Bretylium abolishes neurotransmitter release without necessarily abolishing the nerve terminal action potential in sympathetic terminals

• Published in: British journal of pharmacology 2008-02, Vol.153 (4), p.831-839
• Main Authors: Brain, K L, Cunnane, T C
• Format: Article
• Language: English
• Subjects: Action Potentials - drug effects; Adenosine Triphosphate - metabolism; Animals; Biological and medical sciences; bretylium; Bretylium Compounds - pharmacology; calcium; Calcium Signaling - drug effects; Catecholamines - metabolism; confocal; Immunohistochemistry - methods; Male; Medical sciences; Mice; Mice, Inbred BALB C; Microscopy, Confocal - methods; mouse; Neuropeptide Y - metabolism; Neurotransmitter Agents - metabolism; Pharmacology. Drug treatments; Presynaptic Terminals - drug effects; Presynaptic Terminals - metabolism; Research Papers; sympathetic; Sympathetic Nervous System - drug effects; Sympathetic Nervous System - metabolism; Synaptic Transmission - drug effects; Time Factors; vas deferens; Vas Deferens - innervation
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1038/sj.bjp.0707623

Background and purpose: The antidysrhythmic bretylium is useful experimentally because it selectively abolishes neurotransmitter release from sympathetic peripheral nerve terminals. Its mechanism of action seemed settled, but recent results from optical monitoring of single terminals now suggests a new interpretation. Experimental approach: Orthograde transport of a dextran‐conjugated Ca2+ indicator to monitor Ca2+ in nerve terminals of mouse isolated vas deferens with a confocal microscope. In some experiments, local neurotransmitter release was detected by monitoring neuroeffector Ca2+ transients (NCTs) in adjacent smooth muscles, a local measure of purinergic transmission. Sympathetic terminals were identified with catecholamine fluorescence (UV excitation) or post‐experiment immunohistochemistry. Key results: Bretylium (10 μM) abolished NCTs at 60/61 junctions over the course of 2 h, indicating effective abolition of neurotransmitter release. However, bretylium did not abolish the field stimulus‐induced Ca2+ transient in most nerve terminals, but did increase both action potential delay (by 2±0.4 ms) and absolute refractory period (by 4±2 ms). Immunohistochemistry demonstrated that 85–96% of terminals orthogradely filled with a dextran‐conjugated fluorescent probe contained Neuropeptide Y (NPY). A formaldehyde–glutaraldehyde‐induced catecholamine fluorescence (FAGLU) technique was modified to allow sympathetic terminals to be identified with a Ca2+ indicator present. Most terminals contained catecholamines (based on FAGLU) or secrete ATP (as NCTs in adjacent smooth muscle cells are abolished). Conclusions and implications: Bretylium can inhibit neurotransmitter release downstream of Ca2+ influx without abolishing the nerve terminal action potential. Bretylium‐induced increases in the absolute refractory period permit living sympathetic terminals to be identified. British Journal of Pharmacology (2008) 153, 831–839; doi:10.1038/sj.bjp.0707623; published online 10 December 2007