Characterization of imidazoline receptors by radioligand binding.
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Animals↗
Biomedical subjects
A L Hudson
Publications and source records attributed to A L Hudson.
Characterisation and localisation of [3H]2-(2-benzofuranyl)-2-imidazoline binding in rat brain: a selective ligand for imidazoline I2 receptors.
In rat whole brain homogenates, saturation binding analysis revealed that both [3H]2-BFI (2-(2-benzofuranyl)-2-imidazoline) and [3H]idazoxan (in the presence of 5 microM rauwolscine) bound with high affinity to an apparent single population of sites. However, the Kd for [3H]2-BFI (1.74+/-0.14 nM) was significantly (P < 0.01) less than that for [3H]idazoxan (10.4+/-2.68 nM). In competition studies idazoxan, 2-BFI, BU224 (2-(4,5-dihydroimidaz-2-yl)-quinoline), amiloride and guanabenz displayed high affinity (Ki values = 7.32, 1.71, 2.08, 21.80 and 14.90 nM, respectively) for 70-80% of sites, and low microM affinity for the remaining 20-30% of sites labelled by [3H]2-BFI. In contrast, several alpha2-adrenoceptor, imidazoline I1 receptor and histamine receptor ligands exhibited only micromolar affinity for the [3H]2-BFI labelled site. Quantitative receptor autoradiography revealed high binding by [3H]2-BFI to discrete brain nuclei, notably the area postrema, interpeduncular nucleus, arcuate nucleus, mammillary peduncle, ependyma and pineal gland. These data indicate that [3H]2-BFI recognises imidazoline I2 receptors in rat brain with higher affinity and selectivity than [3H]idazoxan and thus represents a superior radioligand to [3H]idazoxan for the study of imidazoline I2 receptors.
Animals↗
'Seeing through a glass darkly': casting light on imidazoline 'I' sites.
Although imidazoline sites have been the subject of research for several years, there is still controversy about their structure, diversity and physiology. The I1 site is thought to exist principally as a binding site and is widely purported to play a role in controlling systemic blood pressure, although this is still unclear. The majority of I2 sites are widely accepted as being allosteric sites on monoamine oxidase; however, even with selective ligands, their exact function remains to be determined. A putative I3 site modulates insulin secretion and could represent the first functional site to be pharmacologically defined with selective agonists and antagonists. The structure and relevance of the proposed endogenous ligand 'clonidine-displacing substance' remains elusive. A potential candidate for this substance is agmatine; however, although it is capable of displacing bound clonidine from imidazoline sites, it lacks the functionality ascribed to the clonidine-displacing substance. In this review, Richard M. Eglen and colleagues assess our knowledge of imidazoline sites in the light of recent data.
Adrenergic alpha-Agonists↗
Potentiation of barbiturate-induced alterations in presynaptic noradrenergic function in rat frontal cortex by imidazol(in)e alpha2-adrenoceptor agonists.
1. In order to resolve the extent to which presynaptic noradrenergic mechanisms contribute to the anaesthetic-sparing effects of alpha2-adrenoceptor agonists in vivo microdialysis was used to investigate the combined effects of sodium pentobarbitone and imidazol(in)e alpha2-adrenoceptor agonists on extracellular levels of noradrenaline (NA) in the rat frontal cortex. 2. Dialysate levels of NA were markedly reduced by the addition of TTX (2 microM) or by the removal of calcium in the perfusate. These data imply that dialysate NA levels are ultimately dependent on exocytotic release mechanisms from afferent coeruleo-cortical neurones. 3. Systemic administration of sodium pentobarbitone (85 mg kg(-1), i.p.) induced general anaesthesia and reduced NA levels by 92% after 30 min. The restoration of basal levels 90 min later was closely associated with a return of the corneal blink reflex. 4. Basal NA levels in conscious animals were not affected by an intravenous infusion of equally radioactive solutions of either imidazoline (clonidine) or imidazole (mivazerol) alpha2-adrenoceptor agonists. The dose rate employed for each compound was 2 microg kg(-1) h(-1) over 2 h. 5. The co-administration of intravenous clonidine or mivazerol, each at 2 microg kg(-1) h(-1) for 2 h, with sodium pentobarbitone (85 mg kg(-1), i.p.), produced a marked and prolonged reduction in NA efflux. After 2 h, NA levels remained suppressed by 95% (clonidine) and 80% (mivazerol) and animals remained deeply anaesthetized. 6. The accumulation of tritium in brain tissue was 42-73% lower across all brain regions examined after [3H]-mivazerol administration than after [3H]-clonidine administration. Sodium pentobarbitone did not alter the accumulation of tritium in brain tissue after the administration of either alpha2-adrenoceptor agonist. 7. These data demonstrate that alpha2-adrenoceptor agonists potentiate the inhibitory effects of sodium pentobarbitone on extracellular levels of NA in the frontal cortex. Further studies will be necessary to establish a causal role of noradrenergic mechanisms in the potentiation of anaesthesia by selective alpha2-adrenoceptor agonists.
Adrenergic alpha-Agonists↗
Identification of ligands selective for central I2-imidazoline binding sites.
Using radioligand binding techniques, several compounds selective for mammalian brain imidazoline 2 receptors have been identified. In rabbit brain membranes, a series of 6 and/or 7 aromatic-substituted derivatives of the alpha 2-adrenoceptor antagonist idazoxan were found to show moderate affinity for I2 receptors over alpha 2-adrenoceptors, in particular 6,7-dichloroidazoxan, which was 41 fold selective in favour of I2 receptors. Modification of the benzodioxan ring of idazoxan could also result in affinity and selectivity, which was moderate (2.7 nM, 161 fold) in the case of the 1,3-benzodioxan isomer of idazoxan (2-(1,3-benzodioxanyl)-2-imidazoline), and high (1.3 nM, 2873 fold) in the case of 2-(2-benzofuranyl-2-imidazoline) (2-BFI). Analogues of 2-BFI with halogenic substitutions of the aromatic ring were also found to retain high affinity and moderate to high selectivity for I2-sites. In particular, the 7-chloro (Ki 2.8 nM, 2192 fold) and the 4,6-dibromo (Ki 6.1 nM, 361 fold) analogues of 2-BFI. These new ligands should prove invaluable for investigating the pharmacology and physiology of I2 receptors.
Adrenergic alpha-Antagonists↗
A series of novel imidazoline I2-receptor selective Schiff bases of 1-(benzylidenamino)-3,3-dimethylguanidine.
A series of 12 Schiff bases of 1-(benyzylidenamino)-3,3-dimethylguanidine were evaluated for their affinities at imidazoline I2-receptors and alpha 2-adrenoceptors in the guinea pig cerebral cortex by using a 2-curve assay with [3H]idazoxan as ligand and the simultaneous computer analysis of (-)adrenaline masked and non-masked competition curves. All of the dimethylguanidines were selective for I2-receptors with the selectivity ranging 6-110-fold, and the most selective being WAP8 (2-bromobenzylidenamino-3,3-dimethylguanidine) closely followed by FLA151 (2-chlorobenzylidenamino-3, 3-dimethylguanidine). Some of the dimethylguanidines were also tested for affinity at bovine kidney cortex I2-receptors and guinea pig alpha 1-adrenoceptors. These substances were found to have similar affinities for bovine kidney I2-receptors as for guinea pig cerebral cortex I2-receptors, but their affinities for the alpha 1-adrenoceptors were considerably lower.
Animals↗
Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes. 1983.
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Animals↗
Noradrenergic mechanisms in the prefrontal cortex.
There is growing evidence that noradrenergic inputs to the prefrontal cortex (PFC) play an important role in regulating its function. This paper reviews the pharmacological control of noradrenaline (NA) release in this region, with particular reference to our studies using brain microdialysis, and also describes how NA levels are modulated by antidepressant and antipsychotic drugs. The suggestion that atypical antipsychotics such as clozapine and risperidone may produce clinical benefits by their ability to increase NA release is discussed. Finally, a new class of drugs, which show selectivity for imidazoline receptors is described. These compounds are shown to similarly increase extracellular NA in the PFC. Their potential utility as clinical treatments is discussed.
Animals↗
Antisense oligonucleotides in psychopharmacology and behaviour: promises and pitfalls.
Antisense oligonucleotides are used to study the expression and function of a diverse range of proteins. Areas for which antisense has been used for pharmacological investigation include receptors, neuropeptides and immediate early genes, particularly when specific ligands or markers are not yet available. Antisense oligonucleotides target a specific mRNA and block the expression of the protein by sequence specific hybridization. This technique has not only been shown to be a valuable pharmacological tool but also to have potential therapeutic applications. In this review we discuss the technology behind the technique including developments in methodology employed in antisense experiments. Although antisense provides a novel and highly specific tool, the reliability of the technique and many of the problems associated with antisense experiments are discussed. The main focus of this article is the use of antisense in psychopharmacology to investigate behavioural changes following antisense-mediated inhibition of the expression of specific brain proteins and receptors.
Animals↗
[3H]2-(2-benzofuranyl)-2-imidazoline: a new selective high affinity radioligand for the study of rabbit brain imidazoline I2 receptors.
This is the first study characterising the binding of the new imidazoline I2 receptor selective radioligand [3H]2-(2-benzofuranyl)-2-imidazoline (2-BFI) to rabbit brain membranes. [3H]2-BFI binding was found to be saturable and of high affinity identifying two binding sites with KD1 = 0.27 nM, Bmax = 111.2 fmol mg-1 protein and KD2 = 8.97 nM, Bmax = 268 fmol mg-1 protein. Specific binding represented greater than 90% of total binding. Kinetic studies revealed that the binding was rapid and reversible and also showed [3H]2-BFI interacted with these two sites or two affinity states. In competition binding studies against [3H]2-BFI (0.3-InM) idazoxan, 2-BFI, cirazoline, guanabenz, naphazoline, amiloride and BU224 (2-(4,5-dihydroimidaz-2-yl-quinoline) displaced with high affinity. In contrast the alpha 2-adrenoceptor antagonists efaroxan and rauwolscine, the I1 site selective drug moxonidine, the monoamine oxidase-A inhibitor clorgyline and the proposed endogenous imidazoline receptor ligand, agmatine, were weak at displacing [3H]2-BFI binding. These findings are consistent with [3H]2-BFI recognising imidazoline receptors of the I2 subtype in rabbit brain.
Affinity Labels↗
Imidazoline binding sites in Huntington's and Parkinson's disease putamen.
Binding of [3H]2-(2-benzofuranyl)-2-imidazoline ([3H]BFI) to the imidazoline I2 receptor was determined in putamen taken post mortem from patients with two extrapyramidal motor disorders, Parkinson's and Huntington's diseases, and age-matched control subjects. No deficit of binding was apparent in Parkinson's disease, indicating that the receptors are not present on nigrostriatal terminals. A significant loss (by 56%) in imidazoline I2 receptor binding was observed in Huntington's disease, consistent with the receptors being sited on degenerating neurons.
Adrenergic alpha-Antagonists↗
Localization of high affinity [3H]glibenclamide binding sites within the substantia nigra zona reticulata of the rat brain.
The rat substantia nigra zona reticulata contains a high density of binding sites for glibenclamide, an adenosine triphosphate-sensitive potassium channel inhibitor, but the precise location of glibenclamide binding sites within this area has not previously been examined. By combining neurochemical lesion and autoradiographical studies we have shown that high affinity [3H]glibenclamide binding sites are located on striatonigral terminals. Unilateral injections of 6-hydroxydopamine into the medial forebrain bundle or of quinolinic acid into the striatum were performed in anaesthetized adult rats to lesion the nigrostriatal and striatonigral pathways respectively. Autoradiography was performed on coronal sections of midbrain with [3H]glibenclamide, [3H]YM-09151-2 (dopamine D2 receptor antagonist) and [3H]SCH 23390 (dopamine D1 receptor antagonist) at three rostrocaudal levels of the substantia nigra. Under the conditions of the incubation [3H]glibenclamide binds primarily to the high affinity site. Following the 6-hydroxydopamine nigrostriatal lesion, D2 receptor binding was reduced (by up to 67%) on the lesioned side at all three levels of the substantia nigra whereas D1 receptor and glibenclamide binding were not significantly affected. In contrast, following striatonigral pathway lesion with quinolinic acid D2 receptor binding was unchanged on the lesioned side, but both D1 receptor and glibenclamide binding were reduced at all three levels (by up to 85% and 63% in the area of maximum lesion, respectively). In adjacent sections, the pattern of D1 binding loss was closely paralleled by the loss of glibenclamide binding. These results demonstrate that the high affinity glibenclamide binding sites of the substantia nigra zona reticulata are, at least in part, located on the terminals of striatonigral projection neurons.
Adenosine Triphosphate↗
Quantification of in vivo binding of [3H]RX 821002 in rat brain: evaluation as a radioligand for central alpha 2-adrenoceptors.
On the basis of its established in vitro characteristics, [3H]RX 821002 was evaluated in rats as an in vivo radioligand for central alpha 2-adrenoceptors. Estimates for in vivo binding potential, obtained by compartmental analyses of time-radioactivity data, ranged between 1.9 for hypothalamus and 0.2 for cerebellum, with a regional distribution in brain which was similar to that observed in vitro. Selectivity and specificity of the signal were checked by predosing with either the alpha 2-antagonists, idazoxan or yohimbine, the alpha 2-agonist, clonidine, or the alpha 1-antagonist, prazosin. Pretreatment of the rats with the selective neurotoxin, DSP-4, had no significant effect on [3H]RX 821002 binding, suggesting that the majority of labelled sites were situated post-junctionally. The studies indicate that [3H]RX 821002 can be used experimentally as an in vivo marker for central alpha 2-adrenoceptors. The size and rate of expression of the specific signal encourage the development and assessment of [11C]RX 821002 for clinical PET studies.
Adrenergic alpha-Antagonists↗
Characterization and autoradiographical localization of non-adrenoceptor idazoxan binding sites in the rat brain.
1. In rat whole brain homogenates, saturation analysis revealed that both [3H]-idazoxan and [3H]-RX821002, a selective alpha 2-adrenoceptor ligand, bound with high affinity to an apparent single population of sites. However, the Bmax for [3H]-idazoxan was significantly (P less than 0.01) greater than that for [3H]-RX821002. 2. In competition studies, (-)-adrenaline displaced 3 nM [3H]-idazoxan binding with an affinity consistent with [3H]-idazoxan labelling alpha 2-adrenoceptors. However, this displacement was incomplete since 23.68 +/- 1.11% of specific [3H]-idazoxan binding remained in the presence of an excess concentration (100 microM) of (-)-adrenaline. In contrast, unlabelled idazoxan promoted a complete displacement of [3H]-idazoxan binding with a Hill slope close to unity and an affinity comparable with its KD determined in saturation studies. 3. Displacement of [3H]-idazoxan binding by the alpha 2-adrenoceptor antagonists yohimbine, RX821002 (2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline) and RX811059 (2-(2-ethoxy-1,4-benzodioxan-2-yl)-2-imidazoline) was more complex, with Hill slopes considerably less than unity, and best described by a two-site model of interaction comprising a high and low affinity component. The proportion of sites with high affinity for each antagonist was similar (60-80%). 4. The rank order of antagonist potency for the high affinity component in each displacement curve (RX821002 greater than RX811059 greater than yohimbine) is similar to that determined against the binding of [3H]-RX821002 to rat brain, suggesting that these components reflect the inhibition of [3H]-idazoxan binding to alpha 2-adrenoceptors.The remaining component in each displacement curve exhibiting low affinity towards these antagonists is attributable to the displacement of [3H]-idazoxin from a non-adrenoceptor idazoxan binding site (NAIBS) since a comparable amount of [3H]-idazoxan binding was not displaced by an excess concentration of (-)-adrenaline.5. The displacement of [3H]-idazoxan binding by RX801023 (6-fluoro-(2-(1,4-benzodioxan-2-yl)-2-imidazoline) was also best described by a model assuming a two site interaction with 20.07 +/- 3.11% of the sites labelled displaying high affinity for RX801023. The Ki of RX801023 for the remainder of the sites labelled was similar to its Ki versus [3H]-RX821002, indicating that this drug displays improved affinity and NAIBS/z2-adrenoceptor selectivity compared with idazoxan.6. In autoradiographical studies, the distribution of 5 nM [3H]-idazoxan binding to sections of rat whole brain was consistent with that reported from previous studies and resembled the distribution ofM2-adrenoceptors. However, when sections of brain were coincubated with concentrations of alpha2-adrenoceptor agonists or antagonists predicted to saturate alpha2-adrenoceptors, there remained distinct areas of binding corresponding to discrete brain nuclei. This remaining binding was however displaced by unlabelled idazoxan (3 microM) or RX801023 (3 microM) indicative of the labelling of NAIBS.7. Quantitative autoradiography of NAIBS revealed several brain nuclei which contained higher densities of these sites than alpha2-adrenoceptors, notably the area postrema, interpeduncular nucleus,arcuate nucleus, ependyma and pineal gland.
Adrenergic alpha-Antagonists↗
Quantitative autoradiography of [3H]-MK-801 binding sites in mammalian brain.
1. An in vitro receptor autoradiography procedure is described for visualizing binding sites for the excitatory amino acid antagonist radiolabelled MK-801, in rat and gerbil brain sections. 2. Ten micron sections were labelled by incubation at room temperature for 20 min in 30 nM [3H]-MK-801. This was followed by 2 rinses for 20 s in fresh buffer solution. Specifically bound ligand determined with 100 microM unlabelled MK-801 amounted to 55-60% of total. 3. Phencyclidine, (+/-)-SKF 10047, ketamine and 2-aminophosphonovaleric acid (APV) (all 100 microM) prevented the specific binding of [3H]-MK-801. L-Glutamate and N-methyl D-aspartate (NMDA) (100 microM) had no effect. However, L-glutamate prevented the inhibition by APV. 4. The highest concentrations of [3H]-MK-801 binding sites occurred in the hippocampal formation, cerebral cortex, olfactory bulb and thalamus. Very low levels were detected in the brain stem and cerebellum. 5. The distribution of [3H]-MK-801 binding sites was comparable to that of NMDA sites and phencyclidine sites (labelled with [3H]-TCP) but not with high-affinity sigma sites labelled with [3H]-3-PPP. 6. The density of [3H]-MK-801 binding sites in the gerbil hippocampus was examined 1, 2, 6 and 22 days after unilateral carotid artery occlusion for 10 min. Only at 6 and 22 days was the binding reduced (by 36% and 46% respectively) in the CA1 region whereas a significant neuronal loss was apparent at day 2. In CA2 a decrease in binding was only evident at day 22. 7. These results indicate that binding sites for [3H]-MK-801 can be detected in mammalian brain sections by receptor autoradiography. Their distribution supports an association with the NMDA receptor complex and the loss in the hippocampus after carotid artery occlusion indicates their presence on pyramidal cells is vulnerable to ischaemic insult.
Animals↗
GABAA and GABAB receptor site distribution in the rat central nervous system.
An autoradiographic procedure has been used to determine the quantitative distributions of gamma-aminobutyric acid (GABAA and GABAB) receptor subtypes in rat brain. Although the concentrations of both receptor binding sites were similar in some brain regions GABAA sites generally outnumbered GABAB sites. The highest concentration of GABAA sites were detected in the frontal cortex, the granule cell layer of the cerebellum, the olfactory bulb and the thalamic medial geniculate. The highest concentration of GABAB sites occurred in the molecular layer of the cerebellum, the interpeduncular nucleus, frontal cortex, anterior olfactory nucleus and thalamic nuclei. In addition the globus pallidus, temporal cortex, lateral posterior thalamus, superior colliculus, pontine nucleus, raphe magnus, spinal trigeminal tract and substantia gelatinosa contained significantly more GABAB sites than GABAA sites. The physiological and pharmacological significance of this heterogeneity has yet to be determined.
Animals↗
[3H](-)Baclofen: an improved ligand for GABAB sites.
[3H](-)Baclofen, the radiolabelled form of the active isomer of baclofen, has been used as a ligand for GABAB (gamma-aminobutyric acid) receptors on synaptic membranes from whole brain of rat. The pharmacological profile for displacement of this ligand was observed to be identical with that for the racemic ligand [3H](+/-)baclofen and [3H]GABA under conditions where GABAB, but not GABAA sites, were labelled. The displaceable (specific) portion of membrane-bound [3H](-)baclofen was 47.5 +/- 2.3% of the total which was twice that obtained previously with [3H](+/-)baclofen. Two binding components were observed with affinities of 19 and 304 nM and binding capacities of 0.37 and 1.58 pmol/mg protein respectively. It is suggested that [3H](-)baclofen is an improvement over the labelled racemic form and binds to the same sites. It should provide a more reliable tool for studying GABAB receptors.
Animals↗
GABA receptor multiplicity. Visualization of different receptor types in the mammalian CNS.
Receptors for GABA in the mammalian brain are not homogeneous. A clear separation exists between receptors which recognize the antagonist bicuculline and a population which does not. These two classes have been designated GABAA and GABAB sites respectively. Within the GABAA category there may also be many subtypes which exhibit subtle pharmacological differences. Numerous centrally-active agents influence GABAA site function generally via an allosteric interaction. By comparison very few substances currently available interact with GABAB sites. The GABAA and GABAB sites show many contrasting characteristics not least of which is their distribution pattern within the rat brain. Autoradiographic analysis has indicated that although both receptors may be present within many regions, in some areas only one type is present. For example, GABAA sites only are present in the lamina molecularis of the olfactory bulb and granule cell layer of the cerebellum whereas GABAB sites are present in the interpeduncular nucleus without any evidence of GABAA sites. In the spinal cord GABAB sites are unevenly distributed with high densities in laminae I-IV. GABAA sites are more uniformly distributed throughout the dorsal and ventral horns. In conclusion, there is now good evidence for multiple GABA receptors and the way is open to determine the functional significance of the GABAB receptor in relation to the now classical GABAA site.
Animals↗