Ane M. Gabilondo

HDAC2 regulates atypical antipsychotic responses through the modulation of mGlu2 promoter activity.

Histone deacetylases (HDACs) compact chromatin structure and repress gene transcription. In schizophrenia, clinical studies demonstrate that HDAC inhibitors are efficacious when given in combination with atypical antipsychotics. However, the molecular mechanism that integrates a better response to antipsychotics with changes in chromatin structure remains unknown. Here we found that chronic atypical antipsychotics downregulated the transcription of metabotropic glutamate 2 receptor (mGlu2, also known as Grm2), an effect that was associated with decreased histone acetylation at its promoter in mouse and human frontal cortex. This epigenetic change occurred in concert with a serotonin 5-HT2A receptor–dependent upregulation and increased binding of HDAC2 to the mGlu2 promoter. Virally mediated overexpression of HDAC2 in frontal cortex decreased mGlu2 transcription and its electrophysiological properties, thereby increasing psychosis-like behavior. Conversely, HDAC inhibitors prevented the repressive histone modifications induced at the mGlu2 promoter by atypical antipsychotics, and augmented their therapeutic-like effects. These observations support the view of HDAC2 as a promising new target for schizophrenia treatment.

Increased density of μ-opioid receptors in the postmortem brain of suicide victims

The biochemical status of mu-opioid receptors in suicide was evaluated by [3H]DAGO specific binding in postmortem human brains from 15 suicide victims and 15 controls. The density (Bmax) in frontal cortex and thalamus was directly correlated with age. In the frontal cortex and caudate but not in the thalamus of suicide victims the density of mu-opioid receptors was 36-39% higher than in controls. KD values in suicide victims were similar to those in control group (range 1.4-2.2 nM). The results suggest an increase of mu-opioid receptors in suicide associated to some brain areas showing the mu2-opioid receptor subtype.

μ-Opioid receptor and α2-adrenoceptor agonist binding sites in the postmortem brain of heroin addicts

The biochemical status of human brain μ-opioid receptors and α2-adrenoceptors during opiate dependence was studied by means of the binding of [3H] [D-Ala2, MePhe4, Gly-ol5] enkephalin (DAGO) and [3H]clonidine, respectively, in postmortem brains of heroin addicts who had died by opiate overdose or other causes. In the frontal cortex, thalamus and caudate of heroin addicts the density (Bmax) and affinity (KD) of μ-opioid receptors were similar to those in controls. In contrast, the density of α2-adrenoceptors in heroin addicts was found to be significantly decreased in frontal cortex (Bmax 31% lower), hypothalamus (Bmax 40% lower) and caudate (Bmax 32% lower) without changes in KD values. When heroin addicts were divided into two subgroups according to the presence or absence of morphine in body fluids, only the group with positive screening for morphine showed relevant decreases in brain α2-adrenoceptor density (Bmax 36–48% lower), whereas the decreases in receptor density observed in the subgroup with negative screening for morphine did not reach statistical significance. The results suggest that desensitization of brain α2A-adrenceptors is a relevant adaptative receptor mechanism during opiate addiction in humans.

Characterization of receptor-mediated [35S]GTPγS binding to cortical membranes from postmortem human brain

The [35S]GTPgammaS binding assay represents a functional approach to assess the coupling between receptors and G-proteins. The optimal conditions for [35S]GTPgammaS binding to human brain homogenates were established in postmortem samples of prefrontal cortex. The influence of protein content, incubation time, GDP, Mg(2+), and NaCl concentrations on the [35S]GTPgammaS binding were assessed in the absence and presence of the alpha(2)-adrenoceptor agonist UK14304 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine). In conditions of 50 microM GDP and 100 mM NaCl, UK14304 increased the apparent affinity of the specific [35S]GTPgammaS binding without changing the apparent density. Concentration-response curves to agonists of alpha(2)-adrenoceptors, mu-opioid, 5-HT(1A), cholinergic muscarinic, and GABA(B) receptors displayed, in the presence of NaCl, maximal stimulations between 24% and 61% with EC(50) values in the micromolar range. Selective antagonists shifted to the right the agonist-induced stimulation curves. The G(i)/G(o)-protein alkylating agent N-ethylmaleimide decreased basal [35S]GTPgammaS binding in a concentration-dependent manner and inhibited the stimulation induced by the different agonists. In cortical sections, [35S]GTPgammaS binding to gray matter was stimulated by the agonist UK14304. The present study demonstrates that functional studies of the receptor coupling to G(i)/G(o)-proteins can be performed in postmortem human brain samples.

Mutations of Tyr326 in the β2-adrenoceptor disrupt multiple receptor functions

Abstract A tyrosine residue at the cytoplasmic end of the seventh transmembrane helix is conserved in many G-protein-coupled receptors. In the human β2-adrenoceptor, this tyrosine (Tyr326) has been proposed to be a specific determinant for agonist-induced receptor sequestration. In order to probe its contribution to the sequestration process we have replaced this tyrosine by alanine (Y326A) or phenylalanine (Y326F). Wild-type and mutant receptors were stably expressed in Chinese hamster ovary cells. Agonist-induced sequestration was essentially abolished in Y326A receptors and only slightly reduced in Y326F receptors. However, cells expressing Y326A receptors displayed a high percentage of internal receptors under basal conditions while cells expressing wild-type receptors did not. In addition, high-affinity agonist binding and the ability to activate adenylyl cyclase were markedly reduced in Y326A receptors and slightly reduced in Y326F receptors. We conclude that Tyr326 is required for the functional integrity of the β2-adrenoceptor and that it may be involved in multiple agonist-induced effects.

[3H]RX821002 (2-methoxyidazoxan) binds to α2-adrenoceptor subtypes and a non-adrenoceptor imidazoline binding site in rat kidney

The binding of [3H]RX821002 (2-methoxyidazoxan) was evaluated in rat kidney membranes. [3H]RX821002 (0.13-16 nM) recognized a single, saturable binding site with high affinity. Different binding site densities were calculated depending on non-specific binding as defined by (-)-adrenaline or RX821002 (10 microM). Competition assays using (-)-adrenaline and the subtype-selective drugs ARC 239 (2-[2-[4-(o-methoxyphenyl)-piperazin-1-yl]-ethyl]-4,4-dimethyl-1,3 (2H,4H)-isoquinolindione), BRL 44408 (2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidaz ole), oxymetazoline or prazosin for [3H]RX821002 binding sites revealed the presence of alpha 2B-adrenoceptors (33-51%), alpha 2D-adrenoceptors (15-28%) and an adrenaline-insensitive population (34-40%), sensitive to imidazolines. After the addition of (-)-adrenaline (3 microM) to mask alpha 2-adrenoceptors, [3H]RX821002 specifically identified a saturable binding site with high affinity (Kd = 4.9 +/- 1.5 nM). The pharmacological profile of this non-adrenoceptor, [3H]RX821002 binding site (potencies: efaroxan > clonidine > guanabenz > BRL 44408 > ARC 239 > BU 224 (2-(4,5-dihydroimidaz-2-yl)quinoline) > moxonidine > (-)-nor-adrenaline > cimetidine) is different to that of imidazoline I1 or imidazoline I2 binding sites. Alternative incubation in the presence of ARC 239 (50 nM) to mask alpha 2B-adrenoceptors or BRL 44408 (100 nM) to mask alpha 2D-adrenoceptors confirmed the existence of both alpha 2-adrenoceptor subtypes and a non-adrenoceptor imidazoline binding site.

Increased 3H. raclopride binding sites in postmortem brains from schizophrenic violent suicide victims

The specific binding of the D2-dopamine receptor antagonist radioligand [3H] raclopride was quantitated in the postmortem caudate and frontal cortex from schizophrenic suicide victims and control subjects. In schizophrenic suicides the density of binding sites (Bmax) was higher (40%,P<0.05) in the caudate, whereas it did not change in the cortex as compared to those in controls. The apparent dissociation constants (Kd) were also found increased both in caudate (24%) and cortex (75%) from schizophrenics, but these apparent decreases in receptor affinity did not reach statistical significance. The mean Bmax value in drug-free schizophrenic suicides (n=3) did not differ from the Bmax value in neuroleptic drug-treated schizophrenics (n=7) but it was found increased in respect to control subjects (n=9). No differences in [3H] raclopride binding were observed between non-schizophrenic suicide victims (n=4) and matched controls (n=4), suggesting that the modifications of D2-dopamine receptors in schizophrenia are not related to suicide.

Spontaneous Withdrawal from Long‐Term Treatment with Morphine Accelerates the Turnover of α2‐Adrenoceptors in the Rat Brain: Up‐Regulation of Receptors Associated with Increased Receptor Appearance

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.

Monoamine oxidase B activity is increased in human gliomas

Glial tumours are the most common type of brain neoplasm in humans. Tumour classification and grading represent key factors for patient management. However, current grading schemes are still limited by subjective histological criteria. In this context, gliosis has been linked to increases in monoamine oxidase B (MAO-B) activity. Thus, in the present study, MAO-B activity in membranes of glial tumours (n=20), meningiomas (n=12) and non-pathological human brains (n=15) was quantified by [14C]PEA oxidation. MAO-B activity was significantly greater in glioblastoma multiformes than in postmortem control brains (p<0.01) or meningiomas (p<0.001). There were no significant differences in MAO-B activity between glioblastoma multiformes (n=11) and low-grade astrocytomas (n=3) or anaplastic astrocytomas (n=6). In conclusion, the present results demonstrate a significant and selective increase in MAO-B activity in human gliomas when compared with meningiomas or non-tumoural tissue. These results suggest that the quantification of MAO-B activity may be a useful diagnostic tool for differentiating glial tumours from other types of brain tumours or surrounding normal brain tissue.

Increased α2- and β1-adrenoceptor densities in postmortem brain of subjects with depression: Differential effect of antidepressant treatment

BACKGROUND Brain α2- and β-adrenoceptor alterations have been suggested in suicide and major depressive disorder. METHODS The densities of α2-, β1- and β2-adrenoceptors in postmortem prefrontal cortex of 26 subjects with depression were compared with those of age-, gender- and postmortem delay-matched controls. The effect of antidepressant treatment on α2- and β-adrenoceptor densities was also evaluated. α2- and β-adrenoceptor densities were measured by saturation experiments with respective radioligands [(3)H]UK14304 and [(3)H]CGP12177. β1- and β2-adrenoceptor subtype densities were dissected by means of β1-adrenoceptor selective antagonist CGP20712A. RESULTS Both, α2- and β1-adrenoceptors densities were higher in antidepressant-free depressed subjects (n=14) than those in matched controls (Δ~24%, p=0.013 and Δ~20%, p=0.044, respectively). In antidepressant-treated subjects (n=12), α2-adrenoceptor density remained increased over that in controls (Δ~20%), suggesting a resistance of α2-adrenoceptors to the down-regulatory effect of antidepressants. By contrast, β1-adrenoceptor density in antidepressant-treated depressed subjects was not different from controls, suggesting a possible down-regulation by antidepressants. The down-regulation of β1-adrenoceptor density in antidepressant-treated depressed subjects differs from the unaltered β1-adrenoceptor density observed in citalopram-treated rats and in a group of non-depressed subjects also treated with antidepressants (n=6). β2-adrenoceptor density was not altered in depressed subjects independently of treatment. LIMITATIONS Antidepressant-treated subjects had been treated with a heterogeneous variety of antidepressant drugs. The results should be understood in the context of suicide victims with depression. CONCLUSIONS These results show the up-regulation of brain α2- and β1-adrenoceptors in depression and suggest that the regulation induced by chronic antidepressant treatment would be altered in these subjects.