Expression of rat NK-2 (neurokinin A) receptor in E. coli

With the goal of obtaining sufficient functional protein for structural analysis, rat neurokinin-2 receptor was produced in Escherichia coli by linking it to the periplasmic maltose-binding protein. As a first step, we present a biochemical and pharmacological investigation of the recombinant receptor. Western-blots showed that the fusion protein was associated with the membranes. The agonist [4,5-3H-Leu9]neurokinin A and the NK-2 antagonist [3H]SR48,968 bound to the receptor in a highly specific manner. Saturation binding of the [3H]agonist demonstrated a single class of receptors (KD = 10.5 nM, Bmax = 2.5 pmol/mg protein). The [3H]antagonist bound with higher affinity to a larger receptor population (KD = 0.2 nM, Bmax = 7.2 pmol/mg protein). Competition of [3H]agonist binding with other agonists demonstrated a potency order of: neurokinin A > [Nle10]NKA(4-10) = [beta-Ala8]NKA(4-10) > substance P > senktide Against the [3H]antagonist, agonists were only partially inhibitory. Selective NK-2 antagonists inhibited binding of both [3H]ligands with an identical order of potency: SR48,968 > R396 > MEN10,376, which is consistent with NK-2 receptor pharmacology in rat tissue.     

Influence of chronic opioid treatment on low Km GTPase activity in rat brain: evidence for the involvement of G-proteins in opioid tolerance

To investigate G protein function during the initial state of opioid tolerance, low Km GTPase activity was measured following chronic treatment with morphine (mu agonist) and butorphanol (mu/delta/kappa mixed agonist) in rats. Chronic opioid administration (20 mg/kg, IP) was performed once a day for 7 consecutive days. Under these conditions, antinociceptive tolerance to morphine but not butorphanol was developed. Chronic morphine treatment enhanced basal low Km GTPase activity in the pons/medulla, but not in the cortex and midbrain. On the other hand, chronic butorphanol treatment had no effect on basal low Km GTPase activity. These results suggest that chronic in vivo treatment of rats with mu agonists leads to an increase in the hydrolysis of GTP to GDP, by a basal low Km GTPase activity of G-proteins in the pons/medulla and that an enhancement of GTPase activity in this specific area may contribute to the development of antinociceptive tolerance to mu agonists.     

Characterization of [3H]staurosporine binding in protein kinase C-II purified from rat brain

The type II protein kinase C (PKC-II) densely present in mammalian brain plays functional roles in CNS. We examined the characteristics of [3H]staurosporine binding to PKC-II purified from rat brain, compared to [3H]phorbol 12, 13-dibutyrate (PDBu) binding. In brief, [3H]staurosporine binding increased by phosphatidylserine (PtdSer) in a concentration-dependent manner and the binding was enhanced by Ca2+ and phorbol 12-myristate 13-acetate (PMA). In the presence of Ca2+, PMA and PtdSer, Bmax of these bindings markedly increased, but KD did not change. These characteristics of binding were similar to [3H]PDBu binding to PKC-II. Although [3H]PDBu binding was not affected by protein kinase inhibitors such as staurosporine, H-7, K-252a and K-252b, [3H]staurosporine binding was inhibited by these inhibitors. [3H]staurosporine binding was inhibited by several ATP analogues, but was not by guanine nucleotides. PtdSer-induced increase in [3H]PDBu binding was inhibited by Zn2+, but Zn2+ induced increase in [3H]staurosporine binding as well as PtdSer and/or Ca2+. Staurosporine would thus appear to bind to a domain different from phorbol ester-binding one in PKC, interactions between both domains may regulate kinase activity, and 1 mol staurosporine and 4 mol phorbol ester may bind to 1 mol PKC-II.     

Ligand binding profiles of U-69, 593-sensitive and-insensitive sites in human cerebral cortex membranes: evidence of kappa opioid receptors heterogeneity

Receptor binding studies were performed to characterize the properties of subtypes of kappa opioid receptors in membrane preparations of human cerebral cortex. [3H]U69,593 ([3H]U69), a selective kappa 1-agonist, and [3H]diprenorphine ([3H]DIP), a non-selective opioid antagonist, in the presence of 1 microM each of DAMGO, DPDPE and U-69 to block mu-, delta-, and kappa 1-sites, labeled single population of binding sites, respectively. [3H]U-69 binding sites (KD = 3.8 +/- 0.2 nM, Bmax = 6.3 +/- 0.2 fmol/mg protein) had a binding profile that correspond to kappa 1-receptor. That is, dynorphin A (1-13) (Dyn A), bremazocine (BZC), U50,488H (U50), (-)ethylketocyclazocine (EKC) and nor-binaltorphimine (nor-BNI) bound to this site with high affinities. [3H]DIP labeled binding sites (Kd = 7.3 +/- 0.2 nM, Bmax = 102 +/- 9 fmol/mg protein) that were not sensitive to U-50, but to BZC, EKC and nor-BNI. These results indicate that kappa 1 and Kappa 2 opioid receptors exist in human cerebral cortex with different ligand binding profiles.     

Naloxone-precipitated changes in biogenic amines and their metabolites in various brain regions of butorphanol-dependent rats

Influence of a naloxone (an opioid receptor antagonist) challenge (5 mg/kg, IP) on levels of biogenic amines and their metabolites in various brain regions of rats infused continuously with butorphanol (a mu/delta/kappa mixed opioid receptor agonist; 26 nmol/microliter/h) or morphine (a mu-opioid receptor agonist; 26 nmol/microliter/h) was investigated using high-performance liquid chromatography with electrochemical detection (HPLC-ED). Naloxone precipitated a withdrawal syndrome and decreased the levels of: dopamine (DA) in the cortex and striatum, 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum, homovanilic acid (HVA) in the striatum, limbic, midbrain, and pons/medulla regions in butorphanol-dependent rats. However, the levels of norepinephrine (NE), serotonin (5-hydroxytryptamine; 5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) in the regions studied were not affected by naloxone-precipitated withdrawal. In addition, naloxone increased the HVA/DA ratio in the cortex, while this ratio was reduced in the limbic, midbrain, and pons/medulla. The reduction of 5-HIAA/5-HT ratio was also detected in the limbic area. In the animals rendered dependent on morphine, the results obtained were similar to those of butorphanol-dependent rats except for changes of 5-HIAA levels in some brain regions. These results suggest that an alteration of dopaminergic neuron activity following a reduction of DA and its metabolites in specific brain regions (e.g., striatum, limbic, midbrain, and pons/medulla) play an important role in the expression of the opioid withdrawal syndrome.     

Opposite effects of CCK(B) agonists in grooming behaviour in rats: further evidence for two CCK(B) subsites

Opioid receptor binding properties of [3H]Tyr-D-Ala-Phe-Phe-NH2 (TAPP) were characterized in rat brain and Chinese hamster ovary (CHO) cells expressing the rat mu-receptor. In rat brain, [3H]TAPP labeled a single class of opioid sites with a dissociation constant (Kd) of 0.31 nM and maximal number of binding sites (Bmax) of 119 fmol/mg protein. In CHO-mu/1 cell membranes, the Kd and Bmax values were 0.78 nM and 1806 fmol/mg protein, respectively. Binding to rat brain was demonstrated to be pharmacologically identical to that obtained with CHO-mu/1 cell membranes and modulated by Na+ ions and guanine nucleotides. The high affinity and selectivity of [3H]TAPP together with its low non-specific binding make this radioligand a useful tool for labeling the native and cloned mu-opioid receptor.     

3H]5,7-dichlorokynurenic acid recognizes two binding sites in rat cerebral cortex membranes

Binding of [3H]5,7-dichlorokynurenic acid ([3H]DCKA), a competitive antagonist of the strychnine-insensitive glycine site of the N-methyl-D-aspartate (NMDA) receptor channel complex, was characterized in synaptic plasma membranes from rat cerebral cortex. Non linear curve fitting of [3H]DCKA saturation and homologous displacement isotherms indicated the existence of two binding sites: a specific, saturable, high affinity site, with a pKD value of 7.24 (KD = 57.5 nmol/l) and a maximum binding value (Bmax) of 6.9 pmol/mg of protein and a second site, with micromolar affinity. The pharmacological profile of both binding components was determined by studying the effect on [3H]DCKA and [3H]glycine binding of a series of compounds known to interact with different excitatory and inhibitory amino acid receptors. These studies confirmed the identity of the high affinity site of [3H]DCKA binding with the strychnine-insensitive glycine site of the NMDA receptor channel complex. 3-[2-(Phenylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carb oxylic acid sodium salt (GV 150526A), a new, high affinity, selective glycine site antagonist (1), was the most potent inhibitor of this component of binding (pKi = 8.24, Ki = 5.6 nmol/l). The low affinity component of [3H]DCKA binding was insensitive to the agonists glycine and D-serine and the partial agonist (+/-)-3-amino-1-hydroxy-2-pyrrolidone (HA 966), though recognised by glycine site antagonists. The precise nature of this second, low affinity [3H]DCKA binding site remains to be elucidated.     

Competitive inhibition of delta8-tetrahydrocannabinol and its active metabolites for cannabinoid receptor binding

In vitro binding characteristics of delta8-tetrahydrocannabinol (delta8-THC) and its metabolites, 11-hydroxy-delta8-THC (11-OH-delta8-THC) and 11-oxo-delta8-THC, as well as an inactive metabolite, delta8-THC-11-oic acid, as a cannabinoid receptor site from bovine cortex were examined using the specific agonist [3H]CP-55940. 11-OH-delta8-THC and 11-oxo-delta8-THC strongly inhibited the specific binding of [3H]CP-55940. The Ki values of 11-OH-delta8-THC and 11-oxo-delta8-THC for the specific binding of [3H]CP-55940 were 52 and 143 nM, respectively, whereas that of delta8-THC-11-oic acid was 917 nM. Scatchard plot analyses indicated that 11-OH-delta8-THC and 11-oxo-delta8-THC caused a significant increase in the apparent KD value without changing the apparent Bmax. These results reveal that active metabolites of delta8-THC also competitively bind to the cannabinoid receptor as agonists.     

Binding profile of the novel 5-HT1B/1D receptor antagonist, [3H]GR 125,743, in guinea-pig brain: a comparison with [3H]5-carboxamidotryptamine

The aim of the research was to characterize muscarinic receptors of bovine ciliary muscle and to investigate the desensitization process. The role of protein kinase C was analyzed. The results show that muscarinic receptors of bovine ciliary muscle have the pharmacological characteristics of the M3 subtype. Acute exposure to phorbol esters (1 microM phorbol 12,13-dibutyrate, PDB, or 0.1 microM phorbol 12-myristate 13-acetate, PMA, for 15 and 5 min, respectively) resulted in antagonism of muscarinic receptor-mediated contraction. Long-term pretreatment (18 h) with PMA to down-regulate protein kinase C resulted in potentiation of carbachol-induced contraction, reduction of agonist-induced desensitization and loss of phorbol ester-induced desensitization. Staurosporine (3 microM) and H7 [1-(5-isoquinolinesulfonyl)-2-methyl-piperazine] (1 microM), protein kinase C inhibitors, produced a significant potentiation of the contractile effect of carbachol, reduced the desensitization produced by repeated addition of carbachol and suppressed that induced by phorbol esters. In vitro incubation with carbachol, PDB or PMA did not cause any modification of the binding of labeled [3H]quinuclidinyl benzilate. In vitro incubation with PDB and PMA produced, as expected, a significant translocation of protein kinase C from the cytosol to the membrane. The incubation of the ciliary muscle with carbachol, using the protocol of exposure that induced maximal desensitization of contractile responses, produced a significant redistribution of the enzyme from the cytosol to the membrane. These findings suggest that agonist-induced modulation of functional cholinergic sensitivity in ciliary muscle is correlated, at least partially, to the translocation of protein kinase C from the cytosol to the membrane. The desensitization by phorbol esters is completely due to protein kinase C activation; during the desensitization process, direct modification of the density and affinity of muscarinic receptors is not involved.     

Binding characteristics of a potent AMPA receptor antagonist [3H]Ro 48-8587 in rat brain

A new AMPA receptor antagonist, Ro 48-8587, was characterized pharmacologically in vitro. It is highly potent and selective for AMPA receptors as shown by its effects on [3H]AMPA, [3H] kainate, and [3H] MK-801 binding to rat brain membranes and on AMPA- or NMDA-induced depolarization in rat cortical wedges. [3H]Ro 48-8587 bound with a high affinity (KD = 3 nM) to a single population of binding sites with a Bmax of 1 pmol/mg of protein in rat whole brain membranes. [3H]Ro 48-8587 binding to rat whole brain membranes was inhibited by several compounds with the following rank order of potency: Ro 48-8587 > 6-nitro-7-sulphamoylbenzo[f] quinoxaline-2,3-dione (NBQX) > YM 90K > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) > quisqualate > AMPA > glutamate > kainate > NMDA. The distribution and abundance of specific binding sites (approximately 95% of total) in sections of rat CNS, revealed by quantitative receptor radioautography and image analysis, indicated a very discrete localization. Highest binding values were observed in cortical layers (binding in layers 1 and 2 > binding in layers 3-6), hippocampal formation, striatum, dorsal septum, reticular thalamic nucleus, cerebellar molecular layer, and spinal cord dorsal horn. At 1 nM, the values for specific binding were highest in the cortical layers 1 and 2 and lowest in the brainstem (approximately 2.6 and 0.4 pmol/mg of protein, respectively). Ro 48-8587 is a potent and selective AMPA receptor antagonist with improved binding characteristics (higher affinity, selectivity, and specific binding) compared with those previously reported.     

Beauty is in the soul of the beholder: psychological implications of beauty and African American women

An in vitro receptor binding and in vivo microdialysis study was performed to further investigate the modulation of dopamine (DA) D2 receptors by neurotensin (NT) peptides. Saturation experiments with the D2 agonist [3H]NPA (N-propylnorapomorphine) showed that 10 nM of NT, 10 nM of neuromedin N (NN) and 1 nM of the C-terminal NT-(8-13) fragment significantly increased the KD values by 125%, 181%, and 194%, respectively without significantly affecting the Bmax value of the [3H]NPA binding sites in coronal sections of rat ventral forebrain mainly containing the nucleus accumbens (Acb) and the olfactory tubercle. In line with the previous findings that NT can increase GABA release in the Acb and that NT receptors are not found on DA terminals in this brain region, the present in vivo microdialysis study demonstrated that local perfusion of NT (1 nM) counteracted the D2 agonist pergolide (2 mu M) induced inhibition of GABA, but not of DA release in the rat Acb. This result indicates that NT counteracts the D2 agonist induced inhibition of GABA release in the rat Acb, via an antagonistic postsynaptic NT/D2 receptor interaction as also suggested by the inhibitory regulation of D2 receptor affinity in the Acb by the NT peptides demonstrated in the present receptor binding experiments. Thus, the neuroleptic and potential antipsychotic profile of the NT peptides may involve an antagonistic NT/D2 receptor regulation in the ventral striatum.     

Increase in serotonin-1A autoreceptors in the midbrain of suicide victims with major depression-postmortem evidence for decreased serotonin activity

It has been hypothesized that a deficit in serotonin may be a crucial determinant in the pathophysiology of major depression. Serotonin-1A receptors are located on serotonin cell bodies in the midbrain dorsal raphe (DR) nucleus, and the activation of these receptors inhibits the firing of serotonin neurons and diminishes the release of this neurotransmitter in the prefrontal cortex. Repeated treatment with some antidepressant medications desensitizes serotonin-1A receptors in the rat midbrain. The present study determined whether the binding of [3H]8-hydroxy-2-(di-n-propyl)aminotetralin (8-OH-DPAT), an agonist at serotonin-1A receptors, is altered in the midbrain of suicide victims with major depression. Radiolabeling of the serotonin-1A receptor in the DR varied significantly along the rostral-to-caudal extent of the human midbrain. The binding of [3H]8-OH-DPAT to serotonin-1A receptors was increased significantly in the midbrain DR of suicide victims with major depression as compared with psychiatrically normal control subjects. In suicide victims with major depression, the increase in the binding of [3H]8-OH-DPAT to serotonin-1A receptors was detected in the entire DR and specifically localized to the dorsal and ventrolateral subnuclei. Enhanced radioligand binding of an agonist to inhibitory serotonin-1A autoreceptors in the human DR provides pharmacological evidence to support the hypothesis of diminished activity of serotonin neurons in suicide victims with major depression.     

Selective labelling of bradykinin receptor subtypes in WI38 human lung fibroblasts

1. Binding of the B1 bradykinin receptor radioligand, [3H]-des-Arg10-kallidin (-KD) and the B2 receptor radioligand [3H]-bradykinin (-BK) was investigated in membranes prepared from WI38 human foetal lung fibroblasts. 2. One-site analysis of the saturation data for [3H]-des-Arg10-KD gave an equilibrium dissociation constant (KD) value of 0.51 +/- 0.12 nM and a maximum receptor density (Bmax) of 260 +/- 49 fmol mg-1 of protein. [3H]-des-Arg10-KD binding was displaced by ligands in the order: des-Arg10-KD > KD > > des-Arg9[Leu8]-BK > des-Arg9-BK > Hoe 140 > > BK, implying that it was binding selectively to B1 receptors. 3. One-site analysis of the binding of [3H]-BK to W138 membranes indicated that it had a KD value of 0.25 +/- 0.06 nM and a Bmax of 753 +/- 98 fmol mg-1 of protein. The potencies for displacement of [3H]-BK binding were: Hoe 140 > > BK = KD > > > des-Arg10-KD = des-Arg9[Leu8]-BK = des-Arg9-BK, which was consistent with binding to B2 receptors. 4. This is the first characterization of [3H]-des-Arg10-KD binding to include both kinetic and equilibrium data, and demonstrates that [3H]-des-Arg10-KD has a high affinity for human B1 bradykinin receptors and is sufficiently selective to be used as a radioligand for B1 receptors in human cells or tissues expressing an excess of B2 BK receptors.     

Regulation of opioid receptors in rat sensory neurons in culture

To determine whether opioid receptors in sensory neurons are regulated by chronic exposure to opioids, we assessed the binding of various opioid ligands to membranes derived from isolated rat dorsal root ganglia neurons grown in culture. Equilibrium binding of [3H]diprenorphine onto membranes from cells grown for 13-15 days revealed a saturable binding site with a Kd value of 0.3 +/- 0.2 nM and an approximate Bmax value of 1300 +/- 200 fmol/mg of protein. [3H]Diprenorphine binding increased 3-fold from 1-15 days in culture. The mu receptors represent approximately 70 +/- 11% of the [3H]diprenorphine binding sites, as indicated by saturation binding of [3H]DAMGO. The kappa and delta receptors represent approximately 10 +/- 3% and approximately 5 +/- 2% of the [3H]diprenorphine binding sites, respectively. Preexposure of neurons to 10 microM naloxone for 48 hr up-regulated the receptors by 40%, whereas incubation with 100 nM to 10 microM DAMGO for 48 hr resulted in a significant decrease in the Bmax value of opioid receptors, with a maximum reduction of 70%. The identification of a high level of opioid receptors expressed in isolated sensory neurons and their modulation by opioids demonstrates that cultured sensory neurons are an excellent model with which to study opioid receptor regulation.     

Liposomal amikacin: improved treatment of Mycobacterium avium complex infection in the beige mouse model

1. Binding of D,L-(E)-2-amino-4-[3H]-propyl-5-phosphono-3-pentenoic acid ([3H]-CGP 39653), a high affinity, selective antagonist at the glutamate site of the N-methyl-D-aspartate (NMDA) receptor, was investigated in rat brain by means of receptor binding and quantitative autoradiography techniques. 2. [3H]-CGP 39653 interacted with striatal and cerebellar membranes in a saturable manner and to a single binding site, with KD values of 15.5 nM and 10.0 nM and receptor binding densities (Bmax values) of 3.1 and 0.5 pmol mg-1 protein, respectively. These KD values were not significantly different from that previously reported in the cerebral cortex (10.7 nM). 3. Displacement analyses of [3H]-CGP 39653 in striatum and cerebellum, performed with L-glutamic acid, 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and glycine showed a pharmacological profile similar to that reported in the cerebral cortex. L-Glutamic acid and CPP produced complete displacement of specific binding with Ki values not significantly different from the cerebral cortex. Glycine inhibited [3H]CGP 39653 binding with shallow, biphasic curves, characterized by a high and a low affinity component. Furthermore, glycine discriminated between these regions (P < 0.005, one-way ANOVA), since the apparent Ki of the high affinity component of the glycine inhibition curve (KiH) was significantly lower (Fisher's protected LSD) in the striatum than the cortex (33 nM and 104 nM, respectively). 4. Regional binding of [3H]-CGP 39653 to horizontal sections of rat brain revealed a heterogeneous distribution of binding sites, similar to that reported for other radiolabelled antagonists at the NMDA site (D-2-[3H]-amino-5-phosphonopentanoic acid ([3H]-D-AP5) and [3H]-CPP). High values of binding were detected in the hippocampal formation, cerebral cortex and thalamus, with low levels in striatum and cerebellum. 5. [3H]-CGP 39653 binding was inhibited by increasing concentrations of L-glutamic acid, CPP and glycine. L-Glutamic acid and CPP completely displaced specific binding in all regions tested, with similar IC50 values throughout. Similarly, glycine was able to inhibit the binding in all areas considered: 10 microM and 1 mM glycine reduced the binding to 80% and 65% of control (average between areas) respectively. The percentage of specific [3H]-CGP 39653 binding inhibited by 1 mM glycine varied among regions (P < 0.05, two-ways ANOVA). Multiple comparison, performed by Fisher's protected LSD method, showed that the inhibition was lower in striatum (72% of control), with respect to cortex (66% of control) and hippocampal formation (58% of control). 6. The inhibitory action of 10 microM glycine was reversed by 100 microM 7-chloro-kynurenic acid (7-CKA), a competitive antagonist of the glycine site of the NMDA receptor channel complex, in all areas tested. Moreover, reversal by 7-CKA was not the same in all regions (P < 0.05, two-ways ANOVA). In fact, in the presence of 10 microM glycine and 100 microM 7-KCA, specific [3H]-CGP 39653 binding in the striatum was 131% of control, which was significantly greater (Fisher's protected LSD) than binding in the hippocampus and the thalamus (104% and 112% of control, respectively). 7. These results demonstrate that [3H]-CGP 39653 binding can be inhibited by glycine in rat brain regions containing NMDA receptors; moreover, they suggest the existence of regionally distinct NMDA receptor subtypes with a different allosteric mechanism of [3H]-CGP 39653 binding modulation through the associated glycine site.     

The non-peptide neuropeptide Y Y1 receptor antagonist BIBP3226 blocks the [Leu31,Pro34]neuropeptide Y-induced modulation of alpha 2-adrenoceptors in the nucleus tractus solitarii of the rat

The potential blockade of the neuropeptide Y (NPY) Y1 receptor agonist [Leu31,Pro34]NPY-induced modulation of the characteristics of alpha 2-adrenoceptor agonist [3H]p-aminoclonidine binding sites by a selective non-peptide NPY Y1 receptor antagonist BIBP3226, was studied in the nucleus tractus solitarii of the rat by means of quantitative receptor autoradiography. [Leu31,Pro34]NPY at a concentration of 10 nM significantly increased the Kd value of [3H]p-aminoclonidine binding sites in the nucleus tractus solitarii without influencing the Bmax, suggesting the existence of an antagonistic modulation by NPY Y1 receptors of alpha 2-adrenoceptors in the nucleus tractus solitarii. BIBP3226 at 100 nM fully blocked the [Leu31,Pro34]NPY-induced increase in Kd of the [3H]p-aminoclonidine binding sites. The present results therefore provide evidence, by use of a NPY Y1 receptor antagonist, for the existence of a NPY Y1/alpha 2 receptor interaction in the nucleus tractus solitarii.     

Characterization of (S)-des-4-amino-3-[125I]iodozacopride ([125I]DAIZAC), a selective high-affinity radioligand for 5-hydroxytryptamine3 receptors

The 5-hydroxytryptamine(HT)3 receptor subtype is present in the central nervous system (CNS) in low abundance, and few selective radiolabeled antagonists with high specific activity are available to study these sites. DAIZAC [desamino-3-iodo-(S)-zacopride; (S)-5-chloro-3-iodo-2-methoxy-N-(1-azobicyclo-[2.2. 2]oct-3-yl)benzamide] is a compound with high affinity and selectivity for the 5-HT3 receptor. Scatchard analysis of specific binding to NCB-20 cell membranes gave a Bmax of 340 +/- 58 fmol/mg protein and a KD of 0.14 +/- 0.03 nM, which is in agreement with the value previously reported in rat brain (KD = 0.15 nM). Nonspecific binding of [125I]DAIZAC in NCB-20 cells was <1% of total binding at the KD for DAIZAC compared with 17% in the rat brain preparation. Unlabeled DAIZAC (10 microM) showed minimal ability to displace binding of radiolabeled ligands selected for their affinities for other CNS receptor and uptake carrier binding sites. The discrimination ratio of DAIZAC for the 5-HT3 receptor over the M1 muscarinic binding site, the non-5-HT3 site at which it was most potent, was >2800. Serotonergic antagonists at every other known CNS serotonergic binding sites (3-30 microM) were ineffective in displacing [125I]DAIZAC binding in rat brain membranes. Similarly, antagonists (3-30 microM) for other nonserotonergic receptors and uptake sites were ineffective in displacing [125I]DAIZAC binding. Autoradiographic studies showed highest specific binding in area postrema and nucleus solitarius, with intermediate levels of binding in entorhinal cortex and hippocampus. DAIZAC inhibited 5-HT3 receptor-mediated inward cation current in NCB-20 cells with an IC50 of 0.24 nM. [125I]DAIZAC is a potent and highly selective ligand for in vitro studies of the 5-HT3 receptor.     

The effect of prolonged treatment with imipramine on the biosynthesis and functional characteristics of D2 dopamine receptors in the rat caudate putamen

1. The present study shows the effects of imipramine in a single dose (10 mg kg(-1), p.o.) or following repeated (14 days, twice a day) treatment on the level of mRNA coding for D2 dopamine receptors in the rat caudate putamen (CP). Repeated administration of imipramine resulted in the increase of the level of mRNA coding for D2 dopamine receptors. 2. Radioligand binding studies with the D2 receptor agonist, [3H]-N-0437, indicated, that following imipramine administration, the affinity of the agonist for the D2 dopamine receptor significantly increased, though without any alterations in the Bmax. 3. Pharmacological manipulations (by use of forskolin, GppNHp and quinpirole) of the cyclic AMP generating system, ex vivo following administration of imipramine indicated that an up-regulation of factors inhibiting cyclic GMP formation takes place. 4. Most probably it is the D2 dopamine receptor which undergoes functional up-regulation, resulting from the enhancement of its biosynthesis.     

Autoradiographic visualization of group III metabotropic glutamate receptors using [3H]-L-2-amino-4-phosphonobutyrate

1. In vitro receptor autoradiography using [3H]-L-2-amino-4-phosphonobutyrate ([3H]-L-AP4) binding to sections of rat brain has been characterized and shown to most likely represent labelling of group III metabotropic glutamate receptors. 2. Specific [3H]-L-AP4 binding to rat brain sections was observed at high densities in the molecular layer of the cerebellar cortex and the outer layer of the superior colliculus. Moderate levels were observed throughout the cerebral cortex, in the molecular layer of the hippocampal dentate gyrus, in thalamus, striatum, substantia nigra and in the medial geniculate nucleus. Low levels of [3H]-L-AP4 binding were found in other regions of the hippocampal formation, in the entorhinal cortex and the granule cell layer of cerebellum. 3. Inhibitors of sodium- or calcium/chloride-dependent glutamate uptake did not displace [3H]-L-AP4 binding to rat brain sections indicating that the observed binding does not represent [3H]-L-AP4 uptake via these carriers. Furthermore, in contrast to [3H]-L-AP4 uptake into cerebellar membranes, [3H]-L-AP4 binding to brain sections was sensitive to guanosine-5'-O-(3-thio)trisphosphate-gamma-S. 4. In the molecular layer of the cerebellar cortex, [3H]-L-AP4 binding showed a maximal binding density (Bmax) of 0.52+/-0.06 pmol mg(-1) tissue and an affinity (Kd) of 346 nM. The rank order of affinity for displacement of [3H]-L-AP4 binding to rat brain sections was: L-AP4 > L-serine-O-phosphate > glutamate > (L)-2-aminomethyl-4-phosphonobutanoate > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate which is in agreement with a group III metabotropic glutamate receptor pharmacology.