Binding properties of [3H]gacyclidine (cis(pip/me)-1-[1-(2-thienyl)-2-methylcyclohexyl]piperidine) enantiomers in the rat central nervous system

Gacyclidine (cis(pip/me)-1-[1-(2-thienyl)-2-methylcyclohexyl]piperidine) is a TCP derivative, which exhibits potent neuroprotective properties against glutamate-induced neurotoxicity in vitro and in vivo. In order to better understand gacyclidine pharmacological properties, the binding parameters of its enantiomers ((-) and (+)[3H]GK11) were determined in the rat central nervous system (CNS). An autoradiographic study has shown that their binding distributions are correlated with those of N-methyl-D-aspartate (NMDA) receptors throughout the CNS. Globally, the labeling was the highest with (-)[3H]GK11. In the cerebellum, both radioligands similarly labeled the molecular layer. For both radioligands, on telencephalic, cerebellum and spinal cord homogenates, the association and dissociation kinetics were accounted for by multiphasic process. In all regions, (-)[3H]GK11 exhibited the highest affinity in the nanomolar range. The pharmacological study revealed that both enantiomers labeled both high and low affinity sites in all regions. The pharmacological profile of high affinity sites was correlated with those of NMDA receptors. Those of low affinity sites were different in telencephalic and cerebellar homogenates. Overall, this study showed that low affinity sites might constitute a heterogeneous population, which could include sigma receptors in the cerebellum. The autoradiographic study has shown that these sites may be located in the molecular layer. The contribution of low affinity sites to the neuroprotective properties of gacyclidine remains to be investigated.     

Re-evaluation of phencyclidine low-affinity or "non-NMDA" binding sites

TCP and its derivative gacyclidine (+/- GK11) are high-affinity non-competitive antagonists of N-methyl-D-aspartate (NMDA) receptors (NMDARs) and as such exhibit significant neuroprotective properties. These compounds also bind with a low affinity to binding sites whose pharmacological profiles are different from that of NMDARs. With the intention to develop new strategies of neuroprotection, we found it mandatory to investigate whether 1-[1-(2-thienyl)cyclohexyl]piperidine (TCP) and gacyclidine low-affinity sites are similar. The effects of several drugs selective for either NMDARs or the [(3)H]TCP low-affinity site (or PCP(3) site) on (+), (-)[(3)H]GK11 and [(3)H]TCP specific binding were investigated. Competition experiments on cerebellum homogenates revealed substantial differences between the pharmacological profiles of the PCP(3) site and that of gacyclidine's enantiomers low-affinity sites. Under experimental conditions preventing the interaction of the radioligands with NMDARs, the autoradiographic study showed, however, that the distributions of both [(3)H]TCP and (-)[(3)H]GK11 specific binding were similar. The specific labelling was low and uniform in telencephalic structures, whereas in the cerebellum it was higher in the molecular than in the granular layer. Finally, the analysis of competition experiments performed on tissues slices demonstrated that PCP(3) selective ligands were unable to prevent [(3)H]TCP or (-)[(3)H]GK11 binding to "non-NMDA" binding sites. As a whole, our data suggest that: (1) the different pharmacological profiles of [(3)H]TCP and [(3)H]gacyclidine enantiomers on low-affinity sites are due to their selectivity for specific NMDARs subpopulations; (2) the pharmacological isolation of TCP and gacyclidine "non-NMDA" binding sites is the most appropriate way to further study the low-affinity component of their specific binding. Obtaining reliable and specific pharmacological tools for those binding sites is of particular interest, since it is likely that they play a substantial role in the low neurotoxicity, and therefore tolerability, of gacyclidine, a new neuroprotective drug currently evaluated in clinical trials for the treatment of brain and spinal cord injuries.     

Autoradiographic distribution of α2 adrenoceptors, NAIBS, and 5-HT1A receptors in human brain using [H]idazoxan and [H]rauwolscine

The regional distribution of [³H]idazoxan and [³H]rauwolscine was studied autoradiographically in human brain. [³H]Idazoxan binds with high affinity to α₂ adrenoceptors as well as to non-adrenergic sites (NAIBS). [³H]Rauwolscine, besides binding to α₂ adrenoceptors, also binds to 5-HT_1A receptors. Both radioligands labelled the same population of α₂ adrenoceptors, defined as the epinephrine-displaceable binding component. The highest densities of α₂ adrenoceptors occur in the leptomeninges, cerebral cortex and claustrum; lower densities were visualised in the basal ganglia, thalamus, pons, substantia nigra, cerebellum and medulla oblongata; no α₂ adrenoceptors were detected in amygdala and nucleus ruber. NAIBS were present in all the examined brain areas, with the highest densities found in the basal ganglia and substantia nigra. The finding that certain brain regions, such as the amygdala, contained NAIBS but no detectable α₂ adrenoceptors, suggests that the binding sites are independent from each other. The regional distribution of 5-HT_1A receptors labelled by [³H]rauwolscine is in agreement with previous studies using [³H]8-OH-DPAT.     

Autoradiographic localization and quantification of dopamine D2 receptors in normal human brain with [H]N-n-propylnorapomorphine

The precise distribution of dopamine receptors has been studied autoradiographically in the normal human brain using [³H]N-n-propylnorapomorphine ([³H]NPA) as a ligand. Preliminary experiments aimed at optimizing the binding assay conditions revealed that preincubation washing of caudate nucleus sections was a prerequisite to obtain a good ratio of specific to non-specific binding. The binding of [³H]NPA to caudate-putamen sections was saturable, stereospecific, reversible, of high affinity (K_d = 0.27–0.35 nM) and occurred at a single population of sites. Competition experiments with various drugs indicated that in the caudate-putamen the specific [³H]NPA binding sites possess the pharmacological features of the dopamine D₂ receptor. The highest levels of [³H]NPA binding sites were found in the caudate nucleus, putamen, globus pallidus and nucleus accumbens. There were also intermediate to low concentrations of the ³H-ligand in the hippocampus, the insular and cingular cortices and in the occipito-temporal gyrus, while almost undetectable levels of binding were found in the anteior frontal cortex. Thorough examination of the subregional distribution of [³H]NPA binding sites in the caudate-putamen-pallidum complex revealed heterogenous patterns of radioactivity. In these brain regions, the distribution of autoradiographic grains was punctate and islands of high and low densities were observed. Moreover, in the caudate nucleus, there was a subtle high lateral to low medial gradient in the topography of the [³H]NPA binding sites and a more pronounced gradient along the rostrocaudal axis; the highest levels of binding being located at the midbody of the nucleus. No gradients of [³H]NPA binding were observed in the putamen. The present data indicate that [³H]NPA is a suitable ligand for accurate autoradiographic labeling of dopamine D₂ receptors in human postmortem brain tissue and that dopamine receptors are heterogeneously distributed and topographically organized in patches and gradients in the basal ganglia regions.     

Quantitative autoradiographic studies of dopamine D3 receptors in rat cerebellum using [I]S(−)5-OH-PIPAT

Recently, [¹²⁵I]S(−)5-OH-PIPAT (5-hydroxy-2-(N-n-propyl-N-3′-iodo-2′-propenyl)amino-tetralin) was reported to be a selective radioiodinated ligand for dopamine D₂-like receptors. This ligand displayed a high binding affinity (K_d=0.3–0.4 nM) and an agonist binding profile to dopamine D₂ and D₃ receptors expressed in HEK293 cells and dopamine D₄ receptors expressed in CHO cells. Herein, a series of studies to characterize D₃ receptors in native tissues is presented. Based on studies of the distribution of receptor mRNA, D₃, but not D₂, receptors are present in the rat cerebellum. Quantitative autoradiographic experiments using [¹²⁵I]S(−)5-OH-PIPAT to label molecular layers 9 and 10 of rat cerebellum were conducted. Saturation experiments demonstrated that [¹²⁵I]S(−)5-OH-PIPAT bound with high affinity (K_d=0.1 nM) to a low density (∼3 fmol/mg protein) of sites in molecular layers 9 and 10 of rat cerebellum. Increasing concentrations of Gpp(NH)p, but not ATP, decreased the specific binding of [¹²⁵I]S(−)5-OH-PIPAT in rat cerebellum slices. In comparison studies, binding of [¹²⁵I]NCQ298, a dopamine D₂/D₃ receptor antagonist, with a similar affinity (K_d=0.2 nM) for D₃ receptors as [¹²⁵I]S(−)5-OH-PIPAT, was not sensitive to Gpp(NH)p. Analysis of inhibition by S(−)5-OH-PIPAT of [¹²⁵I]NCQ298 binding to rat cerebellum resulted in two-site binding with IC₅₀ values of 0.07 nM and 6.0 nM. In the presence of GTP (300 μM), the data best fit a one-site model with an IC₅₀ value of 1.6 nM. Agonists and antagonists inhibited the binding of [¹²⁵I]S(−)5-OH-PIPAT in the cerebellum with a rank order of potency consistent with an interaction at D₃ receptors. These results indicate that [¹²⁵I]S(−)5-OH-PIPAT binds to D₃ receptors in rat cerebellum. Furthermore, [¹²⁵I]S(−)5-OH-PIPAT binds to GTP sensitive and GTP insensitive states of D₃ receptors with distinctive high and low affinity states, respectively.     

Effects of chronic treatment with (+)- and (−)-nicotine on nicotinic acetylcholine receptors and N-methyl-d-aspartate receptors in rat brain

In this study, the effects of chronic treatment with (+)-nicotine on brain nicotinic acetylcholine receptors (nAChRs) andN-methyl-d-aspartate (NMDA) receptors, as well as on animal body weight were compared with those of chronic treatment with (−)-nicotine. Male Sprague-Dawley rats were s.c. injected with saline, (+)-nicotine (2.0 mg free base/kg b.w.) or (−)-nicotine (0.45 mg free base/kg b.w.) for 18 days. Brain nAChRs were investigated by (-)-[³H]nicotine binding. A significant increase in the high-affinity (−)-[³H]nicotine (5 nM)-binding sites was observed the cortex, hippocampus, midbrain and striatum but not in the cerebellum of the rats treated with either (+)- or (−)-nicotine. The displacement curves of (−)-[³H]nicotine/(−)-nicotine in the cortices of rats treated with either (+)- or (−)-nicotine showed only one population of high-affinity binding sites, whereas both high- and low-affinity binding sites were observed in the cortices of control animals. Brain NMDA receptors were studied by [³H]MK-801, which binds to the NMDA receptor-ion channel complex. A significant decrease in theB_max, but not in theK_D for [³H]MK-801 binding in the cortices of rats treated with either (+)- or (−)-nicotine was only detected under certain experimental conditions where the NMDA receptors seem not to be maximally activated. The body weight of the animals treated with (−)-nicotine was significantly lower than that of the control animals, whereas there was no difference in body weight between (+)-nicotine- and saline-treated animals. These results show that (+)-nicotine treatment has an effect on nAChRs in rat brains which is similar to that of (−)-nicotine treatment, but an effect on body weight which differs from (−)-nicotine, and also suggest that nicotinic agonists may interact with brain NMDA receptors in vivo.     

[I]4-Aminobenzyl-5′-N-methylcarboxamidoadenosine ([I]AB-MECA) labels multiple adenosine receptor subtypes in rat brain

Adenosine modulates neuronal activity and neurotransmitter release through interaction with cell surface receptors. Four adenosine receptor subtypes, A₁, A_2A, A_2B, and A₃ receptors, have been cloned and characterized. The agonist ligand, [¹²⁵I]AB-MECA ([¹²⁵I]4-aminobenzyl-5′-N-methylcarboxamidoadenosine) has high affinity for recombinant A₁ and A₃ receptors [Olah et al., Mol. Pharmacol., 45 (1994) 978–982]. Rodent A₃ receptors are relatively insensitive to xanthines; inhibition of A₁ receptors with xanthines allows selective detection of A₃ receptors despite the lack of selectivity of the ligand. We studied whether [¹²⁵I]AB-MECA is useful for localization and characterization of A₃ receptors in rat brain. The autoradiographic distribution of total [¹²⁵I]AB-MECA (400 pM) binding closely resembled the pattern of A₁ receptor binding, with highest levels in cerebellum, hippocampus, and thalamus, and moderate levels in cortex and striatum. Drug competition studies confirmed that almost all [¹²⁵I]AB-MECA binding could be attributed to labeling of A₁ receptors. Xanthine amine congener (1 μM) reduced specific [¹²⁵I]AB-MECA binding by >95%, indicating that xanthine-resistant A₃ receptors represent a quantitatively minor subtype. Despite the use of a radioligand with high affinity and high specific activity, the low density of A₃ receptors in rat brain appears insufficient to allow localization, or even consistent detection, of this receptor subtype. In the presence of DPCPX (50 nM, to block A₁ receptors), residual [¹²⁵I]AB-MECA binding to A_2A receptors was observed in the striatum. Thus, [¹²⁵I]AB-MECA labels primarily A₁ and A_2A adenosine receptors in rat brain.     

Specific Binding of [H] phencyclidine in rat central nervous tissue: further characterization and technical considerations

The interaction of phencyclidine (PCP) with its specific receptor sites in the central nervous system has been further characterized. Kinetic association and dissociation rate constants of 2.9 × 10⁶ M⁻¹ and 4.8 × 10⁻¹ were determined, yielding a kinetic K_D of 1.6 × 10⁻⁷ M, in agreement with the K_D previously determined at equilibrium. Permissible separation time of 13 s was calculated from the kinetic data, well above the actual separation time of less than 10 s in the rapid filtration assay. Presoaking of filters in 0.01% poly-l-lysine eliminated displacable [³H]PCP adsorption to filter material. Binding data obtained via centrifigation assays was identical to that obtained with the rapid filtration method. Stereospecificity of the PCP receptor was demonstrated by the finding that (+)-ketamine is four-fold more potent than (−)-ketamine in displacing specifically bound [³H]PCP. Several proteolytic enzymes including trypsin, papain and thermolysin potently inactivated PCP receptors. Detailed regional distribution studies showed highest density of PCP receptors in subicular cortex and hippocampus, intermediate levels in hypothalamus, striatum, frontal cortex and cerebellum, lower levels in brainstem and spinal cord, and negligible levels in corpus callosum, a white- matter control area. Benzomorphan opiates with PCP-like behavioral effects interact with the PCP receptor. These data support the pharmacological relevance of the PCP receptor site as demonstrated by the rapid filtration method.     

In vivo binding of [123I]4-(2′-methoxy phenyl)-1-[2′-(N-2″-pyridinyl)-P-iodobenzamido-]ethyl-piperazine,p-MPPI,to 5-HT1A receptors in rat brain

The in vivo regional distribution and pharmacological profile of a novel iodinated phenylpiperazine derivative, [¹²³I]p-MPPI (4-(2′-methoxy-)phenyl-l-[2′-(N-2″pyridinyl)-p-iodobenzamido-]ethyl-piperazine), in the rat brain were evaluated for use as a potential in vivo imaging agent for 5-HT_1A receptors. The new ligand penetrated the blood-brain barrier quickly and efficiently, with 1.2% of the injected dose found in the whole brain at 2 min post i.v. injection. The rate of radioactivity washout was slowest from the hippocampal region, followed by the hypothalamus, cortex, striatum, and cerebellum. The maximum ratio of hippocampus/cerebellum was 3.3 at 30 min postinjection. The specific binding of the radioligand in the hippocampal region, an area rich in 5-HT_1A receptor density, was blocked by pretreatment with a dose of (±) 8-OH-DPAT (2 mg/kg, i.v.) or WAY 100635 (1 mg/kg, i.v.), whereas the regional distribution of [¹²³I]p-MPPI was unaffected by pretreatment with non-5-HT_1A agents such as ketanserin or haloperidol. Ex vivo autoradiographic studies further confirmed that the specific binding of [¹²³I]p- MPPI is associated with 5-HT_1A receptor sites. These results indicate that [¹²³I]p-MPPI may be a useful candidate for noninvasive single photon emission computed tomography (SPECT) imaging of 5-HT_1A receptor sites in the living human brain. © 1994 Wiley-Liss, Inc.     

Synthesis and characterization of two pet radioligands for the metabotropic glutamate 1 (mGlu1) receptor

The metabotropic glutamate 1 receptor (mGlu1) is an important protein in the regulation of glutamate transmission in the brain, and believed to be involved in disorders such as ischemia, epilepsy, neuropathic pain, anxiety, and schizophrenia. The goal of this study was to evaluate two selective mGlu1 antagonists [¹¹C] 3 and [¹⁸F] 4 as potential PET radioligands for the in vivo imaging of the mGlu1 receptor. Biodistribution studies in rats indicated high uptake of [¹¹C] 3 and [¹⁸F] 4 in the brain. The highest activity level was found in the cerebellum, followed by striatum, hippocampus, frontal cortex, and medulla, in a pattern consistent with the distribution of mGlu1 receptor in rat. At 30 min postinjection, the activity ratio of cerebellum to medulla was 4.5 for [¹¹C] 3 , indicating a high degree of specific binding, while specific binding was lower for [¹⁸F] 4 (cerebellum to medulla activity ratio of 2.0). Moreover, binding of the radioligands [¹¹C] 3 and [¹⁸F] 4 in mGlu1 receptor‐rich region such as cerebellum was blocked by pretreatment of the rats with their respective unlabeled compound or the selective mGlu1 antagonist (compound 5 , 2 mg/kg each), but not by the selective mGlu2 antagonist LY341495, or the selective mGlu5 antagonist MPEP (2 mg/kg), thus indicating the binding specificity and selectivity of [¹¹C] 3 and [¹⁸F] 4 to the mGlu1 receptor. However, in imaging experiments in baboons [¹¹C] 3 displayed a small specific binding signal only in the cerebellum, while the specific binding of [¹⁸F] 4 was difficult to detect. Species differences in receptor density and affinity of the radioligands in large part account for the differences in the behavior of [¹¹C] 3 and [¹⁸F] 4 in rats and baboons. Radioligands with higher affinity and/or lower lipophilicity are needed to successfully image the mGlu1 receptor in humans. Synapse, 2012. © 2012 Wiley Periodicals, Inc.     

Differential radioligand binding properties of [H]5-hydroxytryptamine and [H]mesulergine in a clonal 5-hydroxytryptamine1C cell line

[³H]5-Hydroxytryptamine ([³H]5-HT) and [³H]mesulergine were used to label 5-HT_1C receptors expressed in NIH 3T3 mouse fibroblast cells. Using a rapid filtration assay, saturation analysis of the [³H]5-HT radioligand data indicate that the binding is biphasic. Based on computerized analysis of the data, a 2-site model of radioligand binding is significantly more consistent with the data than a one-site model (P < 0.01). The K_D values of [³H]5-HT for the 2 populations are0.5±0.1nM and31±15nM, while the B_max values are400±90pmol/g protein and 3,000±600 pmol/g protein, respectively. A biphasic binding pattern is also observed with [³H]5-HT using a centrifugation assay (K_D1 = 0.6±0.06nM, K_D2 = 60±10nM;B_max1 = 740±90pmol/g, B_max2 = 4,000±700pmol/g). By contrast, saturation analysis of [³H]mesulergine binding is monophasic (K_D = 4.7±0.7nM) with a B_max value (6,800±1,000pmol/g protein) that is significantly greater than that obtained using [³H]5-HT (P < 0.01). Drug competition studies confirm that both [³H]5-HT and [³H]mesulergine label at least 2 subpopulations of expressed 5-HT_1C receptors in NIH 3T3 cells. 10⁻⁴ M GTP eliminates the high affinity [³H]5-HT-labeled binding sites with minimal effect on the low affinity [³H]5-HT-labeled sites and no effect on [³H]mesulergine-labeled sites. These data demonstrate that at least 2 distinct subpopulations of 5-HT_1C receptors in NIH 3T3 cells can be differentiated using radioligand binding techniques.     

Autoradiographic comparison of D1-specific binding of [H]SCH39166 and SCH23390 in the primate cerebral cortex

Quantitative autoradiography was used to compare the binding of the novel dopamine D₁ receptor antagonist, [³H]SCH39166, with that of the widely used radioligand, [³H]SCH23390 (in the presence of ritanserin), in the primate cerebral cortex. Specific binding of both radioligands, determined using SCH23390 or cis-flupentixol as displacing agents, had very similar densities and distributions throughout the cortex. However, the specific binding of [³H]SCH39166 obtained with SCH39166 as a blank was significantly higher than that obtained using SCH23390 or cis-flupentixol as displacing agents in some layers of motor, somatosensory and occipital cortices. In addition, the non-specific binding of [³H]SCH39156 obtained in the presence of an excess of SCH23390 of cis-flupentixol displayed a complex laminar pattern very different from that of the specific binding. These observations suggest that [³H]SCH39166 may have a high affinity to more than the D₁ receptor subtype bound by SCH23390 or cis-flupentixol. Also, these additional sites are likely to be different from 5-HT₂ or 5-HT_1C receptors since the latter sites were not displaced by 1 μM SCH23390.     

RTI-4793-14, a new ligand with high affinity and selectivity for the (+)-MK801-insensitive [3H]1-[1-(2 thienyl)cyclohexyl]piperidine binding site (PCP site 2) of guinea pig brain

[³H]TCP, an analog of the dissociative anesthetic phencyclidine (PCP), binds with high affinity to two sites in guinea pig brain membranes, one that is MK-801 sensitive and one that is not. The MK-801-sensitive site (PCP site 1) is associated with NMDA receptors, whereas the MK-801-insensitive site (PCP site 2) may be associated with biogenic amine transporters (BAT). Although several “BAT ligands” are known that bind selectively to PCP site 2 and not to PCP site 1 (such as indatraline), these compounds have low affinity for site 2 (K_i values > 1 μ). Here we demonstrate that the novel pyrrole RTI-4793-14 is a selective, high affinity ligand for PCP site 2. We determined the IC₅₀ values of RTI-4793-14 and several reference compounds [PCP, (+)-MK801 and indatraline] for PCP site 1 (assayed with [³H](+)-MK801), PCP site 2 (assayed with [³H]TCP in the presence of 500 nM (+)-MK801) and a variety of BAT-related measures ([³H]CFT binding to the DA transporter, [³H]nisoxetine binding to the norepinephrine transporter, [³H]dopamine uptake, [3H]serotonin uptake). In addition, we determined the ability of RTI-4793-14 to block NMDA responses in cultured hippocampal neurons under voltage clamp. (+)-MK801 had high affinity for PCP site 1 (4.6 nM) and potently inhibited NMDA-induced responses, but was much less potent in the BAT-related measures (IC₅₀s > 10 μ). PCP had high affinity at PCP site 1 (IC₅₀ = 92 nM) and PCP site 2 (IC₅₀ = 117 nM), and was moderately potent in all BAT-related measures except [³H]nisoxetine binding. Indatraline was potent in BAT-related measures (IC50s, 2 to 5 nM), but weak in other measures (IC₅₀s > 1 μ). In contrast, RTI-4793-14 had high affinity for PCP site 2 (38 nM), low affinity for PCP site 1 (> 36 μ), moderate IC₅₀s for all BAT-related measures, and negligible activity at NMDA receptors. Viewed collectively, these data indicate that RTI-4793-14 binds with high affinity and selectivity to PCP site 2 and provide further support for an association between PCP site 2 and the BATs. © 1994 Wiley-Liss, Inc.     

ß1-Adrenergic receptors in human neuroblastoma

ß-Adrenergic receptors and adenylate cyclase activity were identified and partially characterized in 10 human neuroblastoma tumors obtained from direct biopsy, from human cell lines grown in the athymic, nude mouse or from established tumor cell lines in tissue culture. Particulate membrane fractions of eight of the tumors contained ß-adrenergic receptor sites identified by the ß-adrenergic antagonists (−)-[³H]dihyroalprenolol ((−)-[³H]DHA) and (−)-[¹²⁵I]iodohydroxybenzylphindolol ((−)-[¹²⁵I]HYP). Specific binding of high affinity sites (B_max) varied greatly among the tumors ranging from non-detectable concentrations to approximately 130 fmol/mg of crude membrane protein. Analysis of the saturation experiments with (−)-[³H]DHA resulted in curvilinear Scatchard plots in the tumors, suggesting the presence of multiple classes of sites and there was no evidence of cooperativity in dissociation experiments with (−)-[³H]DHA. The high affinity class of sites were inhibited by catecholamines, in contrast to the ineffectiveness of catecholamines in inhibiting (−)-[³H]DHA binding at the low affinity class of sites. TheK_D of the high affinity site was approximately 1–2 nM for (−)-[³H]DHA and was similar in all the tumors studied. A single class of sites was demonstrated with (−)-[¹²⁵I]HYP, in several of the tumors studied,K_D approximately 120 pM, and the number of sites determined with (—)-[¹²⁵I]HYP was identical to the number of high affinity sites determined with (−)-[³H]DHA. The properties of the receptor were characterized in several tumor lines: SK-N-MC tumor line contained both a ß-adrenergic receptor site and catecholamine sensitive adenylate cyclase activity. Agonists competed for the ß-adrenergic sites in the order of potency (−)-isoproterenol > (−)-norepinephrine = (−)-epinephrine > > (−)-dopamine characteristic of a ß-adrenergic receptor subtype. The affinity of the receptor for the selective agents, metropol (ß₁-selective antagonist) and zinterol (ß₂-selective agonist) was typical of a homogeneous ß₁-adrenergic site. Guanine nucleotides, guanine triphosphate and guanyl-5′-yl-imidophosphate, decreased catecholamine affinity for the receptor site in several tumors studied and enhanced maximal catecholamine stimulated adenylate cyclase activity in the sensitive cell line. Adenylate cyclase activity in particulate fractions of most of the neuroblastoma tumors was responsive to prostaglandins (PGE₁), guanine nucleotide and NaF. While most tumors contained ß-adrenergic receptor sites only SK-N-MC was clearly catecholamine responsive as assessed by catecholamine-sensitive adenylate cyclase activity. Human neuroblastomas, a homogenous tissue of neural crest origin, contained ß-adrenergic receptor sites which varied greatly in number (B_max) and in their ability to stimulate adenylate cyclase activity. Guanine nucleotides decreased agonist affinity for the receptor in the lines tested and enhanced prostaglandin and catecholamine sensitive activity in responsive tumor lines.     

Autoradiographic study of NMDA-displaceable [H]glutamate and [H]MK-801 binding during butorphanol withdrawal in the rat brain

Influences of continuous administration of butorphanol on the autoradiography of [³H]glutamate binding and [³H]MK-801 binding were investigated to study the effects of butorphanol withdrawal on NMDA receptors. Rats were administered butorphanol (26 nmol μl⁻¹ h⁻¹) by continuous intracerebroventricular (i.c.v.) infusion through pre-implanted cannula connected to osmotic mini-pumps for 3 days. Rats were then sacrificed at 2, 7, and 24 h after discontinuation of butorphanol infusion. [³H]MK-801 binding was slightly increased in the cortical area, hippocampus, and cerebellum in 2, 7, and 24 h withdrawal groups and was shown most significant increase in the 7 h withdrawal group. NMDA-displaceable [³H]glutamate binding was markedly increased in the cortical area, striatum, septum, hippocampus, thalamus, and cerebellum in 7 h withdrawal group and was significantly increased in the striatum, hippocampus, and thalamus in 24 h withdrawal group. These results demonstrate that the development of butorphanol withdrawal is more prominent by 7 h after discontinuation of butorphanol infusion and suggest that NMDA binding sites at NMDA receptors may play more important role in the development of butorphanol withdrawal than that of channel blocking sites.     

A single (−)-nicotine injection causes change with a time delay in the affinity of striatal D2 receptors for antagonist, but not for agonist, nor in the D2 receptor mRNA levels in the rat substantia nigra

The in vitro and in vivo effects of (−)-nicotine on dopamine D₂ receptors in the rat neostriatum have been studied using biochemical binding, in situ hybridization and immunocytochemistry. A single i.p. injection (1 mg/kg) of (−)-nicotine resulted in a reduction of theK_D value of the D₂ antagonist [³H]raclopride binding sites in rat neostriatal membrane preparations at 12 h without any significant change in theB_max value. This action of (−)-nicotine was counteracted by pretreatment 15 min earlier with the nicotine antagonist mecamylamine (1 mg/kg, i.p.). However, theK_D and theB_max values of the D₂ agonist [³H]NPA binding sites in the rat neostriatal membrane preparations were not significantly affected 0.5–48 h after a single i.p. injection with 1 mg/kg of (−)-nicotine. No significant change in neostriatal D₂ receptor mRNA levels was observed at any time interval after the (−)-nicotine injection. No significant change was observed in tyrosine hydroxylase (TH) immunoreactivity in either the substantia nigra or the neostriatum, nor in nigral TH mRNA levels during the time interval studied (4–24 h posttreatment). Furthermore, addition of low (10 nM) or high (1 μM) concentrations of (−)-nicotine in vitro to rat neostriatal membranes did not alter the characteristics of [³H]raclopride or [³H]NPA binding. These results indicate that a single (−)-nicotine injection can produce a selective and delayed increase in the affinity of D₂ receptors for the antagonist, but not for the agonist without modifying the levels of D₂ receptor mRNA, probably via the activation of central nicotinic receptors.     

Autoradiographic localization of D1 dopamine receptors in the rat brain with [H]SCH 23390

The regional distribution of D₁ dopamine (DA) receptors in the rat brain has been studied by quantitative autoradiography using the specific D₁ antagonist [³H]SCH 23390 as a ligand. The binding of [³H]SCH 23390 to striatal sections was saturable, stereospecific, reversible and of high affinity (K_d = 2.05nM); it occurred at single population of sites and possessed the pharmacological features of the D₁ DA receptor. The highest densities of [³H]SCH 23390 binding sites were found in the caudate-putamen, olfactory tubercle, nucleus accumbens and substantia nigra (especially in the pars compacta). High densities were also observed in the nucleus interstitialis striae terminalis, the anterior olfactory nucleus, the entopeduncular nucleus, the subthalamic nucleus, the claustrum and the amygdalohippocampal area. An intermediate labelling was found in the anteromedial and suprarhinal DA terminal fields of the cerebral cortex, the basolateral, medial and lateral amygdaloid nuclei, the endopiriform nucleus, the primary olfactory cortex, the globus pallidus, the superior colliculus (especially the superficial layer), the nucleus amygdaloideus corticalis and the dorsal hippocampus (molecular layer of the CA₁ and dentate gyrus). In the anteromedial and suprarhinal cortices, [³H]SCH 23390 binding was more concentrated in layers V and VI. Moderate levels of [³H]SCH 23390 were found in the thalamus, hypothalamus, the habenula, the ventral tegmental area, the posterior cingulate and entorhinal cortices, the supragenual dopamine terminal system and the cerebellum (molecular layer). This regional distribution of [³H]SCH 23390 closely correlated (except for the cerebellum) with the reported distribution of dopaminergic terminals. The topographical distribution of [³H]SCH 23390 has also been studied in detail in striatal subregions. The density of D₁ receptors was much greater in the ventrolateral sector and medial margin of the striatum than in the ventromedial and dorsolateral sectors. A rostrocaudal decrease in the densities of D₁ sites was also found along the rostrocaudal axis of the caudate-putamen. These lateral to medial and anteroposterior gradients overlapped with the density of the dopaminergic afferents.     

I-BH[Sar,Met(O2)]-SP, a new selective ligand for the NK-1 receptor in the central nervous system

The selective agonist [Sar⁹,Met(O₂)¹¹]-SP was radioiodinated with¹²⁵I-Bolton Hunter in order to study its binding to rat brain membranes and for further comparison with¹²⁵I-BH.SP. Specific binding of¹²⁵I-BH[Sar⁹,Met(O₂¹¹]-SP was temperature-dependent, saturable and reversible. In brain homogenates,¹²⁵I-BH[Sar⁹,Met(O₂)¹¹]-SP interacted with a single class of high affinityk_d = 1.0nM) non-interacting binding sites (B_max of 15 fmol/mg protein). In the central nervous system,¹²⁵I-BH-[Sar⁹,Met(O₂)¹¹]-SP apparently labeled the same number of binding sites as¹²⁵I-BH.SP (19 fmol/mg protein). Competition studies with tachykinins, neurokinins and selective neurokinin agonists indicated that the pharmacological profile of the site labeled by¹²⁵I-BH[Sar⁹,Met(O₂¹¹]-SP is identical with that of NK-1 receptors. In dose-displacement studies made with radiolabeled SP and [Sar⁹,Met(O₂¹¹)]-SP, an excellent correlation (r = 0.96) was found for theK_i values of the different compounds tested; these findings suggest that both radioligands recognize the same receptor in rat brain. The affinity (K_i of various neurokinin-related peptides for the brain site were compared with their biological activities on various isolated organs (dog carotid artery, guinea-pig ileum, rat portal vein). NK-1 binding sites characterized in rat brain homogenates appear to be identical with those present on the dog carotid artery, a preparation known to possess exclusively the NK-1 receptor type.     

Quantitative autoradiography of angiotensin II AT2 receptors with [I]CGP 42112

Most radiolabeled ligands for angiotensin II (Ang II) receptors do not discriminate between the AT₁ and AT₂ receptor subtypes, which must be distinguished by displacement with selective AT₁ or AT₂ ligands. We compared [¹²⁵I]CGP 42112 with the non-selective agonist [¹²⁵I]Sar¹Angiotensin II. We studied the inferior olive, medial geniculate nucleus and the adrenal medulla, areas rich in AT₂ receptors, using both ligands with quantitative autoradiography and membrane binding techniques. [¹²⁵I]CGP 42112 bound with high affinity (K_d = 0.07−0.3nM, depending on the area studied). [¹²⁵I]CGP 42112 binding was selective for AT₂ receptors, as determined by lack of competition with the AT₁ ligand losartan, and competition by the AT₂ ligands PD 123177 and unlabeled CGP 42112 and the non-selective peptides Ang II and angiotensin III (Ang III). Using [¹²⁵I]CGP binding, we found the same order of potency: CGP 42112 > Ang II = Ang III > PD 123177 using both quantitative autoradiography or membrane binding methods. Our results demonstrate that [¹²⁵I]CGP 42112 is the most selective, highest affinity ligand available for AT₂ receptors. Because of these characteristics, and low non-specific binding, quantitative autoradiography with [¹²⁵I]CGP 42112 is the method of choice to selectively characterize AT₂ receptors, especially in tissues like the brain, with a highly heterogeneous distribution of receptor subtypes.