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.