Imaging of the super high affinity binding sites for [3H]Ro15-4513 in rat hippocampus: comparison between in vitro and in vivo binding

Using fluorescence optical and electron spin resonance spectroscopy, we have investigated the production of superoxide by bovine endothelial nitric oxide synthase (NOS). In contrast to neuronal NOS, the heme moiety is identified as the exclusive source of superoxide production by endothelial NOS. Thus, calmodulin-mediated enzyme regulation affects production of nitric oxide and superoxide simultaneously and inseparably. The balance between the nitric oxide/superoxide reaction pathways may be shifted by addition of exogenous heme-specific agents, such as tetrahydrobiopterin. Our results have direct relevance for the pathophysiology of atherosclerosis.     

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.     

Attenuation of the discriminative stimulus effects of ethanol by the benzodiazepine partial inverse agonist Ro 15-4513

The aim of the present study was to investigate whether ethanol training affects the ability of Ro 15-4513 to block the discriminative stimulus effects of ethanol dose differentially. Three different groups of rats were trained to discriminate 1.0 g/kg ethanol (n = 8), 1.5 g/kg ethanol (n = 7) or 2.0 g/kg ethanol (n = 8) from water in a two-lever, food-reinforced procedure. Ethanol and water were administered by gavage 20 min before the onset of the session. When the discrimination performance was stable, rats were pretreated with Ro 15-4513 (1-17 mg/kg; i.p.) 5 min before the administration of ethanol. Ro 15-4513 attenuated the discriminative stimulus effects of 1.0 and 1.5 g/kg ethanol but not 2.0 g/kg ethanol in each of the ethanol training groups. Overall, blockade of the discriminative stimulus effects of 1.0 and 1.5 g/kg ethanol by 5.6 mg/kg Ro 15-4513 occurred without significantly altering response rates or blood ethanol concentrations. A decrease in blood ethanol concentration was, however, found with 17 mg/kg Ro 15-4513 in combination with 2.0 g/kg ethanol. These results suggest that the benzodiazepine partial inverse agonist, Ro 15-4513, can attenuate the discriminative stimulus effects associated with low to moderate doses of ethanol (1.0-1.5 g/kg).     

Effects of buprenorphine and Ro 15-4513 on delayed death and brain beta-endorphin levels in rats treated with cocaine or cocaine-ethanol

The present study was aimed at elucidating the relationship between brain beta-endorphin, which was estimated by the immunofluorescence method, and fatal drug toxicities due to cocaine and combined cocaine-ethanol administration, including the late fatal toxicities clinically noted. beta-endorphin is an endogenous opioid peptide, and its secretion has been suggested to be influenced by physiological stresses. Furthermore, since protection against these fatal toxicities has been previously reported to be provided by buprenorphine (a ligand for opioid receptors) and Ro 15-4513 (a ligand for benzodiazepine receptors), this study also focused on the relationship between the effects of these two ligands and the changes in brain beta-endorphin immunoreactivity. In the fatal toxicity study, a toxic dose (75 mg/kg, i.p.) of cocaine combined with and without ethanol (3 g/kg, i.p.) was administered to the rats, with and without buprenorphine (0.25, 0.5, 1 mg/kg, i.p.) or Ro 15-4513 (5, 10, 15 mg/kg, i.p.). All of the deaths that occurred in these animals were divided into two groups: early deaths with early toxic symptoms in which the drugs were detected in the tissue samples, and late deaths with late toxic symptoms in which no drugs were detected in the samples. Without the administration of buprenorphine or Ro 15-4513, the frequency of late deaths was higher in the cocaine group as compared to the cocaine-ethanol group. The total mortality rate was effectively attenuated by treatment with 0.25 mg/kg buprenorphine or 10 mg/kg Ro 15-4513. Following treatment with 1 mg/kg buprenorphine or 15 mg/kg Ro 15-4513, the frequency of late deaths was significantly enhanced in the cocaine group. The brain and liver cocaethylene concentrations were also attenuated in those groups in which the total mortality rates were attenuated. In the brain beta-endorphin immunoreactivity study, the number of beta-endorphin immunoreactive nerve cells at the arcuate nucleus was counted at 3 minutes or 24 hours after the drug treatment. At 3 minutes after the drug treatment, the number of weakly immunoreactive cells with photographic light absorption values greater than 50% was enhanced in the groups in which the frequency of late deaths had been increased. In the cocaine-ethanol groups treated with buprenorphine or Ro 15-4513, this enhancement of weakly immunoreactive cells was observed when the total mortality rate was increased, regardless of the type of death. At 24 hours after the drug treatment (50 mg/kg cocaine), an enhancement of the weakly immunoreactive cells only was observed in all of the groups in which the occurrence of toxicities had been enhanced, regardless of the type of toxicity. Therefore, it can be concluded that the enhancement of total brain beta-endorphin immunoreactivity was closely correlated with the increase in the frequency of total fatal toxicities, and that the enhancement of weakly immunoreactive cells was closely correlated with the increase in the frequency of delayed fatal toxicities.     

Different functional effect of Ro 15-4513 and Ro 19-4603 on synaptic responses of Purkinje cells in the rat cerebellar slice

The Sugiura operation has been reported to have low operative mortality, rebleeding, and encephalopathy rates when carried out in a predominantly nonalcoholic Japanese population with good liver function. A literature review of reports of the Sugiura procedure outside Japan reveals a high complication and mortality rate when it is used as an emergency procedure in patients with advanced liver disease, especially in those with alcoholic cirrhosis. Uncontrolled studies report results that differ little from the Japanese series when the operation is confined to good-risk patients in the elective situation. Our experience with the Sugiura operation supports its role in these circumstances, especially in patients with portal vein thrombosis and normal liver function. The only good prospective controlled trial has been carried out in patients with schistosomiasis and suggests that the Sugiura operation is far superior to total shunt and may have a slight advantage over the Warren shunt because of its low incidence of postoperative encephalopathy. More controlled trials are required to establish its role in good- to moderate-risk patients with alcoholic cirrhosis.     

Selective in vivo binding of [3H]naltriben to delta-opioid receptors in mouse brain

Naltriben (NTB) is a selective antagonist for the putative delta2-opioid receptor. We have determined the regional kinetics and pharmacological profile of [3H]naltriben in vivo in mouse brain. After i.v. administration to CD1 mice, [3H]naltriben uptake and retention were high in striatum, cortical regions and olfactory tubercles, and low in superior colliculi and cerebellum. Robust rank order correlation was found between [3H]naltriben uptake in discrete brain regions and prior delta-opioid receptor binding determinations in vitro and in vivo. [3H]Naltriben binding in vivo was saturable, and was blocked by the delta-opioid receptor antagonist naltrindole, but not by the mu-opioid receptor antagonist cyprodime or the K-opioid receptor agonist (trans)-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]ben zeneacetamide mesylate (U50,488H). (E)-7-Benzylidenenaltrexone (BNTX), a selective antagonist for the putative delta1-opioid receptor, was 9.6- to 12.9-fold less potent than naltriben as an inhibitor of [3H]naltriben binding. Thus, the sites labeled by [3H]naltriben in vivo may correspond to the delta2-opioid receptor subtype. Such assignment is not definitive, particularly considering the 4-fold higher brain uptake of naltriben as compared to (E)-7-benzylidenenaltrexone. Moreover, the regional distribution of [3H]naltriben in brains from CXB-7/BY (CXBK) mice, a strain that shows supraspinal delta1- but not delta2-opioid receptor agonist effects, was quite similar to that found for CD1 mice.     

Identification of dihydropyridine (DHP) binding sites on cultured monkey renal cells (GMRC) with a photoaffinity probe (-)-[3H]-azidopine

The low affinity binding sites identified in crude membranes from different excitable tissues with the dihydropyridine (DHP) calcium (Ca2+) channel ligands have confused researches in the field of Ca2+ channels as they can represent low affinity state(s) of the DHP receptor, or they can be labelled with DHP-type Ca2+ channel ligands. The aim of this communication was to provide more evidence for the existence of separate DHP binding sites on the surface of cultured green monkey renal cells (GMRC). The saturation ligand binding experiments with [3H]-nitrendipine (NTP) and photoaffinity labelling studies with (-)-[3H]-azidopine (AZI) were performed in order to identify and further characterize the DHP receptor on cultured GMRC. Specific high affinity sites identified on GMRC with [3H]-NTP (Bmax = 0.78 +/- 0.03 pmol/mg protein and KD = 0.06 +/- 0.1 nmol/l in native cells) and photolabelled with AZI represent DHP receptor on L-type Ca2+ channels. The low affinity binding sites photolabelled with AZI on GMRC (9.84 +/- 2.4 pmol/mg protein and KD = 3.21 +/- 1.25 nmol/l in native cells) were significantly increased after preincubation of GMRC with low concentrations of DHPs nitrendipine and nisoldipine. Preincubation of GMRC with Ca2+ channel agonist (-)BAYK 8644 significantly reduced specific photolabelling with AZI on GMRC and increased low affinity labelling. Preincubation of (+)BAYK 8644 was without any effect. Niguldipine (DHP with the voluminous substituent on the port side of the DHP ring) partially inhibited specific photolabelling with AZI on GMRC and also partially reduced the maximal number of low affinity binding sites labelled with AZI. Our results support the hypothesis of separate subsites in the region of DHP receptor of GMRC and the existence of the "marginal" photolabelling of specific DHP binding sites identified on Ca2+ channels.     

Autoradiographic study on [3H]-[D-Ala2]-deltorphin-I binding sites in the rat brain

Previous biochemical and pharmacological studies have shown that [D-Ala2]-deltorphin-I (DADTI) has a high affinity and selectivity for delta-opioid receptors. In this study, designed to provide morphological details, the distribution of DADTI binding sites was examined by autoradiography on coronal, sagittal and horizontal frozen sections of adult rat brain. The sections were incubated with tritiated DADTI solution and exposed for 12 weeks to a 3H-sensitive film. DADTI labelling clearly demonstrated selective and high affinity binding sites of delta-opioid type in several brain regions, including olfactory system, neostriatum, nucleus accumbens, and cortical layers I-II and V-VI.     

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

The recently developed 5-HT2A receptor selective antagonist [3H]MDL100,907 ((+/-)2,3-dimethoxyphenyl-1-[2-(4-piperidine)-methanol]) has been characterized as a radioligand for the autoradiographic visualization of these receptors. [3H]MDL100,907 binding to rat brain tissue sections was saturable, had sub-nanomolar affinity (Kd = 0.2-0.3 nM), and presented a pharmacological profile consistent with its binding to 5-HT2A receptors (rank order of affinity for [3H]MDL100,907-labelled receptors: MDL100,907 > spiperone > ketanserin > mesulergine). The distribution of receptors labelled by [3H]MDL100,907 was compared to the autoradiographical patterns obtained with [3H]Ketanserin, [3H]Mesulergine, and [3H]RP62203 (N-[3-[4-(4-fluorophenyl)piperazin-1-y1]propyl]-1,8-naphtalenes ultam) and to the distribution of 5-HT2A receptor mRNA as determined by in situ hybridization. As opposed to the other radioligands, [3H]MDL100,907 labelled a single population of sites (5-HT2A receptors) and presented extremely low levels of non-specific binding. The close similarity of the distributions of [3H]MDL100,907-labelled receptors and 5-HT2A mRNA further supports the selectivity of this radioligand for 5-HT2A receptors and suggests a predominant somatodendritic localization of these receptors. The present results point to [3H]MDL100,907 as the ligand of choice for the autoradiographic visualization of 5-HT2A receptors.     

Selective in vivo labelling of brain 5-HT1A receptors by [3H]WAY 100635 in the mouse

The novel selective 5-HT1A receptor antagonist radioligand [3H]WAY 100635 ([O-methyl-3H]N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2- pyridyl)cyclohexane-carboxamide) was injected i.v. to mice in an attempt to label in vivo central 5-HT1A receptors. Although 5 min after the i.v. injection of [3H]WAY 100635 (4-7.6 muCi per mouse) the amount of tritium found in the whole brain only accounted for 1.5-1.8% of the injected radioactivity, regional differences in 3H accumulation already corresponded to those of 5-HT1A receptor density. Optimal data were obtained 1 h after [3H]WAY 100635 injection as the distribution of 3H in brain was exactly that of 5-HT1A receptor binding sites in mouse brain sections labelled in vitro with [3H]WAY 100635. In particular, high level of labelling was found in the lateral septum, gyrus dentatus and CA1 area of Ammon's horn in the hippocampus, dorsal raphe nucleus and entorhinal cortex. No labelling was found in he substantia nigra, and 3H accumulated in the cerebellum represented only 12-14% of that found in the hippocampus. Pretreatment with various drugs indicated that only 5-HT1A receptor ligands were able to decrease the accumulation of 3H in all the brain areas examined except in the cerebellum. Assuming that only non-specific binding took place in the latter structure, it was possible to calculate the ID50 values of 5-HT1A receptor agonists (8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin), S 14506 (1-[2-(4-fluorobenzoylamino)ethyl]-4-(7-methoxynaphthyl+ ++)piperazine) and S 20499 ((+)-4-[N-(5-methoxy-chroman-3-yl)-N-propylamino]butyl-8- azaspiro-(4,5)-decane-7,9-dione)) and antagonists (spiperone, (-)-tertatolol, (+)-WAY 100135 (N-tert-butyl-3,4-(2-methoxyphenyl)piperazin-1-yl-2-phenyl- propanamide)) as inhibitors of 3H accumulation in the hippocampus of [3H]WAY 100635-injected mice. Comparison of these values with the in vitro affinity of the same ligands for hippocampal 5-HT1A receptors revealed marked variations in the capacity of 5-HT1A receptor agonists and antagonists to reach the brain when injected via the subcutaneous route in mice.     

Direct determination of dopamine D4 receptors in normal and schizophrenic postmortem brain tissue: a [3H]NGD-94-1 study

Normal speed videography was used to determine the angular parameters of 28 Spanish Thoroughbreds at trot. Horses were divided into 3 groups: Group UT, comprising 9 animals (provided by the VII National Stud, Cordoba, Spain) which had undergone no specific training programme and which were hand led at the trot; Group T, formed by 19 horses considered to be highly bred and trained, and which were also hand led; and Group RT, comprising the same horses as the latter group but this time trotted by a rider. Each animal was filmed 6 times from the right-hand side, using a Hi8 (25 Hz) video camera. Angular parameters for fore- and hindlimb joints were measured in each stride from computer-grabbed frames and entered into a spreadsheet for calculation; parameters included maximum and minimum angles, range of motion, and angles at landing, lift off and maximum hoof height; the times at which maximum angle, minimum angle, lift off and maximum hoof height occurred were calculated as percentages of total stride duration. Stride velocity (mean [s.d.]) was 4.01 (0.62), 3.60 (0.34) and 3.07 (0.36) m/s for Groups UT, T and RT, respectively. Data were then compared between Groups UT-T and Groups T-RT. Compared with Group UT, horses from Group T featured a shorter stance percentage (P<0.001) in both fore- and hindlimbs. The range of motion in forelimbs was smaller (P<0.05), due to lower retraction (P<0.001); moreover, maximum retraction appeared earlier (P<0.05). Greater scapular inclination was in evidence (P<0.05) and the shoulder joint extended further (P<0.05). Fore- and hind fetlock joints revealed a relatively shorter hyperextension period during the stance phase (P<0.01). Compared with Group T, horses from Group RT had a longer stance percentage, with belated maximum retraction of the fore- and hindlimbs. The range of movement in scapular inclination was greater (P<0.05), due to a smaller minimum angle (P<0.01), and the shoulder joint flexed more (P<0.05). The elbow joint extended more and for longer during the stance phase. Initial extension of the hip joint (P<0.05) and tarsus (P<0.001) lasted longer. The carpal and fore and hind fetlock joints recorded relatively longer hyperextension times, in addition to greater hyperextension during the stance phase. The results from the present study suggest that rider-effect must be taken in consideration when well gaited horses are selected for dressage purposes.     

Age-related changes of sodium-dependent D-[3H]aspartate and [3H]FK506 binding in rat brain

We investigated age-related changes in excitatory amino acid transport sites and FK506 binding protein (FKBP) in 3-week-, and 6-, 12-, 18- and 24-month-old Fischer 344 rat brains using receptor autoradiography. Sodium-dependent D-[3H]aspartate and [3H]FK506 were used to label excitatory amino acid transport sites and immunophilin (FKBP), respectively. In immature rats (3-week-old), sodium-dependent D-[3H]aspartate binding was lower in the frontal cortex, parietal cortex, striatum, nucleus accumbens, whole hippocampus, thalamus and cerebellum as compared to adult animals (6-month-old), whereas [3H]FK506 binding was significantly lower in only the hippocampus, thalamus and cerebellum. 3[H]FK506 binding exhibited no significant change in the brain regions examined during aging. However, sodium-dependent D-[3H]aspartate binding showed a conspicuous reduction in the substantia nigra in 18-month-old rats. Thereafter, a significant reduction in sodium-dependent D-[3H]aspartate binding was found in the thalamus, substantia nigra and cerebellum in 24-month-old rats. Other regions also showed about 10-25% reduction in sodium-dependent D-[3H]aspartate binding. The results indicate that excitatory amino acid transport sites are more susceptible to aging process than immunophilin. Further, our findings demonstrate the conspicuous differences in the developmental pattern between excitatory amino acid transport sites and immunophilin in immature rat brain.     

Modulation of [3H]quinpirole binding to dopaminergic receptors by adenosine A2A receptors

Alteration of ligand binding to dopamine D2 receptors through activation of adenosine A2A receptors in rat striatal membranes has been studied by means of kinetic analysis. The binding of dopaminergic agonist [3H]quinpirole to rat striatal membranes was characterized by the constants Kd = 1.50+/-0.09 nM and Bmax = 115+/-2 fmol/mg of protein. The kinetic analyses revealed that the binding had at least two consecutive and kinetically distinguishable steps, the fast equilibrium of complex formation between receptor and agonist (KA = 5.9+/-1.7 nM), followed by a slow isomerization equilibrium (Ki = 0.06). Activation of adenosine A2A receptors by CGS 21680 caused enhancement of the rate [3H]quinpirole binding, altering mainly the formation of the receptor-ligand complexes (KA) as well as the isomerization rate of this complexes (ki), while the deisomerization rate (k[-i]) and the apparent dissociation rate remained unchanged.     

Characterization of [3H]clozapine binding sites in rat brain

We examined the characteristics of [3H]clozapine binding sites in four rat brain regions (frontal cortex, limbic area, hippocampus and striatum) in order to elucidate the pharmacological profile of this unique atypical antipsychotic drug. The specific [3H]clozapine binding was found to be saturable and reversible in all these brain regions. Scatchard analysis of the saturation data indicated that the specific binding consisted of high- and low-affinity components. Displacement experiments showed that the muscarinic cholinergic receptor represented about 50% of [3H]clozapine binding in each brain area. Serotonin 5-HT2 and dopamine D4 receptor binding sites could also be detected by displacement experiments using ketanserin and nemonapride, respectively, in frontal cortex and limbic area, but not in hippocampus or striatum. Alpha-1, alpha-2, histamine H1, dopamine D1, D2, or D3 receptor components could not be determined within the high-affinity [3H]clozapine binding sites in any brain region. It is possible that the atypical property of clozapine may depend on the modulatory effect on dopaminergic function via 5-HT2 receptor blockade and/or may be mediated via D4 receptor blockade in the mesocortical and mesolimbic area.     

The major site of photoaffinity labeling of the gamma-aminobutyric acid type A receptor by [3H]flunitrazepam is histidine 102 of the alpha subunit

The alpha subunit of the gamma-aminobutyric acid type A (GABA(A)) receptor is known to be photoaffinity labeled by the classical benzodiazepine agonist, [3H]flunitrazepam. To identify the specific site for [3H]flunitrazepam photoincorporation in the receptor subunit, we have subjected photoaffinity labeled GABA(A) receptors from bovine cerebral cortex to specific cleavage with cyanogen bromide and purified the resulting photolabeled peptides by immunoprecipitation with an anti-flunitrazepam polyclonal serum. A major photolabeled peptide component from reversed-phase high performance liquid chromatography of the immunopurified peptides was resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The radioactivity profile indicated that the [3H]flunitrazepam photoaffinity label is covalently associated with a 5.4-kDa peptide. This peptide is glycosylated because treatment with the enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, reduced the molecular mass of the peptide to 3.2 kDa. Direct sequencing of the photolabeled peptide by automated Edman degradation showed that the radioactivity is released in the twelfth cycle. Based on the molecular mass of the peptides that can be generated by cyanogen bromide cleavage of the GABA(A) receptor alpha subunit and the potential sites for asparagine-linked glycosylation, the pattern of release of radioactivity during Edman degradation of the photolabeled peptide was mapped to the known amino acid sequence of the receptor subunit. The major site of photoincorporation by [3H]flunitrazepam on the GABA(A) receptor is shown to be alpha subunit residue His102 (numbering based on bovine alpha 1 sequence).