Selective visualization of rat brain 5-HT2A receptors by autoradiography with [3H]MDL 100,907

The recently developed 5-HT_2A receptor selective antagonist [³H]MDL100,907 ((+/–)2,3-dimethoxyphenyl-1-[2-(4-piperidine)-methanol]) has been characterized as a radioligand for the autoradiographic visualization of these receptors. [³H]MDL100,907 binding to rat brain tissue sections was saturable, had sub-nanomolar affinity (Kd=0.2–0.3nM), and presented a pharmacological profile consistent with its binding to 5-HT_2A receptors (rank order of affinity for [³H]MDL100,907-labelled receptors: MDL100,907 > spiperone > ketanserin > mesulergine). The distribution of receptors labelled by [³H]MDL100,907 was compared to the autoradiographical patterns obtained with [³H]Ketanserin, [³H]Mesulergine, and [³H]RP62203 (N-[3-[4-(4-fluorophenyl)-piperazin-1-y1]propyl]-1,8-naphtalenesultam) and to the distribution of 5-HT_2A receptor mRNA as determined by in situ hybridization. As opposed to the other radioligands, [³H]MDL100,907 labelled a single population of sites (5-HT_2A receptors) and presented extremely low levels of non-specific binding. The close similarity of the distributions of [³H]MDL100,907-labelled receptors and 5-HT_2A mRNA further supports the selectivity of this radioligand for 5-HT_2A receptors and suggests a predominant somatodendritic localization of these receptors. The present results point to [³H]MDL100,907 as the ligand of choice for the autoradiographic visualization of 5-HT_2A receptors. Received: 7 April / Accepted: 18 May 1997     

Evidence for presynaptic location of inhibitory 5-HT1D\-like autoreceptors in the guinea-pig brain cortex

The effects of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on tritium overflow evoked by high K⁺ were determined in superfused synaptosomes and slices, preincubated with [³H]5-HT, from guinea-pig brain cortex. In addition, we estimated the potencies of 5-HT receptor ligands in inhibiting specific [³H]5-HT binding (in the presence of 8-hydroxy-2(di-n-propylamino)tetralin and mesulergine to prevent binding to 5-HT_1A and 5-HT_2C sites) to guinea-pig cortical synaptosomes and membranes.5-HT receptor agonists inhibited the K⁺-evoked tritium overflow from synaptosomes and slices. In synaptosomes the rank order of potencies was 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indole-3-yl] ethylamine (L-694,247) >5-carboxamidotryptamine (5-CT) > oxymetazoline (in the presence of idazoxan) 5-HT > sumatriptan 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (RU 24969). The potencies of the agonists in inhibiting tritium overflow from slices correlated with those in synaptosomes, suggesting that the same site of action is involved in both preparations. In synaptosomes the nonselective antagonist at cloned human 5-HT_1D, and 5-HT_1D receptors, methiothepin, shifted the concentration-response curve for 5-CT to the right (apparent pA₂: 7.87). In contrast, ketanserin at a concentration which should block the 5-HT_1D, but not the 5-HT_1D\, receptor did not alter the inhibitory effect of 5-CT on tritium overflow. In cortical synaptosomes and membranes, [³H]5-HT bound to a single site with high affinity. In competition experiments, 5-HT receptor agonists and antagonists inhibited specific [³H]5-HT binding. In synaptosomes the rank order was L-694,247 > methiothepin >5-CT >5-methoxytryptamine >5-HT sumatriptan oxymetazoline > RU 24969 > ketanserin > ritanserin. A very similar rank order was obtained in cerebral cortical membranes. The potencies of the 5-HT receptor agonists in inhibiting tritium overflow from synaptosomes and slices correlated with their potencies in inhibiting [³H]5-HT binding to synaptosomes and membranes.In conclusion, the 5-HT receptors mediating inhibition of 5-HT release in the guinea-pig cortex are located on the serotoninergic axon terminals and, hence, represent presynaptic inhibitory autoreceptors. The [³H]5-HT binding sites in cerebral cortical synaptosomes and membranes exhibit the pharmacological properties of 5-HT_1D receptors. The correlation between the functional responses and the binding data confirms the 5-HT_1D character of the presynaptic 5-HT autoreceptors. According to the results of the interaction experiment of ketanserin and methiothepin with 5-CT on 5-HT release, the presynaptic 5-HT autoreceptors can be subclassified as 5-HT_1D\-like.     

[3H] GR67330, a very high affinity ligand for 5-HT3 receptors

Summary GR67330 potently inhibited 5-hydroxytryptamine (5-HT)-induced depolarizations of the rat isolated vagus nerve. At the higher concentrations used (0.3 nmol/l–1 nmol/l) this was accompanied by a marked reduction in the maximum response to 5-HT. The calculated pK_B value was 10.2.The binding of the tritiated derivative of GR67330 to homogenates of rat entorhinal cortex was examined. Kinetic analysis revealed that specific [³H] GR67330 (0.1 nmol/l) binding was rapid and reversible. Association and dissociation rate constants were 1.48 ± 0.36 × 10⁸ mol/l^–1 s^–1 and 7.85 ± 0.41 × 10^–3 s^–1 respectively. Equilibrium saturation analysis revealed specific binding was to a single site (Bmax 22.6±0.21 fmol/mg protein) of high affinity (Kd 0.038±0.003 nmol/l). At low ligand concentrations, specific binding was up to 90% of total binding. If unlabelled GR67330 was used to define non-specific binding two sites were evident (Kd₁ 0.066 ± 0.007 nmol/l, Kd₂ 20.1 ± 9.7 nmol/l; Bmax₂ 31.5 ± 3.2 fmol/mg protein, Bmax₂ 1110 ± 420 fmol/mg protein). [³H] GR67330 binding was inhibited potently by 5-HT₃ antagonists and agonists. Ligands for other 5-HT receptors and other neurotransmitter receptors were either only weakly active or inactive at inhibiting binding. Hill numbers for antagonist inhibition of binding were close to unity, except for quipazine which was significantly greater than one. In common with other 5-HT₃ binding studies, all 5-HT₃ agonist tested had Hill numbers greater than one (1.51–1.71). GR38032 and GR65630 inhibited a greater proportion of binding than other 5-HT₃ antagonists, this additional binding was interpreted as inhibition from a second saturable site unrelated to the 5-HT₃ receptor.Homogenates of five areas of rat brain were examined for specific [³H]-GR67330 binding (entorhinal cortex, cingulate cortex, parietal cortex, hippocampus and nucleus accumbens/olfactory tubercle). In each brain area a site of very high affinity was labelled. Drug inhibition profiles were also very similar in each brain area. It is concluded that, because of its high affinity, [³H] GR67330 will be a useful ligand to label 5-HT₃ receptors especially in tissues with low receptor densities and to map 5-HT₃ receptors autoradiographically.Abbreviations 5-HT 5-Hydroxytryptamine - 8-OH-DPAT 8-hydroxy-2-di-N-propylaminotetralin - 5-CT 5-carboxyamidotryptamine - GR38032 (±) 1,2,3,9-tetrahydro-9-methyl-3[(2-methyl-1H-imidazol-1-yl)methyl]-4H-carbazol-4-one - GR65630 3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl) -1-propanone - GR67330 (±)1,2,3,9 - tetrahydro-9-methyl-3-[(5-methyl-1H-imidazol-4-yl)methyl]-4H-carbazol-4-one - MDL72222 1H,3,5H-tropan-3-yl-3,5-dichlorobenzoate - ICS 205–930 (3-tropanyl)-1H-indole-3-carboxylic acid ester - BRL24924 endo-4-amino-5-chloro-2-methoxy-N-(1-azabicyclo[3,3,1]non-4-yl)benzamide - BRL43694 endo-N-(9-methyl-9-azabicyclo[3,3,1]non-3-yl)-1-methyl-indazole-3-carboxamide - SDZ 206-830 (3-homotropanyl)-1-methyl-5-fluoro-indole-3-carboxylic acid ester - mCPP meta-chlorophenylpiperazine Send offprint requests to G. J. Kilpatrick at the above address     

The modulation of [3H]noradrenaline and [3H]serotonin release from rat brain synaptosomes is not mediated by the α2B-adrenoceptor subtype

Summary The present study aimed at relating the presynaptic ₂-adrenoceptors, known to modulate noradrenaline and serotonin release, with the recently described _2A- and _2B-adrenoceptor subtypes. The effects of the agonist oxymetazoline (selective for _2A subtype) and of three adrenoceptor antagonists (idazoxan, 1-(2-pyrimidinyl)piperazine (PmP) and prazosin, the last one known to be _2B selective) were evaluated on [³H]noradrenaline and [^H]serotonin release in superfused synaptosomes from rat brain cortex. These drugs were also tested in [³H]yohimbine binding to human platelet membranes (containing only _2A receptors) and to neonatal rat lung membranes (containing only _2B receptors).The affinity pattern of these compounds at _2A-adrenoceptors in binding studies was oxymetazoline > = idazoxan > PmP > prazosin; at _2B-adrenoceptors it was idazoxan > = prazosin > PmP = oxymetazoline. Oxymetazoline inhibited with high and similar potencies the K⁺-evoked [³H]noradrenaline and [³H]serotonin release, IC₅₀ 18 and 7 nM, respectively; in the same conditions, the IC₅₀ values of noradrenaline were 42 and 168 nM, respectively. The antagonist affinity pattern (antagonism against noradrenaline) was idazoxan > PmP > prazosin, either on [³H]serotonin release.These results indicate that presynaptic ₂ auto- or heteroreceptors do not belong to the _2B subtype and suggest that the modulation of noradrenaline and serotonin release may be mediated by the _2A-adrenoceptor subtype. Send offprint requests to M. Gobbi at the above address     

Autoradiographic evidence for G-protein coupled A2-receptors in rat neostriatum using [3H]-CGS 21680 as a ligand

Summary Recently [³H]-CGS 21680 (2-[p-(2-carbonylethyl)-phenylethylamino]-5-N-ethylcarboxamidoadeno-sine) has been identified as a selective adenosine A₂-receptor agonist. In this study the binding of [³H]-CGS 21680 to 10 m sections of rat neostriatum was investigated with quantitative autoradiography. Specific, saturable binding was detectable, and Scatchard analysis of saturation experiments gave estimates for K _D and B _max of 1.7 nM and 322 fmol/mg protein, respectively. The rank order of potency for inhibition of [³H]-CGS 21680 binding was 5-N-ethylcarboxamidoadenosine (1.9 nM) > 2-chloroadenosine (18 nM) > R-N⁶-phenylisoprop-yladenosine (59 nM) > S-N⁶-phenylisoprophyladeno sine (460 nM) > 1,3-dipropyl-8-cyclopentylxanthine (700 nM). The binding of [³H]-CGS 21680 was sensitive to GTP, since 1 M GTP reduced binding to 4.7% of control. These data support the identity of CGS 21680 as an agonist at high affinity adenosine A₂-receptors and indicate these receptors in rat striatum are coupled to guanine nucleotide binding proteins. Send offprint requests to F. E. Parkinson at the above address     

Inhibition of 5-HT3 receptor function by imidazolines in mouse neuroblastoma cells: potential involvement of σ2 binding sites

The influence of several imidazolines and -site ligands on cation influx through the 5-HT₃ receptor channel in NIE-115 mouse neuroblastoma cells was studied by measuring the 2-min influx of the organic cation [¹⁴C] guanidinium induced by 1 M 5-HT (in the presence of 10 M substance P in all experiments). In addition, we determined specific binding of [³H]DTG (1,3-di(2-tolyl)-guanidine), a selective -site radioligand, and [³H] GR65630 (3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)-1-propanone), a selective 5-HT₃ receptor antagonist, to membranes prepared from NIE-115 cells.The 5-HT-induced [¹⁴C]guanidinium influx was inhibited by the imidazolines, ondansetron, antazoline, idazoxan, BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline), cirazoline, naphazoline, clonidine and by the guanidine agmatine, but not by the catecholamine adrenaline. The inhibitory effect of the imidazolines on cation influx through the 5-HT₃ receptor channel was mimicked by the -site ligands, (±)-ifenprodil, (+)-3-PPP ((R)-3-(3-hydroxyphenyl)-N-propylpiperidine), DTG (1,3-di-tolyl-guanidine), haloperidol, dizocilpine, and ketamine as well as by the polyamines, arcane and spermidine. — Ondansetron inhibited [³H]GR65630 binding with high affinity, whereas inhibition of binding of this radioligand to the 5-HT₃ receptor by antazoline, BDF 6143, idazoxan, cirazoline, (±)-ifenprodil, (+)-3-PPP, DTG and haloperidol occurred in the high micromolar range. In the competition experiments with [³H]DTG, (±)-ifenprodil, haloperidol, unlabelled DTG, BDF 6143 and (+)-3-PPP inhibited binding of the radioligand at moderate affinity (K_i values in the range of 1 M or lower), whereas ondansetron, amazoline, idazoxan, cirazoline, naphazoline, clonidine, tolazoline, efaroxan, RX821002 (2-[2-(2-methoxy-1,4-benzodioxanyl)]imidazoline), ketamine and spermidine exhibited affinity in the high micromolar or millimolar range only. Comparison of the potencies of the ligands (pIC_50% values) in inhibiting 5-HT-induced [¹⁴C]guanidinium influx with their affinities (pK_i values) at the 5-HT recognition sites of the 5-HT₃ receptor and at the ₂-sites of the N1E-115 cells by means of multiple regression analysis revealed a significant correlation with the affinities at both sites.In conclusion, our data suggest that imidazolines and -ligands, which as a rule possess low affinity for the 5-HT recognition site of the 5-HT₃ receptor, may be assumed to exert their inhibitory effect on cation influx through the 5-HT₃ receptor channels, at least in part, by interacting with ₂-binding sites.     

Binding properties of the naturally occurring human 5-HT1A receptor variant with the Ile28Val substitution in the extracellular domain

The cDNA from a schizophrenic patient heterozygous for a mutation of the 5-HT_1A receptor gene was used to clone the variant and wild-type DNA into a eukaryotic expression vector. The mutation was characterized by a base pair substitution (A G) at the first position of codon 28, leading to an Ile Val amino acid exchange. COS-7 cells were transfected with the cDNA of either the wild type or the variant 5-HT_1A receptor. The potencies of the 5-HT_1A receptor agonists 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT), 5-HT and roxindole, and of the antagonists methiothepin and spiperone in inhibiting specific binding of [³H]8-OH-DPAT of the mutant and wild-type 5-HT_1A receptor were determined. All five 5-HT_1A receptor ligands concentration-dependently inhibited specific [³H]8-OH-DPAT binding to both the wild-type and the variant 5-HT_1A receptor. The rank order of potency of the ligands in inhibiting [³H]8-OH-DPAT binding was identical at both receptors and was roxindole > 8-OH-DPAT > 5-HT > methiothepin > spiperone. This rank order is characteristic for 5-HT_1A receptors. The negative logarithms of the concentrations required for 50% inhibition (pIC₅₀ values) of the ligands at the mutant 5-HT receptor correlated highly significantly with those at the wild-type receptor (r = 0.995). It is concluded that the pharmacological profile of the mutant 5-HT_1A receptor does not differ from that of the wild-type 5-HT_1A receptor.     

Mu and kappa opioid receptor modulation of 5-HT3 and NK-3 receptor-evoked release of acetylcholine from the guinea-pig ileum myenteric plexus

Summary The effects of three different opioid agonists on contractions and [³H]-acetylcholine (ACh) release evoked by 5-hydroxytryptamine₃ (5-HT₃) and neurokinin-3 (NK-3) receptor activation were examined in the guinea-pig ileum longitudinal muscle-myenteric plexus strip (LMMP) preparation. The selective mu ()-opioid receptor agonist (d-Ala²,NMe-Phe⁴,Gly-ol]-enkephalin) (DAMGO; 1 nM–100 nM) and the selective kappa ()-opioid receptor agonist U50488 (10 nM -1 M) inhibited contractile responses to 5-HT and to the selective NK-3 receptor agonist senktide, producing a concentration-related progressive flattening of their concentration-response curves. IC₅₀ estimates for DAMGO and U50488 were somewhat higher for inhibition of 5-HT-evoked as compared to senktide-evoked contractions, and overall lay in the range 6 nM – 51 nM. The selective delta ()-opioid receptor agonist [d-Pen^2,5]-enkephalin (DPDPE) inhibited contractile responses only at the highest concentration used (1 M). ³H-overflow from LMMP preparations preincubated with [³H]-choline was measured as an indicator of [³H]-ACh release. DAMGO (1 nM –100 nM) and U50488 (10 nM -1 M) inhibited the increases in release of [³H]-ACh evoked by 5-HT (10 M) and by senktide (10 nM) in a concentration-dependant manner. IC₅₀ estimates for DAMGO and U50488 were not significantly different for inhibition of 5-HT as compared to senktide-evoked increases in [³H]-ACh release and lay in the range 6 nM –23 nM. DPDPE again only inhibited these responses at the maximum concentration used (1 M). The inhibitory effects of DAMGO, U50488 and DPDPE on contractions and [³H]-ACh release evoked by 5-HT and senktide were completely reversed by naloxone (10 M).These results show that ACh release in the guinea-pig ileum evoked by 5-HT and senktide can be modulated to a similar extent by the opioid agonists DAMGO and U50488, but not by DPDPE. This suggests that the pathways of excitation for 5-HT₃ and NK-3 receptors converge at some level susceptible to opioid inhibition, which may be mediated by - and -, but not -, opioid receptors.     

[3H]Idazoxan binding to bovine adrenal medullary membranes: identification and pharmacological characterization of I2-imidazoline sites

Bovine adrenal chromaffin cells, which have been shown to lack ₂-adrenoceptors, were used to investigate the pharmacological characteristics of [³H]idazoxan binding sites. The binding of [³H]idazoxan was very rapid, reversible, partly specific (as defined by cirazoline 0.1 mmol/l; 50% specific binding at [³H]idazoxan 10 nmol/l), saturable and of high affinity (K_D 13 nmol/l) and, hence, was compatible with the criteria for the identification of an imidazoline binding site (IBS). Since in competition experiments rauwolscine and (–)-adrenaline showed only negligible affinity for these adrenal medullary binding sites, the lack of ₂-adrenoceptors was confirmed. Histamine and amiloride also did not inhibit [³H]idazoxan binding or caused only negligible inhibitor. In contrast, the specific binding of [³H]idazoxan was concentration-dependently inhibited by several imidazolines and guanidines with the following rank order of potency which conforms to the characteristics of the previously defined I₂-IBS (in parentheses: K_i, nmol/l): idazoxan (4) = cirazoline (4) clonidine (272) = BDF 6143 (299; 4-chloro-2-(2-imidazoline-2-ylamino) isoindoline) > BDF 6100 (563; 2-(2-imidazolin-2-ylamino)-isoindoline) BDF 7579 (868; (4-chloro-2-isoindolinyl)guanidine) > phentolamine (1424) = naphazoline (1451). Equilibrium [³H]idazoxan binding was reduced by K⁺ but not by Na⁺ or the non-hydrolysable GTP-analogue Gpp(NH)p (5-guanylylimidodiphos-phate; 100 mol/l). In conclusion, membranes of the bovine adrenal medulla are endowed with non-adrenergic high-affinity [³H]idazoxan sites which exhibit the pharmacological properties of the amiloride-insensitive subtype of I₂-IBS and probably are not coupled to a G-protein. Correspondence to: G. J. Molderings at the above address     

Subtype specificity of γ-aminobutyric acid type A receptor antagonism by clozapine

Clozapine, an atypical neuroleptic, functionally antagonizes the -aminobutyric acid-induced chloride uptake via the main central inhibitory receptor, -aminobutyric acid type A (GABA_A) receptor, in brain vesicles. GABA_A antagonism by micromolar concentrations of clozapine is more efficient in rat cerebrocortical and hippocampal membranes than in cerebellar membranes, as evidenced by clozapine reversal GABA-inhibition of [³⁵S]t-butylbicyclophosphorothionate ([³⁵S]TBPS) binding. A typical neuroleptic, haloperidol, failed to antagonize GABA in any of these brain regions, while the specific GABA_A antagonist 2-(3-carboxy-2,3-propyl)-3-amino-6-p-methoxyphenylpyrazinium bromide (SR 95531) was efficient in all three brain regions. Clozapine action on [³⁵S]TBPS binding was unaffected by the benzodiazepine receptor antagonist flumazenil. Clozapine inhibited the binding of [³H]muscimol and [³H]SR 95531 to the GABA recognition site, but this effect only partially correlated with the regional differences in and the potency of clozapine antagonism of GABA-inhibition of [³⁵S]TBPS binding, suggesting that also other than GABA sites may mediate clozapine actions. Autoradiography of [35S]TBPS binding revealed GABA antagonism by clozapine in most brain regions. Main exceptions were cerebellar granule cell and molecular layers, olfactory bulb external plexiform and glomerular layers and primary olfactory cortex, where clozapine antagonized GABA inhibition less than average, and lateral hypothalamic and preoptic areas where its antagonism was greater than average. Recombinant 622 receptors, the predominant 6 subunit-containing receptor subtype in cerebellar granule cells, failed to show GABA antagonism by clozapine up to 100 M. In contrast, recombinant 122 receptors, forming the predominant receptor subtype in the brain, were clozapine sensitive. Recombinant 622 and 632 receptors resulted in clozapine-insensitive receptors, whereas 612 receptors were clozapine sensitive. The efficacy of clozapine to antagonize GABA in 1x2 receptors decreased in the order of 112>122>132. The results indicate that clozapine antagonizes the function of most GABA_A receptor subtypes, and that the interaction is determined by the interaction of the and subunit variants. GABA antagonism is a unique property of clozapine, not shared by haloperidol, which might be involved in the pharmacological mechanism for the increased seizure susceptibility associated with clozapine treatment.     

Stimulus-evoked release of tritiated monoamines from rat periaqueductal gray slices in vitro and its receptor-mediated modulation

Summary The periaqueductal gray is a brain region of considerable interest. It is innervated by monoamine-containing neurons as well as by a variety of peptidergic fiber systems, and it participates in the regulation of various functions. Virtually nothing is known about monoamine release in the periaqueductal gray and its receptor-mediated modulation. We therefore studied the release of radioactivity from periaqueductal gray slices preloaded with tritriated monoamines, using an in vitro superfusion method.The release of radioactivity from superfused periaqueductal gray slices after preloading of the tissue with [³H]noradrenaline increased upon electrical stimulation in a frequency-dependent manner. The stimulus-evoked release of radioactivity was Ca²⁺-dependent. Clonidine reduced and yohimbine enhanced the release. The inhibition curve for the effect of clonidine was shifted to the right in the presence of 10^–6 M yohimbine. While phenylephrine, isoprenaline, SK&F 38393, quinpirole, carbachol, [Arg⁸]vasopressin, -MSH and ACTH-(1-24), at a concentration of 10^–6 M, did not influence the electrically evoked release of radioactivity, [Leu⁵]enkephalin reduced it. The selective -opioid receptor agonists [d-Ala²,NMePhe⁴,Gly-ol⁵]enkephalin and [d-Arg²,Lys⁴]-dermorphin-(1–4)-amide reduced the release of radioactivity, whereas the selective opioid receptor agonist [d-Pen²,d-Pen⁵]enkephalin and the selective K opioid receptor agonist U-69593 had no effect. In the presence of naloxone, which by itself had no effect on the release of radioactivity, the effect of [d-Arg²,Lys⁴]dermorphin-(1–4)-amide was abolished. These results show that the release of noradrenaline from periaqueductal gray slices is via a Ca²⁺-dependent. exocytotic process, and that it is modulated through ₂-adrenoceptors as well as via -opioid receptors. Though the overflow of radioactivity from slices preloaded with [3H]dopamine in the presence of desipramine was measurable, there are reasons to assume that we are dealing here with the release of tritiated catecholamines from a population of nerve endings consisting of noradrenergic and dopaminergic terminals.The release of radioactivity from periaqueductal gray slices preloaded with [³H]5-hydroxytryptamine upon elevation of the K⁺ concentration in the superfusion medium was much more pronounced than that induced by electrical stimulation. The K⁺-evoked release of radioactivity was almost completely abolished in the absence of Cat²⁺; showing that the release is via a Ca²⁺-dependent process. 5-Hydrotryptamine reduced the K⁺-evoked release of radioactivity in a concentration-dependent manner.Some of these data were presented at the XIth International Congress of Pharmacology, 1–6 July 1990, Amsterdam, The Netherlands (Eur J Pharmacol 183:408) Send offprint requests to D. H. G. Versteeg at the above address     

Acute ethanol intoxication may not alter α2-adrenoceptors in the human brain

The specific binding of the ₂-adrenoceptor agonists [³H]clonidine and [³H]bromoxidine (UK 14304) was measured in the postmortem brain of ethanol intoxicated nonalcoholic subjects (blood ethanol concentration: 1.37±0.30 g/l) and matched controls. In the frontal cortex, the density of binding sites for [³H]clonidine (B_max=58±7 fmol/mg protein) and [³H]bromoxidine (UK 14304) (B_max=49±7 fmol/mg protein) in ethanol intoxicated subjects did not differ from those in the control groups (B_max=68±4 and 56±8 fmol/mg protein for the respective radioligand). The dissociation constants (K_D) were also similar in both groups. The binding capacities (B_max) and K_D values for both radioligands also were found unchanged in the hypothalamus, amygdala, head of caudate, hippocampus and cerebellum. The results demonstrate that, contrary to the -adrenoceptor, the presence of ethanol in the human brain does not alter the high-affinity state of the ₂-adrenoceptor in the frontal cortex and possibly also in other brain regions.     

Pharmacological and molecular discrimination of brain I2-imidazoline receptor subtypes

I₂-imidazoline receptors labelled with [³H]-idazoxan in the rabbit and rat brains displayed high and low affinity, respectively, for the guanidide amiloride; reinforcing the previous definition of I_2A-imidazoline receptors expressed in the rabbit brain and Its-imidazoline receptors expressed in the rat brain. Other drugs tested displayed biphasic curves in competition experiments, indicating the existence of high and low affinity sites for both subtypes of I₂-imidazoline receptors. Among the drugs studied, bromoxidine, moxonidine, (+)- and (-)-medetomidine and clorgyline were more potent on the high and/or low affinity sites of 1_2B- than on their corresponding of I_2A-imida-zoline receptors (K _iH ratios 20 to 65). No correlation was found for the potencies of the drugs tested at the low affinity sites of both I₂-imidazoline receptor subtypes. Preincubation (30 min at 25°°C) with 10^-6 M clorgyline reduced by 60% the B _max of [³H]-idazoxan binding to I₂ B-imidazoline receptors in the rat brain, but it did not affect the binding parameters of the radioligand saturation curves to I_2A-imidazoline receptors in the rabbit brain. These results indicated that I_2A- and I_2B-imidazoline receptor subtypes differ in the pharmacological profiles of their high and low affinity sites and in the ability to irreversibly bind clorgyline. In rat cortical membranes western blot detection of immunoreactive imidazoline receptor proteins revealed a double band of 29/30 kDa and two less intense bands of 45 and 66 kDa. In rabbit cortical membranes the antibody used detected proteins of 30, 57 and 66 kDa. It is suggested that different imidazoline receptor proteins (45 vs 57 kDa) may account for the different pharmacological profiles of I2-imidazoline receptor subtypes.     

ATP and endogenous agonists inhibit evoked [3H]-noradrenaline release in rat iris via A1 and P2y-like purinoceptors

Summary Effects of ATP, adenosine and purinoceptor antagonists on field stimulation-evoked (3 Hz, 2 min) [³H]-noradrenaline overflow were investigated in the rat isolated iris.ATP and adenosine inhibited the evoked overflow of [³H]-noradrenaline. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) shifted the concentration-response curve of ATP to the right in a concentration-dependent manner, but with a potency (–log K_B = 7.88) much lower than expected for an A1 adenosine receptor. In the continuous presence of DPCPX, the ATP-induced prejunctional inhibition was unaffected by suramin (100 mol/l) and DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 50 mol/l) but was antagonized by the P_2Y-receptor antagonist cibacron blue ( = reactive blue 2;30 and 100 mol/l, –log K_B = 4.7)and ,-methylene-ATP (10 mol/l). Whereas the evoked [³H]-noradrenaline overflow was unaffected by suramin and DIDS, cibacron blue and ,-methylene-ATP caused a small and transient increase. Cibacron blue at 30 mol/l failed to antagonize the inhibition of evoked [³H]-noradrenaline overflow that adenosine produced in the absence of DPCPX. Basal [³H]-noradrenaline overflow was enhanced by cibacron blue, not changed by ,-methylene-ATP and DIDS, and decreased by suramin.The results show that exogenous ATP inhibits sympathetic neurotransmission in the rat iris via A₁ and P_2Y-like purinoceptors. The latter have a low apparent affinity for cibacron blue and probably are blocked by ,-methylene-ATP. Under the present conditions, endogenous purines exert a tonic inhibition not only via A₁- but also via these P_2Y-receptors. Correspondence to: H. Fuder at the above address     

Evidence for the existence of two forms of α2A-adrenoceptors in the rat

Summary The _2A-adrenoceptors in rat spleen, kidney, spinal cord and cerebral cortex were studied using [³H]-RX821002 radioligand binding. In the spleen, spinal cord and cerebral cortex, the ligand bound to saturable sites with a K _d of about 1 nmol/l and capacities of 134, 240 and 290 fmol/mg protein, respectively. Computer modelling competition curves for 39 drugs, including those for _2A-, _2B- or _2C-adrenoceptor selective drugs, indicated that the sites labelled by [³H]-RX821002 in the spleen consisted of a single population of _2A-adrenoceptors. However, the competition curves for guanoxabenz were definitely biphasic and resolved into two site fits, indicating that guanoxabenz was binding to both high affinity (K _d = 35 nmol/1) and low affinity (K _d = 8900 nmol/1) _2A-adrenoceptor sites in the proportions 57% and 43%, respectively. The K _d ^Sfor a number of ₂-adrenoceptor subtype selective drugs, measured in competition with [³H]-RX821002 in cerebral cortex and spinal cord, were highly correlated with those obtained in the spleen indicating their _2A-adrenoceptor nature. However, by contrast to the results with the spleen, the guanoxabenz competition curves for the spinal cord and cerebral cortex were monophasic and resolved only into one site fits, the K _d of guanoxabenz being about 4000 nmol/l for both tissues. Drug K _d ^Sfor kidney _2A-adrenoceptors were also determined using [³H]-RX821002. For nearly all drugs tested, the K _d ^Swere highly correlated with those found for the _2A-adrenoceptors in the other rat tissues. However, for guanoxabenz, the data indicated that it competed with [³H]-RX821002 at a single _2A-adrenoceptor site with a K _d of 39 nmol/1. When the rat _2A-adrenoceptor gene RG20 was transiently expressed in COS-7 cells and its ligand binding properties probed using [³H]-RX821002, the drug K _d ^Sobtained were also highly correlated with those found for the _2A-adrenoceptors in the spleen, cerebral cortex, spinal cord and kidney of the rat. For the RG20 encoded receptor, the guanoxabenz competition curves were steep and monophasic and modelled best into one site fits, with the Kd of guanoxabenz being 5200 nmol/1.It is suggested that guanoxabenz can differentiate between two forms of _2A-adrenoceptors in the rat: _2A1 and _2A2. The _2A1-form is present in the spleen and kidney where it shows a high apparent affinity for guanoxabenz. The _2A2-form shows a low apparent affinity for guanoxabenz and is present in the spleen, cerebal cortex and spinal cord. The _2A2-form of the rat ₂-adrenoceptor appears to be encoded by the RG20 gene. The _2A, and _2A2-adrenoceptor forms do not represent high and low affinity receptor forms for agonists because assays included EDTA, Gpp(NH)p and Na⁺, which eliminated the high affinity receptors for agonists.     

Binding of [3H]clonidine to I1-imidazoline sites in bovine adrenal medullary membranes

Summary Imidazolines bind with high affinity not only to -adrenoceptors but also to specific imidazoline binding sites (IBS) labelled by either [³H]clonidine or [³H]idazoxan and termed I₁- and I₂-IBS, respectively. Since bovine adrenal chromaffin cells lack ₂-adrenoceptors, we investigated the pharmacological characteristics of [³H]clonidine binding sites in the bovine adrenal medulla. The binding of [³H]clonidine was rapid, reversible, partly specific (as defined by naphazoline 0.1 mmol/l; 55% specific binding at [³H]clonidine 10 nmol/l), saturable and of high affinity. The specific binding of [³H]clonidine to bovine adrenal medullary membranes was concentration-dependently inhibited by various imidazolines, guanidines and an oxazoline derivative but not, or with negligible affinity, by rauwolscine and (–)-adrenaline. In most cases, the competition curves were best fitted to a two-site model. The rank order of affinity for the high affinity site (in a few cases the single detectable site) was as follows: naphazoline >- BDF 7579 (4-chloro-2-isoindolinyl guanidine) >-clonidine>- cirazoline >_ BDF 6143 (4-chloro-2-(2-imidazolin-2-ylamino)isoindoline hydrochloride) > BDF 7572 (4,7-chloro-2-(2-imidazolin-2-ylamino)-isoindoline) > moxonidine = rilmenidine > BDF 6100 (2-(2-imidazolin-2-ylamino)-isoindoline) = idazoxan > phentolamine > aganodine = guanabenz > amiloride > histamine. This rank order is compatible with the pharmacological properties of the I₁-IBS. The non-hydrolysable GTP-analogue Gpp(NH)p (5guanylylimidodiphosphate; 100 mol/l) inhibited specific [³H]clonidine binding by about 50%. Equilibrium [³H]clonidine binding was also significantly reduced by K⁺ and Mg²⁺ In conclusion, [³H]clonidine labels non-adrenergic high-affinity sites in plasma membranes of the bovine adrenal medulla; these sites exhibit the pharmacological properties of I₁-IBS, but not of I₂-IBS. Furthermore, the IBS in the adrenal medulla appear to be coupled to a G-protein.Correspondence to G. J. Molderings at the above address     

Autoradiographic characterisation and localisation of 5-HT1D compared to 5-HT1B binding sites in rat brain

Summary The regional distribution and the pharmacology of the binding sites labelled with the novel 5-hydroxytryptamine (serotonin) 5-HT_1B/1D selective radioligand serotonin-O-carboxy-methyl-glycyl-[¹²⁵I]tyrosinamide (abbreviated [¹²⁵I]GTI for the sake of simplicity) was determined using quantitative autoradiography in rat brain. The distribution of [¹²⁵I]GTI binding sites was largely comparable to that of [¹²⁵I] iodocyanopindolol ([¹²⁵I] ICYP) which labels 5-HT_1B binding sites (in the presence of 8-OH-DPAT (8-hydroxy-[2N-dipropylamino]tetralin) and isoprenaline, to prevent binding to 5-HT_1A and -adrenoceptor binding sites), although a detailed analysis revealed differences.The pharmacology of the [¹²⁵I]GTI binding sites was analysed using compounds known to display high affinity for and/or distinguish between 5-HT_1B and 5-HT_1D sites: 5-carboxamidotryptamine (5-CT), sumatriptan, CP 93129 (5-hydroxy-3(4-1,2,5,6-tetrahydropyridyl)-4-azaindole), (–)pindolol, PAPP (4[2-[4-[3-(trifluoromethyl)phenyl]-1-piperazinyl]ethyl] benzeneamine), rauwolscine, and 8-OH-DPAT. The displacement of [¹²⁵I]GTI by 5-CT was monophasic. By contrast, the selective 5-HT_1B compound CP 93129 and (–)pindolol produced biphasic curves showing a majority of high affinity sites in the globus pallidus and the substantia nigra, whereas PAPP and sumatriptan (which are somewhat 5-HT_1D selective) produced biphasic curves indicating a minority of high affinity sites in these areas. In addition, by blocking the 5-HT_1B sites with 100 nM CP 93129, the remaining population of [¹²⁵I]GTI binding sites could be studied and was found to have high affinity for PAPP, rauwolscine and 8-OH-DPAT. The pharmacological profile of the major binding component was typical of the 5-HT_1B type: 5-CT > CP 93129 (–)pindolol > sumatriptan >/ PAPP > rauwolscine. The profile of the minor component of [¹²⁵I] GTI binding is best characterised as that of a 5-HTID site: 5-CT > PAPP sumatriptan > rauwolscine > (–)pindolol CP 93129.The localisation of the non 5-HT_1B [¹²⁵I]GTI binding sites was characterised by blocking the 5-HT_1B receptors with 100 nM CP 93129. Low densities of the 5-HT_1D recognition sites were found to be present in globus pallidus, ventral pallidum, caudate-putamen, subthalamic nucleus, entopeduncular nucleus, substantia nigra (reticular part), nuclei of the (normal and accessory) optic tract, different nuclei of the geniculate body and frontoparietal cortex, although higher densities of 5-HT_1B sites were always observed in the same structures. Thus, in agreement with the recent cloning of a rat 5-HT_1D receptor cDNA, the presence and the distribution of 5-HT_1D sites could be documented in rat brain. However, when compared to 5-HT_1B sites, 5-HT_1D sites represent only a minor component of the [¹²⁵I]GTI binding in the rat brain structures studied.Correspondence to: D. Hoyer at the above address     

Evidence that the presynaptic A2a-adenosine receptor of the rat motor nerve endings is positively coupled to adenylate cyclase

The action of the A_2a-adenosine analogue, CGS 21680C, on electrically evoked [³H]acetylcholine ([³H]-ACh) release, and its interaction with forskolin (an activator of adenylate cyclase), MDL 12,330A (an irreversible inhibitor of adenylate cyclase), rolipram (an inhibitor of cyclic AMP specific phosphodiesterase), dibutyryl- (db-cAMP) and 8-bromo- (8-Br-cAMP) cyclic AMP analogues (substances that mimic intracellular actions of cyclic AMP), were investigated using rat phrenic nerve-hemidiaphragm preparations.CGS 21680C facilitated [³H]ACh release. Forskolin (but not 1,9-dideoxy forskolin), rolipram, db-cAMP and 8-Br-cAMP also increased evoked neurotransmitter release in a concentration-dependent manner. When the evoked [³H]-ACh release that is dependent on stimulation of the adenylate cyclase/cyclic AMP transduction system was supramaximally stimulated by these compounds, CGS 21680 C (3 mol/l) could not further increase [³H]-ACh release. Phosphodiesterase inhibition with low concentrations ( 30 mol/l) of rolipram significantly potentiated the augmenting effect of CGS 21680C (1 mol/l) on evoked [³H]ACh release. MDL 12,330A (an irreversible inhibitor of adenylate cyclase) decreased evoked [³H]-ACh release. The irreversible blocking action of MDL 12,330A on [³H]-ACh release was overcome by by-passing cyclase activation with db-cAMP and 8-Br-cAMP, but could not be overcome with FSK or CGS 21680 C. The inhibitory effect of MDL 12,330A on evoked [³H]-ACh release was not mimicked by nifedipine.It is concluded that the increase in [³H]-ACh release caused by CGS 21680C results from activation of an A_2a-adenosine receptor positively linked to the adenylate cyclase/cyclic AMP system.     

An increase in cardiac alpha1-adrenoceptors following chronic clonidine treatment

Summary Chronic treatment (22 days) of rats with clonidine (0.5 mg/kg s.c. twice a day followed by 20 h of withdrawal) resulted in a significant increase in the specific [³H]WB4101 binding to ventricular and intraventricular septal ₁-adrenoceptors but no alteration of the atrial ₁-adrenoceptors. Scatchard analysis indicated that the increase in the [³H]WB4101 binding to the clonidine-treated cardiac tissues was due to an enhancement of the ₁-adrenoceptor density since there was a significant increase in the B _max value for the [³H]WB4101 binding to the treated ventricles without a change in the K _d value. The specific [³H]WB4101 binding to cardiac ₁-adrenoceptors was not altered by the acute (1 day) or 7 days treatment with clonidine. Chronic treatment with clonidine had no significant effect on the specific [³H](–)DHA binding to the atrial and ventricular -adrenoceptors. The noradrenaline (NA) concentrations in the clonidine-treated ventricles and intraventricular septae were decreased by 16–20%. These data provide biochemical evidence compatible with a significant reduction of sympathetic outflow to the ventricular myocardium by clonidine.     

[3H]5′-N-ethylcarboxamide adenosine binds to both Ra and Ri adenosine receptors in rat striatum

Summary Adenosine analogs such as 5-N-ethylcarboxamide adenosine and N⁶-cyclohexyladenosine stimulate or inhibit adenosine cyclase activity in preparations of rat striatum depending on the assay conditions. N⁶-cyclohexyladenosine inhibits but does not stimulate adenosine cyclase activity in preparations of hippocampus. These findings suggest that the striatum contains both R _a (stimulatory) and R _i (inhibitory) adenosine receptors while the hippocampus contains only R _i receptors. We have previously shown that [³H]N⁶-cyclohexyladenosine binds to R _i receptors in rat hippocampus (Yeung and Green 1983). Comparisons of the characteristics of [³H]5-N-ethylcarboxamide adenosine and [³H]N⁶-cyclohexyladenosine binding to hippocampus show that [³H]5-N-ethylcarboxamide adenosine also binds to R _i receptors with high affinity. [³H]5-N-ethylcarboxamide adenosine binds to R _i receptors in the striatum and to a second site that is present in striatum but not hippocampus. High affinity binding of both ligands to R _i receptors can be blocked by treatments with N-ethylmaleimide that do not markedly affect [³H]5-N-ethylcarboxamide adenosine binding to the second site in the striatum. The pharmacological characteristics of the second site indicate that it is the R _a adenosine receptor.The abbreviations used are NEM N-ethylmaleimide - Gpp(NH)p 5-guanylylimidodiphosphate - NECA 5-N-ethylcarboxamide adenosine - l-PIA N⁶-(l-phenylisopropyl)adenosine - d-PIA N⁶-(d-phenylisopropyl) adenosine - DPX 1,3-diethyl-8-phenylxanthine