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.
Adenine and uracil nucleotides release noradrenaline from rat postganglionic sympathetic neurones by activation of P2X-receptors and distinct receptors for uracil nucleotides, respectively. The present study on cultured neurones of rat thoracolumbal paravertebral ganglia has analysed the involvement of action potentials and calcium influx in the nucleotide-induced transmitter release. ATP and UDP (100 microM each) caused a marked release of previously incorporated [3H]noradrenaline. The P2-receptor antagonists suramin (300 microM) and cibacron blue 3GA (3 microM) decreased the ATP-induced but not the UDP-induced release. The response to ATP was decreased by the sodium channel blocker tetrodotoxin (0.5 microM; by 47%), by the N-type calcium channel blocker omega-conotoxin GVIA (100 nM; by 35%), and by the alpha2-adrenoceptor agonist UK-14,304 (1 microM; by 45%); it was not changed by the potassium channel blocker tetraethylammonium (10 mM). The response to UDP was abolished by tetrodotoxin, greatly decreased by omega-conotoxin (by 78%), also abolished by UK-14,304, and increased by tetraethylammonium (by 410%). ATP (100 microM) caused a marked increase in intra-axonal free calcium as measured by fura-2 microfluorimetry. Withdrawal of extracellular calcium diminished the calcium response to ATP by 85%, and tetrodotoxin and omega-conotoxin diminished it by about 40%. As studied with the amphotericin B-perforated patch method, ATP (100 microM) induced a large depolarisation and action potential firing. UDP (100 microM) induced only a small depolarisation but it also elicited action potentials. UDP increased the excitability of the neurones. The results indicate that the ATP-induced release of noradrenaline depends on influx of calcium from the extracellular space. Calcium passes through two structures: voltage-gated channels and - probably - the P2X-receptor itself. Only that component of ATP-induced transmitter release which is triggered by opening of voltage-gated calcium channels is sensitive to modulation by alpha2-adrenoceptors. In contrast to ATP, the UDP-induced release of noradrenaline is entirely due to generation of action potentials followed by calcium influx through voltage-gated channels. All of the UDP-induced release is therefore sensitive to alpha2-adrenoceptor modulation.
1 In mouse atria, angiotensin II and bradykinin lose much or all of their noradrenaline release-enhancing effect when presynaptic a 2 -autoinhibition does not operate either because of stimulation with very brief pulse trains or because of treatment with a 2 antagonists. We now studied this operational condition in a 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 a 2A -adrenoceptor (a 2A KO) or the a 2B -adrenoceptor (a 2B KO), the concentration -response curves of angiotensin II and bradykinin (120 pulses at 3 Hz) were unchanged. In tissues from mice lacking the a 2C -adrenoceptor (a 2C KO) or both the a 2A -and the a 2C -adrenoceptor (a 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 a 2A KO and a 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 a 2B KO tissues, three-fold in a 2A KO and a 2C KO tissues, and two-fold in a 2AC KO tissues. 6 Results confirm that angiotensin II and bradykinin require ongoing a 2 -autoinhibition for the full extent of their release-enhancing effect. Specifically, they require ongoing a 2C -autoinhibition. The peptide effects that remain in a 2C -autoreceptor-deficient mice seem to be because of a 2B -autoinhibition. The results hence also suggest that in addition to a 2A -and a 2C -mouse postganglionic sympathetic neurons possess a 2B -autoreceptors.
Cultured neurons from the thoracolumbar sympathetic chain of newborn mice are known to possess release-inhibiting ␣ 2 -autoreceptors. The present study was carried out in a search for release-modulating heteroreceptors on these neurons. Primary cultures were preincubated with [ 3 H]noradrenaline and then superfused and stimulated by single pulses, trains of 8 pulses at 100 Hz, or trains of 36 pulses at 3 Hz. The cholinergic agonist carbachol reduced the evoked overflow of tritium. Experiments with antagonists indicated that the inhibition was mediated by M 2 muscarinic receptors. The cannabinoid agonist WIN 55,212-2 reduced the evoked overflow of tritium through CB 1 receptors. Prostaglandin E 2 , sulprostone, and somatostatin also caused presynaptic inhibition. The inhibitory effects of carbachol, WIN 55,212-2, prostaglandin E 2 , and somatostatin were abolished (at the highest concentration of WIN 55,212-2 almost abolished) by pretreatment of the cultures with pertussis toxin (250 ng/ml). Several drugs, including the  2 -adrenoceptor agonist salbutamol, opioid receptor agonists, neuropeptide Y, angiotensin II, and bradykinin, failed to change the evoked overflow of tritium. These results demonstrate a distinct pattern of presynaptic inhibitory heteroreceptors, all coupled to pertussis toxin-sensitive G proteins. The lack of operation of several presynaptic receptors known to exist in adult mice in situ may be due to the age of the (newborn) donor animals or to the culture conditions. Key Words: Mouse sympathetic neuron cultures-Noradrenaline release -Presynaptic receptors-Muscarinic receptors-Cannabinoid receptors-Prostanoid receptors-Somatostatin receptors. J. Neurochem. 75, 2087Neurochem. 75, -2094Neurochem. 75, (2000.As in intact tissues, various receptors modulate the action potential-evoked release of noradrenaline in cultures of postganglionic sympathetic neurons from, for example, chicken and rats (for review, see Boehm and Huck, 1997). Neurons of mice, however, were not studied in this respect until we recently prepared primary cultures of thoracolumbar sympathetic ganglion cells from this species. The cells possessed release-inhibiting ␣ 2A/D -autoreceptors, but not release-modulating P2, A 1 , or A 2A receptors, which are alternative autoreceptors due to the cotransmitter role of ATP (Trendelenburg et al., 1999a, b). We have now carried out a search for releasemodulating heteroreceptors on these neurons. Most heteroreceptors ever observed were included (see Fuder and Muscholl, 1995;Powis and Bunn, 1995). The study concentrated on receptors modifying action potentialevoked release; receptors that, when activated, elicit exocytotic release of noradrenaline will be described in a subsequent report. Neuronal action potentials were triggered by electrical stimulation, and release of previously incorporated [ 3 H]noradrenaline was measured. MATERIALS AND METHODS Culturing and [ 3 H]noradrenaline releaseThese techniques were modified from our previous work (Trendelenburg et al., 1999a). Thora...
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