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

To determine whether N- or L-type Ca2+ channels mediate acetylcholine (ACh) release from airway parasympathetic nerve endings, we compared t he effects of omega-conotoxin (N-type inhibitor) and nifedipine (L-typ e inhibitor) on electrically evoked release of ACh in guinea pig trach ea. Reconnected segments of guinea pig trachea were mounted in organ b aths containing Krebs-Henseleit buffer, indomethacin (10 muM) to inhib it 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 chromatograph y 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 concentration s of omega-conotoxin or vehicle alone (acetic acid). Thirty minutes wa s allowed between stimulation periods. We found that EFS-evoked releas e 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 releas e was measured for two separate periods (S1 and S2), and between perio ds 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 sh owed no differences, whereas treatment with a subphysiological concent ration of Ca2+ or TTX caused expected declines in evoked release of AC h. We conclude that N-type, omega-conotoxin-sensitive Ca2+ channels me diate EFS-evoked release of ACh in guinea pig trachea.