Subclassification of presynaptic α2-adrenoceptors: α2D-autoreceptors in mouse brain

The study was devised to classify, by means of antagonist affinities, the presynaptic ₂-autoreceptors in mouse cerebral cortex in terms of _2A, _2B, _2C and _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 ₂-autoinhibition as shown by the lack of an increase by rauwolscine of the evoked overflow of tritium. The ₂-selective agonists 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) and -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 -methylnoradrenaline to the right. pK_d values of the antagonists were calculated from the shifts. The pK_d values of rauwolscine against UK 14,304 and -methylnoradrenaline were very similar (8.0 and 7.9, respectively).Comparison with antagonist affinities for prototypic native ₂ binding sites, ₂ binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature — indicates that the ₂-autoreceptors in mouse brain cortex are _2D. This is the first subtype determination of ₂-autoreceptors in the mouse. It supports the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D branch of the ₂-adrenoceptor tree.     

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.     

Subclassification of presynaptic α2-adrenoceptors: α2A-autoreceptors in rabbit atria and kidney

The study was devised to classify, by means of antagonist affinities, the presynaptic ₂-autoreceptors of rabbit atria and kidney in terms of _2A, _2B, _2C and _2D-A set of antagonists was chosen that was able to discriminate between the four subtypes. Small pieces of the left atrium and slices of the kidney cortex were preincubated with ³H-Noadrenaline and then superfused and stimulated electrically.In one series of experiments, tissue pieces were stimulated by relatively long pulse trains (1 or 2 min) leading to ₂-autoinhibition. All 11 (atria) or 10 (kidney) antagonists increased the evoked overflow of tritium. pEC_30% values (concentrations causing 30% increase) were interpolated from concentration-response curves. In a second series of experiments, tissue pieces were stimulated by brief pulse trains (0.4 s) that did not lead to ₂-autoinhibition, and concentration-inhibition curves of the ₂-selective agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) were determined. Most of the 11 (atria) or 8 (kidney) antagonists shifted the concentration-inhibition curve of UK 14,304 to the right. pK_d values of the antagonists were calculated from the shifts. pEC_30% values correlated with pK_d values, both in atria (r = 0.728) and in the kidney (r = 0.930). pEC_30% values in atria correlated with pEC_30% values in the kidney (r = 0.988) and pK_d values in atria correlated with pK_d values in kidney (r = 0.923).It is concluded that the ₂-autoreceptors in atria and the kidney are the same. Comparison with antagonist affinities for prototypic native ₂ binding sites, ₂ binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature — indicates that both autoreceptors are _2A. This conclusion is reached with either of the two independent estimates of autoreceptor affinity, pEC_30% and pK_d. The results are compatible 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 rodent and lagomorph species.     

Estimation of the biophase concentration of noradrenaline at presynaptic α2-adrenoceptors in brain slices

Summary The aim of the present study was to determine the local concentrations of noradrenaline existing at presynaptic ₂-adrenoceptors during electrical pulse train stimulation of brain slices at different frequencies. The experiments are based on the assumption that the concentration of released noradrenaline at the ₂-adrenoceptors exerting a certain autoinhibition should be equal to the concentration of exogenous noradrenaline causing the same inhibition under conditions in which any influence of the released transmitter is excluded. In order to avoid autoinhibition, hippocampus and cortex slices of the rabbit and the rat, prelabelled with [³H]noradrenaline and superfused in presence of an uptake inhibitor, were electrically stimulated using 4 pulses delivered at 100 Hz (POP stimulation). Exogenous noradrenaline diminished the overflow of tritium elicited by POP stimulation in a concentration-dependent manner. In rabbit brain tissues the EC₅₀ value and maximum inhibition of noradrenaline release were found to be approximately 6 nmol/l and more than 95%, respectively, whereas in rat tissues the corresponding values were between 20 and 30 nmol/l and approximately 90%. When electrical stimulation was performed with trains of 36 pulses delivered at 0.1, 0.3 or 3 Hz in absence or presence of an uptake inhibitor, the ₂-adrenoceptor antagonist yohimbine (1 or 10 mol/l) enhanced the evoked tritium overflow in a manner which was dependent on the frequency of stimulation and on blockade of the re-uptake mechanism. The facilitatory effects of yohimbine reflected an extent of autoinhibition which was between 53% (36 pulses/0.1 Hz, no uptake inhibitor) and 85% (36 pulses/3 Hz, uptake inhibitor present) in rabbit and between 16% (36 pulses/0.3 Hz, no uptake inhibitor) and 71% (36 pulses/3 Hz, uptake inhibitor present) in rat brain slices. Accordingly, the corresponding estimated biophase concentrations of noradrenaline were generally higher in rat than in rabbit tissues (they were between 32.5 and 74.5 or 5.1 and 51.6 nmol/l in the presence or absence of an uptake inhibitor, respectively, in the rat, and between 15 and 23.1 or 6.1 and 18.6 nmol/l in the rabbit). The observed frequency dependence of the effect of re-uptake blockade on the calculated biophase concentrations of noradrenaline would be compatible with the idea of a dependence of the effectiveness of the re-uptake mechanism on the firing rate of the neurone in being more effective at lower frequencies. Moreover, the stikingly low biophase concentrations of noradrenaline suggest that also in brain tissue noradrenaline causes lateral inhibition of release as has recently been shown for guinea-pig vas deferens. Send offprint requests to C. Allgaier at the above address     

Subtype classification of presynaptic α2-adrenoceptors

• 1.1. Presynaptic α₂-adrenoceptors in a few tissues have recently been pharmacologically classified in functional studies.
• 2.2. Autoreceptors are of α_2A-subtype in rabbit occipito-perietal cortex, rat cerebral cortex, vas deferens, submandibular gland, kidney, guinea-pig ileum submucosal arterioles and urethra.
• 3.3. Heteroreceptors are of α_2A-subtype in rat cerebral cortex, vas deferens, guinea-pig ileum submucosal plexus and Auerbach's plexus.
• 4.4. In rat atria autoreceptors have been shown to be of α_2B-subtype.
• 5.5. Classification is done mainly with the α_2A-adrenoceptor-selective oxymetazoline, WB 4101 and BRL 44408, and the α_2B-adrenoceptor-selective prazosin, AR-C 239, chlorpromazine and BRL 41992.
• 6.6. With four α₂-adrenoceptor subtypes to consider, a larger number of subtype-selective compounds may have to be characterized in the classification in the many tissues, where presynaptic α₂-adrenoceptors are found.

     

Phentolamine and hypoxia: modulation of contractility and α1-adrenoceptors in isolated rat atria

The hypoxia-induced effects on the binding sites and affinity constant of adrenoceptors, in the presence and absence of phentolamine, were determined for atrial membranes of hearts from normal and genetically hyperlipidaemic Yoshida (YOS) rats. Atrial function was also measured during normoxia and hypoxia, in the presence and absence of phentolamine.Hypoxia increased a1-adrenoceptor density in atrial membranes of normal rats (B_max 10.6 to 26.7 fmoles/mg protein). Phentolamine prevented the increase in the B_max of ₁-adrenoceptors and increased the equilibrium dissociation constant of these receptors (K _D 0.17 to 0.53 nmol/l). Beta-adrenoceptors did not change during hypoxia, but the B_max was slightly increased (26%) in the presence of phentolamine. Thus, the ₁/\ ratio increased from 0.40 in normoxia to 1.06 in hypoxia. In normoxic atria from YOS rats, the ₁/\ ratio was already elevated (0.86) in comparison to control rats (mainly due to a higher density of at-adrenoceptors in atrial membranes from YOS rats). This ratio was not modified by hypoxia (0.84), but decreased when phentolamine was present (0.30).Hypoxia reduced the force of contraction and increased diastolic tension of atria of normal rats, while the sinus rate was not significantly modified. Phentolamine abolished the increase in diastolic tension and reduced the negative effect of hypoxia on contractile force. In YOS rat atria, functional parameters were modified by hypoxia in a qualitatively similar way to that of normal rat atria.The observed increase in ₁-adrenoceptor density during hypoxia is in accordance with the results of experiments with animal models of the ischaemic heart and with findings in human heart failure. The possible therapeutic significance of these data is considered. Correspondence to: G. Fassina at the above address     

Modulation of N-methyl-d-aspartate (NMDA)-stimulated noradrenaline release in rat brain cortex by presynaptic α2-adrenoceptors

Summary Rat brain cortex slices and synaptosomes preincubated with [³H]noradrenaline were used to investigate whether the NMDA-evoked noradrenaline release is modulated by agonists or antagonists at presynaptic ₂-adrenoceptors.In experiments on slices, noradrenaline and the preferential ₂-adrenoceptor agonists talipexole (former B-HT 920) and clonidine inhibited the NMDA evoked tritium overflow whereas the selective ₁-adrenoceptor agonists cirazoline and methoxamine were ineffective. The ₂-adrenoceptor antagonists rauwolscine and idazoxan facilitated the NMDA-evoked tritium overflow whereas the preferential ₁-adrenoceptor antagonist prazosin was ineffective. The concentration-response curve of talipexole for its inhibitory effect on NMDA-evoked overflow was shifted to the right by idazoxan (apparent pA₂ = 7.5). The EC₅₀ of NMDA (97 mol/l) for its stimulating effect on tritium overflow was not substantially changed by blockade of ₂-autoreceptors with 1 mol/l rauwolscine (EC₅₀ of NMDA in the presence of the ₂-adrenoceptor antagonist, 155 mol/l), but the maximal overflow of tritium was increased 2.5 fold by this rauwolscine concentration. In experiments on synaptosomes, talipexole and noradrenaline inhibited the NMDA-evoked tritium overflow. The inhibitory effect of talipexole was abolished by idazoxan which, given alone, was ineffective, as was prazosin. Talipexole did also not produce an inhibition when tritium overflow was evoked by NMDA in the presence of -conotoxin GVIA 0.1 mol/l; the latter, by itself, decreased the response to NMDA by about 55%. It is concluded that the NMDA-evoked noradrenaline release in the cerebral cortex is modulated via presynaptic ₂-adrenoceptors on the noradrenergic neurones. Stimulation of these autoreceptors in slices by endogenous noradrenaline does not result in a decreased potency of NMDA, but in a decreased maximum effect, in stimulating noradrenaline release. The inhibitory effect of ₂-adrenoceptor agonists on the NMDA-evoked release is at least partially due to a functional interaction between the NMDA receptors and ₂-autoreceptors at the level of the same varicosities. The results obtained with -conotoxin GVIA suggest that Ca²⁺ influx via the N-type voltage-sensitive calcium channel (VSCC) occurs in response to NMDA receptor stimulation and contributes substantially to the induction of NMDA-evoked noradrenaline release. The inhibitory effect of ₂-adrenoceptor stimulation on this release appears to be ultimately due to an inhibition of the influx of Ca²⁺ via the N-type VSCC. Correspondence to: M. Göthert at the above address     

Inhibition of a peripheral sympathetic-cholinergic system by presynaptic α2-adrenoceptors

Summary Intravenous administration of catecholamines produced dose-related inhibition of electrodermal responses (EDRs) evoked by electrical stimulation of the post-ganglionic sciatic nerve in anaesthetized rats, with relative potencies being (–)-adrenaline > (±)-adrenaline > (–)-noradrenaline = (+)-adrenaline. The suppression of EDRs by (±)-adrenaline and (–)-noradrenaline was blocked by pretreatment with yohimbine (0.75 mg/kg i.v.) but not by prazosin (0.3 mg/kg i. v.). The selective ₂-adrenoceptor agonist B-HT 920 also inhibited neurally evoked skin potential responses. This effect of B-HT 920 was antagonized by the selective ₂-adrenoceptor antagonist idazoxan (0.1 mg/kg i. v.) but was insensitive to prazosin. Idazoxan was more potent than yohimbine in blocking (±)-adrenaline-induced suppression of EDRs. Methacholine administered into the femoral artery evoked EDRs by an atropine-sensitive mechanism. Methacholine-induced EDRs were not suppressed by intravenous administration of (–)-adrenaline (1 g/kg or 3 g/ kg) whereas EDRs evoked by the sciatic nerve stimulation on the other hindpaw were inhibited. Increase in the endogenous catecholamines by asphyxia strongly inhibited EDRs by a mechanism which was also sensitive to yohimbine but not to prazosin. These results suggest that peripheral presynaptic ₂-adrenergic mechanisms are involved in inhibition of transmitter release in this sympatheticcholinergic system. Send offprint requests to M. C. Koss     

Autoreceptors and α2-adrenoceptors at the serotonergic axons of rabbit brain cortex

Summary Slices of the rabbit occipito-parietal cortex were preincubated with ³H-serotonin and then superfused and stimulated electrically (2 min at 3 Hz). In the absence of drugs, the stimulation-evoked overflow of tritium was approximately 3% of the tritium content of the tissue. Unlabelled serotonin and 5-carboxamido-tryptamine, when administered in the presence of 6-nitroquipazine, reduced the evoked overflow of tritium. Their effects were antagonized by metitepin (apparent pA₂ value 8.1) and (±)-cyanopindolol (apparent pA₂ value 6.4). Metitepin, but not cyanopindolol, increased evoked tritium overflow; the effect of metitepin was greater in the presence than in the absence of nitroquipazine. The evoked overflow of tritium was also depressed by clonidine, an effect antagonized by idazoxan (apparent pA₂ value 7.0) but not by prazosin. Phenylephrine caused a decrease only at high concentrations that simultaneously accelerated basal tritium efflux. Prazosin and idazoxan did not change evoked tritium overflow, and phentolamine increased it significantly only when administered in the presence of (+)-oxaprotiline. Rauwolscine produced an inhibition that was prevented by metitepin. It is concluded that the serotonergic axons of the rabbit occipitoparietal cortex possess presynaptic, release-inhibiting serotonin autoreceptors and ₂-adrenoceptors. The receptors appear to receive an input of endogenous serotonin and, to a lesser extent, noradrenaline, under the conditions of these in vitro experiments.     

Estimation ofpA2 values at presynaptic α2-autoreceptors in rabbit and rat brain cortex in the absence of autoinhibition

Summary An attempt was made to determinepA₂ values of antagonists at the presynaptic, release-inhibiting ₂-autoreceptorsof rabbit and rat brain cortex under conditions when there was very little released noradrenaline in the autoreceptor biophase and, hence,pA₂ values were not distorted by endogenous autoinhibition. Cortex slices were preincubated with³H-noradrenaline and then superfused and stimulated by trains of 4 pulses delivered at 100 Hz or, in a few cases, by trains of 36 pulses at 3 Hz. The -adrenoceptor agonists clonidine, noradrenaline, and -methylnoradren-aline concentration-dependently decreased the stimulation-evoked overflow of tritium. The a-adrenoceptor antagonists yohimbine, rauwolscine and idazoxan did not increase the overflow of tritium elicited by 4 pulses/100 Hz in rabbit brain slices and increased it only slightly in rat brain slices. In contrast, the antagonists increased markedly the overflow at 36 pulses/3 Hz. All antagonists caused parallel shifts to the right of the concentration-response curves of clonidine, noradrenaline, and -methylnoradrenaline.pA₂ values were calculated either from linear regression of log [agonist concentration ratio – 1] on log [antagonist concentration] or from sigmoid curve fitting. The slopes of the linear regression lines were close to unity, and thepA₂ values calculated by the two methods agreed well. There was no consistent preferential antagonism of any antagonist to any agonist.pA₂ values determined with stimulation by 4 pulses/100 Hz were by 0.53–0.80 log units higher than those determined with stimulation by 36 pulses/3 Hz. ThepA₂ values (4 pulses/100 Hz) of yohimbine and rauwolscine in rabbit brain slices (approximately 7.9 and 8.2, respectively), were slightly higher than in rat brain slices (approximately 7.6 and 7.7, respectively), whereas thepA₂ value of idazoxan in the rabbit. (about 7.1) was lower than itspA₂ value in the rat (about 8.0). The experiments confirm thatpA₂ values determined under conditions of autoinhibition are too low. Stimulation with short (30 ms) bursts of pulses permits the estimation ofpA₂ values at presynaptic a2-autoreceptors without (rabbit) or almost without (rat) the complication of autoinhibition. The values suggest that ₂-adrenoceptors in rabbit brain cortex differ slightly from those in rat brain cortex. Send offprint requests to E. A. Singer at the above address     

Modulation of 5-hydroxytryptamine release by presynaptic inhibitory α2-adrenoceptors in the human cerebral cortex

Summary Slices and synaptosomes from human cerebral cortex (which had to be removed to reach deeply located tumours) and, for comparison, synaptosomes from guinea-pig and rat cerebral cortex were preincubated with [³H]5-hydroxytryptamine and superfused with physiological salt solution containing an inhibitor of 5-hydroxytryptamine uptake. The effects of -adrenoceptor agaonists and antagonists on the electrically (slices) or potassium-evoked (synaptosomes) tritium overflow were studied.In human cerebral cortical slices, the electrically-evoked [³H] overflow was inhibited by noradrenaline (pIC₂₅ value: 6.35); the non-selective -adrenoceptor antagonist phentolamine, at a concentration of 0.32 mol/l, strongly antagonized the inhibitory effect of noradrenaline (apparent pA₂ value: 8.19) but did not affect the evoked overflow by itself. In synaptosomes from humans, guinea-pigs and rats, noradrenaline also inhibited the K⁺-evoked[³H] overflow in a concentration dependent manner; the ₂-adrenoceptor clonidine (1 mol/l), but not the ₁-adrenoceptor agonist methoxamine (1 mol/l), mimicked the effects of noradrenaline; the effect of noradrenaline (0.3 mol/l) was abolished by the ₂-but not by the ₁-adrenoceptor antagonist prazosin (1 mol/l).It is concluded that release-inhibiting adrenoceptors of the ₂-subtype exist on 5-hydrpxytryptamine terminals innervating the cerebral cortex in human and guinea-pig brain.Send offprint requests to M. Raiteri at the above address     

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.     

Blockade of presynaptic α-receptors and of amine uptake in the rat brain by the antidepressant mianserine

Summary Mianserine (Org GB 94, Tolvon^®) is 1, 2, 3, 4, 10, 14b-hexahydro-2-methyl-dibenzo [c, f] pyrazino [1, 3-a] azepine hydrochloride, a new antidepressant drug. Its effect on noradrenaline release and its capacity to inhibit amine uptake were investigated. Mianserine increased the release of ³H-noradrenaline from field-stimulated cortical slices previously labelled with the tritiated transmitter. The assumption that this effect is due primarily to the blockade of the presynaptic noradrenergic -receptors is supported by the fact that mianserine failed to augment ³H-noradrenaline release further after blockade of the presynaptic -receptors by phentolamine. In the reciprocal experiment, phentolamine failed to augment ³H-noradrenaline release after exposure of the slices to mianserine. The hypothesis is further reinforced by the fact that mianserine antagonized the reduction of ³H-noradrenaline release by clonidine in the same manner as the -blocking drugs phentolamine and phenoxybenzamine. Mianserine inhibited noradrenaline uptake in vitro and in vivo (in the rat heart and midbrain-diencephalon synaptosomes from pretreated rats.) Only a marginal inhibition of serotonin uptake was observed.It therefore appears that mianserine increases the concentration of noradrenaline in the synaptic cleft by blocking the presynaptic -receptors and inhibiting uptake. Whether or not this increase has functional consequences at postsynaptic noradrenergic receptor sites is unknown. It is possible, however, that postsynaptic receptor blockade counteracts the increase in available noradrenaline.A part of these results was presented at the 16th Spring Meeting of the German Pharmacological Society, Mainz, March 4–7, 1975     

Regulation of ß-adrenoceptors in the guinea-pig sinoatrial node

This study examined the changes of ß-adrenoceptors in the guinea-pig sinoatrial nodal region following 7 day (–)-isoprenaline (400 g/kg/h s.c.) infusion and the relationship between ß-adrenoceptor desensitization and receptor down-regulation. Changes in ß₁-and ß₂-adrenoceptor density were measured using quantitative autoradiography and function in organ bath studies.(–)-Isoprenaline treatment produced a marked decrease in total (from 57.5 to 33.9 fmol/mg protein), ß₁-(from 49.4 to 32.8 fmol/mg protein), and ß₂-adrenoceptor density (from 8.1 to 1.05 fmol/mg protein) in the sinoatrial node. In adjacent right atrium, treatment produced no change in total (39.5 and 36.7 fmol/mg protein) or ß₁-adrenoceptors (35.9 and 36.4 fmol/mg protein) but did decrease ß₂-adrenoceptors (from 3.7 to 0.3 fmol/mg protein). Chronotropic effects were measured in spontaneously beating right atrium. Procaterol, a selective ß₂-adrenoceptor agonist, caused a biphasic chronotropic response in control right atria, the first part of which was abolished in the tissue from treated animals. The maximum increase in right atrial rate to R0363, a ß₁-adrenoceptor selective partial agonist, was reduced from 114 bpm in control to 43 bpm in treated animals. In electrically driven right atrium with the sinoatrial node removed procaterol failed to produce a positive inotropic response via ß₂-adrenoceptors, but the maximum response to RO 363 was reduced from 0.75 g in the control tissue to 0.12 g in the treated tissue.This study showed that changes in ß₂-adrenoceptor density following 7 day (–)-isoprenaline infusion are compatible with reduced functional responsiveness in the SA node. The reduction of al-adrenoceptor number in the SA node was also compatible with the reduced chronotropic response in this tissue. However the lack of effect on ß₁-adrenoceptor density in the right atrium was not consistent with the decrease in Qt-adrenoceptor mediated inotropic response in this tissue. This suggests that ß-adrenoceptor desensitization is not always associated with receptor down-regulation but depends also on the changes in the cell signalling system beyond the level of the receptor which differ according to the cardiac location.This work was supported by grants from the National Health and Medical Research Council and the National Heart Foundation of Australia Correspondence to: R. J. Summers at the above address     

Supersensitivity of 5-HT1A-autoreceptors and α2-adrenoceptors regulating monoamine synthesis in the brain of morphine-dependent rats

The sensitivity of 5-HT1A serotonin receptors and alpha2-adrenoceptors (autoreceptors and heteroreceptors) modulating brain monoamine synthesis was investigated in rats during morphine treatment and after naloxone-precipitated withdrawal. The accumulation of 5-hydroxytryptophan (5-HTP) and 3,4-dihydroxyphenylalanine (DOPA) after decarboxylase inhibition was used as a measure of the rate of tryptophan and tyrosine hydroxylation in vivo. Acute morphine (3-100 mg/kg, 1 h) increased the synthesis of 5-HTP/5-HT in various brain regions (15%-35%) and that of DOPA/dopamine (DA) in striatum (28%-63%), but decreased the synthesis of DOPA/noradrenaline (NA) in hippocampus and cortex (20%-33%). Naloxone (2-60 mg/kg, 1 h) did not alter the synthesis of 5-HTP or DOPA in brain. Tolerance to the inhibitory effect of morphine on DOPA/NA synthesis and a sensitization to its stimulatory effects on DOPA/DA and 5-HTP/5-HT synthesis were observed after chronic morphine and/or in morphine-withdrawn rats. In morphine-dependent rats (tolerant and withdrawn states) the inhibitory effects of the 5-HT1A agonists 8-OH-DPAT and buspirone (0.1 mg/kg, 1 h), and that of the alpha2-adrenoceptor agonist clonidine (0.1 mg/kg, 1 h), on the synthesis of 5-HTP/5-HT were potentiated (25%-50%). Moreover, the effect of 8-OH-DPAT was antagonized by WAY 100135, a selective 5-HT1A antagonist. In morphine-dependent rats (tolerant state), the inhibitory effects of clonidine on the synthesis of DOPA/NA (hippocampus, hypothalamus) and DOPA/DA (striatum) also were potentiated (35%-55%). In summary, we conclude that morphine addiction is associated with supersensitivity of 5-HT1A serotonin receptors and alpha2-adrenoceptors (autoreceptors and heteroreceptors) that modulate the synthesis of monoamines in brain.     

Release-inhibiting α2-adrenoceptors at serotonergic axons in rat and rabbit brain cortex: evidence for pharmacological identity with α2-autoreceptors

The pharmacological properties of the presynaptic ₂-adrenoceptors modulating the release of serotonin in rat and rabbit brain cortex (₂-heteroreceptors) were compared with the properties of presynaptic ₂-autoreceptors in the same brain area. Brain cortex slices were preincubated with [³H]-serotonin or [³H]-noradrenaline and then superfused and stimulated by brief high-frequency pulse trains.The ₂-adrenoceptor agonist bromoxidine reduced the electrically evoked overflow of tritium in experiments with both [³H]-noradrenaline and [³H]-serotonin and in brain slices from either species. The antagonists phentolamine, idazoxan, (+)-mianserin, rauwolscine, 5-chloro-4(1-butyl-1,2,5,6-tetrahydropyridin-3-yl)-thiazole-2-amine (ORG 20350), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), (–)-mianserin and corynanthine caused parallel shifts of the concentration-inhibition curves of bromoxidine to the right. Negative logarithms of antagonist dissociation constants pK_d were calculated from the shifts. In the rat, the ₂-autoreceptor pK_d value of each single antagonist was similar to its ₂-heteroreceptor pK_d value, maximal difference 0.4, giving a close correlation, r = 0.97 (P<0.001). In the rabbit equally, the ₂-autoreceptor pK_d value of each single antagonist was similar to its ₂-heteroreceptor pK_d value, maximal difference 0.4, again yielding a close correlation, r = 0.96 (P < 0.001). However, antagonist pK_d values at rat ₂-autoreceptors differed from those at rabbit ₂-autoreceptors, r = 0.70 (P > 0.05), and antagonist pK_d values at rat ₂-heteroreceptors differed from those at rabbit ₂-heteroreceptors, r = 0.64 (P > 0.05). Comparison with radioligand binding experiments from the literature indicated that, in the rat, both auto- and heteroreceptors conformed best to the _2D subtype (r 0.97, P < 0.01 for pK_d correlation with binding sites in rat submaxillary gland) whereas, in the rabbit, they conformed best to the _2A subtype (r 0.93, P < 0.01 for pK_d correlation with binding sites in HT29 cells).It is concluded that, in both the rat and the rabbit, the ₂-adrenoceptors modulating the release of serotonin are pharmacologically identical with the presynaptic ₂-autoreceptors. However, rat ₂-autoreceptors and -heteroreceptors differ pharmacologically from rabbit ₂-autoreceptors and -heteroreceptors. Presynaptic ₂-auto-as well as -heteroreceptors are _2D in the rat and _2A in the rabbit. Correspondence to: N. Limberger at the above address     

Occurrence, pharmacology and function of presynaptic α2-autoreceptors in α2 A/D-adrenoceptor-deficient mice

The occurrence, pharmacological properties and function of alpha2-autoreceptors were studied in hippocampal slices, occipito-parietal cortex slices, segments of heart atria and segments of the vas deferens of wildtype (WT) mice and mice in which the alpha2 A/D-adrenoceptor gene had been disrupted (alpha2 A/D(KO)). Tissues were preincubated with [3H]-noradrenaline and then superfused and stimulated electrically. Stimulation periods for brain slices consisted either of 1 pulse or of 2-64 pulses delivered at 1-s intervals; stimulation periods for peripheral tissues consisted either of 1 POP (pseudo-one-pulse; brief burst of 20 pulses/50 Hz) or of 2-4 POPs delivered at 1-s intervals. Single pulses or POPs were used to study the effect of medetomidine and its interaction with antagonists. One or more pulses or POPs per stimulation period were used to study alpha2-autoinhibition. Medetomidine decreased the evoked overflow of tritium in WT tissues. In alpha2 A/D(KO) tissues, the inhibition was slightly (peripheral tissues) or greatly (brain slices) attenuated but not abolished. Phentolamine, rauwolscine, spiroxatrine, 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), tolazoline and prazosin antagonized the effect of medetomidine in all tissues. Their pKd values against medetomidine were compared with pKd values at prototypical alpha2 binding sites by means of a correlation analysis. For WT brain and atrial autoreceptors, the correlations indicated an alpha2D pharmacology, whereas for WT vas deferens autoreceptors they favoured an alpha2B pharmacology. In the KO tissues, any correlation with alpha2D was lost, and the non-alpha2 A/D-autoreceptors displayed alpha2B or alpha2C pharmacology. When 2 or more pulses or POPs were applied to WT tissues per stimulation period, the pulse number-overflow curve (POP number-overflow curve) was flat, indicating that overflow elicited by p pulses (POPs) was much smaller than p times the overflow elicited by a single pulse (POP); moreover, rauwolscine caused a pulse (POP) number-dependent and, at high pulse (POP) numbers, large increase in evoked tritium overflow. In alpha2 A/D(KO) tissues, the pulse (POP) number-overflow curve was much steeper, indicating that overflow elicited by p pulses (POPs) was closer to p times the overflow elicited by a single pulse (POP); moreover, rauwolscine caused no (atria) or only a small increase in overflow, and did so in brain slices only at high pulse numbers (16 and 64). In conclusion, the predominant alpha2D pharmacology of the autoreceptors in WT tissues supports the idea that the main mammalian presynaptic alpha2-autoreceptors belong to the alpha2 A/D subtype. However, alpha2 A/D-deficient animals also possess autoreceptors. As expected, these non-alpha2 A/D-autoreceptors display alpha2B or alpha2C pharmacology. In WT animals, alpha2B- or alpha2C-autoreceptors or both may coexist with alpha2 A/D-autoreceptors, at least in peripheral tissues. Little autoinhibition by released noradrenaline in trains of pulses remains when the alpha2 A/D-adrenoceptor is lacking, again in accord with a predominance of alpha2 A/D-autoreceptors.     

Electrically evoked noradrenaline release from cultured chick sympathetic neurons: modulation via presynaptic α2-adrenoceptors and lack of autoinhibition

Summary Sympathetic neurons from twelve day old chick embryos were plated on polystyrol discs and kept in culture for five days. After incubation with ³H-noradrenaline the discs were transferred to small chambers and superfused. Electrical field stimulation (36 pulses at 3 Hz) increased the outflow of tritium. The evoked overflow of tritium was abolished in the absence of extracellular calcium and was diminished by about 90% in the presence of tetrodotoxin (1 mol/l). The ₂-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK-14,304) caused a concentration-dependent decrease in overflow, whereas the ₁-adrenoceptor agonist methoxamine was ineffective at up to 1 mol/l. The concentration-response curve of UK-14,304 was shifted to the right by the ₂-adrenoceptor antagonist yohimbine (0.03 mol/l). Yohimbine on its own caused no significant change. The noradrenaline reuptake inhibitor cocaine (10 mol/l) caused a small (20%) increase in evoked overflow. The results indicate that cultured chick sympathetic neurons possess release-modulating ₂-adrenoceptors and that the electrically induced overflow of transmitter occurs under conditions virtually free of autoinhibition. Send offprint requests to E.A. Singer at the above address     

Facilitation of serotonin (5-HT) release in the rat brain cortex by cAMP and probable inhibition of adenylate cyclase in 5-HT nerve terminals by presynaptic α2-adrenoceptors

Summary Stimulation-evoked tritium overflow was examined in superfused rat brain cortex slices (stimulus: electrical impulses; 3 Hz) and synaptosomes (stimulus: potassium 12 mmol/l) preincubated with ³H-5-HT. 1. In slices and synaptosomes, the evoked ³H overflow was facilitated by forskolin and 8-Br-cAMP, but was not affected by AH 21-132 (an inhibitor of cAMP phosphodiesterase; cis-6-(p-acetamidophenyl)-1, 2, 3, 4, 4 a,10b-hexahydro-8, 9-dimethoxy-2-methylbenzo [c] [1,6]-naphthyridine). In the presence of AH 21-132, the facilitatory effect of forskolin on evoked overflow was increased. 2. In slices, AH 21-132 or combined administration of forskolin plus AH 21-132 did not change the percentage of basal or evoked ³H overflow represented by unmetabolized ³H-serotonin (about 30% and 60%, respectively). 3. In slices, cocaine or 6-nitroquipazine, an inhibitor of serotonin uptake, did not influence the increase in evoked overflow produced by forskolin plus AH 21-132. Forskolin plus AH 21-132 did not alter the inhibitory effect of serotonin (examined in the presence of 6-nitroquipazine) and the facilitatory effect of metitepin (a serotonin receptor antagonist) on evoked ³H overflow, but considerably decreased the inhibitory effect of clonidine or B-HT 920 (2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo-[5,4-d]-azepine). The present results suggest that the serotoninergic nerve terminals in the rat brain cortex are endowed with an adenylate cyclase, which is negatively coupled to the presynaptic ₂-adrenoceptors, but is not linked to the presynaptic autoreceptors.This study was supported by a grant of the Deutsche Forschungsgemeinschaft. Part of the present results was reported at the 9th International Congress of Pharmacology, London 1984 (Schlicker et al. 1984) Send offprint requests to M. Göthert     

Benzamide action at α2-adrenoceptors modifies catecholamine-induced contraction and relaxation of circular smooth muscle from guinea-pig stomach

Summary Dopamine was shown to act on the circular smooth muscle of the stomach body to cause contraction at a yohimbine-sensitive site (₂) and a relaxation at a prazosin-sensitive site (₁). Metoclopramide and tiapride failed to modify either response, failed to antagonise a relaxation to phenylephrine at 1(₁ sites in the same tissue, and failed to modify the contractions caused by dopamine and phenylephrine at an ₂-adrenoceptor site in the pyloric sphincter. However, (+)- and (–)-sultopride and (+)-sulpiride antagonised the dopamine-induced contractions of the stomach body indicating an ₂-antagonist action. An ability to attenuate the relaxation of this tissue may reflect a displacement of the contraction curve to the right rather than an ₂-antagonist action since the response to phenylephrine was not antagonised either in this tissue or in the pyloric sphincter. Within the central nervous system the (–)-enantiomers of sultopride and sulpiride have a highly selective dopamine receptor blocking action. This cotrasts with the present findings in the stomach musculature of a non-stereospecific antagonism at ₂-type adrenoceptors.