The expression of prostaglandin H synthase-2 and the profile of arachidonic acid metabolism by human fallopian tubes.

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Objective: To determine the expression of prostaglandin H synthase-2 (PGHS-2) and the profile of arachidonic acid (AA) metabolism by human fallopian tubes Design: Prospective study. Materials/Methods: Segments of fallopian tubes were obtained from patients undergoing postpartum tubal ligation (within 18 hours of delivery) or hysterectomy for non-tubal diseases. Samples were placed on ice and brought to the laboratory immediately. Depending on the nature of the experiment, they were used immediately, or stored at −70°C or in formalin. For whole tissue experiments, the samples were minced into 1 × 1 mm pieces. For other experiments, microsomes were prepared. Our institutional review board approved the use of human tissue in this project. The microsome protein thus prepared was used for Western blot analysis using a PGHS-2 monoclonal antibody that does not cross-react to prostaglandin H synthase-1 (PGHS-1). PGHS-2 was immunohistochemically localized in paraffin sections using a rabbit polyclonal antibody, also not cross-reactive to PGHS-1. The metabolism of [1-C14]AA by microsome protein, whole tubes, isolated tubal smooth muscle, and isolated tubal lumen was investigated. The metabolites were separated and identified using high-pressure liquid chromatography (HPLC) equipped with a radio-detector. To confirm that PGHS-2 was functionally active, we used NS-398 (5 μM) to block its activity. To determine the rate of prostaglandin I2 (PGI2) synthesis, samples of microsome protein were incubated with AA (20 μM) at 37°C for 4 minutes and the reaction stopped by adding methanol. The stable metabolite of PGI2, 6-keto prostaglandin F2α, was determined using enzyme immunoassay. The rate of PGI2 synthesis was expressed as pg PGI2/μg microsome protein/minute. Results: Western blot analysis confirmed the presence of a 72 kDa protein, reactive to PGHS-2 antibody, in microsome fractions prepared from fallopian tubes (n = 10). Immunoreactive PGHS-2 was localized in luminal epithelia, smooth muscle cells and vascular endothelial cells (postpartum, follicular and luteal phases, n = 6 each). HPLC analysis showed PGI2 and prostaglandin E2 (PGE2) were the major metabolites, accounting for 55.8 ± 11.4% and 34.5 ± 11.1% (mean ± SD, n = 4), respectively, of total prostaglandins (PGs). On the other hand, prostaglandin D2, prostaglandin F2α, and thromboxane A2 accounted for less than 5% of total PGs. Similar patterns of metabolites were observed in samples obtained from follicular and luteal phases (n = 2 and 3, respectively) of menstrual cycle or immediately postpartum (n = 4). Isolated smooth muscle (n = 3) and tubal lumen (n = 3) metabolized [1-C14] AA the same way as did the whole tube. PGHS-2 inhibitor, NS-398 (5 μM), reduced the PGs converted from [1-C14] AA by 84% (n = 2). The rate of PGI2 synthesis was 77.6 ± 12.3 pg/μg microsome protein/minute (mean ± SD, n = 10). Conclusions: Luminal epithelia and smooth muscle cells of human fallopian tube express functionally active PGHS-2, which is critical for the synthesis of PGs. The major PGs formed from AA are PGI2 and PGE2. Thus, PGHS-2, PGI2 and PGE2 may have important roles in the functions of fallopian tubes. Supported by: J-C Huang is a WRHR scholar (HD 01277).