Subclassification of presynaptic α2-adrenoceptors: α2D-autoreceptors in guinea-pig atria and brain

The study was devised to classify, by means of antagonist and agonist affinities, the presynaptic ₂-autoreceptors in guinea-pig heart atria and brain cortex in terms of _2A, _2B, _2C and _2D. A set of antagonists and agonists was chosen that was able to discriminate between the four subtypes. Small pieces of the atria and slices of the brain cortex were preincubated with ³H-noradrenaline and then superfused and stimulated electrically.In one series of experiments (atria only), tissue pieces were stimulated by relatively long pulse trains (1 min) leading to marked ₂-autoinhibition. All 10 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 (atria and brain slices), tissue pieces were stimulated by brief pulse trains (0.4 s or 40 ms) that led to little (atria) or no (brain slices) ₂-autoinhibition, and antagonist effects against the ₂-selective agonist 5-bromo-6(2-imidazolin-2-ylamino)-quinoxaline UK 14,304 were examined. All 10 (atria) or 8 (brain) antagonists shifted the concentration-inhibition curve of UK 14,304 to the right. pK_d values of the antagonists were calculated from the shifts. In a third series of experiments (brain slices only), also with brief pulse trains (40 ms), pK_a values (negative logarithms of dissociation constants of agonist-₂-adrenoceptor complexes) were determined by comparison of concentration-inhibition curves of UK 14,304, guanoxabenz and oxymetazoline in normal tissue and in tissue in which a fraction of the receptors had been blocked by phenoxybenzamine. pEC_30% values in atria correlated with pK_d values (r = 0.942). pK_d values in atria correlated with pK_d values in the brain cortex (r = 0.970).It is concluded that the ₂-autoreceptors in atria and the brain cortex are the same. 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 autoreceptors are _2D. In atria, this identification is reached with either of the two independent estimates of autoreceptor affinity, pEC_30% and pK_d, and in the brain cortex it is supported by the agonist pK_a values. The results are compatible with the hypothesis that at least the majority of ₂-autoreceptors belong to the _2AD branch of the ₂-adrenoceptor tree.     

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: 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.     

Increase by α-adrenolytic drugs of acetylcholine release evoked by field stimulation of the guinea-pig ileum

Summary The release of acetylcholine evoked by field stimulation of the guinea-pig ileum (3 Hz) is increased by yohimbine and tolazoline but not affected by phentolamine. It is proposed that yohimbine and tolazoline by blocking -adrenoceptors of the cholinergic nerves abolish the inhibition caused by endogenous noradrenaline, and thus facilitate the output of acetylcholine.     

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.

     

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     

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     

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     

A kinetic study of the release of noradrenaline by electrical stimulation: influence of presynaptic α-adrenoceptors

Summary Dog saphenous vein strips were incubated with 1.4 mol/l ³H-(-)-noradrenaline for 60 min, after inhibition of the noradrenaline-metabolizing enzymes and of extraneuronal uptake. At the end of the incubation period the strips were perifused for 150 min; cocaine (10 mol/l) was added to the perifusion fluid from t=75 min onwards. In some experiments either phentolamine (10 mol/l) or clonidine (0.1 mol/l) was also added at this time. Some strips were subjected to electrical stimulation from t=100 to 150 min of perifusion (t=0 being the start of perifusion), with frequencies ranging from 0.5 to 13.5 Hz. A compartmental analysis of spontaneous or electrically-induced efflux of ³H-noradrenaline was made. The spontaneous efflux had a long half time (t/2=124 min) and most of the ³H-noradrenaline which had accumulated in the strips did not participate in the efflux (bound fraction, representing 90% of tissue activity at t=100 min of perifusion). Neither phentolamine nor clonidine modified the half time or the bound fraction observed for spontaneous efflux. Electrical stimulation (>0.5 Hz) mobilized only one compartment of noradrenaline, which represented about 50% of the noradrenaline accumulated in the strips. The half time of ³H-efflux induced by electrical stimulation decreased when the frequency increased from 0.5 Hz up to 13.5 Hz. Phentolamine increased the rate of efflux for all frequencies of stimulation and decreased the half time of efflux. However, the releasable pool of noradrenaline was only increased by phentolamine at 0.5 Hz, but not at higher frequencies. Clonidine was used only at two frequencies of stimulation, 1.5 and 4.5 Hz. For the low frequency clonidine decreased the releasable pool, but no change was observed at 4.5 Hz.The results support the view that there is a norarenaline pool which is resistant to electrical stimulation and that its magnitude is not dependent on the activity of presynaptic -adrenoceptors.Results presented in part to the 13th Annual Meeting of the Portuguese Pharmacological Society (Porto, December 1982) and to the 5th Meeting on Adrenergic Mechanisms (Porto, October 1983)Work supported by a grant from Instituto Nacional de Investigação Científica (FmPl)     

Binding of 3H-clonidine to an α-adrenoceptor in membranes of guinea-pig ileum

Summary The binding of ³H-clonidine to membrane particles from guinea-pig ileum was investigated. The specific binding, i.e. the binding that could be inhibited by high concentrations of unlabeled clonidine or noradrenaline, was of high affinity, K _D3 nM. The number of sites was approximately 25 fmol/mg protein. Rate constants of association and dissociation were 5.3×10⁷ M^–1 min^–1 and 0.18 min^–1, respectively. Affinites of various drugs to the binding site were determined by measuring their effect on the binding of ³H-clonidine. The affinity of adrenergic agonists decreased in the order clonidine = tramazoline > (–)-erythro--methylnoradrenaline > (–)-noradrenaline (–)-phenylephrine. (–)-Noradrenaline had about 20 times more affinity than the (+)-isomer. The affinity of -adrenoceptor antagonists decreased in the order phentolamine > rauwolscine = yohimbine > WB 4101 > pseudoyohimbine > prazosin = corynanthine. Yohimbine and rauwolscine had about 100 times more affinity than their stereoisomer corynanthine. Serotonin 10 M and metiamide 10 M did not affect the binding, and propranolol inhibited it only at high concentrations. — The results indicate that ³H-clonidine labels an ₂-adrenoceptor in guinea-pig ileum. The orders of affinity of -adrenoceptor agonists and antagonists agree well with their orders of potency in functional tests, namely as modulators of cholinergic transmission in the guinea-pig ileum and as modulators of noradrenaline release in the rabbit pulmonary artery. An -adrenoceptor should be classified as ₂ when the affinities of clonidine, tramazoline and -methylnoradrenaline greatly exceed the affinity of phenylephrine, and when the affinities of rauwolscine and yohimbine exceed those of prazosin and corynanthine.     

Neural ATP release and its α2-adrenoceptor-mediated modulation in guinea-pig vas deferens

Summary Contractions, release of previously stored [³H]-noradrenaline (measured as overflow of total tritiated compounds) and release of ATP elicited by electrical field stimulation (210 pulses, 7 Hz) were studied in the superfused vas deferens of the guinea pig. Prazosin and suramin were used to suppress non-neural ATP release, and effects of bromoxidine and rauwolscine on the neural release thus isolated were examined.Electrical stimulation elicited reproducible contraction, tritium overflow and ATP overflow. Both prazosin (0.03–3 M) and suramin (30–300 M) reduced contractions as well as the evoked overflow of ATP. No visible contraction remained in 21 of 28 tissues exposed to prazosin 0.3 M combined with suramin 300 M. The evoked overflow of ATP under these conditions was about 17% of that observed in the absence of drugs. In the presence of prazosin 0.3 M and suramin 300 M, bromoxidine (0.01–1 M) decreased and rauwolscine (0.1–10 M) increased the evoked overflow of both tritium and ATP. Rauwolscine increased the evoked overflow of tritium to a significantly greater extent than the overflow of ATP.It is concluded that the overflow of ATP elicited by electrical (neural) stimulation in the presence of prazosin 0.3 M and suramin 300 M reflects purely neural release of ATP. This release of ATP, like the release of noradrenaline, is modulated through prejunctional ₂-adrenoceptors. The ₂-adrenoceptor modulation of the release of noradrenaline seems to be more marked than the modulation of the release of ATP. Correspondence to B. Driessen at the above address     

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.     

Effects of some α-adrenoceptor agonists and antagonists on the guinea-pig ileum

Summary The purpose of the present study was to further characterize the -adrenoceptors located on parasympathetic fibres. Segments of guinea-pig ileum were stimulated by transmural electrical pulses, and the ensuing contractions, which are due to the release of acetylcholine from postganglionic parasympathetic fibres, were monitored. Clonidine and tramazoline, which are thought to act preferentially on presynaptic -adrenoceptors, reduced the contractions, whereas phenylephrine and methoxamine, postsynaptic -adrenoceptor agonists, were ineffective. Contractions induced by acetylcholine were not changed by clonidine but were abolished by atropine. Yohimbine, piperoxan, phentolamine and thymoxamine reversed or prevented the inhibitory effect of clonidine. Prazosin and AR-C239 did not antagonize this effect. The inhibitory effect of tramazoline was antagonized by piperoxan but not by AR-C239 or by prazosin. Naloxone did not alter the action of clonidine, and piperoxan did not change the inhibitory effect of morphine.In conclusion, these experiments suggest the presence on cholinergic postganglionic fibres of both opiate receptors and -adrenoceptors. The latter appear to resemble more closely ₁-adrenoceptors than ₁-adrenoceptors.     

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     

κ1- and κ2-opioid receptors mediating presynaptic inhibition of dopamine and acetylcholine release in rat neostriatum

1. The effects of selective opioid receptor agonists and antagonists on N-methyl-D-aspartate (NMDA, 10 μM)-induced release of [³H]-dopamine and [¹⁴C]-acetylcholine (ACh) from superfused neostriatal slices were studied to investigate the possible occurrence of functional κ-opioid receptor subtypes in rat brain.
2. The κ receptor agonists (−)-ethylketocyclazocine ((−)-EKC), {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 and the endogenous opioid peptide dynorphin A_1–13 caused a naloxone-reversible inhibition of NMDA-induced [³H]-dopamine release, with pD₂ values of about 9, 8.5 and 8.2, respectively, whereas both the μ agonist Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO) and theδ agonist D-Pen²-D-Pen⁵-enkephalin (DPDPE) were ineffective in this respect. The inhibitory effect of submaximally effective concentrations of dynorphin A_1–13, {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 and (−)-EKC on NMDA-induced [³H]-dopamine release were not changed by the δ₁/δ₂-opioid receptor antagonist naltrindole (up to a concentration of 1 μM), but reversed by the κ receptor antagonist nor-binaltorphimine (nor-BNI), with an IC₅₀ as low as 0.02 nM, indicating the involvement of {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593-sensitive κ₁-opioid receptors.
3. NMDA-induced [¹⁴C]-ACh release was reduced in a naloxone-reversible manner by DPDPE (pD₂ about 7.2), dynorphin A_1–13 (pD₂ 6.7) and EKC (pD₂ 6.2), but not by {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 and DAMGO. The inhibitory effect of a submaximally effective concentration of DPDPE, unlike those of dynorphin A_1–13 and (−)-EKC, on NMDA-induced [¹⁴C]-ACh release was antagonized by naltrindole with an IC₅₀ of 1 nM, indicating the involvement of δ-opioid receptors in the inhibitory effect of DPDPE. On the other hand, the inhibitory effects of dynorphin A_1–13 and (−)-EKC on [¹⁴C]-ACh release were readily antagonized by nor-BNI with an IC₅₀ of about 3 nM. A 100 fold higher concentration of nor-BNI also antagonized the inhibitory effect of DPDPE, indicating the involvement of {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593-insensitive κ₂-opioid receptors in the inhibitory effects of dynorphin A_1–13 and (−)-EKC.
4. Although naloxone benzoylhydrazone (NalBzoH), displaying high affinity towards the putative κ₃-opioid receptor, antagonized the inhibitory effects of dynorphin A_1–13 and (−)-EKC on [³H]-dopamine and [¹⁴C]-ACh release as well as that of {"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 on [³H]-dopamine release, it displayed a low apparent affinity (IC₅₀ about 100 nM) in each case.
5. In conclusion, whereas activation of κ₁-opioid receptors causes presynaptic inhibition of NMDA-induced dopamine release, κ₂ receptor activation results in inhibition of ACh release in rat neostriatum. As such, this study is the first to provide unequivocal in vitro evidence for the existence of functionally distinct κ-opioid receptor subtypes in the brain.

     

Interaction of the antidepressant mirtazapine with α2-adrenoceptors modulating the release of 5-HT in different rat brain regions in vivo

Mirtazapine (MIR) is a novel antidepressant, reported to raise extracellular noradrenaline (NA) through blockade of alpha2-autoreceptors and serotonin (5-HT) output via (1) indirect activation of facilitatory alpha1-adrenoceptors on the cell bodies of ascending 5-HT neurones and (2) blockade of presynaptic release-modulating alpha2-heteroreceptors on 5-HT terminals in the forebrain. To further assess the effect of MIR on NA/5-HT system interplay, including putative regional differences in the effects of the drug on 5-HT release in rat forebrain, we used in vivo microdialysis in anaesthetised rats. Probes were implanted in the dorsal hippocampus (DH) and frontal cortex (FCx), representing median and dorsal raphe 5-HT projection areas, respectively. In the DH, MIR (10 mg/kg s.c.) completely blocked the 5-HT release-suppressing action of the selective alpha2-adrenoceptor agonist clonidine (0.1 mg/kg s.c.), but had no effect per se on the 5-HT output. Neither drug significantly changed the 5-HT levels in the FCx. MIR perfused locally (10 microM via reverse-dialysis) also failed to significantly elevate 5-HT output, and did not affect the clonidine response in either brain area. Thus, the data confirm the basic alpha2-adrenoceptor-blocking properties of MIR, but are only partly concordant with previous studies reporting an increase of 5-HT output after MIR alone. Moreover, we find no elevation in 5-HT by the reference alpha2-adrenoceptor antagonist idazoxan (0.3-1.0 mg/kg s.c.). The discrepancies encountered, and the potential ability of alpha2-adrenoceptor antagonists in general to raise the output of 5-HT, are discussed with particular reference to methodological and other factors that may influence the experimental outcome (e.g., brain regional aspects, different alpha2-adrenoceptor subtypes, potential differences in adrenoceptor tone under varying experimental conditions).     

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     

Possible involvement of presynaptic α1-adrenoceptors in the effects of idazoxan and prazosin on 3H-noradrenaline release from tail arteries of SHR

Summary The effects of several -adrenoceptor antagonists have been examined on tritium release elicited by electrical stimulation from isolated perfused SHR tail artery preparations prelabelled with ³H-noradrenaline (³H-NA). Phentolamine and yohimbine potently facilitated the stimulation evoked release of tritium at low frequencies of stimulation, but the ₂-adrenoceptor antagonist idazoxan was only weakly active at 1 mol/l, despite antagonising the clonidine-evoked inhibition of ³H-release at a lower concentration of 0.1 mol/l. The ₁-adrenoceptor antagonists prazosin and corynanthine also increased stimulation evoked tritium release in this preparation, suggesting the presence of prejunctional ₁-adrenoceptors. Furthermore, the ₁-adrenoceptor agonist methoxamine (3 mol/l) caused a significant inhibition of tritium-evoked release, an effect which was blocked by prazosin (10 nmol/l).When ₁-adrenoceptors were blocked in the presence of prazosin, idazoxan (0.1 mol/l) produced a significant facilitatory effect on the electrically-evoked release of ³H-transmitter. On the other hand, when ₂-adrenoceptors were blocked in the presence of yohimbine, exposure to idazoxan (0.1 mol/l) reduced significantly the stimulation-evoked release of tritium elicited by electrical stimulation.The results indicate that in the SHR tail arteries, idazoxan has a partial agonist inhibitory activity on transmitter release, which can mask the facilitatory effects due to blockade of presynaptic ₂-adrenoceptors. The inhibitory effects of idazoxan appear to involve presynaptic ₂-adrenoceptors, which when stimulated, reduce ³H-NA release in SHR tail arteries.     

Methoxamine inhibits noradrenaline release through activation of α1- and α2-adrenoceptors in rat isolated kidney: involvement of purines and prostaglandins

Summary The effects of the at-adrenoceptor agonist methoxamine and the ₂-adrenoceptor agonist bromoxidine (UK 14034) on the stimulation induced (S-1) outflow of radioactivity at 100 Hz/6 pulses from rat isolated kidney preincubated with ³H-noradrenaline were investigated. Methoxamine (0.3 – 30 mol/l) inhibited S-1 outflow of radioactivity to a maximum of 83% with a pEC₅₀ of 5.85 (5.71–5.94). UK 14304 (0.0003-0.3 mol/l) inhibited S-I outflow of radioactivity to a maximum of 99% with a pEC₅₀ of 8.35 (8.26–8.47). a Adrenoceptor antagonist affinities (pK_D) against methoxamine and UK 14304 at prejunctional -adrenoceptors were determined. The concentration response curve of methoxamine was shifted to the right by the 1/_2B-adrenoceptor antagonist prazosin (0.1 mol/l) with a pK_D of 7.41 and that of UK 14304 by prazosin (0.3 mol/l) with a pK_D of 6.24. The ₂-adrenoceptor antagonist rauwolscine (0.1 mol/l) shifted the concentration response curve of UK 14304 potently to the right with a p_KD of 8.34. The concentration response curve of methoxamine was shifted also to the right by rauwolscine (0.1 mol/l) and the ₂-adrenoceptor antagonist idazoxan (0.1 mol/l), however, both antagonists suppressed the maximum response of methoxamine to 466% and 56%, respectively. A ten times lower concentration of rauwolscine (0.01 mol/l) did not shift the concentration response curve of methoxamine but the inhibitory effect of methoxamine still reached only a maximum of 59%. The concentration response curve of methoxamine obtained in the presence of rauwolscine (0.01 mol/l) was shifted to the right by further addition of prazosin (0.1 mol/l) with a pK_D of 8.80 but was also shifted to the right by either the purinoceptor antagonist 8-(p-sulfophenyl) theophylline (8-SPT; 100 mol/l) or the prostaglandin synthesis inhibitor indomethacin (20 mol/l). These results suggest that methoxamine inhibits S-1 outflow of radioactivity in rat isolated kidney probably through three different mechanisms. 1. Activation of postjunctional 1-adrenoceptors and prostaglandin mediated transjunctional inhibition. 2. Activation of postjunctional ₂-adrenoceptors and purine mediated transjunctional inhibition. 3. Activation of prejunctional inhibitory ₂-adrenoceptors at which methoxamine seems to be a partial agonist. Correspondence to L. C. Rump at the above address     

Pronounced facilitation of endogenous noradrenaline release by presynaptic β2-adrenoceptors in the vasculature of freely moving rats

Summary The facilitation of the noradrenaline (NA) over-flow by stimulation of the presynaptic -adrenoceptor of the rat portal vein was investigated, using the freely moving unanesthetized permanently cannulated rat as a model. The ₂-selective agonist fenoterol caused a maximal enhancement of about 300% of the basal NA level at a dose of 0.5 mg/kg. Following administration of cocaine (2.5 mg/kg plus 0.05 mg/kg/min) basal NA levels increased to 150% whereas combination of cocaine and fenoterol results in a dose dependant rise up to over 560% of the basal level (at a fenoterol dosage of 0.5 mg/kg). Blockade of the ₂-adrenoceptors with yohimbine (0.5 mg/kg) which enhances the NA level to 486%, followed by 0.125 mg/kg fenoterol results in a further 2.53-fold rise to more than 1,200% of the basal level, indicating the pronounced counterregulatory role of the presynaptic ₂-adrenoceptor. After ganglionic blockade with hexamethonium (3 mg/kg plus 6 mg/kg/h) the effect of yohimbine (0.5 mg/kg) alone was diminished to 162%, but the additional facilitatory effect of 0.125 mg/kg fenoterol still was 1.82-fold, to 294% of the basal level. Combination of cocaine (2.5 mg/kg plus 0.05 mg/kg/min), yohimbine (0.5 mg/kg) and fenoterol (0.125 mg/kg) induced a rise to over 9,000 pg/ml NA (about 40-fold of the basal NA level). During electrical stimulation (2 Hz, 3 ms, 5 mA) of the local portal vein nervous plexus, the role of the inhibitory ₂-adrenoceptor becomes even more pronounced. Both in absence and in presence of cocaine, at the highest dose of fenoterol (0.5 mg/kg), the levels of electrically evoked release reverted to control values. However, after yohimbine (0.5 mg/kg) the evoked release was further enhanced by a much lower dosage of 0.125 mg/kg fenoterol to 450% of the control evoked release. This value was not changed significantly after ganglionic transmission blockade (hexamethonium 3 mg/kg plus 6 mg/kg/h). The results demonstrate that presynaptic -adrenoceptors in the vasculature of the unanesthetized freely moving rat actually possess a pronounced capacity to facilitate NA release from sympathetic varicosities. Send offprint requests to R. Remie at the above address