N-TYPE, OMEGA-CONOTOXIN-SENSITIVE CA-2+ CHANNELS MEDIATE ELECTRICALLY-EVOKED RELEASE OF ACH IN GUINEA-PIG TRACHEA

To determine whether N- or L-type Ca2+ channels mediate acetylcholine (ACh) release from airway parasympathetic nerve endings, we compared the effects of omega-conotoxin (N-type inhibitor) and nifedipine (L-type inhibitor) on electrically evoked release of ACh in guinea pig trachea. Reconnected segments of guinea pig trachea were mounted in organ baths containing Krebs-Henseleit buffer, indomethacin (10 muM) to inhibit cyclooxygenase, neostigmine (1 muM) to inhibit acetylcholinesterase, and atropine (0.3 muM) to inhibit muscarinic autoreceptors, as well as phentolamine and propranolol to inhibit adrenergic receptors. After electrical field stimulation (EFS), aliquots of buffer were removed, and ACh was measured directly by high-performance liquid chromatography with electrochemical detection. Tracheas were stimulated for 10-min periods at a frequency of 5 Hz, and ACh release was measured for five separate periods (S1-S5) after treatment with increasing concentrations of omega-conotoxin or vehicle alone (acetic acid). Thirty minutes was allowed between stimulation periods. We found that EFS-evoked release of ACh was inhibited by omega-conotoxin in a concentration-dependent manner [mean effective concentration (EC50) almost-equal-to 8 nM] but was unaffected by vehicle treatment. In other experiments, ACh release was measured for two separate periods (S1 and S2), and between periods tracheas were treated with omega-conotoxin (1 muM), nifedipine (10-100 muM), tetrodotoxin (TTX), or buffer containing low (0.8 mM) Ca2+. ACh release was 12 +/- 2 (mean +/- SE) and 3 +/- 0.3 pmol.mg protein-1.min-1 before and during omega-conotoxin (n = 5, P < 0.05). Before and during nifedipine (in concentrations that decreased histamine-induced smooth muscle tone), EFS-evoked release of ACh was 9 +/- 2 and 11 +/- 5 pmol.mg protein-1.min-1, respectively. Time-control results also showed no differences, whereas treatment with a subphysiological concentration of Ca2+ or TTX caused expected declines in evoked release of ACh. We conclude that N-type, omega-conotoxin-sensitive Ca2+ channels mediate EFS-evoked release of ACh in guinea pig trachea.