Binding properties of [H]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- -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 σ 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.     

Measurement of glycine binding site of N-methyl-D-asparate receptors in living human brain using 4-acetoxy derivative of L-703,717, 4-acetoxy-7-chloro-3-[3-(4-[11c] methoxybenzyl) phenyl]-2(1H)-quinolone (AcL703) with positron emission tomography

N-methyl-D-aspartate (NMDA) receptors are of major interest in brain functions and neuropsychiatric disorders. However, at present there are few suitable radioligands for in vivo imaging of NMDA receptors. 7-choloro-4-hydroxy-3-[3-(4-methoxybenzyl) phenyl]-2(1H)-quinolone (L-703,717) is one of the potent ligands for the glycine-binding site of NMDA receptors. 4-Acetoxy derivative of L-703,717 (AcL703) is a candidate, as a positron emission tomography (PET) ligand for NMDA receptors, because of its better permeability at the blood-brain barrier compared with L-703,717. After intravenous injection of 624-851 MBq of [11C]AcL703, dynamic PET scan was performed on six healthy males for 90 min. Regions-of-interest were located on the cerebral cortices, cerebellar cortex, and cerebral white matter. The binding potential (BP) was calculated from the ratio of the area under the curve (AUC) of radioactivities from 40 to 90 min in the target region to that in white matter. Regional radioactivities reached close to equilibrium in all regions after about 40 min postinjection. Regional brain uptake of [11C]AcL703 at 40 min after injection was 0.00028-0.00065% of the injected dose/milliliter. Radioactivity concentration of [11C]AcL703 was highest in the cerebellar cortex and lowest in white matter. AUC in the cerebellar cortex was higher than those of cerebral cortices, thalamus, striatum, and white matter. BP in the cerebellar cortex was twofold higher than in the cerebral cortices (cerebellar cortex: BP=2.20+/-0.72; cerebral cortices: BP=1.05+/-0.45). Despite the low brain uptake of [11C]AcL703, regional distributions were in good agreement with our previous studies of rodents. This indicates the possibility of in vivo evaluation of NMDA receptors using PET with [11C]AcL703 in living human brain.     

Autoradiographic distribution of brain neurokinin-1/substance P receptors using a highly selective ligand [3H]-[Sar9,Met(O2)11]-substance P.

The autoradiographic distribution of neurokinin (NK)-1 receptors was visualized in the rat brain using the highly selective ligand, [3H]-[Sar9,Met(O2)11]-substance P. This ligand apparently binds to a single class of high affinity (Kd = 1.4 +/- 0.5 nM), low capacity (Bmax = 160 +/- 3.0 fmol/mg protein) sites in rat brain membrane preparations. The ligand selectivity profile reveals that substance P (SP) and unlabeled [Sar9,Met(O2)11]-SP are potent competitors of [3H]-[Sar9,Met(O2)11]-SP binding while NK-2 and NK-3 analogues are virtually inactive demonstrating the selectivity of this radioligand for the NK-1 receptor class. Autoradiographic data show that [3H]-[Sar9,Met(O2)11]-SP binding sites are broadly but discretely distributed in rat brain, the highest densities of sites being located in the external plexiform layer of the olfactory bulb, striatum, olfactory tubercule, amygdala-hippocampal area, endopiriform and entorhinal cortices, superior colliculus, locus coeruleus and substantia gelatinosa of the spinal cord. This distribution is similar, but not identical, to that previously reported for NK-1 sites using less selective ligands such as [125I]Bolton-Hunter SP. For example, some difference in labelling patterns are observed in the hippocampal formation. This could be explained by the existence of NK-1 receptor subtypes, only one of them being recognized by [3H]-[Sar9,Met(O2)11]-SP or by the greater selectivity of this radioligand for NK-1 over NK-2 and NK-3 receptor classes.     

Further pharmacological characterization of [3H]idazoxan binding sites in rat brain: evidence for predominant labeling of alpha 2-adrenergic receptors.

In addition to binding to alpha 2-adrenergic receptors, the antagonist [3H]idazoxan has been reported to bind to non-adrenergic sites in a number of tissues and species. In the present study, the pharmacological nature of [3H]idazoxan binding sites in rat brain slices has been examined using radioligand binding and autoradiographic techniques. In Na2KHPO4 buffer, four drugs with high affinity for alpha 2-adrenergic binding sites were potent inhibitors of [3H]idazoxan binding, with the rank order of potency being RX821002 greater than phentolamine greater than yohimbine greater than (-)epinephrine. Non-linear regression analysis resolved all competition curves into two components, with the high affinity site representing the majority of total [3H]idazoxan binding. In autoradiographic studies performed in Na2KHPO4 buffer, all alpha 2-selective ligands displaced greater than or equal to 75% of total [3H]idazoxan binding to most brain regions. These findings indicate that the major component of [3H]idazoxan binding was to sites that are alpha 2-adrenergic in nature. [3H]Idazoxan binding was also examined in glycylglycine buffer. In contrast to binding in Na2KHPO4 buffer, the proportion of low affinity sites was significantly increased in glycylglycine buffer. Autoradiographic studies confirmed these findings. These pharmacological data are consistent with our previously reported conclusions that, under appropriate assay conditions, [3H]idazoxan predominantly labels alpha 2-adrenergic binding sites in rat brain. These sites are widely distributed and have pharmacological characteristics consistent with those previously reported for alpha 2A-adrenergic receptors.     

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.     

Interaction of gacyclidine enantiomers with 'non-NMDA' binding sites in the rat central nervous system

Gacyclidine, a channel blocker of N-methyl-D-aspartate receptors (NMDAR), exhibits potent neuroprotective properties and a low self-neurotoxicity. Preventing its interaction with NMDARs we demonstrate, through the use of its enantiomers, that gacyclidine also interacts with other ('non-NMDA') binding sites. The autoradiographic study showed that these sites displayed a uniform specific binding in the forebrain and a more discrete distribution in the molecular layer of the cerebellum. The 'non-NMDA' binding sites could exert a modulatory control on glutamatergic neurotransmission.     

125I-BH[Sar9, Met(O2)11]-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 with 125I-Bolton Hunter in order to study its binding to rat brain membranes and for further comparison with 125I-BH.SP. Specific binding of 125I-BH[Sar9,Met(O2)11]-SP was temperature-dependent, saturable and reversible. In brain homogenates, 125I-BH[Sar9,Met(O2)11]-SP interacted with a single class of high affinity (kd = 1.0 nM) non-interacting binding sites (Bmax 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 as 125I-BH.SP (19 fmol/mg proteins). Competition studies with tachykinins, neurokinins and selective neurokinin agonists indicated that the pharmacological profile of the site labeled by 125I-BH[Sar9,Met(O2)11]-SP is identical with that of NK-1 receptors. In dose-displacement studies made with radiolabeled SP and [Sar9,Met(O2)11)]-SP, an excellent correlation (r = 0.96) was found for the Ki values of the different compounds tested; these findings suggest that both radioligands recognize the same receptor in rat brain. The affinity (Ki) 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.     

Autoradiographic identification and characterization of sigma receptors in guinea pig brain using [3H]1(cyclopropylmethyl)-4-(2'-(4'-fluorophenyl)-2'-oxoethyl) piperidine ([3H]DuP 734), a novel sigma receptor ligand.

The psychotomimetic effects of certain cycloalkyls and benzomorphans that interact with sigma receptors has led to the hypothesis that these sites may be important in the etiology of schizophrenia. DuP 734 [1-(cyclopropylmethyl)-4-(2'-(4'-fluoro-phenyl)-2'-oxoethyl) piperidine HBr] is a novel sigma receptor ligand. The receptor binding specificity and neuroanatomical distribution of [3H]DuP 734-labeled sigma receptors in guinea pig brain were examined using quantitative autoradiography. [3H]DuP 734 binding (10 microM haloperidol displaceable) to slide-mounted sections of guinea pig brain was saturable and of high affinity (Ki = 3.9 nM). Competition studies, under conditions identical to those used to visualize the receptor, yielded the following rank order of potency: DuP 734 > haloperidol > (+)-pentazocine > (-)-butaclamol > DTG > (+)-SKF 10,047 > (+)-3-PPP > (-)-pentazocine > (+)-butaclamol > U50,488H > (-)-SKF 10,047 > cinanserin > PCP > MK801, sulpiride. High densities of [3H]DuP 734 binding sites displaceable by haloperidol were present in the limbic system, in particular the dorsal and ventral bands of Broca as well as the ventral pallidum. Within the hippocampus, the pyramidal layers were sparsely labeled, while higher densities of binding sites were evident in the dentate gyrus. The frontal cortex, the mammillary complex of the hypothalamus, the central gray and red nucleus of the midbrain, the pontine reticular nucleus, the Purkinje cell layer of the cerebellum and dorsal and ventral horns, as well as the central gray matter of the spinal cord, all showed enrichments of [3H]DuP 734 binding sites. Lower levels of binding were present in the other regions of the cerebral cortex including parietal, pyriform, occipital, cingulate cortex, as well as the basal ganglia, and negligible specific binding was present in the white matter tracts. The kinetic and pharmacological characteristics and distribution of [3H]DuP 734 binding sites in brain are similar to those previously reported for sigma receptors.     

The binding properties and regional ontogeny for [3H]glutamic acid Na+-independent and [3H]kainic acid binding sites in chick brain

The binding kinetics, pharmacological properties and regional ontogeny of L-[3H]glutamic acid Na+-independent and [3H]kainic acid binding sites were studied in preparations of chick brain. One binding component was found for L-[3H]glutamic acid with a Kd value of 176 x 10(9) M. For [3H]kainic acid two binding components were found in the hemispheres, optic lobes and brain stem, one with high affinity and a Kd value of 12.5 x 10(9) M and one with low affinity and a Kd value of 260 x 10(9) M. In cerebellum only one binding site was detected for [3H]kainic acid with a Kd value of 144 x 10(9) M. The ontogeny of L-[3H]glutamic acid and [3H]kainic acid binding sites was studied using membrane preparations (48,000 g pellet) of hemispheres, optic lobes, brain stem and cerebellum. Binding of L-[3H]glutamic acid was already significant in all brain regions by embryonic day 11 but major increases in total receptor number per brain region or per mg of protein were apparent by embryonic day 19 and especially after hatching. Cerebral hemispheres, optic lobes and brain stem showed few [3H]kainic acid binding sites by day 13 in ovo. An increase follows which, in hemispheres and optic lobes, continues at the same rate during the first two weeks after hatching. In cerebellum, by contrast, the kainic acid binding site is almost undetectable until embryonic day 15. The appearance of these binding sites in cerebellum takes place during the restricted period between days 15 in ovo and 5 post-hatching. This pattern of development of [3H]kainic acid binding sites almost parallels the developmental patterns of the molecular layer of chick cerebellum and it is consistent with the results of our autoradiographic study showing that the great majority of kainic acid binding sites are localized in the molecular layer.     

Heterogeneous binding of [3H]4-DAMP to muscarinic cholinergic sites in the rat brain: evidence from membrane binding and autoradiographic studies.

The present study shows that [3H]4-DAMP binds specifically, saturably, and with high affinity to muscarinic receptor sites in the rat brain. In homogenates of hippocampus, cerebral cortex, striatum, and thalamus, [3H]4-DAMP appears to bind two sub-populations of muscarinic sites: one class of high-affinity, low capacity sites (Kd less than 1 nM; Bmax = 45-152 fmol/mg protein) and a second class of lower-affinity, high capacity sites (Kd greater than 50 nM; Bmax = 263-929 fmol/mg protein). In cerebellar homogenates, the Bmax of [3H]4-DAMP binding sites was 20 +/- 2 and 141 +/- 21 fmol/mg protein for the high- and the lower-affinity site, respectively. The ligand selectivity profile for [3H]4-DAMP binding to its sites was similar for both the high- and lower-affinity sites; atropine = (-)QNB = 4-DAMP much greater than pirenzepine greater than AF-DX 116, although pirenzepine was more potent (16-fold) at the lower- than at the high-affinity sites. The autoradiographic distribution of [3H]4-DAMP sites revealed a discrete pattern of labeling in the rat brain, with the highest densities of [3H]4-DAMP sites present in the CA1 sub-field of Ammon's horn of the hippocampus, the dentate gyrus, the olfactory tubercle, the external plexiform layer of the olfactory bulb and layers I-II of the frontoparietal cortex. Although the distribution of [3H]pirenzepine sites was similar to that of [3H]4-DAMP sites in many brain regions, significant distinctions were apparent. Thus, both the ligand selectivity pattern of [3H]4-DAMP binding and the autoradiographic distribution of sites suggest that although the high-affinity [3H]4-DAMP sites may consist primarily of muscarinic-M3 receptors, the lower-affinity [3H]4-DAMP sites may be composed of a large proportion of muscarinic-M1 receptors.     

Autoradiographic localization and quantification of dopamine D2 receptors in normal human brain with [3H]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 (Kd = 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 anterior frontal cortex. Thorough examination of the subregional distribution of [3H]NPA binding sites in the caudate-putamen-pallidum complex revealed heterogeneous 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.     

Linkage between phencyclidine (PCP) and N-methyl-D-aspartate (NMDA) receptors in the cerebellum

It has been suggested that phencyclidine (PCP) receptors may not be linked with N-methyl-D-aspartate (NMDA) receptors in all brain areas. We found that NMDA enhanced [3H]TCP (a PCP analog) binding in extensively washed cortical, but not cerebellar membranes. However, PCP potently inhibited NMDA-induced [3H]norepinephrine release from cerebellar slices in a concentration-dependent manner, suggesting that a subtype of cerebellar PCP receptors is functionally linked with NMDA receptors. It is suggested that this subtype cannot be demonstrated by [3H]TCP binding because of the predominance of low affinity PCP receptors in the cerebellum.     

Autoradiographic distribution of alpha 2 adrenoceptors, NAIBS, and 5-HT1A receptors in human brain using [3H]idazoxan and [3H]rauwolscine.

The regional distribution of [3H]idazoxan and [3H]rauwolscine was studied autoradiographically in human brain. [3H]Idazoxan binds with high affinity to alpha 2 adrenoceptors as well as to non-adrenergic sites (NAIBS). [3H]Rauwolscine, besides binding to alpha 2 adrenoceptors, also binds to 5-HT1A receptors. Both radioligands labelled the same population of alpha 2 adrenoceptors, defined as the epinephrine-displaceable binding component. The highest densities of alpha 2 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 alpha 2 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 alpha 2 adrenoceptors, suggests that the binding sites are independent from each other. The regional distribution of 5-HT1A receptors labelled by [3H]rauwolscine is in agreement with previous studies using [3H]8-OH-DPAT.     

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.     

Dog cerebral cortex contains μ-, δ- and κ-opioid receptors at different densities: apparent lack of evidence for subtypes of the κ-receptor using selective radioligands

The biochemical and pharmacological properties of mu (mu), kappa (kappa) and delta (delta) opioid receptors were ascertained in dog cerebral cortex homogenates. The selective peptides, [3H]D-Pen2-D-Pen5enkephalin [( 3H]DPDPE) and [3H]D-Ala2-MePhe4-Glyol5-enkephalin [3H]Glyol; [3H]DAMGO), bound to delta- and mu-opioid receptors with high affinity (dissociation constants, Kd values = 4.7 and 1.6 nM) but to different densities of binding sites (Bmax values of 49.2 and 6.6 fmol/mg protein, respectively) in washed homogenates of dog cerebral cortex. In contrast, the non-peptides, [3H]U69593 [( 3H]U69) and [3H]etorphine [( 3H]ET), labeled a high concentration of kappa-opioid receptors (respective Bmax values of 67.2 and 76.6 fmol/mg protein) of high affinity (respective Kds of 1.4 and 0.47 nM) in the same tissue homogenates. Thus, the relative rank order of opioid receptor densities was: kappa greater than delta much greater than mu. The selective labeling of the kappa-receptors with two different drugs [( 3H]U69 and [3H]ET) failed to reveal the possible existence of multiple kappa-sites based on the relative Bmax values of the two radioligands. This conclusion was further supported by the similarity of the pharmacological specificity of both [3H]U69 and [3H]ET binding, where all the opioids tested produced 100% inhibition of these labels and where the rank order of potency of opioids at inhibiting the binding of these probes was: U50488 greater than U69593 greater than dynorphin-(1-8) greater than naloxone much greater than morphine much greater than Glyol (DAMGO) greater than DPDPE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Visualization of neurokinin-3 receptor sites in rat brain using the highly selective ligand [3H]senktide.

The autoradiographic distribution of the neurokinin (NK)-3 receptor sub-type was visualized in the rat brain using [3H]senktide, a highly selective ligand, [3H]Senktide apparently binds to a single class of high affinity (Kd = 2.8 +/- 1.0 nM), low capacity (Bmax = 31.2 +/- 3.0 fmol/mg protein) sites in rat brain cortex. The ligand selectivity pattern reveals that eledoisin and senktide are potent competitors of both [3H]senktide and [125I]Bolton-Hunter eledoisin binding sites demonstrating the NK-3 nature of these sites. Autoradiographic data show that [3H]senktide binding sites are concentrated in mid-cortical layers, supraoptic nucleus, zona incerta, basolateral nucleus of the amygdala and interpeduncular nucleus. Much lower densities of binding are seen in most other areas such as the caudate-putamen and cerebellum. This distribution is similar, but not identical, to that previously reported for NK-3 sites using less selective ligands. It is most likely because less selective probes also bind to other classes of NK receptors. The higher selectivity of [3H]senktide is thus an important advantage for the precise characterization of NK-3 receptor binding parameters.     

Nicotinic binding in rat brain: autoradiographic comparison of [3H]acetylcholine, [3H]nicotine, and [125I]-alpha-bungarotoxin

Three radioligands have been commonly used to label putative nicotinic cholinoceptors in the mammalian central nervous system: the agonists [3H]nicotine and [3H]acetylcholine ([3H]ACh--in the presence of atropine to block muscarinic receptors), and the snake venom extract, [125I]-alpha-bungarotoxin([125I]BTX), which acts as a nicotinic antagonist at the neuromuscular junction. Binding studies employing brain homogenates indicate that the regional distributions of both [3H]nicotine and [3H]ACh differ from that of [125I]BTX. The possible relationship between brain sites bound by [3H]nicotine and [3H]ACh has not been examined directly. We have used the technique of autoradiography to produce detailed maps of [3H]nicotine, [3H]ACh, and [125I]BTX labeling; near-adjacent tissue sections were compared at many levels of the rat brain. The maps of high affinity agonist labeling are strikingly concordant, with highest densities in the interpeduncular nucleus, most thalamic nuclei, superior colliculus, medial habenula, presubiculum, cerebral cortex (layers I and III/IV), and the substantia nigra pars compacta/ventral tegmental area. The pattern of [125I]BTX binding is strikingly different, the only notable overlap with agonist binding being the cerebral cortex (layer I) and superior colliculus. [125I]BTX binding is also dense in the inferior colliculus, cerebral cortex (layer VI), hypothalamus, and hippocampus, but is virtually absent in thalamus. Various lines of evidence suggest that the high affinity agonist-binding sites in brain correspond to nicotinic cholinergic receptors similar to those found at autonomic ganglia; BTX-binding sites may also serve as receptors for nicotine and are possibly related to neuromuscular nicotinic cholinoceptors.     

Behaviour of [11C]R(-)- and [11C]S(+)-rolipram in vitro and in vivo, and their use as PET radiotracers for the quantificative assay of PDE4

Cyclic AMP (cAMP) is a continually produced nucleotide which is inactivated by hydrolysis to 5'AMP via phosphodiesterase 4 (PDE4) enzymes. Rolipram is a selective PDE4 inhibitor which exists in two enantiomeric forms, R(-) and S(+). Both of these enantiomers have previously been labelled with carbon-11 and used as positron emission tomography (PET) ligands for measuring PDE4 expression and function, and indirectly to explore the function of the cAMP second messenger, in vivo, using PET. The aim of these studies was to relate the in vitro affinities of the two rolipram enantiomers using standard pharmacological assays with the in vivo behaviour of the two enantiomers using PET. In vitro competition assays were performed using rat cortical membranes and [(3)H]R(-)- and [(3)H]S(+)-rolipram with increasing concentrations of either unlabelled R(-)- or S(+)-rolipram. In vivo, a series of PET studies were performed in the porcine brain using [(11)C]R(-)-rolipram with co-administration of increasing doses of either unlabelled R(-)- or S(+)-rolipram. Additional in vivo PET studies were performed using [(11)C]S(+)-rolipram with saturating doses of rolipram. In all studies, R(-)-rolipram exhibited a higher affinity for the PDE4 enzyme than S(+)-rolipram. The calculated affinity ratios were 7.97 from the in vitro studies; 12.5 from the in vivo studies using [(11)C]R(-)-rolipram; and 14.7 from the in vivo studies using [(11)C]S(+)-rolipram. To conclude, the in vitro affinities of R(-)- and S(+)-rolipram predict their apparent in vivo behaviour in the porcine brain, as measured by PET.     

Synthesis and pharmacological characterization of a novel, highly potent, peptidomimetic delta-opioid radioantagonist, [3H]Tyr-Tic-(2S,3R)-beta-MePhe-Phe-OH

[(3)H]Tyr-Tic-(2S,3R)-beta-MePhe-Phe-OH (where Tic: 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) with a specific radioactivity of 53.7 Ci/mmol was synthesized and characterized in receptor binding assays at 25 degrees C in rat brain membranes. The specific binding was saturable and displayed high affinity, with a K(D) of 0.16+/-0.005 nM and B(max) of 85.9+/-6.3 fmol/mg protein. NaCl increased its affinity by about 4-fold in membranes of rat brain and Chinese Hamster Ovary Cells stably transfected with the human delta-opioid receptors (hDOR-CHO) showing that the new ligand is an antagonist. The prototypic delta-opioid ligands were much more potent than mu- or kappa-specific ligands in competition assays. The autoradiographic distribution of the binding sites of the new ligand agreed with the known locations of the delta-opioid receptors in rat brain. The unlabeled new ligand was about 7-fold more potent than the parent peptide in competing for the binding sites of [(3)H]Tyr-Tic-(2S,3R)-beta-MePhe-Phe-OH in rat brain membranes. Likewise, the threo-beta-methyl analog was 3.8-fold more potent than the parent compound in antagonizing the effect of DPDPE in the [(35)S]GTPgammaS functional assay in hDOR-CHO membranes. The new, highly potent, conformationally constrained antagonist may be a valuable pharmacological tool in understanding the structural and topographical requirements of peptide ligand binding to the delta-opioid receptors.