Guinea pig tracheal rings in organ baths

Studies were approved by the Columbia University Animal Care and Use Committee (protocol number AC-AAAH4701). Tracheal rings were isolated from male Hartley guinea pigs under pentobarbital anesthesia. Connective tissue and epithelium were removed and tracheal rings were suspended in 4 ml water-jacketed organ baths as previously described [19]. Organ baths contained Krebs-Henseleit (KH) buffer of the following composition: (in mM): NaCl 118, KCl 5.6, CaCl₂ 0.5, MgSO₄ 0.2, NaHCO₃ 25, NaH₂PO₄ 1.3, D-glucose 5.6, (pH 7.4) with 10 μM indomethacin. The buffer was maintained at 37 °C and continuously bubbled with 95% O₂/5% CO₂. The effects of the anesthetic agents dexmedetomidine, lidocaine and remifentanil were then studied under four paradigms in the absence or presence of these anesthetics; (1) the magnitude of the contractile force induced by cholinergic EFS; (2) the amount of acetylcholine released into the buffer during cholinergic EFS stimulation; (3) the relaxation of a sustained exogenous acetylcholine-induced contraction; and (4) the magnitude of the contractile force induced by exogenous capsaicin which activates transient receptor potential cation channel subfamily V member 1 (TRPV1) receptors on C fibers which releases substance P to cause airway smooth muscle contraction.

In those experiments in which acetylcholine released into the buffer was measured during cholinergic EFS, the buffer was further supplemented with 0.1 μM atropine, 10 μM guanethidine and 0.1 μM neostigmine. Atropine was added to block presynaptic inhibitory muscarinic receptors on peripheral cholinergic nerves to maintain stimulus-evoked release of acetylcholine [20]. Neostigmine was used to prevent the degradation of released acetylcholine. Preliminary studies were performed to determine the optimal concentration of neostigmine in the organ bath buffer that protected released acetylcholine from degradation without inhibiting the subsequent acetylcholine detection assay. Guanethidine was included in the buffer to prevent the release of norepinephrine from the sympathetic nerves within the tracheal rings in response to EFS, since catecholamines can modify airway cholinergic activity [21, 22].