single infusion of tezosentan 5mg/h over 30 min corresponding to 2.5 mg

Aortic valves were obtained from 16 patients at the time of aortic valve replacement surgery. After surgical removal, each valve was divided approximately into 15 pieces. Valve pieces (4 mm diameter) of normal and sclerotic aortic valves were cultured in αMEM (Gibco, Paisley, UK), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Autogen Bioclear, Wiltshire, UK), 20 mM HEPES (Gibco) 100 U/ml penicillin, 0.1 mg/ml streptomycin (Gibco) and 2 mM L-glutamine (Gibco) at 37°C in 5% CO2 and 95% air. For the measurement of radioactivity in cell culture experiments, valve pieces from two normal patients (resulting in 30 valve pieces) as well as six stenotic patients (resulting in 90 valve pieces) were inserted into the 24-well plates, the medium was changed, and the test drugs (125I-ET-1, 10 nmol/l; ET-1, 100 µmol/l; tezosentan, 10 nmol/l) were added on the second day of culture. After 20 min and 3 h of incubation, the valves were washed three times with DPBS (BioWhittaker, Lonza, Belgium). Radioactivity was measured using a Wallac Multigamma counter (Wallac, Turku, Finland) and results were expressed as counts per minute.