A cumulative dose-response technique for the characterization of presynaptic receptors modulating [3H]noradrenaline...

A cumulative dose-response technique was developed for the characterization of presynaptic receptors involved in the modulation of [3H]noradrenaline (NA) release from rat hippocampus slices, using continuous K+ (20 mM) depolarization. The results obtained with this technique were compared with those obtained using a repetitive K+ stimulation procedure. The release of [3H]NA induced by continuous K+ stimulation as well as that caused by repetitive K+ stimulation was strongly Ca2+-dependent and consisted for more than 90% of unmetabolized [3H]NA. Using continuous K+ stimulation it was demonstrated that the presynaptic inhibition of 3H-NA release by exogenous NA reached a maximum 10 min after addition of NA. The inhibitory effect of NA appeared to be independent of the time of addition, suggesting that the sensitivity of the presynaptic alpha-adrenoceptors remained unchanged during the experiment. Cumulative dose-response curves were recorded by the successive addition, at 10 min intervals, of increasing concentrations of NA. It was shown that continuous stimulation and repetitive K+ stimulation were basically similar with regard to the characteristics of the resulting [3H]NA release as well as its presynaptic alpha-adrenoceptor-mediated modulation by exogenous NA. However, the cumulative dose-response technique, which can be carried out only using continuous K+ stimulation, makes it possible to determine more rapidly and also more accurately the apparent affinities and intrinsic activities of drugs towards receptors involved in the modulation of neurotransmitter release from brain slices.