Vasomotor function studies
Vasomotor function in uterine arteries (main branch) and aortas from both non-pregnant and pregnant (E18.5)Gch1fl/fl Tie2cre and wild-type littermates was examined using isometric tension studies in a wire myograph (MultiMyogrph 610M, Danish Myo Technology, Denmark). Briefly, mice were culled by overdose of inhaled isoflurane and vascular rings were isolated from the uterine horns or thoracic aorta. The 2-mm rings were mounted in a wire myograph containing 5 ml of ice-cold Krebs-Henseleit buffer (KHB [in mmol/l]: NaCl 120, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, CaCl2 2.5, NaHCO3 25, glucose 5.5) at 37°C, gassed with 95% O2/5% CO2. After allowing vessels to equilibrate for 30 minutes, the optimal resting tension equivalent to 100 mmHg was set. Concentration-response contraction curves were established using cumulative half-log concentrations of U46619 (thromboxane A2 receptor agonist; uterine artery) and phenylephrine (aorta) respectively. Vessels were washed three times with fresh KHB, equilibrated for 20 minutes, and then precontracted to approximately 80-90% of maximal tension with U46619 for uterine arteries or with phenylephrine for aortas. Acetylcholine was used to stimulate endothelium-dependent vasodilatations in increasing cumulative concentrations. Responses were expressed as a percentage of the pre-contracted tension. Finally, the NO donor sodium nitroprusside (SNP) was used to test endothelium-independent smooth muscle relaxation in the presence of L-NAME. All pharmacological drugs were pre-incubated at least 20 min before the dose-response curves were determined. L-NAME was used at 100 µM, apamin at 50 nM, charybdotoxin at 100 nM, and indomethacin at 10 µM. All drugs used were purchased from Sigma Chemical Company.