Introduction: The quantification of [(3)H]noradrenaline spillover from electrically stimulated, sympathetic nerves is a widely used method to study presynaptic effects of hormones, transmitters and drugs. Although a straightforward approach, the execution of the experiments is not trivial. This holds true mainly for a reliable control of the experimental conditions, a major pitfall of the commonly used superfusion setup, and problems concerning the sampling of the tritium containing probes.
Methods: These difficulties prompted us to develop a variant of this method, which is based on a classical organ bath setup. Rabbit thoracic aortic rings were incubated with [(3)H]-labeled noradrenaline. Instead of being constantly washed away by superfusion, the [(3)H]noradrenaline is allowed to accumulate in the medium.
Results: Electrical field stimulation (EFS) (2 Hz, 3 ms, 150 mA) caused a significant increase of [(3)H]noradrenaline outflow by approximately a factor 4.2 (P<.05). The fractional release of noradrenaline during consecutive periods of stimulation remained unaltered (FR(2)/FR(1) 0.99+/-0.03). The EFS-evoked release could be nearly abolished by the selective sodium channel blocker tetrodotoxin (1 microM) (FR(2)/FR(1) 0.06+/-0.03, P<.05). The N-type calcium antagonist omega-conotoxin GVIA (0.3 microM) abolished the stimulation-evoked outflow (FR(2)/FR(1) 0.01+/-0.06, P<.05), whereas the antisympathotonic agent guanethidine (10 microM) attenuated the EFS-evoked noradrenaline outflow by approximately a factor 2 (FR(2)/FR(1) 0.46+/-0.07, P<.05). Angiotensin II (0.1 and 1 nM) enhanced the EFS-evoked [(3)H]noradrenaline outflow by nearly a factor 1.5 and 2, respectively (FR(2)/FR(1) of 1.43+/-0.11 (0.1 nM) and 2.03+/-0.11 (1 nM); n=6-8, P<.05). All agents failed to influence basal outflow.
Discussion: Our modified experimental approach appears to be suitable to study presynaptic influences on sympathetic transmission in the rabbit thoracic aorta. In addition to optimal control of the experimental conditions, the method offers the advantage of a safe and reliable sampling.