Male Swiss Webster mice from 6 to 10 wk of age were anaesthetized with 50 mg kg−1 intrapertoneally injected pentobarbitone. After each animal was unresponsive to any applied stimulus, the trachea was quickly removed and placed in a prechilled dissociation solution consisting of 135 mM NaCl, 6 mM KCl, 5 mM MgCl2, 0.1 mM CaCl2, 0.2 mM EDTA, 10 mM HEPES, and 10 mM glucose, pH 7.3. This method is in accordance with the guidelines of the Animal Care Committee of the University of Massachusetts Medical School and is consistent with the recommendation of the Panel on Euthanasia of the American Veterinary Medical Association. The tracheas were dissected free from the surface of the connective tissue. The tissue was then incubated in the dissociation medium containing 30 U/ml papain (Sigma-Aldrich), 1 mM dithiothreitol, and 0.5 mg/ml BSA at 35°C for 30 min. The tissue was then transferred to a dissociation medium containing 3 U/ml collagenase F (Sigma-Aldrich) and 0.5 mg/ml BSA and incubated at 35°C for another 15 min to produce isolated ASM cells. Finally, the tissue was agitated with
