Noradrenaline release from rat sympathetic neurones triggered by activation of B 2 bradykinin receptors

1 The role of bradykinin receptors in the regulation of sympathetic transmitter release was investigated in primary cultures of neurones dissociated from superior cervical ganglia of neonatal rats. These cultures were loaded with [ 3 H]‐noradrenaline and the outflow of radioactivity was determined under continuous superfusion. 2 Bradykinin (100 nmol l −1 applied for 10 min) caused a transient increase in tritium outflow that reached a peak within four minutes after the beginning of the application and then declined towards the baseline, despite the continuing presence of the peptide. ATP (100 μmol l −1 ) and nicotine (10 μmol l −1 ) caused elevations in 3 H outflow with similar kinetics, whereas outflow remained elevated during a 10 min period of electrical field stimulation (0.5 ms, 50 mA, 50 V cm −1 , 1.0 Hz). 3 When bradykinin was applied for periods of 2 min, the evoked 3 H overflow was half‐maximal at 12 nmol l −1 and reached a maximum of 2.3% of cellular radioactivity. The preferential B 1 receptor agonist des‐Arg 9 ‐bradykinin failed to alter 3 H outflow. The B 2 receptor antagonists, [ D ‐Phe 7 ]‐bradykinin (1 μmol l −1 ) and Hoe 140 (10 nmol l −1 ), per se did not alter 3 H outflow, but shifted the concentration‐response curve for bradykinin‐evoked 3 H overflow to the right by a factor of 7.9 and 4.3, respectively. 4 Bradykinin‐induced overflow was abolished in the absence of extracellular Ca 2+ and in the presence of either 1 μmol l −1 tetrodotoxin or 300 μmol l −1 Cd 2+ , as was electrically‐induced overflow. Activation of α 2 ‐adrenoceptors by 1 μmol l −1 UK 14,304 reduced both bradykinin‐ and electrically‐triggered overflow. The Ca 2+ ‐ATPase inhibitor thapsigargin (0.3 μmol l −1 ) failed to alter either type of stimulated overflow. Caffeine (10 mmol l −1 ) enhanced bradykinin‐induced overflow, but reduced overflow triggered by electrical field stimulation. 5 Inclusion of Ba 2+ (0.1 to 1 mmol l −1 ) in the superfusion medium enhanced electrically induced overflow by approximately 100% and potentiated bradykinin‐triggered overflow by almost 400%. Application of 1 mmol l −1 Ba 2+ for periods of 2 min triggered 3 H overflow, and this overflow was abolished by 1μmol l −1 tetrodotoxin and enhanced by 10 mmol l −1 caffeine. In contrast, inclusion of tetraethylammonium (0.1 to 1 mmol l −1 ) in the superfusion buffer caused similar increases of bradykinin‐ and electrically evoked 3 H overflow (by about 100%), and tetraethylammonium, when applied for 2 min, failed to alter 3 H outflow. 6 Treatment of cultures with 100 ng ml −1 pertussis toxin caused a significant increase in bradykinin‐, but not in electrically‐, evoked tritium overflow. Treatment with 100 ng ml −1 cholera toxin reduced both types of stimulated 3 H overflow. 7 These data reveal bradykinin as a potent stimulant of action potential‐mediated and Ca 2+ ‐dependent transmitter release from rat sympathetic neurones in primary cell culture. This neurosecretory effect of bradykinin involves activation of B 2 ‐receptors, presumably linked to pertussis‐ and cholera toxin‐insensitive G proteins, most likely members of the Gq family. Results obtained with inhibitors of muscarinic K + (K M ) channels, like caffeine and Ba 2+ , indicate that the secretagogue action of bradykinin probably involves inhibition of these K + channels.British Journal of Pharmacology (1997) 122 , 455–462; doi: 10.1038/sj.bjp.0701404