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Notes: Abstract: The aim of this study was to quantify and compare the turnover of brain α2-adrenoceptors during chronic morphine treatment and after spontaneous morphine withdrawal in rats. The oral administration of increasing doses of morphine (10–90 mg/kg) for 20 days did not alter the specific binding of the agonist [3H]clonidine in the cerebral cortex. However, spontaneous opiate withdrawal (24 h) significantly increased the density of cortical α2-adrenoceptors (Bmax for [3H]clonidine was 21% greater). The recovery of [3H]clonidine binding after irreversible inactivation by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (1.6 mg/kg) was assessed in naive, morphine-dependent, and morphine-withdrawn rats to study the process of α2-adrenoceptor repopulation and to calculate receptor turnover parameters. The simultaneous analysis of receptor recovery curves revealed that the turnover of brain α2-adrenoceptors in morphine-withdrawn rats was accelerated [appearance rate constant (r) = 21 fmol/mg of protein/day; disappearance rate constant (k) = 0.25 day−1] compared with those in morphine-dependent (r = 13 fmol/mg of protein/day; k = 0.14 day−1) and naive (r = 15 fmol/mg of protein/day; k = 0.16 day−1) rats. Moreover, this analysis also indicated that the increased density of cortical α2-adrenoceptors observed during morphine withdrawal was due to a significantly higher receptor appearance (Δr = 37–57%) and not to a decreased receptor disappearance, which in fact showed also an increase (Δk = 56–79%). It is proposed that the increased rate of α2-adrenoceptor production in the brain of morphine-dependent rats during spontaneous withdrawal is most probably mediated by the overactivity of the adenylyl cyclase/cyclic AMP system induced by opiate addiction.

Notes: Abstract: In the postmortem human brain (27 specimens of frontal cortex, Brodmann area 9), the specific binding of the antagonists [3H]RX 821002 (2-methoxyidazoxan) to α2A-adrenoceptors and that of [3H]idazoxan to l2-imidazo-line sites (a nonadrenoceptor mitochondrial site) were determined in parallel to study the effect of aging (range, 4–89 years) on both brain proteins. The density of α2A-adrenoceptors and age were negatively correlated (r=-0.71; p 〈 0.001). In contrast, the density of l2-imidazo-line sites was positively correlated with aging (r= 0.59; p 〈 0.005). The ratio of receptor densities (α2A/l2) also showed a marked negative correlation with age (r=-0.76; p 〈 0.001). In an age-selected group (range, 10–89 years), the density of monoamine oxidase (MAO)-B sites labeled by [3H]Ro 19–6327 (lazabemide) also showed a positive correlation with age (r= 0.80; p 〈 0.005). In these subjects, the density of l2-imidazoline sites correlated well with the density of MAO-B sites (r= 0.70; p 〈 0.005). The ratio of the density of these sites (MAO-B/l2) did not correlate with the age of the subject at death (r=-0.15). In the human frontal cortex, idazoxan displayed very low affinity (Ki= 89 μM) against the binding of [3H]Ro 19–6327 to MAO-B, which discounted a direct interaction of [3H]idazoxan with the active center of the enzyme and indicated that the l2-imidazoline site cannot be identified with MAO-B. However, l2-imidazoline sites and MAO-B show a clear coexpression not only in the human frontal cortex during the process of aging, but also in various brain regions of the human and rat brains. It is suggested that the l2-imidazoline site has a specific location on glial (astrocyte) cells.

Notes: Abstract The full agonist [3H]UK 14304 [5-bromo-6-(2-im-idazolin-2-yl-amino)-quinoxaline] was used to characterize α2-adrenoceptors in postmortem human brain. The binding at 25°C was rapid (t1/2, 4.6 min) and reversible (t1/2, 14.1 min), and the Kd determined from the kinetic studies was 0.48 nM In frontal cortex, the rank order of potency of adrenergic drugs competing with [3H]UK 14304 or [3H]clonidine showed the specificity for an α2A-adrenoceptor: UK 14304 ≅ yohimbine ≅ oxymetazoline ≅ clonidine 〉 phentolamine ≅ (–)-adrenaline 〉 idazoxan ≅ (–) -noradrenaline 〉 phenylephrine 〉 (±)-adrenaline ≫ corynanthine 〉 prazosin ≫ (±)-propranolol. GTP induced a threefold decrease in the affinity of [3H]UK 14304, with no alteration in the maximum number of binding sites, suggesting that the radioligand labelled the high-affinity state of the α2-adreno-ceptor. In the frontal cortex, analyses of saturation curves indicated the existence of a single population of noninter-acting sites for [3H]UK 14304 (Kd= 0.35 ± 0.13 nM; Bmax= 74 ± 9 fmol/mg of protein). In other brain regions (hypothalamus, hippocampus, cerebellum, brainstem, caudate nucleus, and amygdala) the Bmax ranged from 68 ± 7 to 28 ± 4 fmol/mg of protein. No significant changes in the Kd values were found in the different regions examined. The binding of [3H]UK 14304 was not affected by age, sex or postmortem delay. [3H]UK 14304 should be a useful tool to assess brain α2-adrenoceptor density in a variety of neuropsychiatric disorders.

Notes: Abstract: To examine directly in the brain the status of α2-adrenoceptors in major depression, the specific binding of the agonist [3H]UK 14304 was measured by quantitative receptor autoradiography in the hippocampus and frontal cortex of suicide victims (n = 17) with a retrospective diagnosis of depression (n = 7) or other psychiatric disorders (n = 10) as well as of matched control subjects (n = 9). In suicide victims, a significant increase in the number of α2-adrenoceptors was found in the CA1 field (40%) and dentate gyrus (20%) of the hippocampus and in the external layers I (33%) and II (31%) of the frontal cortex, compared with that in matched controls. In depressed suicide victims, the increase in α2-adrenoceptors in the CA1 field (57%) was significantly greater (24%, p 〈 0.05) than that observed in the group of suicide victims with other diagnoses (26%). In the same depressed suicide victims, the increase in cortical α2-adrenoceptors was restricted to layer I (34%) and it was equivalent to that found in layer I (33%) of suicide victims with other diagnoses. The results indicate that suicide is associated with increases in the high-affinity state of brain α2-adrenoceptors and that there is a pronounced localized increase of this inhibitory receptor in the hippocampus of depressed suicide victims.

Notes: Abstract: The biochemical status of human brain protein kinase C (PKC)-αβ during opiate dependence was studied by means of immunoblotting techniques in postmortem brain of heroin addicts who had died by opiate overdose. In the frontal cortex, a marked decrease (53%, p 〈 0.05) in the immunoreactivity of PKC-αβ was found in heroin addicts compared with matched controls. The loss of PKC-αβ in the brain of human addicts paralleled that observed in the frontal cortex of rats after chronic treatment with morphine (10–100 mg/kg i.p. for 5 days) (PKC-αβ decreased by 34%, p 〈 0.05). Chronic treatment with naloxone (1 mg/kg i.p. every 12 h for 5 days) did not alter PKC-αβ immunoreactivity in the rat brain. However, in morphine-dependent rats, naloxone-precipitated withdrawal induced a rapid and strong behavioral reaction with a concomitant up-regulation of PKC-αβ immunoreactivity to control values. These results indicated that the decrease of brain PKC-αβ induced by heroin/morphine is a μ-opioid receptor-mediated effect. The chronic administration of opiates has been associated with a marked sensitization of the adenylyl cyclase/cyclic AMP system, although this phenomenon is not exclusive of the opioid system but the general cellular adaptation to chronic inhibition of adenylyl cyclase. In this context, chronic treatment of rats with other inhibitory agonists (e.g., clonidine, 1 mg/kg i.p. every 12 h for 14 days) acting through receptors (e.g., α2-adrenoceptors) also coupled to adenylyl cyclase did not alter brain PKC-αβ immunoreactivity. Together these findings suggest that the brain PKC system might play a major role in opiate addiction.

Notes: : The specific binding of [3H]idazoxan in the presence of 10−6M(−)-adrenaline was used to evaluate the density of imidazoline receptors in the brain of spontaneously hypertensive (SHR) rats and sex-and age-matched normotensive Wistar-Kyoto (WKY) rats. In SHR rats the density of imidazoline receptors (cerebral cortex, hypothalamus, and medulla oblongata) was not different from that in normotensive (WKY) rats. However, repeated treatment with idazoxan consistently increased (23–80%) the density of imidazoline receptors in the various brain regions of WKY rats but not in SHR rats. In normotensive Sprague-Dawley rats, repeated treatment with the imidazoline drugs idazoxan and cirazoline also increased (33–37%) the density of imidazoline receptors in the cerebral cortex. The lack of regulation by idazoxan of the density of imidazoline receptors in the brain of SHR rats might reflect the existence of a relevant abnormality of these receptors in this genetic model of hypertension.

Notes: Abstract: Competition [3H]RX821002 ([3H]2-methoxyidazoxan) binding experiments with α2-adrenoceptor subtype-specific antagonists—BRL 44408 (α2A selective), ARC 239 (α2B selective), and others—were performed to delineate through rigorous computer modeling receptor subtypes in the postmortem human brain. In the hippocampus, hypothalamus, cerebellum, and brainstem the whole population of α2-adrenoceptors appears to belong to the α2A subtype (100%; Bmax = 34–90 fmol/mg of protein). In the frontal cortex, the predominant receptor was the α2A subtype (87%; Bmax = 53 fmol/mg of protein), although a small population of the α2B/C subtype (13%; Bmax = 8 fmol/mg of protein) was also detected. In the caudate nucleus, a mixed population of α2A (64%; Bmax = 9 fmol/mg of protein) and α2B/C (36%; Bmax = 5 fmol/mg of protein) subtypes was detected. In the cortex and caudate and in the presence of ARC 239 (to mask the α2B/C-adrenoceptors), competition experiments with the agonist guanoxabenz clearly modeled the high- and low-affinity states of the α2A subtype. In the presence of ARC 239 and the GTP analogue guanylyl-5′-imidodiphosphate together with NaCl and EDTA (to eliminate the high-affinity α2A-adrenoceptor) guanoxabenz only recognized the low-affinity α2A-adrenoceptor. The results indicate that in the human brain the predominant α2-adrenoceptor is of the α2A subtype and that this functionally relevant receptor subtype is not heterogeneous in nature.

Notes: Abstract— The alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) is a peptide-coupling agent that is being used to inactivate irreversibly α2-adrenoceptors and other receptors. The aim of the present study was to assess the in vitro and in vivo effects of EEDQ on the newly discovered brain l2-imidazoline sites, located mainly in mitochondria. Preincubation of rat cortical membranes with EEDQ (10−8-10−5M) markedly decreased (20–90%) the specific binding of the selective antagonist [3H]R821002 to α2-adrenoceptors without affecting that of [3H]idazoxan (in the presence of adrenaline) to l2-imidazoline sites. In EEDQ-pretreated membranes (10−5M, 30 min at 25°c), the density of l2-imidazoline sites (Bmax= 80 ± 4 fmol/mg of protein) was not different from that determined in untreated membranes in the presence of 10−6M (-)-adrenaline (Bmax= 83 ± 4 fmol/mg of protein), and both densities were lower (24%, p 〈 0.05) than the total native density of [3H]idazoxan binding sites (Bmax= 107 ± 6 fmol/mg of protein) (l2-imidazoline sites plus a2-adrenoceptors). Treatment of rats with an optimal dose of EEDQ (1.6 mg/kg, i.p., for 2 h to 30 days) reduced maximally at 6 h (by 95 ± 1%) the specific binding of [3H]-R821002 to α2-adrenoceptors, but also the binding of [3H]idazoxan to l2-imidazoline sites (by 44 ± 5%). Pretreatment with yohimbine (10 mg/kg, i.p.) fully protected against EEDQ-induced α2-adrenoceptor inactivation. In contrast, pretreatment with cirazoline (1 mg/kg, i.p.), did not protect against EEDQ-induced inactivation of l2-imidazoline sites. Treatment with EEDQ (1.6 mg/kg, i.p., for 6 h) did not alter the density of brain monoamine oxidase-A sites labeled by [3H]Ro 41–1049 or that of monoamine oxidase-B sites labeled by [3H]Ro 19–6327 (lazabemide), two relevant mitochondrial markers. Competition experiments with cirazoline against the specific binding of [3H]idazoxan to l2-imidazoline sites demonstrated the presence of the expected two affinity states for the drug in EEDQ-pretreated membranes as well as in rats treated with EEDQ. The results indicate that EEDQ in vitro is a useful tool for quantitating l2-imidazoline sites when using [3H]-imidazoline ligands that also recognize α2-adrenoceptors. In vivo, however, EEDQ is also able to inactivate partially brain l2-imidazoline sites probably by an indirect mechanism. Key Words: Brain l2-imidazoline sites—[3H]-Idazoxan—α2-Adrenoceptors—[3H] R821002—N -Ethoxycarbonyl-2-ethoxy-li2-dihydroquinoline—Monoamine oxidase-A—[3H]Ro 41–1049—Monoamine oxidase-B—[3H]Ro 19–6327.

Notes: Abstract: The α2A- and α2C-adrenoceptor subtypes were evaluated in postmortem brains from suicides with depression (n = 22), suicides with other diagnoses (n = 12), and controls (n = 26). Membrane assays with the antagonist [3H]RX821002 (2-[3H]methoxyidazoxan) suggested the presence of α2A-adrenoceptors in the frontal cortex and both α2C-adrenoceptors and α2A-adrenoceptors in the caudate. The proportions in caudate were similar in controls (α2A, 86%; α2C, 14%), depressed suicides (α2A, 91%; α2C, 9%), and suicides with other diagnoses (α2A, 88%; α2C, 12%). Autoradiography of [3H]RX821002 binding under α2B/C-adrenoceptor-masking conditions confirmed the similar densities of α2A-adrenoceptors in the cortex, hippocampus, and striatum from controls and suicides. In the frontal cortex of depressed suicides, competition of [3H]RX821002 binding by (−)-adrenaline revealed a greater proportion (61 ± 9%) of α2A-adrenoceptors in the high-affinity conformation for agonists than in controls (39 ± 5%). Simultaneous analysis with the agonists [3H]clonidine and [3H]UK14304 and the antagonist [3H]RX821002 in the same depressed suicides confirmed the enhanced α2A-adrenoceptor density when evaluated by agonist, but not by antagonist, radioligands. The results indicate that depression is associated with a selective increase in the high-affinity conformation of the brain α2A-adrenoceptors.

Notes: Abstract : Suicide and depression are associated with an increaseddensity of α2-adrenoceptors (radioligand receptor binding) inspecific regions of the human brain. The function of these inhibitoryreceptors involves various regulatory proteins (Gi couplingproteins and G protein-coupled receptor kinases, GRKs), which work in concertwith the receptors. In this study we quantitated in parallel the levels ofimmunolabeled α2A-adrenoceptors and associated regulatoryproteins in brains of suicide and depressed suicide victims. Specimens of theprefrontal cortex (Brodmann area 9) were collected from 51 suicide victims and31 control subjects. Levels of α2A-adrenoceptors,Gα1/2 proteins, and GRK 2/3 were assessed by immunoblottingtechniques by using specific polyclonal antisera and the immunoreactiveproteins were quantitated by densitometry. Increased levels ofα2A-adrenoceptors (31-40%), Gα1/2 proteins(42-63%), and membrane-associated GRK 2/3 (24-32%) were found in theprefrontal cortex of suicide victims and antidepressantfree depressed suicidevictims. There were significant correlations between the levels of GRK 2/3(dependent variable) and those of α2A-adrenoceptors andGα1/2 proteins (independent variables) in the same brainsamples of suicide victims (r = 0.56, p = 0.008) anddepressed suicide victims (r = 0.54, p = 0.041). Antemortemantidepressant treatment was associated with a significant reduction in thelevels of Gα1/2 proteins (32%), but with modest decreases inthe levels of α2A-adrenoceptors (6%) and GRK 2/3 (18%) inbrains of depressed suicide victims. The increased levels in concert ofα2A-adrenoceptors, Gα1/2 proteins, and GRK2/3 in brains of depressed suicide victims support the existence ofsupersensitive α2A-adrenoceptors in subjects with major depression.