In vivo and in vitro imaging of I2 imidazoline receptors in the monkey brain.

I2 imidazoline receptors (I2Rs) are associated with depression, Alzheimer's disease, and Huntington's disease. However, in vivo imaging of I2Rs in the monkey brain has not been reported until now. We performed in vitro and in vivo imaging of (I2Rs) in the monkey brain using ¹¹C-labeled 2-(3-fluoro-4-tolyl)-4,5-dihydro-1H-imidazole ([¹¹C]FTIMD) which has high and selective affinity of I2Rs. In an auto-radiography (ARG) study, the distribution pattern of [¹¹C]FTIMD in the monkey brain was similar to that of [³H]idazoxan binging to I2Rs in the human brain, which was previously described. The specific binding of [¹¹C]FTIMD accounted for >97% of total binding in brain regions existing I2 Rs. In positron emission tomography (PET) studies, the radioactivity was accumulated in brain regions existing I2Rs ligand BU224, the accumulated radioactivity was decreased to approximately 66%-75% of the baseline measurement at 15-45 min after injection of [¹¹C]FTIMD. These results suggest that [¹¹C]FTIMD shows the specific-binging to I2Rs in the monkey brain as depicted by PET and ARG. We performed the first in vivo imaging of I2Rs using [¹¹C]FTIMD in the monkey brain.

The alpha(2) adrenoceptor antagonist idazoxan alleviates L-DOPA-induced dyskinesia by reduction of striatal dopamine levels: an in vivo microdialysis study in 6-hydroxydopamine-lesioned rats.

L-DOPA-induced dyskinesia is characterised by debilitating involuntary movement, which limits quality of life in patients suffering from Parkinson's disease. Here, we investigate effects of the a2 adrenoceptor antagonist idazoxan on L-DOPA-induced dyskinesia as well as on alterations of extracellular L-DOPA and dopamine (DA) levels in the striatum in dyskinetic rats. Male Wistar rats were unilaterally lesioned with 6-hydroxydopamine and subsequently treated with L-DOPA/benserazide to induce stable dyskinetic movements.Administration of idazoxan [(9 mg/kg, intraperitoneal (i.p.)]significantly alleviated L-DOPA-induced dyskinesia, whereas idazoxan (3 mg/kg, i.p.) did not affect dyskinetic behaviour.Bilateral in vivo microdialysis revealed that idazoxan 9 mg/kg reduces extracellular peak L-DOPA levels in the lesioned and intact striatum as well as DA levels in the lesioned striatum. In parallel, the exposure to idazoxan in the striatum was monitored.Furthermore, no idazoxan and L-DOPA drug-drug interaction was found in plasma, brain tissue and CSF. In conclusion, the decrease of L-DOPA-derived extracellular DA levels in the lesioned striatum significantly contributes to the anti-dyskinetic effect of idazoxan.

Changes in adrenoreceptors in the prefrontal cortex of subjects with dementia: evidence of compensatory changes.

In Alzheimer's disease (AD) there is a significant loss of locus coeruleus (LC) noradrenergic neurons. However, recent work has shown the surviving noradrenergic neurons to display many compensatory changes, including axonal sprouting to the hippocampus. The prefrontal cortex (PFC) is a forebrain region that is affected in dementia, and receives innervation from the LC noradrenergic neurons. Reduced PFC function can reduce cognition and disrupt behavior. Because the PFC is an important area in AD, we determined if noradrenergic innervation from the LC noradrenergic neurons is maintained and if adrenoreceptors are altered postsynaptically. Presynaptic PFC alpha2-adrenoreceptor (AR) binding site density, as determined by 3H-RX821002, suggests that axons from surviving noradrenergic neurons in the LC are sprouting to the PFC of subjects with dementia. Changes in postsynaptic alpha1-AR in the PFC of subjects with dementia indicate normal to elevated levels of binding sites. Expression of alpha1-AR subtypes (alpha1A- and alpha1D-AR) and alpha2C-AR subtype mRNA in the PFC of subjects with dementia is similar to what was observed in the hippocampus with one exception, the expression of alpha1A-AR mRNA. The expression of the alpha1A-AR mRNA subtype is significantly reduced in specific layers of the PFC in subjects with dementia. The loss of alpha1A-, alpha1D- and alpha2C-AR mRNA subtype expression in the PFC may be attributed to neuronal loss observed in dementia. These changes in postsynaptic AR would suggest a reduced function of the PFC. Consequence of this reduced function of the PFC in dementia is still unknown but it may affect memory and behavior.

Pharmacological characterization and autoradiographic distribution of alpha2-adrenoceptor antagonist [3H]RX 821002 binding sites in the chicken brain.

Knowledge about the noradrenergic system in birds is very scarce even though their biological diversity and complex social behavior make them an excellent model for studying neuronal functions and developmental biology. While the role of norepinephrine has been described in depth in a large number of central and peripheral functions in mammals, reports for avian species are limited. The radioligand [(3)H]RX 821002 ([(3)H]1,4-[6,7(n)3H]-benzodioxan-2-methoxy-2-yl)-2-imidazol) has been used to map and characterize alpha(2)-adrenoceptors through the chicken brain using in vitro autoradiography and membrane homogenates binding assays. [(3)H]RX 821002 showed a saturable and high affinity binding to a site compatible with alpha(2)-adrenoceptor, and to a serotonergic component. The autoradiographic assays displayed a similar alpha(2)-adrenoceptor distribution than those previously reported in birds using other radioligands such as [(3)H]UK 14304 ([(3)H]5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine) or [(3)H]clonidine. [(3)H]RX 821002 binding pharmacological characterization was carried out in different chicken brain regions using membrane homogenates for competition assays with different alpha(2)-adrenoceptor agonists and antagonists drugs (oxymetazoline, BRL 44408 [2-(2H-(1-methyl-1,3-dihydroisoindole)methyl)-4,5-dihydroimidazole] ARC 239 [2-(2-4-(O-methoxyphenyl)-piperazin-1-yl)-ethyl-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione], prazosin, UK 14304 and RX 821002). The results showed alpha(2A) as the predominant alpha(2)-adrenoceptor subtype in the chicken brain while alpha(2B)- and/or alpha(2C)-adrenoceptor subtypes were detected only in the telencephalon. RX 821002, serotonin (5-HT) and 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin] competition assays, and competition binding assays performed in the presence of serotonin demonstrated that [(3)H]RX 821002 binds with higher affinity to a serotonergic component, probably 5-HT(1A) receptors, than to the alpha(2)-adrenoceptors. Similar pharmacological properties for the alpha(2)-adrenoceptor component were observed both in rat and chicken brain. The results demonstrate that the different alpha(2)-adrenoceptor subtypes are present in chicken brain and suggest that these receptors are highly conserved through evolution.

Receptor subtype-induced targeting and subtype-specific internalization of human alpha(2)-adrenoceptors in PC12 cells.

The three alpha(2)-adrenergic receptor subtypes have distinct tissue distributions, desensitization properties, and, in some cell types, subtype-specific subcellular localization and trafficking properties. The subtypes also differ in their neuronal physiology. Therefore, we have investigated the localization and targeting of human alpha(2)-adrenoceptors (alpha(2)-AR) in PC12 cells, which were transfected to express the alpha(2)-AR subtypes A, B, and C. Inspection of the receptors by indirect immunofluorescence and confocal microscopy showed that alpha(2A)-AR were mainly targeted to the tips of the neurites, alpha(2B)-AR were evenly distributed in the plasma membrane, and alpha(2C)-AR were mostly located in an intracellular perinuclear compartment. After agonist treatment, alpha(2A)- and alpha(2B)-AR were internalized into partly overlapping populations of intracellular vesicles. Receptor subtype-specific changes in PC12 cell morphology were also discovered: expression of alpha(2A)-AR, but not of alpha(2B)- or alpha(2C)-AR, induced differentiation-like changes in cells not treated with NGF. Also alpha(2B)-AR were targeted to the tips of neurites when they were coexpressed in the same cells with alpha(2A)-AR, indicating that the targeting of receptors to the tips of neurites is a consequence of a change in PC12 cell membrane protein trafficking that the alpha(2A)-subtype induces. The marked agonist-induced internalization of alpha(2A)-AR observed in both nondifferentiated and differentiated PC12 cells contrasts with earlier results from non-neuronal cells and points out the importance of the cellular environment for receptor endocytosis and trafficking. The targeting of alpha(2A)-AR to nerve terminals in PC12 cells is in line with the putative physiological role of this receptor subtype as a presynaptic autoreceptor.

Opposite changes in imidazoline I2 receptors and alpha2-adrenoceptors density in rat frontal cortex after induced gliosis.

Opposite age-dependent changes in alpha2-adrenoceptor and imidazoline I2 receptor (I2-IRs) density have been related to brain gliosis development with aging. To check this hypothesis we applied in rats a model of reactive gliosis induced by heat. The specific binding of [3H]idazoxan (0.5-20 nM) in the presence of (-)adrenaline (5 x 10(-6) M) to membranes from rat brain cortex showed that the density of I(2)-IRs was significantly higher in membranes of injured cortex (Bmax=60+/-6 fmol/mg protein; n=9) than in control (Bmax=38+/-3 fmol/mg protein; n=9; p=0.0053). Conversely, the density of alpha2-adrenoceptors, measured by [3H]clonidine (0.25-16 nM), in the injured cortex (Bmax=75+/-4 fmol/mg protein; n=9) was significantly lower than in sham membranes (Bmax=103+/-7 fmol/mg protein; n=9; p=0.0035). No significant differences in receptor's affinity were observed between both groups. These results support the hypothesis that gliosis induces opposite changes in alpha2-adrenoceptor and I2-IR density.

Electroconvulsive shocks decrease α2-adrenoceptor binding in the Flinders rat model of depression.

Despite years of drug development, electroconvulsive therapy (ECT) remains the most effective treatment for severe depression. The exact therapeutic mechanism of action of ECT is still unresolved and therefore we tested the hypothesis that the beneficial effect of ECT could in part be the result of increased noradrenergic neurotransmission leading to a decrease in α2-adrenoceptor binding. We have previously shown that both the Flinders sensitive line (FSL) and Flinders resistant line (FRL) rats had altered α2-adrenoceptor binding compared to control Sprague-Dawley (SD) rats. In this study, we treated female FSL, FRL and SD rats with electroconvulsive shock (ECS), an animal model of ECT, or sham stimulation for 10 days before brains were removed and cut into 20µm thick sections. Densities of α2-adrenoceptors were measured by quantitative autoradiography in the hippocampus, thalamic nucleus, hypothalamus, amygdala, frontal cortex, insular cortex, and perirhinal cortex using the α2-adrenoceptor antagonist, [(3)H]RX 821002. ECS decreased the binding of α2-adrenoceptors in cortical regions in the FSL and cortical and amygdaloid regions in the control FRL rats compared to their respective sham treated group. The normal SD controls showed no significant response to ECS treatment. Our data suggest that the therapeutic effect of ECS may be mediated through a decrease of α2-adrenoceptors, probably due to a sustained increase in noradrenaline release. These data confirm the importance of the noradrenergic system and the α2-adrenoceptor in depression and in the mechanism of antidepressant treatments.

Gender differences in brain metabolic and plasma catecholamine responses to alpha 2-adrenoceptor blockade.

alpha 2-Adrenergic receptors modulate the release of several neurotransmitters implicated in the treatment and pathophysiology of mood and anxiety disorders. Significant sex differences occur in the prevalence of both disorders. To test whether gender affects alpha 2 function, the plasma catecholamine and brain metabolic responses to alpha 2 blockade were measured in male and female volunteers. Ten female and thirteen male volunteers underwent [18F]-fluorodeoxyglucose (FDG) positron emission tomography (PET) scans before and after infusion of idazoxan (200 micrograms/kg). Measures of plasma catecholamines, blood pressure, and anxiety were obtained. Norepinephrine responses were larger in males. Women showed global increases in metabolism, whereas males had no global changes and some regional decreases in FDG uptake following idazoxan administration. The differences in norepinephrine increases are consistent with previously reported effects of gender on sympathetic activation. The PET data suggest gender differences in responses to alpha 2-receptor blockade in brain as well.

Regulatory effects of reboxetine treatment alone, or following paroxetine treatment, on brain noradrenergic and serotonergic systems.

When patients do not respond to an initial antidepressant, one clinical approach is to switch to an agent in a different pharmacological class. However, few studies have examined the neurochemical consequences of this practice. To study this, we examined changes in binding sites in rat brain for norepinephrine (NET) and serotonin transporters (SERT), alpha1, alpha2, and beta1 adrenergic receptors after chronic administration of paroxetine (PRX), reboxetine (RBX), or PRX followed by RBX. We also examined the effects of these treatments on mRNA expression for tyrosine hydroxylase (TH). RBX treatment for 3 weeks reduced NET binding significantly, by approximately 40% in terminal field areas, and 6 weeks of RBX reduced it even more, by approximately 60%. RBX treatment for 3 and 6 weeks reduced beta1 adrenergic receptor-binding sites equally, by 50-60%. At no time did RBX treatment reduce SERT-binding sites. PRX treatment had no effect on beta1 adrenergic or NET-binding sites, but reduced SERT-binding sites by 75-80%. Neither treatment altered mRNA for TH, alpha1, or alpha2 adrenergic receptor-binding sites. When 3 weeks of RBX treatment followed 3 weeks of PRX treatment, NET-binding sites were reduced to the same extent as measured after 6 weeks of RBX treatment alone, indicating that PRX pretreatment may have 'primed' the subsequent regulatory effect of RBX on the NET. Thus, pretreatment of rats with PRX actually enhanced at least one regulatory effect of RBX treatment on the noradrenergic system, and did not interfere with any other pharmacological effect caused by RBX treatment.

Attenuation of alpha2A-adrenergic receptor expression in neonatal rat brain by RNA interference or antisense oligonucleotide reduced anxiety in adulthood.

Brain alpha2-adrenergic receptors (alpha2-ARs) have been implicated in the regulation of anxiety, which is associated with stress. Environmental treatments during neonatal development could modulate the level of brain alpha2-AR expression and alter anxiety in adults, suggesting possible involvement of these receptors in early-life programming of anxiety state. The present study was undertaken to determine whether the reduction of the expression of A subtype of these receptors most abundant in the neonatal brain affects anxiety-related behavior in adulthood. We attenuated the expression of alpha2A-ARs during neonatal life by two different sequence specific approaches, antisense technology and RNA interference. Treatment of rats with the antisense oligodeoxynucleotide or short interfering RNA (siRNA) against alpha2A-ARs on the days 2-4 of their life, produced a marked acute decrease in the levels of both alpha2A-AR mRNA and [3H]RX821002 binding sites in the brainstem into which drugs were injected. The decrease of alpha2A-AR expression in the neonatal brainstem influenced the development of this receptor system in the brain regions as evidenced by the increased number of [3H]RX821002 binding sites in the hypothalamus of adult animals with both neonatal alpha2A-AR knockdown treatments; also in the frontal cortex of antisense-treated, and in the hippocampus of siRNA-treated adult rats. These adult animals also demonstrated a decreased anxiety in the elevated plus-maze as evidenced by an increased number of the open arm entries, greater proportion of time spent in the open arms, and more than a two-fold increase in the number of exploratory head dips. The results provide the first evidence that the reduction in the brain expression of a gene encoding for alpha2A-AR during neonatal life led to the long-term neurochemical and behavioral alterations. The data suggests that alterations in the expression of the receptor-specific gene during critical periods of brain development may be involved in early-life programming of anxiety-related behavior.

Influence of neonatal short-term reduction in brainstem alpha2A-adrenergic receptors on receptor ontogenesis, acoustic startle reflex, and prepulse inhibition in rats.

Neonatal treatments can disrupt prepulse inhibition (PPI) of startle response later in life. Alpha2A-adrenergic receptors (alpha2A-ARs) regulate the release of brain neurotransmitters that may influence PPI. The authors examined the effects of short-term reduction in the neonatal brainstem alpha2A-ARs on subsequent development of this receptor system and acoustic startle reflex in rats. Administration of antisense oligodeoxynucleotide complementary to the alpha2A-ARs on Days 2-4 of life reduced receptor expression in the brainstem by Day 5. The treatment increased alpha2-AR numbers in the cortex, hippocampus, and amygdala at 40 days of age, and in cortex and hypothalamus at 90 days of age. Transient increases in hippocampal and amygdalar alpha2-ARs were accompanied by attenuation of acoustic startle response and impairment of PPI.

Identification of human I1 receptors and their relationship to alpha 2-adrenoceptors.

I1 imidazoline receptors (I1R) were defined as receptors insensitive to catecholamines and highly sensitive to [3H]clonidine and analogs. By contrast, the I2R subtype is more sensitive to [3H]idazoxan. [3H]clonidine and [3H]idazoxan imidazoline specific binding sites (IBS) have been detected in crude human membranes. Pharmacologic characterization by binding assays clearly differentiates IBS from alpha 2-adrenoceptors, whereas differences between [3H]clonidine and [3H]idazoxan IBS are less clear in crude preparations. In fact, only moderate affinity for [3H]clonidine was detectable in such preparations. However, purification procedures allowed detection of high affinity [3H]clonidine IBS in the human brain, corresponding to the I1R. Difficulties in the characterization of the I1R in crude membranes are due to multiple factors including heterogeneity of IBS, their low Bmax value, the existence of allosteric modulation, and possibly the presence of natural binding inhibitors. Immunologic studies with specific anti-idiotypic antibodies revealed a 43-kD protein as the best candidate for I1R as binding activity coincides with immunodetection. No cross-reaction was found with anti-monoamine oxidase (MAO) A/B antibodies and the 43-kD protein, ruling out the possibility of this protein being an MAO-associated I2R. Neither anti-alpha 2A- nor anti-alpha 2B-specific antibodies were able to immunodetect the 43-kD protein in crude membrane preparations or in purified fractions. These results and further biochemical characterization (pHi, N-glycosylation) of the 43-kD protein definitely assessed that human brain I1R and alpha 2-adrenoceptors clearly differ physically. However, coexpression of I1R and alpha 2-adrenoceptors in synaptic plasma membranes of the bovine brainstem reinforce the possibility of a functional relationship between the two types of receptor.

Pharmacologic and molecular discrimination of I2-imidazoline receptor subtypes.

I2-imidazoline receptors (I2-IR) are characterized by their high affinity for imidazolines and guanidines and medium affinity for imidazolidines. The differential recognition of I2-IR by amiloride led to subtype these sites as amiloride-sensitive (I2A-IR) and amiloride-insensitive (I2B-IR). I2-IR labeled with [3H]idazoxan or [3H]2-BFI in the rabbit cerebral cortex (I2A-IR) displayed higher affinities for amiloride and amiloride analogs than in the rat cerebral cortex (I2B-IR). Other drugs tested displayed biphasic curves in competition experiments, indicating the existence of high and low affinity sites for both I2-IR subtypes. The drugs (+)- and (-)-medetomidine, bromoxidine, moxonidine, and clorgyline were more potent on the high and/or low affinity sites of I2B-IR than on I2A-IR. Preincubation (30 min at 25 degrees C) with 10(-6) M isothiocyanatobenzyl imidazoline (IBI) or with 10(-6) M clorgyline reduced by 40% and 26%, respectively, the binding of [3H]2-BFI to I2B-IR, but it did not alter the binding of the radioligand to I2A-IR. These results indicated that the I2-IR subtypes differ in their pharmacologic profiles and in the nature of the imidazoline binding site involved in clorgyline and IBI alkylation. In rat cortical membranes, western blot detection of immunoreactive imidazoline receptor proteins revealed a double band of approximately 29/30 kD and three less intense bands of approximately 45, approximately 66, and approximately 85 kD. In rabbit cortical membranes the antibody detected proteins of approximately 30, approximately 57, approximately 66, and approximately 85 kD. It is suggested that I2-IR may be related to more than one receptor protein and that I2-IR subtypes differ in the nature of the proteins implicated.

Effects of tramadol on alpha2-adrenergic receptors in the rat brain.

In recent years, it has been postulated that tramadol, used mainly for the treatment of moderate to severe pain, might display a potential as an antidepressant drug. The present study investigated the effects of acute and repeated tramadol administration on the binding of [3H]RX 821002, a selective alpha2-adrenergic receptor ligand, in the rat brain. Male Wistar rats were used. Tramadol (20 mg/kg, i.p.) administered acutely (single dose), at 24 h after dosing, induced a significant decrease in the alpha2-adrenergic receptors in all brain regions studied. The most pronounced effects were observed in all subregions of the olfactory system, nucleus accumbens and septum, thalamus, hypothalamus, amygdala, and cerebral cortex. Repeated treatment with tramadol (20 mg/kg, i.p., once daily for 21 days) also induced statistically significant downregulation of [3H]RX 821002 binding sites in the rat brain. However, the effect--although statistically significant--was less pronounced than in the group treated acutely with the drug. Since drugs such as mianserin and mirtazapine are potent antagonists of central alpha2-adrenergic receptors and are effective antidepressants, it is tempting to suggest that, in addition to other alterations induced by tramadol, downregulation of these receptors may represent a potential antidepressant efficacy. On the other hand, one should be careful to avoid the treatment of chronic pain with tramadol in patients already receiving antidepressant drugs. Tramadol-induced downregulation of alpha2-adrenergic receptors--when combined with ongoing antidepressant therapy with drugs, which themselves inhibit serotonin reuptake or are antagonists of alpha2-adrenergic receptors--might cause threatening complications.

Different apparent modes of inhibition of alpha2A-adrenoceptor by alpha2-adrenoceptor antagonists.

The inhibition of alpha2A-adrenoceptor-mediated Ca2+ elevation by alpha2-adrenoceptor antagonists was measured in HEL human erythroleukemia cells. The antagonists could be divided in two classes: those that displayed surmountable inhibition (right-shift of the agonist dose-response curve), and those that displayed different degrees of insurmountable inhibition (depression of the maximum signal and a possible right-shift of the agonist dose-response curve). The degree of surmountability of the inhibition correlated well with the measured antagonist dissociation rates, suggesting that the hypothesis of the antagonist dissociation rate governing the mode of inhibition of fast responses, holds true. HEL cells thus provide a useful model system for the investigation of physiological consequences of different dissociation rates. Also, the dissociation rates of antagonists not available in radiolabelled form can be predicted from the functional data. The data stresses the importance of measurement of kinetic parameters of the drug-receptor interaction in addition to the equilibrium binding constants.

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.

Human adipocytes express alpha 2-adrenergic receptor of the alpha 2A-subtype only: pharmacological and genetic evidence.

In the present study we have reinvestigated the subtype of alpha 2-adrenoceptors expressed in human adipocytes (from subcutaneous and internal fat deposits) by means of radioligand binding using subtype-selective antagonists, and RNase mapping using a set of specific probes prepared from human alpha 2-adrenoceptors genes (alpha 2C2, alpha 2C4 and alpha 2C10). Comparison of the pharmacological properties of the human adipocyte alpha 2-adrenoceptors with those of the different human adrenoceptors expressed in COS-7 cells demonstrated that: i) human adipocyte alpha 2-adrenoceptors displays a KD for [3H]RX821002 and [3H]MK912 identical to that found in COS-7 cells transfected with the alpha 2C10 gene; ii) yohimbine and oxymetazoline is 1,000-fold more potent than prazosin to inhibit [3H]antagonist binding. RNase protection assays on cellular RNA prepared from the three fat deposits showed the presence of substantial amounts of alpha 2C10 transcripts: in contrast, mRNAs from alpha 2C2 and alpha 2C4 genes were undetectable. Altogether these results definitively establish that human adipocytes express only one alpha 2-adrenoceptor which is of the alpha 2A-subtype and encoded by the alpha 2C10 gene.

[3H]RX 821002 in human dorsolateral prefrontal cortex: no changes in postmortem tissue from subjects with schizophrenia.

One of the major differences between the atypical antipsychotic drugs clozapine and olanzapine is that clozapine has a two-fold higher affinity for the alpha(2)-adrenoreceptors. As clozapine can have therapeutic benefits in individuals that do not respond to other antipsychotic drugs, this raises the possibility that changes in the alpha(2)-adrenoreceptors could be a marker for a predisposition to treatment resistance. A methodology has been optimised to measure the binding of [3H]RX 821002 to alpha(2)-adrenoreceptors in human postmortem CNS and has shown that these receptors are not altered in Brodmann's area 9 from subjects with schizophrenia. These data add to those of one other study that showed the alpha(2)-adrenoreceptors were not altered in Brodmann's area 10 and the hippocampus from subjects with schizophrenia, and do not support the hypothesis that changes in alpha(2)-adrenoreceptors are a marker for treatment resistance in schizophrenia.

Identification and characterization of the imidazoline I2b-binding sites in the hamster brown adipose tissue as a study model for imidazoline receptors.

The imidazoline-type compound, MPV-1743, has been found to activate nonshivering thermogenesis (NST) in brown adipose tissue (BAT) of the genetically obese Zucker rats. The regulation of NST in BAT is linked to the catecholamine metabolism, and the imidazoline I2-binding sites have been found on the monoamine oxidase, a catecholamine metabolising enzyme. In this study, the I2-binding sites of hamster BAT have been characterised using a receptor binding assay with 3H-idazoxan as a radioligand, and the interaction of MPV-1743 with these I2-binding sites has been studied using the enantiomers of MPV 1743, that is, MPV 2088 and MPV 2089. Cirazoline was used to determine the specific binding of 3H-idazoxan to the imidazoline I2-binding sites. Rauwolscine was added in the 3H-idazoxan binding assay in order to inhibit any binding to potential alpha2-adrenergic sites. In the presence of rauwolscine mask 3H-Idazoxan labelled a population of non-adrenergic binding sites expressing the properties of the imidazoline I2b-receptor subtype similar to that found in the rat liver (cirazoline >> guanabenz = amiloride >> clonidine). The binding of 3H-idazoxan to the I2b-binding sites could be displaced by the imidazole compounds with the following affinities: detomidine (KiHigh 9.2 nM; KiLow 3200 nM), MPV-2088 (KiHigh 19 nM; IKiLow 760 nM) and MPV-2089 (KiHigh 190 nM; KiLow 1300 nM), atipamezole (3500 nM) and dexmedetomidine (Ki 8400 nM). These results have shown that the hamster BAT contains the imidazoline I2b-binding sites with heterogeneous binding properties for some test compounds. In addition, the enantiomers of MPV 1743, that is, MPV 2088 and MPV 2089, had high affinity to these BAT imidazoline I2b-binding sites. Therefore, it is suggested that the regulation of NST in the hamster BAT may be an attractive model to study the role of imidazoline I2b-binding sites.

Effects of clonidine and idazoxan on tetrathiomolybdate-induced copper and lysosomal enzyme excretion into sheep bile.

This study investigated the effects of intravenous (IV) administration of tetrathiomolybdate (TTM), and α(2)-adrenergic agonist clonidine (CLO) and α(2)-antagonist idazoxan (IDA), alone or in combination with TTM, on sheep fed low (LCu) and high (HCu) copper diets. Effects on bile flow, biliary Cu concentration and excretion, plasma Cu concentration, and lysosomal enzyme ß-glucuronidase (ß-GLU) activity in bile and plasma were determined. Tetrathiomolybdate alone or with CLO or IDA significantly enhanced biliary Cu excretion most likely by removing Cu from hepatocyte lysosomes as evidenced by a significant increase in ß-GLU enzyme activity in bile. A significant increase in plasma ß-GLU concentration occurred only in sheep treated with CLO in combination with TTM. Because of the lytic nature of the lysosomal enzymes, caution is advocated in use of drugs, especially α(2)-adrenergic agonists, to further enhance TTM-induced biliary Cu excretion in the treatment of chronic Cu poisoning in sheep.