The Effects of Exogenous Arachidonic Acid Oncyclooxygenase Activity and Isoforms Expressed in Cultured Endothelial Cells

Abstract

Cyclooxygenase (COX) which exist as COX-1 and COX-2 isoform, is the first enzyme in thepathway in which arachidonic acid (AA) is converted to several prostaglandins (PGs) such asprostacyclin (PGI₂). PGE₂ and thromboxane (TX) A2. AA is released from cell membrane(endogenous AA) by several agonists such as histamine, bradykinins, angiotensin, cytokines and growth factors, including shear stress. However, exogenous AA can directly activate PG synthesis viaCOX enzyme (COX activity). Here, we have investigated the effects of AA on COX activity and isoform expression in human umbilical vein endothelial cell (HUVEC). HUVEC was obtained from babies born to normal pregnancy and grown as standard technique. The cells were grown to confluent and replaced with fresh medium containing AA (0.1, I, I 0 and 20 μM). Cells were then incubated at 37°C under 5 % C02 concentration in the CO₂-incubator for variable periods of times (5, 10, 20, and 30 minutes). After which time, the release of 6-keto-PGF_1a(a stable metabolite of PGI₂) in supernatant medium was measured by using enzyme immunoassay (EIA). The remained cells were extracted todetect COX protein expression using specific antibody to COX-1and COX-2. The effects of AA on cell viability were also investigated using MIT assay. Neither various concentrations (0.1-20 μM) norvariable periods of times (5-30 min) of AA had any effects on cell viability. Control HUVEC without AA released undetectable amount of 6-keto-PGF_1α(<3 pg/ml). The incubation of exogenous AA (0.1-20 μM) in HUVEC can release significantly higher amount of 6-keto-PGF_1αwhich could be referredto activity of the COX enzyme. However, the release of 6-keto-PGF_1αin AA activated HUVEC didnot depend on incubation of AA when cells were incubated with AA up to 24 h. In our model study wefound that the incubation of AA 10μMfor 10 min is the most appropriate concentration and time todetermine COX activity in HUVEC. Moreover, AA did not effect on COX-I protein which wasconstitutively expressed in HlNEC while COX-2 protein was undetectable in AA (10 μM) treatedHUVEC for up to 24 h. Thus the increase release of 6-keto-PGF_1αfrom exogenous AA (10μM) was presumably due to the increase in COX activity but not from the increase amount of COX-1 or the induction ofCOX-2