Inducible COX-2 dominates over COX-1 in prostacyclin biosynthesis: Mechanisms of COX-2 inhibitor risk to heart disease

Aim: Our aim is to understand the molecular mechanisms of the selective nonsteroidal anti-inflammatory drugs (NSAID), cyclooxygenase-2 (COX-2) inhibitors', higher priority to reduce synthesis of the vascular protector, prostacyclin (PGI2), compared to that of nonselective NSAIDs. Main methods: COX-1 or COX-2 was co-expressed with PGI2 synthase (PGIS) in COS-7 cells. The Km and initial velocity (1/2t Vmax) of the coupling reaction between COX-1 and COX-2 to PGIS were established. The experiment was further confirmed by a kinetics study using hybrid enzymes linking COX-1 or COX-2 to PGIS. Finally, COX-1 or COX-2 and PGIS were respectively fused to red (RFP) and cyanic (CFP) fluorescence proteins, and co-expressed in cells. The distances between COXs and PGIS were compared by FRET. Key findings: The Km for converting arachidonic acid (M) to PGI2 by COX-2 coupled to PGIS is similar to 2.0 mu M; however, it was 3-fold more (similar to 6.0 mu M) for COX-1 coupled to PGIS. The Km and 1/2t Vmax for COX-2 linked to PGIS were similar to 2.0 mu M and 20 s, respectively, which were 2-5 folds faster than that of COX-1 linked to PGIS. The FRET study found that the distance between COX-2-RFP and PGIS-CFP is shorter than that between COX-1-RFP and PGIS-CFP. Significance: The study provided strong evidence suggesting that the low Km, faster 1/2t Vmax, and closer distance are the basis for COX-2 dominance over COX-1 (coupled to PGIS) in PGI2 synthesis, and further demonstrated the mechanisms of selective COX-2 inhibitors with higher potential to reduce synthesis of the vascular protector, PGI2. (C) 2010 Elsevier Inc. All rights reserved.