Iloprost

N^ω-nitro-L-arginine methyl ester (L-NAME), ACh, phenylephrine (PE) and the COX substrate, AA were purchased from Sigma (St Louis, MO). PGI₂, a stable PGI₂ analogue and IP receptor agonist iloprost, the COX-1 selective inhibitor FR122047, which has an IC₅₀ value of 0.03 or 65 μM for COX-1 and COX-2, respectively [39 (link)], the TP receptor antagonist SQ29548, the IP receptor antagonist CAY10441, and the TxA₂ synthase (TXAS) inhibitor ozagrel, which has an IC₅₀ value of 11 nM [40 (link)], were bought from Cayman Chemicals (Ann Arbor, MI). L-NAME, PE, ACh, iloprost, AA, and FR122047 were dissolved in distilled water (purged with N₂ for dissolving AA), while PGI₂ was dissolved in carbonate buffer (50 mM, pH 10.0). SQ29548, CAY10441, and ozagrel were dissolved in DMSO at 2,000-fold working concentration (the final concentration of DMSO was 0.05/100, v/v).
The compositions of physiological salt solution (PSS; pH 7.4 with 95%O₂-5% CO₂) were as follows (in mM): NaCl 123, KCl 4.7, NaHCO₃ 15.5, KH₂PO₄ 1.2, MgCl₂ 1.2, CaCl₂ 1.25, and d-glucose 11.5. The 60 mM K⁺-PSS (K⁺) was prepared by replacing an equal molar of NaCl with KCl.

In order to study the effect of PGI2 on the abundance of mRNA transcripts of selected genes, myometrial explants were used. Briefly, a 10 to 20 cm long fragment was cut off from each of six uterine horns, which were transported to the laboratory, and opened longitudinally. Then, the myometrial tissue was separated from the endometrium using scissors, washed with sterile PBS, and cut with a scalpel blade into small pieces (20 to 25 mg). A total of 110–120 mg of the myometrial tissue was placed into glass vials (5 explants per vial) containing 2 mL of incubation medium (Dulbecco’s Modified Eagle’s Medium [D2902, Sigma-Aldrich] supplemented with antibiotics and 25 mM HEPES [H4034, Sigma-Aldrich]) and pre-incubated for 2 h. Then, the pre-incubation media were removed, and myometrial explants were incubated for 8 h in an incubation medium enriched with 0.1% BSA (81-003-3, Millipore, Kankakee, IL, USA) with vehicle (0.01% ethanol; control) or with 1 µM of iloprost (a stable analog of PGI2; 18215, Cayman Chemicals, Ann Arbor, MI, USA). After incubation, myometrial explants were washed with ice-cold PBS, snap-frozen in liquid nitrogen, and maintained at −80 °C until RNA isolation. The treatment was carried out in duplicate using myometrial tissue from six gilts.