Isometric Contraction Measurement

Isometric contraction experiments were performed essentially as described previously (Roscioni et al., 2011c). Briefly, ASM strips were mounted for isometric recording in 20 ml organ-baths, containing Krebs-Henseleit (composition in mM: NaCl 117.5, KCl 5.60, MgSO₄ 1.18, CaCl₂ 2.50, NaH₂PO₄ 1.28, NaHCO₃ 25.00, and glucose 5.50) buffer at 37°C. During a 90 min equilibration period with wash-outs every 30 min, resting tension was adjusted to 1 g, followed by pre-contractions with 10 μM methacholine. Following wash-out, maximal relaxation was established by the addition of 0.1 μM (-)-isoprenaline. Tension was readjusted to 1 g, followed by refreshing of the Krebs-Henseleit buffer twice. After another equilibration period of 30 min, cumulative concentration–response curves were constructed with methacholine (0.1 nM – 1 mM). When maximal tension was reached, strips were washed several times and maximal relaxation was established using 10 μM (-)-isoproterenol. Contractions were corrected for tissue weight and expressed as percentage of the maximal methacholine-induced contraction in vehicle-treated strips. Curves were fitted using Prism 5.0. After the contraction protocol, strips were collected and tissue homogenates were prepared as previously described (Roscioni et al., 2011c) for western blot measurement of α-sm-actin, calponin and PCNA.