Life sciences · 2010
Inducible COX-2 dominates over COX-1 in prostacyclin biosynthesis: mechanisms of COX-2 inhibitor risk to heart disease.
СтатьяПоддержка NIH: внешняя программаПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Ruan Cheng-Huai, So Shui-Ping, Ruan Ke-He
Аннотация
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.
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.