K L Brain1, T C Cunnane. 1. Department of Pharmacology, University of Oxford, Oxford, UK. keith.brain@pharm.ox.ac.uk
Abstract
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 Ca(2+) indicator to monitor Ca(2+) in nerve terminals of mouse isolated vas deferens with a confocal microscope. In some experiments, local neurotransmitter release was detected by monitoring neuroeffector Ca(2+) 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 microM) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) influx without abolishing the nerve terminal action potential. Bretylium-induced increases in the absolute refractory period permit living sympathetic terminals to be identified.
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 Ca(2+) indicator to monitor Ca(2+) in nerve terminals of mouse isolated vas deferens with a confocal microscope. In some experiments, local neurotransmitter release was detected by monitoring neuroeffector Ca(2+) 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 microM) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) influx without abolishing the nerve terminal action potential. Bretylium-induced increases in the absolute refractory period permit living sympathetic terminals to be identified.
Authors: Alexander B Hansen; Gilbert Moralez; Steven A Romero; Christopher Gasho; Michael M Tymko; Philip N Ainslie; Florian Hofstätter; Simon L Rainer; Justin S Lawley; Christopher M Hearon Journal: Am J Physiol Heart Circ Physiol Date: 2020-06-05 Impact factor: 4.733