[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     

Species variations in 5-HT3 recognition sites labeled by 3H-quipazine in the central nervous system

Summary The specific binding of ³H-quipazine to putative 5-HT₃ receptors was analyzed in multiple species. Specific and saturable binding of the radioligand could be detected in both rat (K _D = 1.2 nM; B _max = 3.0 pmol/g) and pig (K _D = 1.3 ± 0.2 nM; B _maX = 1.5 ± 0.2 p/mol/g) cortical membranes. By contrast, no significant specific binding of ³H-quipazine could be detected in human, cow, dog, turtle, mouse, guinea pig, chicken or rabbit brain membranes. These data indicate that marked species variations exist in the presence and/or density of 5-HT₃ membrane recognition sites in the central nervous system. Send offprint requests to Stephen J. Peroutka at the above address     

[3H]MDL 100,907: a novel selective 5-HT2A receptor ligand

In studies using standard radioligands, unlabeled MDL 100,907 (R-(+)--(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol) has been shown to have a high degree of selectivity for the 5-HT_2A receptor. The present study was undertaken to investigate the receptor binding characteristics of [³H]MDL 100,907 in rat cortical homogenates. [³H]MDL 100,907 was found to reach equilibrium at 37°C after 15 min. Saturation experiments indicated binding to a single site with a K_D of 0.56 nM, Hill slope of 1.15, and a B_max of 512 fmol/mg protein. In parallel experiments with the standard 5-HT_2A receptor radioligand, [³H]ketanserin, with prazosin added to block ₁ receptors, a similar Hill slope and B_max was noted but a two-fold higher K_D was found. In competition binding studies using 0.5 nM [³H]MDL 100,907, some 19 standard ligands to various receptors including the 5HT_1A, D₂, ₁, and receptors resulted in estimated K_I values that were consistent with [³H]MDL 100,907 selectively binding to the 5-HT_2A receptor. A comparison of the K_I values for 17 standard 5-HT_2A receptor agonists and antagonists displacing [³H]MDL 100,907 versus [³H]ketanserin resulted in a highly significant linear correlation (R² = 0.96, P<0.001). Taken together these results suggest that [³H]MDL 100,907 is binding to the 5-HT_2A receptor with a sub-nanomolar affinity without the use of secondary blocking agents.     

[3H]ICS 205-930 labels 5-HT3 recognition sites in membranes of cat and rabbit vagus nerve and superior cervical ganglion

Summary The binding characteristics of [³H]ICS 205-930, a 5-hydroxytryptamine 5-HT₃ receptor antagonist, were investigated in membranes prepared from cat and rabbit vagus nerve (VN) and superior cervical ganglion (SCG). The autoradiographic localisation of 5-HT₃ recognition sites was also assessed using [³H]ICS 205-930 in slices from cat medulla oblongata, nodose ganglion and vagus nerve.[³H]ICS 205-930 bound to a homogeneous population of high affinity recognition sites in cat VN: B_max = 201 ± 43 fmol/mg protein, pK_D = 9.26 ± 0.17 and SCG: B_max = 291 ± 40 fmol/mg, pK_D = 9.35 ± 0.80 (n = 3). Competition experiments performed in membranes from cat VN and SCG with agonists and antagonists suggested the presence of a homogeneous population of [³H]ICS 205-930 recognition sites. Competition curves were steep and monophasic and were best fitted by a 1 receptor site model. The following rank order of affinity for [³H]ICS 205-930 binding sites was observed with antagonists: SDZ 206-830 = ICS 205-930 > BRL 43694 > SDZ 206–792 > quipazine > MDL 72222 > metoclopramide > mCPP and agonists: 2-methyl-5-HT = 5-HT > phenylbiguanide. A similar profile was observed for a limited series of compounds in rabbit membranes. Drugs acting at 5-HT₁, 5-HT₂ and dopamine receptors (domperidone, spiperone and metergoline) showed very low affinities for [³H]ICS 205-930 recognition sites. The sites labelled with [³H]ICS 205-930 in vagus nerve and superior cervical ganglion of both species displayed the pharmacological profile of a 5-HT₃ receptor. There was a significant correlation between the rank order of affinity of the tested compounds for [³H]ICS 205-930 recognition sites in cat and rabbit membranes and their rank order of affinity for 5-HT₃ receptors from neuroblastoma-glioma NG 108-15 cells. Autoradiographic studies suggest that [³H]ICS 205-930 binding sites are present over and around the nodose ganglion cell somata, along certain fibers of the vagus nerve and in the terminal areas of this nerve in the medullar nucleus of the vagus.The present data demonstrate that [³H]ICS 205-930 identifies 5-HT₃ receptors in preparations of cat and rabbit vagus nerve and superior cervical ganglion.Send offprint requests to D. Hoyer at the above addressThe present results have been presented in part at the Winter Meeting of the British Pharmacological Society, London, December 20–22, 1988 (Hoyer et al. 1989)     

5.HT1 receptors in the vertebrate brain

Summary The regional distribution of high affinity [³3H]5-HT recognition sites in the brain of several vertebrates (pigeon, rat, mouse, guinea-pig, cat, dog, monkey and human) was analyzed using in vitro autoradiography. The presence of subtypes of 5-HT₁ binding sites was investigated by selective displacements with 8-OH-DPAT, mesulergine and (±)SDZ 21-009 at appropriate concentrations to block 5-HT_1A, 5-HT_1c and 5-HT_1B sites respectively. In addition, 5-HT_1A, and 5-HT_1c sites were directly visualized with the more selective radioligands [³H]8-OH-DPAT and [³H]mesulergine, respectively. In the pigeon brain, total [³H]5-HT binding sites were enriched in all telencephalic areas. Densely labelled regions were also present in the optic tectum and the brainstem. No binding was observed in the cerebellum. 8-OH-DPAT and mesulergine only displaced a small proportion of [³H]5-HT binding in most of the areas where high concentrations of 5-HT₁ sites were found. (±)SDZ 21-009 did not affect [³H]5-HT binding in the regions examined. Taking into account our pharmacological studies, these results suggest that the majority of 5-HT₁ sites belong to the 5-HT_1D subtype in the pigeon brain. In the mammalian species investigated high levels of [³H]5-HT binding were found in the neo-cortex, hippocampal formation, basal ganglia and related structures (substantia nigra), raphe dorsalis, nucleus superior colliculus and choroid plexus. However, these brain areas were differentially enriched in subtypes of 5-HT₁ recognition sites. 5-HT_1A sites were observed in the neo-cortex, the hippocampal formation and the raphe nucleus, whereas 5-HT_1C sites accounted for all 5-HT₁ binding in the choroid plexus. In the mouse and rat brain, 5-HT_1B binding sites were enriched in the basal ganglia and associated regions (substantia nigra). These areas were enriched in 5-HT_1D sites in the brain of the other mammals studied. In these animals, no site with a 5-HT_1B pharmacological profile were detected.These results indicate that 5-HT_1A 5-HT_1c and 5-HT_1D sites are present already in the lower vertebrate species investigated and that 5-HT_1B appear to be exclusive of the myomorph rodents (mouse, rat). Furthermore, the different subtypes of the 5-HT₁, receptors present a conserved regional distribution with the 5-HT_1D sites being enriched in the basal ganglia and the 5-HT_1A sites predominating in the hippocampal formation.     

Pharmacological comparison of the sigma recognition site labelled by [3H]haloperidol in human and rat cerebellum

Summary The radioligand binding characteristics of [³H]haloperidol (in the presence of spiperone, 25 nmolL^–1) were investigated in rat and human cerebellar membranes.In both rat and human cerebellar membrane preparations saturation studies with [³H]haloperidol (non-specific binding defined by pentazocine, 10 molL^–1) demonstrated high affinity saturable specific binding to a homogenous population of binding sites (rat, Bmax 6693 ± 1242 fmol mg^–1 protein, pK_D 8.33 ± 0.08; human, Bmax 2550 ± 437 fmol mg^–1 protein, pK_D 8.59 ± 0.11; mean ± SEM, n = 3–6). Competition studies employing a wide range of structurally diverse competing compounds displayed that the [³H]haloperidol binding site was pharmacologically similar in both preparations and comparable to sigma recognition sites previously identified in various tissues originating from different species. In addition, with reference to the potential subtypes of sigma recognition sites, the labelling of these sites by low nanomolar concentrations of [³H]haloperidol provides evidence that they belong to the sigma-1 recognition site subtype.The present findings suggest that the pharmacology of the rat and human cerebellar sigma recognition site are directly comparable and provides further supporting evidence towards the use of [³H]haloperidol radioligand binding studies in the rat to detect sigma receptor ligands with potential therapeutic activity. Send offprint requests to: N.M. Barnes at the above address     

Binding studies with [3H]cis-methyldioxolane in different tissues

Summary Special conditions - tricine buffer containing Ca²⁺ and Mg²⁺, 22°C (TCM) — allow to label a much higher proportion of muscarinic receptors by [³H]cis-methyldioxolane (CD) than hitherto described (Vickroy et al. 1984 a). Taking the maximum number of binding sites, B _max, of [³H]QNB as 100%, B _max of [³H]CD amounts to 83% in the rat heart instead of the reported 17%, 33% in the cerebral cortex instead of 6%, 20% in hippocampus and 55% in pons/medulla. In the salivary glands specific binding was negligible. The affinities of a number of muscarinic agonists and antagonists to [³H]CD and [³H]QNB binding sites in different tissues of the rat are compared. Apparent affinities of agonists are much higher in the [³H]CD system, affinities of antagonists are slightly higher in the [³H]QNB system. In both assay systems receptors of heart and pons/ medulla membranes seem to have similar drug specificity. They differ somewhat from those in the cortex. Receptors in the salivary glands, however, seem to be completely different from those in the other three tissues. In the heart [³H]CD binding can be abolished almost completely by GppNHp. In the cortex about half of the [³H]CD binding is susceptible to GppNHp. The reduction of binding in the cortex is due to a change in B _max and not in the dissociation constant K _D. Competition of unlabelled pirenzepine with [³H]CD: In heart and pons/medulla only low affinity sites for pirenzepine (M₂-receptors) are labelled by [³H]CD. In regions rich in M₁ receptors like hippocampus (80% M₁ receptors) or cortex (65–70% M₁ receptors) the proportion of M₁ receptors labelled by [³H]CD is smaller than expected considering the concentration of M₁ receptors present in these tissues. Thus [³H]CD, under the conditions described in this paper, seems to label preferentially but not exclusively M₂ receptors in their agonist high affinity form. Send offprint requests to A. Closse at the above address     

Quantitative [3H] dipyridamole autoradiography: evidence for adenosine transporter heterogeneity in guinea pig brain

Summary [³H] Dipyridamole binding in guinea pig brain slices has been characterized. Binding of [³H] dipyridamole to guinea pig forebrian slices was found to be rapid, reversible and saturable. Saturation experiments revealed a class of high affinity binding sites with a B _max value of 592 ± 118 fmol/mg protein and K _d value of 10.8 nM ± 2.1 nM in the analysed concentration range. In competition experiments, the adenosine transport inhibitors hexobendine and dipyridamole itself were the most potent displacers (inhibition constants of 4.6 nM ± 1 nM and 11.5 nM ± 3 nM) with pseudo-Hill coefficients close to 1. Competition curves with nitrobenzylthioinosine, another adenosine transport inhibitor, however, showed a biphasic profile with a pseudo-Hill coefficient of 0.33 ± 0.04. Just 42% ± 4% of [³H] dipyridamole binding were inhibited by nanomolar concentrations of nitrobenzylthioinosine and only micromolar concentrations displaced the remainder. Subsequent quantitative autoradiography demonstrated regional differences in the inhibition of [³H] dipyridamole binding by submicromolar concentrations of nitrobenzylthioinosine. While in cortical areas of cerebrum and cerebellum 500 nM nitrobenzylthioinosine displaced binding of [³H] dipyridamole to only about one-third of its sites (in the Purkinje cell layer less than 10%), it showed similar potency as dipyridamole in various areas of the brainstem and hypothalamus. This biphasic and regionally heterogenous interaction of nitrobenzylthioinosine with [³H] dipyridamole binding sites in guinea pig brain slices strongly suggests heterogeneity of adenosine transporters.     

The Antidepressant Metapramine is a Low-affinity Antagonist at N-methyl-d-aspartic Acid Receptors

Metapramine, a pharmacological compound with antidepressant activity in humans, was tested for possible antiglutamatergic activity, in vitro. We investigated the effects of metapramine on the N-methyl-d-aspartic acid (NMDA) receptor complex, by determining whether this compound would interfere with the binding of [³H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([³H]TCP) to rat cortical membranes in the presence of either glycine NMDA, or both. Metapramine in the micromolar range inhibited the binding of [³H]TCP in the presence of both NMDA and glycine (ic₅₀ = 1.4 ± 0.2 μM). That very similar affinities were observed when either NMDA or glycine was present suggests that metapramine exerted a direct action at the PCP site. The affinity of metapramine for this site was about 25 and 350 times lower than that of PCP and MK-801, respectively. Metapramine inhibited the NMDA-evoked increase in guanosine 3′,5′-cyclic monophosphate (cGMP) levels of neonatal rat cerebellar slices (ic₅₀ = 13 μM). These results suggest that metapramine is a low-affinity antagonist of the NMDA receptor complex channel. This paper discusses the potential application of metapramine to the treatment of diseases linked to excessive stimulation of glutamatergic NMDA receptors. © 1997 Elsevier Science Ltd. All rights reserved.     

In vivo affinity of [<Superscript>18</Superscript>F]fallypride for striatal and extrastriatal dopamine D<Subscript>2</Subscript> receptors in nonhuman primates

Rationale [¹⁸F]Fallypride is a new and promising radiotracer, suitable for imaging D₂ receptors with Positron Emission Tomography (PET) in both striatal and extrastriatal regions. The high signal to noise ratio of [¹⁸F]fallypride has been attributed to its high affinity for D₂ receptors (K_D of 0.03 nM, measured in vitro at room temperature).Objectives We sought to further characterize this tracer in terms of its in vivo affinity, possible affinity differences between brain regions and dependence of in vitro affinity on temperature.Methods PET scans were performed in baboons over a wide range of concentrations to measure the in vivo K_D of [¹⁸F]fallypride in striatal and extrastriatal regions. Several analytical approaches were used, including nonlinear kinetic modeling and equilibrium methods. Also, in vitro assays were performed at 22 and 37°C.Results No significant differences in the in vivo K_D were detected between regions. In vivo K_D of [¹⁸F]fallypride was 0.22±0.05 nM in striatum, 0.17±0.05 nM in thalamus, and 0.21±0.07 nM in hippocampus. These values were intermediate between in vitro K_D measured at 22 (0.04±0.03 nM) and 37 degrees (2.03±1.07 nM).Conclusion The in vivo affinity of [¹⁸F]fallypride was not as high as previously estimated from in vitro values. This property might contribute to the favorable kinetic properties of the tracer. The in vivo affinity was similar between striatal and extrastriatal regions. This result indicates that the measured regional in vivo affinities of this tracer are not affected by putative regional differences in endogenous dopamine, and that [¹⁸F]fallypride is an appropriate tool to provide unbiased estimates of the occupancy of D₂ receptors by antipsychotic drugs in striatal and extrastriatal regions.     

5-HT4 receptor antagonist affinities of SB207710, SB205008, and SB203186 in the human colon,rat oesophagus,and guinea-pig ileum peristaltic reflex

Functional 5-HT₄ receptors have been reported to be present in numerous isolated tissue preparations including the rat oesophagus, guineapig ileum, and human colon. The pharmacological properties of the novel, potent and selective 5-HT₄ receptor antagonists SB203186 (1-piperidinyl)ethyl 1H-indole 3-carboxylate, SB205008 (1-butyl-1-methyl4-piperidinylmethyl)-8-amino-7-chloro-1,4-benzodioxan-5-carboxylate iodide, and SB207710 (1-butyl-4-piperidinylmethyl)-8-amino-7-iodo-1,4 benzodioxan-5-carboxylate) were studied in these tissues. The nature of antagonism of the 5-HT-induced effects was investigated on the above isolated tissue preparations.5-HT produced its effect with the following EC₅₀ values: 400 ± 0.4 nM (rat oesophagus, n = 20), 154 ±14 nM (guinea-pig ileum, n = 9) and 144 ± 0.1 nM (hu man colon, n = 9). SB207710 (0.03–1 nM), SB205008 (1.0–10 nM), and SB203186 (10–100 nM) antagonised the 5-HT₄ receptor-mediated relaxations of the carbachol-contracted rat isolated oesophagus against 5-HT with pK_B values of 10.9 ± 0.1, 9.5 ± 0.1, and 9.0 ± 0.1 respectively without effecting the maximum response. On the guinea-pig ileum peristaltic reflex preparation, SB207710 (0.01–1 nM) did not modify the reflex but it behaved as an antagonist of the 5-HT-induced facilitation with a pA₂ value of 9.9 ± 0.2. The agonists DAU 6236 (endo-8-methyl-8-azabicyclo [3.2.1] oct-3-yl 2,3-dihydro-3-ethyl-2-oxo-1H-benzimidazole-1-carboxylate hydrochloride) at 2 M, and SC 53116 (1-S,8-S)-4-amino-5-chloro-N- [(hexahydro-1H-pyrrolizin-1-yl)methyl]-2-methoxy-benzamide hydrochloride at 500 nM facilitated the peristaltic reflex. SB207710 (0.25–5 nM), SB205008 (1–100 nM) and SB203186 (10–1000 nM) failed to modify the spontaneous activity of the circular muscle of the human colon, but caused parallel, dextral shifts in the concentration-effect curves to 5-HT with apparent pK_B values of 10.0 ± 0.3 (0.25 nM), 9.8 ± 0.2 (1 nM), and 8.6 ± 0.3 (10 nM) respectively. Higher concentrations of each antagonist shifted the concentration-effect curves to 5-HT to the right but this antagonism did not appear to be simple competitive. Methysergide (10 M) and ondansetron (10 M) increased the activity of SB207710 (5 nM) in the human colon giving rise to a 4 point (0.25, 0.5, 1,5 nM) Schild slope not significantly different from unity and a pK_B of 10.1 ± 0.1. Cocaine (30 M). did not significantly affect the activity of SB207710.The compounds SB207710, SB205008 and SB 203186 were shown to be high affinity 5-HT₄ receptor antagonists. SB207710 is the most potent 5-HT₄ receptor antagonist described so far and as such, is a potentially useful tool for 5-HT₄ receptor characterisation in functional studies of tissue from man and other species.     

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     

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.     

Comparison of 5-HT4 receptors in guinea-pig colon and rat oesophagus: effects of novel agonists and antagonists

5-HT₄ receptors in isolated distal colon myenteric plexus of guinea-pig, mediating contraction of longitudinal smooth muscle, have been further characterized by selective agonists and antagonists. The indole agonists, 5-HT and 5-methoxytryptamine (5-MeOT), were full agonists (relative to 5-HT) with potency values (pEC₅₀) of 8.0 ± 0.1 (n = 50) and 7.8 ± 0.1 (n = 12), respectively. 5-HT₄ receptor agonists of other structural classes, including benzimidazolones (BIMU 1 and BIMU 8), and benzamides ((S)-zacopride, (R)-zacopride, renzapride, SC 49518) were partial agonists with intrinsic activities less than that of 5-HT. In general, the potencies for these compounds at 5-HT₄ receptors in guinea-pig colon were similar to the potencies seen in the rat isolated oesophagus, where 5-HT₄ receptors mediate relaxation.GR 113808 {[1-[2-[(methylsulfonyl)amino]ethyl]-4-piperidinyl]methyl1-methyl-1H-indole-3-carboxylat}, RS 39604 {1-[4-amino-5-chloro-2-(3,5-dimethoxybenzyloxy)phenyl]-3-[1-[2-[(methylsulfonyl)amino] ethyl]-4-piperidinyl]-1-propanone hydrochloride and SB 204070 {(1-n-butyl-4-piperidinyl)methyl 8-amino-7-chloro-1,4-benzodioxane-5-carboxylate} antagonized 5-HT responses with pA₂ values of 9.1 ± 0.1, 9.0 ± 0.2 and 11.0 ± 0.1, respectively. These affinity values were similar to those obtained at 5-HT₄ receptors in isolated rat oesophagus (9.0 ± 0.4, 9.3 ± 0.1 and 10.6 ± 0.1, respectively).Despite these operational similarities between 5-HT₄ receptors in guinea-pig colon and rat oesophagus, several novel compounds have revealed important differences between 5-HT₄ receptors in the two tissues. For example, the substituted benzoate, RS 23597 {3-(piperidine-1-yl) propyl-4-amino-5-chloro-2-methoxybenzoate hydrochloride, acted as a partial agonist (intrinsic activity 0.5) in guinea-pig colon with a potency of 7.6 ± 0.1 (n = 16). In isolated rat oesophagus, however, this compound was a surmountable antagonist (pA₂ = 7.8 ± 0.1) with no intrinsic activity. In contrast, the substituted naphthalimide (S)RS 56532 {(S)6-amino-5-chloro-2-(1-azabicyclo[2, 2, 2]octan-3-yl)2,3-dihydro-1H-benz isoquinoline-1,3-dione hydrochloride}, was a potent (pEC₅₀ = 7.9 ± 0.1), efficacious partial agonist (intrinsic activity = 0.8) in the rat oesophagus. However, in guinea-pig colon, it was a surmountable antagonist with an affinity (pK_B) of 9.4 ± 0.1. Furthermore, several novel, selective, 5-HT₄ compounds also showed opposing patterns of intrinsic activities similar to those described for RS 23597 and (S)RS 56532.It is concluded that these differences are inconsistent with differences in 5-HT₄ receptor reserves, and may suggest that 5-HT₄ receptors in the guinea-pig colon and the rat oesophagus can be operationally distinguished.     

Allosteric modulation of 5-HT(1A) receptors by zinc: Binding studies

5-HT_1A receptors were studied via [³H]WAY-100635 and [³H]8-OH-DPAT binding to rat brain cortical membranes. We characterized the effect of zinc (Zn²⁺) on the binding properties of the 5-HT_1A receptor. The allosteric ternary complex model was applied to determine the dissociation constant (K_A) of Zn²⁺ and their cooperativity factors (α) affecting the dissociation constants (K_D, K_i) of [³H]WAY-100635, [³H]8-OH-DPAT, and serotonin (5-HT), the endogenous neurotransmitter. Zn²⁺ (5 μM-1 mM) inhibited the binding of agonist/antagonist to 5-HT1A receptors, mostly by decreasing both the ligands' affinity and the maximal number of sites. In [³⁵S]GTPγS binding assays Zn²⁺ behaved as insourmountable antagonist of 5-HT1A receptors, in agreement with radioligand binding assays. The residues involved in the formation of the inhibitory binding site on the 5-HT1A receptor were assessed by using N-ethyl-maleimide (NEM) or diethylpyrocarbonate (DEPC) which modify preferentially cysteine and histidine residues, respectively. Exposure to both agents did not block the negative allosteric effects of Zn²⁺ on agonist and antagonist binding. Our findings represent the first quantitative analysis of allosteric binding interactions for 5-HT_1A receptors. The physiological significance of Zn²⁺ modulation of 5-HT_1A receptors is unclear, but the colocalization of 5-HT_1A receptors and Zn²⁺ in the nervous system (e.g. in the hippocampus and cerebral cortex) suggests that Zn²⁺ released at nerve terminals may modulate signals generated by the 5-HT_1A receptors in vivo. Finally, these findings suggest that synaptic Zn²⁺ may be a factor influencing the effectiveness of therapies that rely on 5-HT_1A receptor activity.     

Binding of [H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes — a new, selective antagonist radioligand for A2A adenosine receptors

The present study describes the preparation and binding properties of a new, potent, and selective A_2A adenosine receptor (AR) antagonist radioligand, [³H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine ([³H]MSX-2). [³H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [³H]MSX-2 labeled a single class of binding sites with high affinity (K_d=8.0 nM) and limited capacity (B_max=1.16 fmol·mg⁻¹ of protein). The presence of 100 μM GTP, or 10 mM magnesium chloride, respectively, had no effect on [³H]MSX-2 binding. AR agonists competed with the binding of 1 nM [³H]MSX-2 with the following order of potency: 5′-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS-21680)>2-chloroadenosine (2-CADO)>N⁶-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3,7-dimethyl-1-propargylxanthine (BS-DMPX)>1,3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5,6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K_i values for antagonists were in accordance with data from binding studies with the agonist radioligand [³H]CGS21680, while agonist affinities were 3–7-fold lower. [³H]MSX-2 is a highly selective A_2A AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20–30%, at 1 nM.     

Increasing effect of ethanol on 5-HT3 receptor-mediated 14C-guanidinium influx in N1E-115 neuroblastoma cells

NlE-115 mouse neuroblastoma cells were used to study the influence of ethanol on the 5-HT- and veratridine-induced influx of ¹⁴C-guanidinium via the 5-HT₃ receptor channel and the fast sodium channel, respectively.Ethanol (10–100 mM) concentration-dependently increased the 5-HT-induced ¹⁴C-guanidinium influx, leaving the basal and veratridine (100 M)-induced influx unaffected. The increasing effect of ethanol (100 mM) was observed at all 5-HT concentrations investigated; accordingly, ethanol increased the maximum response to 5-HT. Whereas in the absence of ethanol the concentration-response curve for 5-HT was bell-shaped, this was no longer the case when ethanol (100 mM) was present in the incubation buffer; the descending branch of the concentration-response curve for 5-HT at concentrations above 300 M was virtually no longer observed. When, in the presence of substance P 10 M the 5-HT-induced ¹⁴C-guanidinium influx was already enhanced, the ability of ethanol (100 mM) to increase the 5-HT-induced influx was considerably diminished (by 72%). Preincubation of N1E-155 cells with 5-HT caused a decay of the subsequent 5-HT response (desensitization) which was dependent on the duration of preincubation; ethanol (100 mM) did not affect the rate of this decay of the 5-HT response. The 5-HT (30M)-induced ¹⁴C-guanidinium influx was also increased by methanol (100 mM) and n-propanol (100 mM). The rank order of the increasing effect of the n-alkanols (at 100 mM) was: methanol < ethanol < n-propanol; i.e. the degree of enhancement increased with the lipophilicity of the alcohols. Ethanol (100 mM) did not alter the time-course of non-specific and specific binding of the selective 5-HT₃ receptor antagonist ³H-GR 65630 to intact N1E-155 cells. In competition binding experiments, inhibition of ³H-GR 65630 binding by 5-HT was characterized by a high affinity (pIC₅₀ = 4.71 ± 0.19) and a low affinity component (pIC₅₀ = 2.65 ± 0.03). Ethanol did not affect the 5-HT-induced inhibition of ³H-GR 65630 binding.These results indicate that ethanol does not interfere with the 5-HT recognition site of the 5-HT₃ receptor. Since the enhancement of cation influx by alcohols increased in proportion to their lipophilicity, it is suggested that ethanol and other n-alkanols interact with a hydrophobic region of the 5-HT₃ receptor channel. In analogy to its effect on the nicotinic receptor channel, ethanol may stabilize the open state of the 5-HT₃ receptor channel.     

Properties of agonist binding at theβ-adrenoceptor of the rat reticulocyte

Summary To study the fundamental differences between agonist and antagonist interaction with the -adrenoceptor of the rat reticulocyte the radiolabeled agonist³H hydroxybenzylisoprenaline (³H HBI) and the radiolabeled antagonist³H dihydroalprenolol (³H DHA) were used.Equilibrium binding experiments with³H HBI revealed all characteristics expected to a -adrenoceptor site, i. e. high affinity binding (K _{D high}=7.4±0.9×10^–9 M), saturability (B _{max high}=230±24 fmoles/mg protein), and stereoselectivity. The rank order of potency for competing agonists was isoprenaline > adrenaline > noradrenaline > dopamine.³H HBI high affinity binding sites amounted to about 25% of -adrenoceptor sites detectable with³H DHA.In competition experiments with³H HBI and (-)isoprenaline[(-)Ipn]aK _{D high}-value for (-)Ipn of 3.1±0.6×10^–8M was obtained corresponding to theK _{D high}-value of (-)Ipn obtained from competition experiments using³H DHA. For (-)propranololK _D-values of 0.9±0.5×10^–8 M and 1.0 ±1.0×10^–8 M were measured using³H HBI and³H DHA respectively.Agonist affinity derived from competition experiments with (-)Ipn versus³H DHA was not affected by temperature changes.Guanylyl-imidodiphosphate [Gpp(NH)p] decreased concentration dependently the number of high affinity binding sites of³H HBI not affecting the respectiveK _D-value. Similar effects were observed after omission of Mg²⁺ from the binding assay or inclusion of Na⁺ in the Mg²⁺-free incubation mixture.The association reaction of³H HBI at the -adrenoceptor revealed two different velocities. The slower phase of the association reaction which represents high affinity binding (80% of equilibrium binding) is not observed in the presence of Gpp(NH)p.A biphasic dissociation of³H HBI binding was induced by 10^–4 M (±)propranolol: 25% dissociated with at _1/2 of 1.3 min whereas the high affinity binding was reversed with at _1/2 of 150 min. This slowly reversible binding of³H HBI however was rapidly reversed by Gpp(NH)p (t _1/2<1 min).It is concluded that the agonist ligand³H HBI permits a direct qualitative and quantitative characterization of the agonist induced high affinity state of the -adrenoceptor. In particular, the kinetic studies strongly support a two step binding model for the agonist--adrenoceptor interaction.This work was supported by a grant from the Deutsche ForschungsgemeinschaftParts of this work were presented at the Spring Meetings of the German Pharmacological Society (Wiemer et al. 1978, 1981 b)Herrn Professor Dr. med. Hans Herken, Pharmakologisches Institut der Freien Universität Berlin, zum 70. Geburtstag gewidmet.     

Topographical distribution of 5-HT3 receptor recognition sites in the ferret brain stem

Summary The distribution of [³H]zacopride (1.0 nM) to putative 5-HT₃ receptor recognition sites in the ferret hindbrain was assessed using autoradiography. Specific binding (defined by the inclusion of granisetron, 1.0 M) was heterogeneously distributed with highest density within the dorsal vagal complex (area postrema, nucleus tractus solitarius and dorsal motor nucleus of the vagus nerve). Lower densities were detected in the spinal trigeminal nerve complex whilst no other significant specific binding was detected ventral to the dorsal vagal complex. The location of 5-HT₃ receptor recognition sites within the dorsal vagal complex may provide sites of action for zacopride and other 5-HT₃ receptor antagonists to inhibit the emesis induced by cancer chemotherapeutic agents and x-radiation. Send offprint requests to N. M. Barnes at the above address     

The 5-HT3 receptor ligand, [H]BRL 43694, binds to presynaptic sites in the nucleus tractus solitarius of the rat

This study has employed receptor autoradiography to localise the distribution of binding sites for the 5-HT₃ receptor ligand [³H]BRL 43694 in sections of the brain of the rat (using a concentration of 10 nM [³H]BRL 43694 with 100 μM GR38032F to define non-specific binding). The highest density of binding sites for [³H]BRL 43694 was observed in the nucleus tractus solitarius and amounted to 652 fmol/mg tissue. The binding of [³H]BRL 43694 was also examined in sections prepared 10 days after unilateral nodose ganglionectomy, in an attempt to determine the neuronal location of these binding sites. Denervation reduced the binding of [³H]BRL 43694 by around 50% in the ipsilateral side of the nucleus tractus solitarius, relative to the contralateral side. This would indicate that the 5-HT₂ binding sites may have a presynaptic location on vagal afferent terminals.