A study of presynaptic α₂-autoreceptors in α_2A/D-, α_2B- and α_2C-adrenoceptor-deficient mice

Abstract.

The function of presynaptic α₂-autoreceptors was studied in the hippocampus, occipito-parietal cortex, atria and vas deferens of NMRI mice, mice in which the α_2A/D-, the α_2B- or α_2C-adrenoceptor gene had been disrupted (α_2A/DKO, α_2BKO and α_2CKO, respectively), and the wildtype mice from which the knockout animals had been generated. Tissue pieces were preincubated with ³H-noradrenaline and then superfused and stimulated electrically.

The α₂-adrenoceptor agonist medetomidine reduced the electrically evoked overflow of tritium in all tissues from all mouse strains (stimulation with single pulses or single high-frequency pulse trains, called POPs, i.e. pulse patterns leading to minimal autoinhibition). The effects of medetomidine did not differ in NMRI, wildtype, α_2BKO and α_2CKO mice but were greatly reduced in α_2A/DKO brain preparations and to a lesser extent in α_2A/DKO atria and vasa deferentia. Six drugs were tested as antagonists against medetomidine. Their pK_d values indicated that the hippocampal and occipito-parietal α₂-autoreceptors in NMRI and wildtype mice were α_2D (the rodent variant of the α_2A/D-adrenoceptor) whereas the atrial and vas deferens α₂-autoreceptors in NMRI and wildtype mice could not be identified with a single α₂ subtype. Deletion of the α_2A/D gene changed the pK_d values in all tissues so that they now reflected α_2C properties, whereas deletion of the α_2C gene changed the pK_d values in atria and vasa deferentia so that they now had α_2D properties (as they had in NMRI and wildtype brain preparations). Autoinhibition by released noradrenaline was created using trains of up to 64 pulses or up to 4 POPs, and the overflow-enhancing effect of the α₂ antagonist rauwolscine was determined. Results did not differ, irrespective of whether preparations were obtained from NMRI, wildtype, α_2BKO or α_2CKO mice: the overflow of tritium elicited by p pulses or POPs was much smaller than p times the overflow elicited by a single pulse or POP, and rauwolscine greatly increased the evoked overflow. Results differed, however, in tissues taken from α_2A/DKO mice: in these tissues, the overflow of tritium elicited by p pulses or POPs was close to p times the overflow elicited by a single pulse or POP, and rauwolscine did not increase the evoked overflow of tritiumor increased it only marginally. When a greater degree of autoinhibition was produced in atria and vasa deferentia by stimulation with 120 pulses, both disruption of the α_2A/D gene and disruption of the α_2C gene but not disruption of the α_2B gene attenuated the overflow-enhancing effects of phentolamine and rauwolscine. In NMRI and wildtype atria and vasa deferentia, the relative potencies of phentolamine and rauwolscine at enhancing the evoked overflow were not easily compatible with a single α₂ subtype. In α_2A/DKO atria and vasa deferentia, the relative potencies of phentolamine and rauwolscine indicated that the autoinhibition-mediating receptors were α_2C, whereas in α_2CKO atria and vasa deferentia the relative potencies indicated that the autoinhibition-mediating receptors were α_2D.

It is concluded that α₂-autoreceptors function identically in NMRI mice and the wildtype mice from which the receptor-deficient animals had been generated. There is no evidence from the experiments for any contribution of α_2B-adrenoceptors to autoreceptor function. The main presynaptic α₂-autoreceptors are α_2A/D, both as sites of action of exogenous agonists and as sites of action of previously released noradrenaline. However, there are in addition non-α_2A/D-, probably α_2C-autoreceptors. They are less prominent in mediating the inhibitory effects of exogenous agonists and the negative feedback effect of released noradrenaline. They operate not only after deletion of the α_2A/D-adrenoceptors but also in normal (NMRI, wildtype) mice without gene deletion.