleurisy

Abstract

1. The profiles of cyclo-oxygenase (COX) and nitric oxide synthase (NOS) isoforms were determined in the rat carrageenin-induced pleurisy model of acute inflammation. 2. The enzymes were assessed in peripheral blood leucocyte (PBL) cell pellets taken from untreated animals and at 2, 6 and 24 h after injection of the irritant in pleural exudate cell pellets and lung homogenates. 3. COX activity was assessed by the generation of prostacyclin (PGI2, measured as the stable metabolite, 6-keto prostaglandin F1 alpha) and prostaglandin E2 (PGE2). Western blot analysis and immunohistochemistry were also carried out. 4. NOS activity was based on the conversion of [3H]-L-arginine to [3H]-L-citrulline in the presence (total NOS activity) or absence of Ca2+ (inducible NOS; iNOS). 5. Peripheral blood leucocyte samples contained low levels of COX activity. In pleural exudate cell pellets, COX activity peaked at 2 to 6 h after injection of the carrageenin. At 24 h, COX activity was significantly reduced. 6. Western blot analysis demonstrated that the inducible isoform of COX (COX-2), was the predominant enzyme at all time points. Low levels of COX-2 were seen in PBLs. In pleural exudate cell pellets maximal COX-2 protein levels were seen at 2 h. 7. Immunohistochemistry confirmed the findings of Western blot studies. Approximately 10% of polymorphonuclear neutrophils (PMNs) in PBLs from untreated animals were immunopositive for COX-2. In cell pellet smears from carrageenin-induced pleurisy taken 2 h after injection of the irritant, PMNs were also the major source of COX-2 immunoreactivity. A small proportion of macrophages and mesothelial cells were also immunolabelled for COX-2.8. Low levels of NOS activity were seen in PBLs. In pleural exudates NOS activity was maximum at 6 h and greatly reduced by 24 h. This activity was solely attributable to iNOS.9. The present results illustrated a similar profile of COX and NOS activity in the carrageenin-induced pleurisy model of acute inflammation. It was demonstrated that COX-2 and iNOS were the predominant isoforms of their respective enzymes.