All three alpha2-adrenoceptor types serve as autoreceptors in postganglionic sympathetic neurons.

Postganglionic sympathetic neurons and brain noradrenergic neurons use alpha(2A)- and alpha(2C)-adrenoceptors as presynaptic autoreceptors. The present experiments were carried out in order to see whether they possess presynaptic alpha(2B)-autoreceptors as well. Pieces of atria, vasa deferentia, the occipito-parietal cortex and the hippocampus were prepared from either wildtype (WT) mice or mice in which both the alpha(2A)- and the alpha(2C)-adrenoceptor gene had been disrupted (alpha(2AC)KO). The pieces were incubated with (3)H-noradrenaline and then superfused and stimulated electrically. In a first series of experiments, single pulses or brief, autoinhibition-poor pulse trains were used for stimulation. The alpha(2)-adrenoceptor agonist UK 14,304 (brimonidine) reduced the evoked overflow of tritium in all four tissues from WT mice but did not change it in any tissue from alpha(2AC)KO mice. A different pattern was obtained with medetomidine as alpha(2 )agonist. Like UK 14,304, medetomidine reduced the evoked overflow of tritium in all four tissues from WT mice and did not affect overflow in brain slices from alpha(2AC)KO mice; however, in contrast to UK 14,304, medetomidine reduced evoked overflow also in atrial and vas deferens pieces from alpha(2AC)KO mice, although with a lower maximum and potency than in WT preparations. The alpha-adrenoceptor antagonists rauwolscine, phentolamine, prazosin, spiroxatrine and WB 4101 shifted the concentration-response curve of medetomidine in alpha(2AC)KO atria and vasa deferentia to the right. The pK(d) values of the five antagonists against medetomidine in alpha(2AC)KO atria and vasa deferentia correlated with pK(d) values at prototypical alpha(2B) radioligand binding sites but not at alpha(2A) or alpha(2C) binding sites. In a second series of experiments, autoinhibition-rich pulse trains were used for stimulation. Under these conditions, rauwolscine and phentolamine increased the evoked overflow of tritium from alpha(2AC)KO atrial and vas deferens pieces but not from alpha(2AC)KO brain slices. The increase was smaller (by 40% in atria and by 70% in the vas deferens) than previously observed in WT preparations (by 200-400%). In a last series of experiments, mRNA for the alpha(2B)-adrenoceptor was demonstrated by RT-PCR in thoracolumbar sympathetic ganglia from WT, alpha(2A)KO, alpha(2C)KO and alpha(2AC)KO mice but not from alpha(2B)KO mice. The results show that brain noradrenergic neurons express only alpha(2A)- and alpha(2C)-adrenoceptors as autoreceptors. Postganglionic sympathetic neurons, however, can express alpha(2B)-adrenoceptors as presynaptic autoreceptors as well. The alpha(2B)-autoreceptors are activated by medetomidine but not by UK 14,304. They are also activated by previously released noradrenaline. The two-alpha(2)-autoreceptor hypothesis has to be replaced by a three-autoreceptor hypothesis for postganglionic sympathetic neurons.

Crosstalk between presynaptic angiotensin receptors, bradykinin receptors and alpha 2-autoreceptors in sympathetic neurons: a study in alpha 2-adrenoceptor-deficient mice.

1. In mouse atria, angiotensin II and bradykinin lose much or all of their noradrenaline release-enhancing effect when presynaptic alpha(2)-autoinhibition does not operate either because of stimulation with very brief pulse trains or because of treatment with alpha(2) antagonists. We now studied this operational condition in alpha(2)-adrenoceptor-deficient mice. Release of (3)H-noradrenaline was elicited by electrical stimulation. 2. In tissues from wild-type (WT) mice, angiotensin II and bradykinin increased the overflow of tritium evoked by 120 pulses at 3 Hz. This enhancement did not occur or was much reduced when tissues were stimulated by 120 pulses at 3 Hz in the presence of rauwolscine and phentolamine, or when they were stimulated by 20 pulses at 50 Hz. 3. In tissues from mice lacking the alpha(2A)-adrenoceptor (alpha(2A)KO) or the alpha(2B)-adrenoceptor (alpha(2B)KO), the concentration-response curves of angiotensin II and bradykinin (120 pulses at 3 Hz) were unchanged. In tissues from mice lacking the alpha(2C)-adrenoceptor (alpha(2C)KO) or both the alpha(2A)- and the alpha(2C)-adrenoceptor (alpha(2AC)KO), the concentration-response curves were shifted to the same extent downwards. 4. As in WT tissues, angiotensin II and bradykinin lost most or all of their effect in alpha(2A)KO and alpha(2AC)KO tissues when rauwolscine and phentolamine were present or trains consisted of 20 pulses at 50 Hz. 5. Rauwolscine and phentolamine increased tritium overflow evoked by 120 pulses at 3 Hz up to seven-fold in WT and alpha(2B)KO tissues, three-fold in alpha(2A)KO and alpha(2C)KO tissues, and two-fold in alpha(2AC)KO tissues. 6. Results confirm that angiotensin II and bradykinin require ongoing alpha(2)-autoinhibition for the full extent of their release-enhancing effect. Specifically, they require ongoing alpha(2C)-autoinhibition. The peptide effects that remain in alpha(2C)-autoreceptor-deficient mice seem to be because of alpha(2B)-autoinhibition. The results hence also suggest that in addition to alpha(2A)- and alpha(2C)- mouse postganglionic sympathetic neurons possess alpha(2B)-autoreceptors.

Heterogeneity of presynaptic muscarinic receptors mediating inhibition of sympathetic transmitter release: a study with M2- and M4-receptor-deficient mice.

1 Presynaptic muscarinic receptors modulate sympathetic transmitter release. The goal of the present study was to identify the muscarinic receptor subtype(s) mediating inhibition of sympathetic transmitter release in mouse atria, urinary bladder and vas deferens. To address this question, electrically evoked noradrenaline release was assessed using tissue preparations from NMRI, M(2)- and M(4)-knockout, and the corresponding M(2)- and M(4)-wildtype mice, after preincubation with (3)H-noradrenaline. 2 The muscarinic agonist carbachol decreased evoked tritium overflow (20 pulses/50 Hz) in each tissue and strain investigated. After deletion of the M(2)-receptor the maximal inhibition by carbachol was significantly reduced (by 41-72%), but not abolished, in all tissues. After deletion of the M(4)-receptor a moderate and significant reduction of the maximal inhibition by carbachol (by 28%) was observed only in the vas deferens. 3 Experiments with the muscarinic antagonists methoctramine and pirenzepine confirmed that the presynaptic muscarinic receptors were predominantly M(2) in atria and bladder and probably a mixture of M(2) and M(4) in the vas deferens. 4 Experiments in the urinary bladder with the cholinesterase inhibitor physostigmine and the muscarinic antagonist ipratropium demonstrated that endogenously released acetylcholine predominantly acted through M(2)-receptors to inhibit noradrenaline release. However, the results do not exclude a minor contribution of M(4)-receptors to this endogenous inhibition. 5 In conclusion, our results clearly indicate that the release-inhibiting muscarinic receptors on postganglionic sympathetic axons in mouse atria, bladder and vas deferens represent mixtures of M(2)- and non-M(2)-receptors. The non-M(2)-receptors remain unknown in atria and the bladder, and may represent primarily M(4)-receptors in the vas deferens. These results reveal an unexpected heterogeneity among the muscarinic receptors mediating inhibition of noradrenaline release.