*2.4. [3H]-Noradrenaline Release Experiments*

Evaluation of [3H]-noradrenaline (NA) release experiments was carried out as previously described [50–52]. Arteries were preincubated in 2 mL of Krebs-Henseleit solution containing 0.1 μmol/L [3H]-NA (for 60 min at 37 ◦C) and transferred into superfusion chambers, superfused with [3H]-NA-free medium (1 mL/min; constant rate: Krebs-Henseleit solution with desipramine 400 nmol/L to inhibit NA's neuronal uptake). Three identical periods of electrical stimulation were applied (Hugo Sachs Elektronik, March-Hugstetten, Germany; constant current mode, rectangular pulses; 1 ms, with current strength 50 mA; 5 Hz, 100 pulses). The first, starting at t = 30 min (S0) was not used for determination

of tritium outflow. The subsequent periods (S1 and S2) were applied at t = 90 min and t = 120 min, respectively. The superfusate was collected each 5-min period, starting from minute 85 of superfusion onwards. At the end of the experiments (t = 130 min), tritium was measured in superfusate samples and solubilized arteries (sonicated 1 h with 2.5 mL of 0.2 mol/L perchloric acid) by liquid scintillation spectrometry (LS 6500, Beckman Instruments, Fullerton, CA, USA) after adding 6 mL of a scintillation mixture to each sample.

Electrically evoked tritium overflow from artery segments incubated with [3H]-NA was shown to reflect action potential-evoked neuronal NA release and evoked tritium overflow are assumed to reflect changes in neuronal NA release [43,50,53]. Values of [ 3H]-NA uptake were estimated as previously described [54]: the tissue tritium content was obtained at the end of each [3H]-NA release experiment and was summed to values of [3H]-NA previously collected in the 5-min superfusate samples (from t = 85 min to t = 130 min, control segments). The final value was considered as the total amount of incorporated [3H]-NA in individual mesenteric artery segments (total tissue tritium content).
