Cheng-Huai Ruan1, Shui-Ping So, Ke-He Ruan. 1. Department of Internal Medicine, New York Hospital Medical Center of Queens/Weil Cornell Medical College Affiliated Hospital, Flushing, NY 11355, United States. chr9062@nyp.org
Abstract
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 (½t 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 (AA) to PGI2 by COX-2 coupled to PGIS is ~2.0μM; however, it was 3-fold more (~6.0μM) for COX-1 coupled to PGIS. The Km and ½t Vmax for COX-2 linked to PGIS were ~2.0μM and 20s, 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 ½t 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.
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 (½t 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 (AA) to PGI2 by COX-2 coupled to PGIS is ~2.0μM; however, it was 3-fold more (~6.0μM) for COX-1 coupled to PGIS. The Km and ½t Vmax for COX-2 linked to PGIS were ~2.0μM and 20s, 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 ½t 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.
Authors: Paul C Norris; Donna Reichart; Darren S Dumlao; Christopher K Glass; Edward A Dennis Journal: J Leukoc Biol Date: 2011-06-07 Impact factor: 4.962
Authors: Yvonne Alexander; Elena Osto; Arno Schmidt-Trucksäss; Michael Shechter; Danijela Trifunovic; Dirk J Duncker; Victor Aboyans; Magnus Bäck; Lina Badimon; Francesco Cosentino; Marco De Carlo; Maria Dorobantu; David G Harrison; Tomasz J Guzik; Imo Hoefer; Paul D Morris; Giuseppe D Norata; Rosa Suades; Stefano Taddei; Gemma Vilahur; Johannes Waltenberger; Christian Weber; Fiona Wilkinson; Marie-Luce Bochaton-Piallat; Paul C Evans Journal: Cardiovasc Res Date: 2021-01-01 Impact factor: 10.787