Sodium dependent 3H-noradrenaline release from rat neocortical slices in the absence of extracellular calcium presynaptic modulation by μ-opioid receptor and adenylate cyclase activation

Summary In Ca²⁺-free EGTA (1 mmol/l)-containing medium veratrine (3 mol/l) and ouabain (100 mol/l) strongly enhanced the efflux of ³H-noradrenaline from superfused rat brain neocortical slices prelabelled with the radioactive amine. In both cases ³H-noradrenaline release was prevented by tetrodotoxin (1 mol/l). These effects of veratrine and ouabain were virtually additive and independent of whether the noradrenaline uptake carrier was blocked with 1 mol/l desipramine or not. The adenylate cyclase activator forskolin (10 nmol/l–10 mol/l) strongly enhanced veratrine- and ouabain-induced ³H-noradrenaline release, without affecting spontaneous tritium efflux. The release induced by both stimuli was profoundly inhibited by the selective -opioid receptor agonist [d-Ala, MePhe⁴, Gly-ol⁵]enkaphalin (DAGO, 3 nmol/l–1 mol/l) in a concentration-dependent manner. The inhibitory effects of 1 mol/l DAGO were abolished by 1 mol/l naloxone. On the other hand, preincubation of the slices for 1 h with the -opioid receptor-selective irreversible ligand fentanyl isothiocyanate (1 pmol/l) did not change the inhibitory effects of DAGO.These data show that veratrine- and ouabain-induced ³H-noradrenaline release from central noradrenergic nerve terminals is facilitated by increasing intracellular cyclic AMP levels and reduced by activation of presynaptic -opioid receptors, indicating the involvement of exocytotic neurotransmitter release. The results provide further evidence for the hypothesis that under these conditions neurotransmitter release from central noradrenergic neurons is triggerred by a Na⁺-induced efflux of Ca²⁺ ions from intracellular stores.Abbreviations DAGO [d-Ala², McPhe⁴, Gly-ol⁵]enkephalin Send offprint requests to A. N. M. Schoffelmeer at the above address     

Pre- and postsynaptic effects of yohimbine stereoisomers on noradrenergic transmission in the pulmonary artery of the rabbit

Summary Effects on noradrenergic neurotransmission of five stereoisomers of yohimbine and of the closely related compound yohimbol were studied in strips of the pulmonary artery of the rabbit. In some experiments the tissue was preincubated with ³H-noradrenaline. Three effects were observed. Firstly, antagonism to the contractile effect of noradrenaline and of sympathetic nerve stimulation; the antagonism reflected competitive blockade of postsynaptic -adrenoceptors. Secondly, an increase in the stimulation-evoked overflow of total tritium and ³H-noradrenaline; the increase appeared to be due to blockade of presynaptic -adrenoceptors. Thirdly, an increase in the basal outflow of ³H-3,4-dihydrophenylglycol, presumably by impairment of the vesicular storage of ³H-noradrenaline. According to their relative potencies in eliciting these effects, the drugs could be divided into three groups. Rauwolscine, -yohimbine and yohimbol preferentially blocked the presynaptic -adrenoceptor; rauwolscine and -yohimbine, like yohimbine, at low concentrations increased the contractile response to sympathetic nerve stimulation. Corynanthine preferentially blocked the postsynaptic -adrenoceptor. Pseudoyohimbine and 3-epi--yohimbine were very weak antagonists at either receptor; they mainly accelerated the basal outflow of ³H-3,4-dihydroxyphenylglycol.From these results and those of a previous study it is concluded that, in a series of twelve -adrenolytic drugs, rauwolscine shows the greatest preference for presynaptic and corynanthine the greatest preference for postsynaptic -adrenoceptors. In view of the chemical similarity of the two compounds these opposite properties are striking. Corynanthine and rauwolscine might be useful tools for the subclassification of -adrenoceptors.     

N-ethylmaleimide (NEM) diminishes α2-adrenoceptor mediated effects on noradrenaline release

Summary The effect of N-ethylmaleimide (NEM), which has been shown to abolish rather selectively inhibition of adenylate cyclase, on the ₂-adrenoceptor modulation of noradrenaline release was studied. Slices of the rabbit hippocampus were loaded with ³H-noradrenaline, superfused continuously and stimulated twice electrically.NEM (30 mol/l) applied for 30 min enhanced both basal and stimulation-evoked tritium overflow significantly. Occupation of the receptor by the ₂-adrenoceptor agonist clonidine prior to and during NEM treatment did not protect the ₂-adrenoceptor-mediated autoinhibitory feedback system from being affected by NEM. Preincubation of the hippocampal slices with NEM was without any influence on ³H-noradrenaline uptake. The inhibitory effect of clonidine on ³H-noradrenaline release was attenuated in a non-competitive manner. In addition, the facilitatory effect of the ₂-adrenoceptor antagonist yohimbine on the stimulusevoked tritium overflow was reduced. The facilitation of the evoked noradrenaline release by yohimbine or yohimbine and NEM converged with increasing concentrations of yohimbine, suggesting that yohimbine and NEM were acting at the same signal-transduction system.These results are compatible with the idea that NEM, by alkylating the N_i-unit of a presynaptically located adenylate cyclase, prevents the ₂-adrenoceptor-mediated modulation of noradrenaline release.Abbreviations NEM N-ethylmaleimide - IAP islet-activating protein     

Subclassification of presynaptic α2-adrenoceptors: α2D-autoreceptors and α2D-adrenoceptors modulating release of acetylcholine in guinea-pig ileum

The study was designed to classify in terms of _2A, _2B, _2C and _2D the presynaptic ₂-autoreceptors, as well as the ₂-receptors modulating the release of acetylcholine, in the myenteric plexus-longitudinal muscle (MPLM) preparation of the guinea-pig ileum. A set of antagonists was chosen that was able to discriminate between the four subtypes. Small pieces of the MPLM preparation were preincubated with ³H-noradrenaline or ³H-choline and then superfused and stimulated electrically.The stimulation periods used (3H-noradrenaline: 3 trains of 20 pulses, 50 Hz, train interval 60 s; ³H-choline: single trains of 30 pulses, 0.2 Hz) did not lead to ₂-autoinhibition or inhibition of ³H-acetylcholine release by endogenous noradrenaline. The ₂-selective agonist 5-bromo6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) reduced the evoked overflow of tritium in both ³H-noradrenaline and ³H-choline experiments. Most (³H-noradrenaline) or all (³H-choline) of the 10 antagonists shifted the concentration-inhibition curves of UK 14,304 to the right. pK_d values of the antagonists were calculated from the shifts. pK_d values from ³H-noradrenaline experiments correlated with pK_d values from ³H-choline experiments (r = 0.981).It is concluded that ₂-autoreceptors and ₂-heteroreceptors modulating the release of acetylcholine in the MPLM preparation are of the same subtype. Comparison with antagonist affinities for prototypic native ₂ binding sites, binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature — indicates that both are _2D. The results are consonant with the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D branch of the ₂-adrenoceptor tree, across mammalian or at least across rodent and lagomorph species. The same may hold true for ₂-adrenoceptors on non-noradrenergic neurones.     

Dopaminergic modulation of hippocampal noradrenaline release

Summary ³H-Noradrenaline release in the rabbit hippocampus and its possible modulation via presynaptic dopamine receptors was studied. Hippocampal slices were preincubated with ³H-noradrenaline, continuously superfused in the presence of cocaine (30 mol/l) and subjected to electrical field stimulation. The electrically evoked tritium over-flow from the slices was reduced by 0.1 and 1 mol/l dopamine and apomorphine, but significantly enhanced by 10 mol/l apomorphine or by 0.1 and 1 mol/l bromocriptine. If the ₂-adrenoceptor antagonist yohimbine (0.1 mol/l) was present throughout superfusion, the inhibitory effects of dopamine and apomorphine were more pronounced and even 10 mol/l apomorphine and 1 mol/l bromocriptine inhibited noradrenaline release. Qualitatively similar observations were made in the presence of another ₂-antagonist, idazoxane (0.1 mol/l). In the presence of the D2-receptor antagonist domperidone (0.1 mol/l) the inhibitory effects of dopamine were almost abolished, whereas both apomorphine (>1 mol/l) and bromocriptine (>0.01 mol/l) greatly facilitated noradrenaline release. The D2-receptor agonist LY 171555 (0.1 and 1 mol/l) significantly reduced the evoked noradrenaline release whereas the D1-selective agonist SK & F 38393 was ineffective at similar concentrations. The effects of LY 171555 were abolished in the presence of domperidone (0.1 mol/l) but remained unchanged in the presence of yohimbine or idazoxane (0.1 mol/l, each).At 1 mol/l the D2-receptor antagonists domperidone and (-)sulpiride significantly increased the evoked noradrenaline release by about 10%. However, at this concentration, domperidone (but not (-)sulpiride) affected also basal tritium outflow. Bulbocapnine and the preferential D1-receptor antagonists SCH 23390 enhanced the evoked noradrenaline release already at 0.1 mol/l. Their marked facilitatory effects (50 to 60% increase at 1 mol/l) were reduced in the presence of idazoxane (0.1 mol/l) and almost abolished in the presence of 0.1 mol/l yohimbine, whereas the increase due to 1 mol/l (-)sulpiride persisted under these conditions.The evoked tritium efflux from rabbit hippocampal slices preincubated with ³H-serotonin was not affected by dopamine receptor agonists.From our results we conclude that hippocampal noradrenaline, but not serotonin release, is modulated via D2-dopamine receptors. In addition, our results provide evidence for more or less pronounced ₂-adrenoceptor agonistic properties of dopamine and ₂-adrenoceptor antagonistic properties of apomorphine, bromocriptine, SCH 23390 and bulbocapnine in this noradrenaline release model from CNS tissue.     

Noradrenaline uptake inhibitors do not reduce the presynaptic action of clonidine on 3H-noradrenaline release in superfused synaptosomes

Summary The interaction between clonidine, as an agonist at the ₂-autoreceptors regulating noradrenaline release, and inhibitors of noradrenaline neuronal uptake was investigated in superfused synaptosomes, i.e. in conditions preventing accumulation of the released transmitter in the vicinity of presynaptic autoreceptors. Desipramine or cocaine did not decrease the inhibitory action of clonidine on the release of ³H-noradrenaline evoked by 15 mM KCl from rat cortex synaptosomes, even when the concentration ratio between uptake inhibitor and clonidine was very high. The present results do not support either the hypothesis of an interaction between imidazolines and noradrenaline uptake inhibitors at the level of ₂-autoreceptors, or that of a functional coupling between presynaptic ₂-autoreceptors and noradrenaline uptake mechanism.     

Opposite modulation of cotransmitter release in guinea-pig vas deferens: increase of noradrenaline and decrease of ATP release by activation of prejunctional β-adrenoceptors

Effects of isoprenaline on contraction, release of noradrenaline and release of ATP elicited by electrical field stimulation (210 pulses, 7 Hz) as well as on contractions elicited by exogenous noradrenaline and ATP were studied in the isolated vas deferens of the guinea pig. Release of noradrenaline was assessed as overflow of total tritium after preincubation with [³H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique.In [³H]-noradrenaline-pretreated tissues, electrical stimulation elicited an overflow of tritium and ATP and a biphasic contraction. Isoprenaline (1–100 nM) reduced the contraction, mainly phase I, and enhanced the evoked overflow of tritium; evoked overflow of ATP was not changed significantly. No, or almost no, contraction remained in [³H]-noradrenaline-pre-treated tissues exposed to both prazosin (0.3 M) and suramin (300 M), and the evoked overflow of ATP was reduced by about 82%. Under these conditions, isoprenaline (1–100 nM) again enhanced the evoked overflow of tritium, but it now decreased the evoked overflow of ATP. Propranolol (1 M), when added on top of prazosin and suramin, prevented the effects of isoprenaline (1–100 nM). In some tissues not pretreated with [³H]-noradrenaline, purinergic and adrenergic components of the neurogenic contraction (again to 210 pulses, 7 Hz) were isolated by exposure to prazosin (0.3 M) and suramin (300 M), respectively. Isoprenaline (1–100 nM) decreased the isolated purinergic component but did not change significantly the isolated adrenergic component. Contractions elicited by ATP (1000 M) were not changed and contractions elicited by noradrenaline (100 M) were slightly increased by isoprenaline (1–100 nM). Isoprenaline (100 nM) did not change the degradation of ATP (100 M) by pieces of the vas deferens.It is concluded that, in the guinea-pig vas deferens, activation of prejunctional -adrenoceptors modulates the neural release of noradrenaline and ATP in opposite directions: release of noradrenaline is enhanced, whereas release of ATP is decreased.     

Pre- and postganglionic stimulation-induced noradrenaline overflow is markedly facilitated by a prejunctional β2-adrenoceptor-mediated control mechanism in the pithed rat

The aim of the study was to further explore the prejunctional -adrenoceptor-mediated control mechanism of noradrenaline release from sympathetic nerves in response to preganglionic nerve stimulation (PNS) and local nerve stimulation of the portal vein, respectively, in the pithed rat.Baseline values as well as the increments of mean arterial blood pressure (-BP), heart rate (-HR) and plasma noradrenaline levels (-NA) in response to four PNS episodes (0.8 Hz, 3 ms, 75 V for 45 s at 20 min intervals), respectively, were evaluated. Fenoterol administration (0.25 mg/kg, i.v.) reduced significantly the basal blood pressure but did not alter d-BP in response to PNS. Basal heart rate markedly increased after fenoterol without any further change in heart rate induced by PNS.The ₁-selective antagonist CGP 20712A attenuated -BP in response to PNS and prevented the fenoterol-induced increase in basal heart rate. The ₂-selective antagonist ICI 118,551 per se did not change the blood pressure and heart rate values, but antagonized the fenoterol-induced decrease in basal blood pressure.Fenoterol enhanced plasma -NA in response to PNS by 105% in comparison to the corresponding control value. This effect of fenoterol could be blocked by pretreatment with ICI 118,551 but not with CGP 20712A (a selective ₁-adrenoceptor antagonist) which per se did not significantly change plasma -NA.Repeated local stimulation of the portal vein (S1–S 3, 2 Hz, 3 ms, 10 mA, for 120 s at 30 min intervals) increased portal plasma noradrenaline without changing mean blood pressure and heart rate in pithed rats. Fenoterol enhanced the increase in portal-vein plasma noradrenaline evoked by nerve stimulation by 110%. Pretreatment with ICI 118,551 antagonized this effect of fenoterol, but had per se no effect on the portal vein nerve stimulation-evoked increase in portal plasma noradrenaline.It is concluded that the increase in plasma noradrenaline evoked both by pre- and postganglionic nerve stimulation can be markedly enhanced by activation of a facilitatory prejunctional ₂-adrenoceptor control mechanism. Correspondence to: V.I. Tarizzo     

Activation of β2-adrenoceptors by isoprenaline and adrenaline enhances noradrenaline release in cortical kidney slices of young spontaneously hypertensive rats

Summary The aim of the present study was to investigate -adrenoceptor modulation of noradrenaline release from sympathetic nerves in superfused cortical kidney slices of 4-week-old spontaneously hypertensive rats (SHR) and age-matched controls (WKY). After preincubation with ³H-noradrenaline the kidney slices were electrically stimulated in superfusion chambers. The stimulation induced (S-I) outflow of radioactivity was mainly composed of unmetabolized 3H-noradrenaline in both strains and thus taken as an index of noradrenaline release. There was a frequency-dependent (1.25–20 Hz) increase in the S-1 outflow of radioactivity. At all stimulation frequencies tested S-I outflow of radioactivity was similar or even slightly lower in SHR than in WKY kidney slices in either the absence or presence of cocaine (10 mol/l). The non-selective -adrenoceptor agonists isoprenaline (0.l gmol/1) and adrenaline (0.01 and 0.1 mol/l) enhanced S-I outflow of radioactivity. The facilitatory effects of isoprenaline (0.1 mol/l) and adrenaline (0.1 mol/l) were blocked by the selective ₂-adrenoceptor antagonist ICI 118551 (0.1 mol/l) but not by the selective ₁-adrenoceptor antagonist atenolol (0.3 mol/l). The cell-permeable CAMP analogue 8-bromo-cAMP (300 mol/l) enhanced S-1 outflow of radioactivity to a similar extent in both SHR and WKY kidney slices. A combination of 8-bromo-cAMP (300 mol/l) and adrenaline (0.1 mol/l) did not enhance S-1 outflow of radioactivity to a greater extent than 8-bromo cAMP (300 mol/l) alone in both strains. However, the facilitatory effects of isoprenaline (0.1 mol/l) and adrenaline (0.1 mol/l) but not that of adrenaline (0.01 mol/l) were significantly greater in SHR than in WKY. The results suggest that stimulation of prejunctional ₂-adrenoceptors by adrenaline even in the absence of a-adrenoceptor blockade enhances noradrenaline release in kidney cortex of young SHR and WKY. This ₂-adrenoceptor mediated effect may possibly be dependent on cAMP formation. The greater facilitatory effects of isoprenaline (0.1 mol/l) and adrenaline (0.1 mol/l) in SHR as compared to WKY are in accord with receptor binding studies which show a higher density of ₂-adrenoceptors in SHR than in WKY kidney cortex.Abbreviations SHR Spontaneously hypertensive rats - WKY WistarKyoto rats - cAMP 3-5-cyclic adenosine monophosphate - S-I stimulation induced Send offprint requests to: L. C. Rump     

Differential effects of α-β-methylene ATP on responses to nerve stimulation in SHR and WKY tail arteries

Summary The effects of ,-methylene-adenosine triphosphate, (,-methylene ATP, a P₂-receptor desensitising agent) have been evaluated on vasoconstrictor responses elicited by exogenous agonists or electrical field stimulation in isolated perfused SHR or WKY tail arteries and on tritium release elicited by electrical field stimulation in SHR-tail arteries pre-labeled with ³H-noradrenaline.Exposure to ,-methylene ATP (0.1 mol/l) significantly inhibited vasoconstrictor responses to electrical field stimulation in SHR tail arteries. These inhibitory effects were not further increased at a higher concentration of ,-methylene ATP (1 mol/l). In WKY tail arteries, ,-methylene ATP (1 mol/l) failed to significantly inhibit vasoconstrictor responses to electrical stimulation.In SHR tail arteries prelabelled with ³H-noradrenaline, ,-methyleneATP (1 mol/l) did not inhibit the stimulation evoked release of tritium. However, at this concentration, ,-methylene ATP significantly antagonized the vasoconstrictor responses of SHR tail arteries induced by exogenous ATP (1 mol/l), ,-methylene ATP (30 mol/l), a stable agonist at P₂-receptors, or 60 mmol/l KCl. These effects of ,-methylene ATP on contractile responses to KCl were not observed in WKY-tail arteries.In tail arteries obtained from reserpine pretreated SHR, despite a 85–95% decrease in endogenous noradrenaline tissue content, the vasoconstrictor responses induced by periarterial field stimulation were greatly diminished, but not abolished. These residual responses to periarterial field stimulation were not antagonized by prazosin (0.1 mol/l), but were practically abolished by the addition of ,-methylene ATP (1 mol/l).In tail arteries from WKY rats pretreated with reserpine, exposure to prazosin (0.1 mol/l) further reduced the residual responses elicited by electrical field stimulation. In these WKY-tail arteries, addition of ,-methylene ATP (1 mol/l) did not further inhibit the remaining vasoconstrictor response obtained in the presence of prazosin.While our results suggest a significantly greater cotransmitter role for ATP with noradrenaline in tail arteries of SHR compared with control normotensive WKY rats, additional effects of ,-methylene ATP not involving P₂ receptors cannot be entirely excluded.     

Influence of neuronal uptake on the presynaptic α-adrenergic modulation of noradrenaline release

Summary Conditions required for the inhibitory feedback modulation of noradrenergic neurotransmission were studied in isolated atria of the rat.The alpha adrenergic antagonist, yohimbine, 0.8 M, or phentolamine, 1 M, did not affect the chronotropic response to 4 or 8 shocks at 0.8 Hz but increased it when a higher number of shocks was applied. When neuronal uptake was inhibited by cocaine, 2.9 M, or desipramine, 0.1 M, the enhancement of neurotransmission by yohimbine or phentolamine was higher than that observed in the presence of -adrenergic antagonists alone.In atria preincubated with ³H-noradrenaline, the effect of the drugs on the ³H-overflow evoked by 240 shocks at 2.0 Hz was studied. Cocaine 2.9 M, did not increase the evoked overflow but yohimbine, 0.8 M, did. The ³H-overflow obtained in the group of yohimbine plus cocaine was significantly higher than was expected from the effects of both drugs alone.It is concluded that yohimbine or phentolamine enhance the chronotropic response in rat atria only when the concentration of noradrenaline in the biophase is sufficiently high to activate presynaptic receptors. In this tissue, the efficiency of the neuronal uptake influences the degree of -adrenergic autoinhibition.     

Blockade of α2-adrenoceptors increases opioid μ-receptor-mediated inhibition of the firing rate of rat locus coeruleus neurones

Summary In pontine slices of the rat brain, the frequency of spontaneous action potentials of locus coeruleus (LC) neurones was recorded extracellularly. Noradrenaline 0.1–100 mol/l, UK 14,304 0.01–100 nmol/l, [Met⁵]-enkephalin 1–10,000 nmol/l and [D-Ala², D-Leu⁵]enkephalin 0.1–1,000 nmol/l, all depressed the firing rate. Rauwolscine 1 mol/l antagonized the effects of both noradrenaline and UK 14,304, but potentiated the effects of [Met']enkephalin and [D-Ala², D-Leu⁵]enkephalin. Idazoxan 1 mol/l acted in a similar manner. Prazosin 1 mol/l did not change the effects of either noradrenaline or [Met⁵]enkephalin. Naloxone 0.1 mol/l antagonized both [Met']enkephalin and [D-Ala², D-Leu⁵]enkephalin, but failed to alter the effects of either noradrenaline or UK 14,304. Rauwolscine, idazoxan and prazosin, all 1 mol/l, as well as naloxone 0.1 mol/l, did not influence the firing rate when given alone. Desipramine 1 mol/l inhibited the discharge of action potentials in a rauwolscine-antagonizable manner. Noradrenaline 10 mol/l produced the same depression of firing, both in the presence of noradrenaline 1 mol/l and [Met⁵]enkephalin 0.03 mol/l. Likewise, the effect of [Met⁵]enkephalin 0.3 mol/l was the same, irrespective of whether it was added to a medium containing [Met⁵]enkephalin 0.03 mol/l or noradrenaline 1 mol/l. The spontaneous activity of LC neurones is inhibited by somatic ₂-adrenoceptors and opioid -receptors. We suggest that the two receptors interact with each other at a site located between themselves and not in the subsequent common signal transduction system.Send offprint requests to: P. Illes at the above address     

Effects of sympathectomy on the in vitro α and β-responses of the parotid gland

Summary Amylase and K⁺ release were measured in slices obtained from innervated and sympathetically denervated rat parotid glands. Amylase release induced by noradrenaline was found to be increased after chronic denervation. The postjunctional component of supersensitivity to noradrenaline was evidenced by an increase in the maximal response to this agonist. The maximal response of denervated glands was blocked by propranolol 10^–5 M. On the other hand, chronic denervation did not modify the -adrenoceptor-mediated response, K⁺ release, either in the presence or in the absence of ouabain. It is concluded that, after sympathetic denervation, postjunctional supersensitivity develops for the -adrenoceptor-mediated but not for the -adrenoceptors-mediated effects of noradrenaline.     

Protection of presynaptic α-adrenoceptors against irreversible blockade by phenoxybenzamine: preservation of the modulatory effects of exogenous noradrenaline and yohimbine

Summary 1. Receptor protection experiments were carried out in order to study the site of action of -adrenoceptor agonists and antagonists on the release of noradrenaline. Cerebrocortical slices from rabbits were preincubated with ³H-noradrenaline. They were then superfused with medium containing cocaine 30 mol/l and stimulated electrically (3 Hz) three times, after 60, 250 and 295 min of superfusion (S₁, S₂, S₃). Phenoxybenzamine 10 mol/1 when used, was added between S₁ and S₂ for 30 min; putative protecting drugs (clonidine 100 mol/1 or yohimbine 10 mol/1) were present 5 min before and during the exposure to phenoxybenzamine and then washed out together with the latter. Either the voltage drop between the electrodes at S₂ and S₃ or the Ca²⁺-concentration of the superfusion medium at S₂ and S₃ was diminished, if necessary, in order to bring the overflow evoked by S₂ close to the overflow at S₁. Blockade by phenoxybenzamine, or protection against the blockade, was examined by addition of the test compounds noradrenaline 0.1 mol/1 or yohimbine 1 mol/1 before S₃. 2. In slices not exposed previously to -adrenoceptor ligands, noradrenaline 0.1mol/1 greatly reduced, whereas yohimbine 1 mol/1 greatly increased the evoked overflow of tritium. Both effects were abolished in slices treated with phenoxybenzamine 10 pmol/1 alone between S₁ and S₂. 3. In contrast to phenoxybenzamine alone, exposure to phenoxybenzamine 10 mol/1 in the presence of either clonidine 100 pmol/1 or yohimbine 10 mol/1 failed to abolish the effects of the test compounds noradrenaline 0.1 mol/1 and yohimbine 1 mol/1, although the effects were reduced. 4. It is concluded that the irreversible antagonist phenoxybenzamine, the protecting agents clonidine and yohimbine, the test compounds noradrenaline and yohimbine, and by inference endogenous noradrenaline as well, all act at the same site, namely the presynaptic -autoreceptor. Send offprint requests to K. Starke at the above address     

Inhibition by dopamine of 3H-noradrenaline release elicited by nerve stimulation in the isolated cat's nictitating membrane

Summary After loading the isolated nerve-muscle preparation of the cat nictitating membrane with ³H-(±)-noradrenaline the effects of exogenous dopamine and (-)-noradrenaline were determined on ³H-transmitter overflow elicited by nerve stimulation in the presence of cocaine, 29 M. Dopamine, 0.20 M, and (-)-noradrenaline, 0.18 M, inhibited ³H-noradrenaline release elicited by nerve stimulation at 4 or 10 Hz. Similar results were obtained with apomorphine 0.03 or 0.1 M. Chlorpromazine, 1 M, or pimozide, 1 M, antagonized selectively the reduction in ³H-noradrenaline release obtained with dopamine or apomorphine, without affecting the inhibition obtained with (-)-noradrenaline. Phentolamine, 1 M, antagonized more effectively the inhibitory effects of (-)-noradrenaline than those of dopamine. Phenoxybenzamine, 0.29 M, prevented the inhibition of ³H-transmitter overflow obtained with (-)-noradrenaline, dopamine or apomorphine. In the absence of cocaine neither chlorpromazine nor pimozide were able to increase ³H-transmitter overflow during nerve stimulation. In contrast to these results, block of -adrenoceptors by phentolamine or phenoxybenzamine resulted in an increase ³H-transmitter overflow during nerve stimulation. Inhibition by dopamine of ³H-transmitter overflow appears to be mediated through dopamine receptors probably located in the outer surface of adrenergic nerve endings. These dopamine receptors differ from the prejunctional -adrenoceptors that mediate the negative feed-back regulatory mechanism for noradrenaline release by nerve stimulation. The prejunctional inhibitory dopamine receptors are not involved in an endogenously mediated regulatory mechanism for noradrenaline release by nerve stimulation under normal conditions. The possibility that these dopamine receptors are involved in the hypotension commonly observed in patients with chronic l-Dopa treatment is discussed.     

Presynaptic α2-autoreceptors in mouse heart atria: evidence for the α2D subtype

Presynaptic ₂-autoreceptors in mouse atria were characterized in terms of the _2A, _2B, _2C and _2D subtypes. Segments of the atria were preincubated with ³H-noradrenaline and then superfused and stimulated electrically. The affinity of up to 16 antagonists for the autoreceptors was assessed as (1) pEC_30% values, i.e. concentrations that increased previously autoinhibited release of ³H-noradrenaline (120 pulses, 3 Hz) by 30%, and (2) pK_d values against the release-inhibiting effect of 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) under conditions of no or little autoinhibition (2 trains of 20 pulses, 50 Hz, train interval 120 s).The pK_d values correlated well with the pEC_30% values (r = 0.98; P<0.001; slope of regression line 0.93), indicating that UK 14,304 and released noradrenaline modulated the release of noradrenaline through pharmacologically identical receptors. Comparison with antagonist affinities for (1) prototypic native ₂ radioligand binding sites, (2) radioligand binding sites in COS cells transfected with ₂ subtype genes, and (3) previously classified presynaptic ₂-autoreceptors — all taken from the literature — indicated that the mouse atrial autoreceptors corresponded to the _2D subtype. For example, the pK_d values at mouse atrial autoreceptors correlated closely with pK_d values at native _2D binding sites in the bovine pineal gland (r = 0.96; P<0.001); with pK_d values at _2D binding sites in COS cells transfected with the rat _2D gene (r= 0.85; P<0.01); and with pK_d values at guinea-pig cerebral and atrial and mouse cerebral _2D-autoreceptors (r=0.96–0.98; P<0.001). The antagonist pK_d values at mouse atrial autoreceptors correlated less with pK_d values at _2A, _2B, and _2C sites.It is concluded that the presynaptic ₂-autoreceptors in mouse atria are _2D This identification supports the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D pair of orthologous ₂-adrenoceptors.     

Activation of presynaptic α2-adrenoceptors attenuates the inhibitory effect of μ-opioid receptor agonists on noradrenaline release from brain slices

Summary ³H-noradrenaline release from rat neocortical slices induced by 15 mM K⁺ was concentration-dependently inhibited by morphine, [D-Ala²-D-Leu⁵] enkephalin (DADLE) and the calcium entry blocker Cd²⁺. Blockade of presynaptic ₂-adrenoceptors with phentolamine, almost doubling K⁺-induced ³H-noradrenaline release, slightly enhanced the relative inhibitory effects of morphine and DADLE, whereas that of Cd²⁺ remained unaffected. In contrast, activation of presynaptic ₂-adrenoceptors with clonidine (1 M) or TL-99 (1 M), inhibiting release by about 50%, completely abolished the inhibitory effects of morphine and DADLE without affecting that of Cd²⁺. When in the presence of 1 M clonidine adenylate cyclase was activated with forskolin (10 M), which restored release to the drug-free control level, the opioids still did not display their inhibitory effects. Therefore, -opioid receptor efficacy appears to be dependent on the degree of activation of ₂-adrenoceptors in central noradrenergic nerve terminals, probably through a local receptor interaction within the nerve terminal membrane.     

Pertussis toxin abolishes the inhibition of Ca2+ currents and of noradrenaline release via α2-adrenoceptors in chick sympathetic neurons

Summary Effects of ₂-adrenoceptor agonists on whole-cell Ca²⁺ currents and ³H-noradrenaline release were investigated by applying the patch-clamp technique and electrical field stimulation to cultured embryonic chick sympathetic neurons. A 24-h exposure of the sympathetic neurons to pertussis toxin (100 ng/ml) abolished both the 2-adrenoceptor-mediated inhibition of Ca ²⁺ currents and the modulation of noradrenaline release caused by noradrenaline (1 mol/l; in the presence of 10 mol/l cocaine) or the ₂-adrenoceptor agonists 5-bromo-6-(2imidazolin-2-ylamino)quinoxaline (UK 14,304, 10 mol/ l) and clonidine (10 mol/l). These results suggest that the ₂-autoreceptor-mediated inhibition of noradrenaline release from chick sympathetic neurons operates through the modulation of Ca²⁺ channels via pertussistoxin-sensitive GTP-binding-proteins. Send offprint requests to S. Boehm at the above address     

Opposite modulation of noradrenaline and ATP release in guinea-pig vas deferens through prejunctional β-adrenoceptors: evidence for the β2 subtype

Activation of prejunctional -adrenoceptors has been suggested to increase the release of noradrenaline but to decrease the neural release of ATP in the guinea-pig vas deferens. Experiments were carried out to determine the subtype of -adrenoceptor involved.In [³H]-noradrenaline-preincubated tissues superfused with medium containing prazosin and suramin, isoprenaline (1–100 nM), salbutamol (0.01–1 M) and terbutaline (0.1–10 M) increased the overflow of tritium but reduced the overflow of ATP elicited by electrical stimulation (210 pulses/7 Hz). The effects of isoprenaline were blocked by the ₂-selective antagonist 1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118,551; 100 nM). In prazosin- and suramin-free medium, isoprenaline (100 nM) did not change the overflow of ATP elicited by exogenous noradrenaline (10 M). Isoprenaline (1–100 nM), salbutamol (0.01–1 M) and terbutaline (0.1–10 M) reduced the initial twitch contraction elicited by electrical stimulation (210 pulses/7 Hz) in prazosin-and suramin-free medium as well as the isolated purinergic neurogenic contraction obtained by exposure to prazosin. They increased or tended to increase the secondary sustained contraction elicited by electrical stimulation in prazosin- and suramin-free medium as well as the isolated adrenergic neurogenic contraction obtained in the presence of suramin. The inhibition by isoprenaline of the isolated purinergic contraction was attenuated by ICI 118,551 (100 nM) but not by the ₁-selective antagonist 1-[2-((3carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol (CGP 20712A; 100 nM).The results confirm the opposite -adrenoceptor-mediated modulation of noradrenaline and neural ATP release in the guinea-pig vas deferens. They show that the prejunctional -adrenoceptor is of the ₂ subtype.     

Further study of prerequisites for the enhancement by α-adrenoceptor antagonists of the release of noradrenaline

Summary Segments of the rabbit ear artery were preincubated with (–)-³H-noradrenaline and then perfused/superfused and stimulated by transmural electrical pulses. The outflow of ³H-noradrenaline and total tritium was determined.In the first series of experiments, stimulation periods of approximately constant length (50 s) were used (cocaine 5 M present). Thirteen pulses (0.25 Hz) elicited an overflow of ³H-noradrenaline of 0.024% of tissue tritium; 26 pulses (0.5 Hz) elicited an overflow of 0.059%, and 52 pulses (1 Hz) of 0.166%. Rauwolscine 1 M did not change the overflow evoked by 13 pulses, increased that evoked by 26 pulses and increased most markedly that evoked by 52 pulses. Phentolamine 1 M decreased the overflow at 13, did not change the overflow at 26, and increased the overflow at 52 pulses. Corynanthine 1 M decreased the overflow at 13, and did not change the overflow at 26 and 52 pulses. The effect of tetraethylammonium (TEA) 100 M was opposite to that of rauwolscine; it increased most markedly the overflow evoked by 13 pulses, increased less that evoked by 26 pulses, and least the overflow at 52 pulses.In the second series of experiments, the frequency of stimulation was kept constant (2 Hz). In the absence of cocaine, 10 pulses elicited an overflow of ³H-noradrenaline of 0.023% of tissue tritium; 20 pulses elicited an overflow of 0.043%, and 40 pulses of 0.089%. Phentolamine 1 M did not change the overflow evoked by 10 pulses, increased that evoked by 20 pulses, and increased most markedly that evoked by 40 pulses. TEA 100 M increased the evoked overflow at all pulse numbers. Similar results were obtained in the presence of cocaine 5 M.The results demonstrate that the enhancement by -adrenoceptor antagonists of the release of noradrenaline depends on the biophase concentration of noradrenaline. Under the present conditions, graded increases in biophase noradrenaline concentration led to graded increases in the effect of the antagonists. A second prerequisite for the release-enhancing effect appears to be a sufficient length of the pulse train. Under the present conditions, graded increases in train length up to about 20s led to graded increases in the effect of the antagonists, even though the average biophase concentration of noradrenaline did not change with the pulse train length. This pattern of effects of the -antagonists is not shared by at least one other release-enhancing drug, namely TEA.