Subclassification of presynaptic alpha 2-adrenoceptors: alpha 2D-autoreceptors in mouse brain

The study was devised to classify, by means of antagonist affinities, the presynaptic alpha 2-autoreceptors in mouse cerebral cortex in terms of alpha 2A, alpha 2B, alpha 2C and alpha 2D. A set of antagonists was chosen that was able to discriminate between the four subtypes. Slices of the cortex were preincubated with 3H-noradrenaline and then superfused and stimulated electrically. The stimulation periods used (4 pulses, 100 Hz) did not lead to alpha 2-autoinhibition as shown by the lack of an increase by rauwolscine of the evoked overflow of tritium. The alpha 2-selective agonists 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) and alpha-methylnoradrenaline reduced the evoked overflow. All 10 antagonists shifted the concentration-inhibition curve of UK 14,304 to the right. Rauwolscine also shifted the concentration-inhibition curve of alpha-methylnoradrenaline to the right. pKd values of the antagonists were calculated from the shifts. The pKd values of rauwolscine against UK 14,304 and alpha-methylnoradrenaline were very similar (8.0 and 7.9, respectively). Comparison with antagonist affinities for prototypic native alpha 2 binding sites, alpha 2 binding sites in cells transfected with alpha 2 subtype genes, and previously classified presynaptic alpha 2-adrenoceptors--all taken from the literature--indicates that the alpha 2-autoreceptors in mouse brain cortex are alpha 2D. This is the first subtype determination of alpha 2-autoreceptors in the mouse. It supports the hypothesis that at least the majority of alpha 2-autoreceptors belong to the alpha 2A/D branch of the alpha 2-adrenoceptor tree.