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

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Abstract
The profiles of cyclo‐oxygenase (COX) and nitric oxide synthase (NOS) isoforms were determined in the rat carrageenin‐induced pleurisy model of acute inflammation. 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. COX activity was assessed by the generation of prostacyclin (PGI2, measured as the stable metabolite, 6‐keto prostaglandin F1α) and prostaglandin E2 (PGE2). Western blot analysis and immunohistochemistry were also carried out. 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). 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. 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 2h. 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. Low levels of NOS activity were seen in PBLs. In pleural exudates NOS activity was maximum at 6h and greatly reduced by 24 h. This activity was solely attributable to iNOS. 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.