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Keywords:

  • α2A-Adrenoceptors;
  • G proteins;
  • G protein-coupled receptor kinase;
  • Human brain;
  • Suicide;
  • Depression

Abstract

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. Density of α2A-adrenoceptors, Gi, and GRK2 proteins in the prefronal cortex of suicide victims
  6. DISCUSSION
  7. Acknowledgements

Abstract : Suicide and depression are associated with an increased density of α2-adrenoceptors (radioligand receptor binding) in specific regions of the human brain. The function of these inhibitory receptors involves various regulatory proteins (Gi coupling proteins and G protein-coupled receptor kinases, GRKs), which work in concert with the receptors. In this study we quantitated in parallel the levels of immunolabeled α2A-adrenoceptors and associated regulatory proteins in brains of suicide and depressed suicide victims. Specimens of the prefrontal cortex (Brodmann area 9) were collected from 51 suicide victims and 31 control subjects. Levels of α2A-adrenoceptors, Gα1/2 proteins, and GRK 2/3 were assessed by immunoblotting techniques by using specific polyclonal antisera and the immunoreactive proteins were quantitated by densitometry. Increased levels of α2A-adrenoceptors (31-40%), Gα1/2 proteins (42-63%), and membrane-associated GRK 2/3 (24-32%) were found in the prefrontal cortex of suicide victims and antidepressantfree depressed suicide victims. There were significant correlations between the levels of GRK 2/3 (dependent variable) and those of α2A-adrenoceptors and Gα1/2 proteins (independent variables) in the same brain samples of suicide victims (r = 0.56, p = 0.008) and depressed suicide victims (r = 0.54, p = 0.041). Antemortem antidepressant treatment was associated with a significant reduction in the levels of Gα1/2 proteins (32%), but with modest decreases in the levels of α2A-adrenoceptors (6%) and GRK 2/3 (18%) in brains of depressed suicide victims. The increased levels in concert of α2A-adrenoceptors, Gα1/2 proteins, and GRK 2/3 in brains of depressed suicide victims support the existence of supersensitive α2A-adrenoceptors in subjects with major depression.

α2-Adrenoceptors are of special interest in subjects with major depression, because this family of receptors controls the activity of noradrenergic and serotonergic neurons in the brain. Thus, when these presynaptic inhibitory α2-adrenoceptors are stimulated, release and synthesis of noradrenaline and serotonin are inhibited (Trendelenburg et al., 1994 ; Esteban et al, 1996) ; therefore, increased α2-autoreceptor/heteroreceptor density/sensitivity could result in insufficient neuronal release of noradrenaline/serotonin and lead to depression. Supersensitivity of α2-adrenoceptors in specific brain regions (e.g., prefrontal cortex) could represent a common feature of the reduced monoaminergic (noradrenergic and/or serotonergic) function postulated in depression. The human brain contains the three subtypes of α2-adrenoceptors (α2A, α2B, and α2C) identified by molecular cloning, although the predominant one, especially in the frontal cortex, is the α2A-subtype (Sastre and García-Sevilla, 1994b ; Grijalba et al., 1996). Furthermore, recent studies have unraveled the crucial role of the α2A-adrenoceptor as mediator of most of the inhibitory effects of α2-adrenoceptor agonists (MacDonald et al., 1997).

Suicide and major depression are associated with an increased density of α2-adrenoceptors (radioligand receptor binding) in specific brain regions including the prefrontal cortex (Meana and García-Sevilla, 1987 ; Meana et al., 1992a ; González et al., 1994 ; Ordway et al., 1994 ; De Paermentier et al., 1997). A selective increase of α2A-adrenoceptor agonist binding sites in the prefrontal cortex of depressed suicide victims has been recently demonstrated (Callado et al., 1998). In the human brain, this α2A-subtype is the autoreceptor that regulates the release of noradrenaline (Raiteri et al., 1992). Reduced noradrenergic function in subjects with suicide/depression may also be associated with a reduced number of noradrenegic neurons in the locus ceruleus (Arango et al., 1996), which in turn could be related to the induction of up-regulation of α2A-autoreceptors in this brain nucleus (Ordway et al., 1994) and of α2A-autoreceptors and postsynaptic α2-adrenoceptors in target brain regions (Meana et al., 1992a ; González et al., 1994 ; Callado et al., 1998). These α2A-autoreceptors on noradrenergic neurons in the locus ceruleus regulate up to 80% of the extracellular levels of noradrenaline in the prefrontal cortex (Van Gaalen et al., 1997). Clearly, enhanced activity of brain α2A-adrenoceptors may represent a major feature in subjects with suicide and major depression.

The α2A-adrenoceptor is regulated in a complex manner. Thus, the agonist (noradrenaline)-induced high-affinity conformation of the receptor leads to inhibition of adenylyl cyclase through activation of specific guanine nucleotide binding (Gi1/2/3) heterotrimeric proteins (α, β, and γ subunits). This results in dissociation of the complex into receptor (low-affinity conformation), Gαi-GTP, and Gβγ components. Then, the largest Gαi subunit is able to modulate the effector system (i.e., adenylyl cyclase/cyclic AMP system) (Manji, 1992) and the Gβγ complex facilitates the translocation to the plasma membrane of a specific kinase for receptor regulation (see below) (Lohse et al., 1996). The prototypical α2A-adrenoceptor of the human platelet was shown to induce the inhibition of adenylyl cyclase through activation of Gαi2 proteins (Simonds et al., 1989). In human platelets and brain tissue, the high-affinity conformation of the α2A-adrenoceptor most probably corresponds to the complex with Gαi2 proteins (Simonds et al., 1989 ; Sastre and García-Sevilla, 1994a). For final receptor regulation, agonist occupancy of α2A-adrenoceptors also leads to activation of specific G protein-coupled receptor kinases (GRK2/3, formerly named β-adrenergic receptor kinases or βARK1/2) that recognize and phosphorylate the agonist-occupied receptor (Eason et al., 1995 ; Premont et al., 1995 ; Jewell-Motz and Liggett, 1996). This process is facilitated by the increased Gβγ complex availability, which bind and activate GRK2/3 (Lohse et al., 1996). GRK-mediated phosphorylation causes receptor desensitization by facilitating the binding to the phosphorylated receptor of other regulatory proteins termed arrestins (Böhm et al., 1997). GRK enzymes have been shown to phosphorylate a wide variety of G protein-coupled receptors (e.g., adrenoceptors and serotonin receptors) (Chuang et al., 1996) implicated in depression and in the effects of antidepressant drugs.

The up-regulation of brain α2-adrenoceptor density (including that of the α2A-subtype) in suicide victims and subjects with depression has been demonstrated mainly through an enhanced binding of imidazoline radioligands (Meana et al., 1992a ; González et al., 1994 ; Ordway et al., 1994 ; Callado et al., 1998), which in addition to α2-adrenoceptors also bind to the newly discovered imidazoline receptors (García-Sevilla et al., 1996). Although the experimental conditions of these studies clearly indicated that the specific binding of imidazoline radioligands to brain membranes was associated with α2-adrenoceptors, it appeared most relevant to assess whether this receptor up-regulation can be demonstrated with other approaches such as the immunodetection of the receptor protein with a specific antibody. The present study was designed to evaluate in parallel the levels of immunolabeled α2A-adrenoceptors and associated regulatory proteins (Gαi1/2 and GRK2/3) in brains of suicide and depressed suicide victims. A preliminary account of part of this study was given at the Sixth World Congress of Biological Psychiatry (García-Sevilla et al., 1997a).

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. Density of α2A-adrenoceptors, Gi, and GRK2 proteins in the prefronal cortex of suicide victims
  6. DISCUSSION
  7. Acknowledgements

Subject selection and brain samples

Human brains were obtained at autopsy from the Institute of Forensic Medicine (HUG series), Geneva, Switzerland, and from the Forensic Anatomical Institute (UIB series), Palma de Mallorca, Spain. This study was approved by the research and ethics review boards of the Department of Psychiatry, Medical School, University of Geneva, Geneva, and the Spanish Research Council, Madrid, Spain.

In the HUG series, specimens of the right prefrontal cortex (Brodmann area 9) were collected from 34 suicide victims [18 men and 16 women ; mean ± SEM : age, 46 ± 3 years ; postmortem delay (PMD), 30 ± 3 h]. As determined by the medical examiner, the suicide victims died of hanging (n = 7), drowning (n = 7), self-inflected gunshot wound (n = 5), trauma caused by jumping from a height (n = 3), carbon monoxide poisoning (n = 2), benzodiazepine/ethanol overdose (n = 3), barbital overdose (n = 1), insulin overdose (n = 1), digoxin overdose (n = 1), electrocution (n = 2), self-inflected stab wound (n = 1), and asphyxia (n = 1). In this group of suicide victims, a retrospective searching for antemortem clinical diagnosis and drug treatment through review of medical histories of psychiatric units and examiner's information was performed. This searching was blind to the biochemical findings and was conducted after the completion of the assays. Suicide victims were then divided into various subgroups [Diagnostic and Statistical Manual of Mental Disorders, third edition, revised (DSM III-R) criteria] (American Psychiatric Association, 1987) as follows : (1) a subgroup of patients (n = 17) with a definitive diagnosis of major depression (recurrent, n = 13 ; bipolar depressed, n = 3) or dysthymic disorder (n = 1), (2) a subgroup with other diagnoses that included alcohol dependence (n = 3), personality disorder (n = 2), schizoaffective disorder (n = 2), and schizophrenia (n = 1), and (3) a subgroup of suicide victims (n = 9) in whom no clear diagnosis could be made because of the absence of past hospital records. Most depressed suicide victims had probably received antidepressant drugs during their illness and shortly before death (within the last month of life), as revealed by prescriptions found in medical histories. The subgroup of suicide victims with other diagnoses had probably received various psychotropic medications, mainly antipsychotic drugs. Possible medications received by the subgroup of suicide victims without a diagnosis were unknown. In suicide victims, a toxicological screening (quantitative assays for antidepressant and other psychotropic drugs, and ethanol) was performed on blood, urine, liver, and kidney samples at the Toxicology Unit, Institute of Forensic Medicine, using standard procedures. In the subgroup of depressed suicide victims, therapeutic concentrations of some of the prescribed antidepressant drugs (clomipramine, citalopram, venlafaxine, trazodone, and moclobemide) were detected in blood samples of only a few subjects (n = 6), indicating that most depressed suicide victims were antidepressant drug free (n = 10). In the subgroup of suicide victims with other diagnoses, the prescribed antipsychotic drugs were not detected in blood samples. In the subgroup of suicide victims without a diagnosis, toxicological screening in five subjects did not reveal the presence of psychotropic drugs in blood samples. Therapeutic plasma concentrations of benzodiazepines were detected in most suicide victims. Toxic plasma concentrations of benzodiazepines (0.5-1.7 μg/ml), barbital (350 μg/ml), and digoxin (102 nmol/L) were detected in those subjects who committed suicide by these means. Ethanol was also detected in some suicide victims (blood concentration, 1.52 ± 0.63 g/L ; n = 11). The control group for this series of suicide victims consisted of similar brain specimens from 20 subjects (eight men and 12 women ; mean ± SEM : age, 46 ± 4 years ; PMD, 31 ± 3 h), without a documented history of neuropsychiatric disorder or drug abuse, who died of various causes, i.e., motor-vehicle accidents (n = 10), pulmonary embolism (n = 4), gunshot wound, homicide (n = 2), myocardial infarction (n = 2), perforated aneurysm (n = 1), and asphyxia (n = 1). A retrospective searching for antemortem clinical diagnosis in these control subjects had excluded two subjects because of past hospital records of alcohol dependence or panic disorder. Toxicological screening in blood samples did not reveal the presence of psychotropic drugs in these subjects. Ethanol was detected in some control subjects (blood concentration : 0.38 ± 0.21 g/L ; n = 5).

In the UIB series, specimens of the right prefrontal cortex (Brodmann area 9) were collected from 17 suicide victims (10 men and seven women ; mean ± SEM : age, 46 ± 5 years ; PMD, 31 ± 4 h). These suicide victims died of hanging (n = 6), trauma caused by jumping from a height (n = 5), carbon monoxide poisoning (n = 3), benzodiazepine/ethanol overdose (n = 1), drowning (n = 1), and exsanguination (n = 1). After a retrospective searching for antemortem clinical diagnosis, these suicide victims were also divided into various subgroups (according to DSM III-R) as follows : (1) a subgroup of patients with a definitive diagnosis of major depression (recurrent, n = 6), (2) a subgroup with schizophrenia (n = 2), and (3) a subgroup of suicide victims (n = 9) in whom no diagnosis could be made. Most depressed suicide victims (n = 4) had probably received antidepressant drugs (clomipramine, fluoxetine, and mianserin) as revealed by medical histories. The rest of the suicide victims with a diagnosis were apparently medication free at the time of death. The control group for this series of suicide victims consisted of similar brain specimens from 11 subjects (nine men and two women ; mean ± SEM : age, 46 ± 6 years ; PMD, 29 ± 3 h), without an apparent history of neuropsychiatric disorder or drug abuse, who died of various causes, i.e., motor vehicle accidents (n = 4), myocardial infarction (n = 4), labor accidents (n = 2), and accidental drowning (n = 1). In this series of suicide victims and controls, only qualitative ethanol screening was performed on blood samples, and ethanol was detected in some suicide victims (n = 5) and controls (n = 3).

The mean age, sex ratio, and PMD (time from death to the storage of the brain specimen) were similar in control subjects and suicide victims and between both series of subjects (see above). In a given experiment, suicide victims were matched with control subjects for sex, age, and PMD (see below). The immunoreactivities of the target proteins did not correlate with the PMD (range, 8-72 h at 4°C ; r = -0.10 to 0.01 ; n = 29-51) (data not shown) (Escribá et al., 1994 ; Sastre and García-Sevilla, 1994a ; Ozaita et al., 1998). Tissue storage (time from freezing to immunoblot analysis ; mean ± SEM, 10 ± 1 month) did not differ between control subjects and suicide victims.

Specimens of prefrontal cortex were dissected on a glass surface cooled with ice and care was used to include only gray matter (no more than a 1-mm white-matter border was included) in cortical samples (150-200 mg). Cortical membranes or the cytosolic plus membrane fractions were then prepared for immunodetection of the target proteins as described in detail previously (Escribá et al., 1994 ; Ozaita et al., 1998). The samples were processed blind for diagnoses. The levels of α2A-adrenoceptors were measured in both series (HUG and UIB) of brain specimens. In the HUG series, the levels of Gαi1/2 proteins, membrane-associated GRK2, and neurofilament [low molecular weight neurofilament (NF-L)] proteins were also determined. In the UIB series, the levels of Gαq/11 proteins, total GRK2, and α-tubulin were also quantitated.

Immunoblot analysis and quantitation of the target proteins

The levels of α2A-adrenoceptors, Gαi1/2, and Gαq/11 proteins, and total and membrane-associated GRK2, were assessed by immunoblotting by using specific polyclonal antisera (see Materials section). The levels of 68-kDa neurofilament (NF-L) protein, a neuron-specific marker, and α-tubulin, a cytoskeletal protein used as a loading negative control, were also assessed by immunoblotting, using monoclonal antibodies (Escribá et al., 1994 ; García-Sevilla et al., 1997b). In brief, solubilized target proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (6 × 8-cm minigels, 15 wells, Mini 2-D cell, Bio-Rad Laboratories, Hercules, CA, U.S.A.), transferred to nitrocellulose membranes (western blotting), labeled with the specific primary antibody (overnight at 4°C), and incubated for 2 h at room temperature with a blocking solution containing the secondary antibody, horseradish peroxidase-linked donkey anti-rabbit IgG or sheep anti-mouse IgG antiserum (1 : 4,000 or 1 : 5,000 dilution). For the primary antibodies, the dilutions were as follows : 1 : 500 (anti-α2A-adrenoceptor), 1 : 5,000 (anti-Gαi1/2, AS/7 ; anti-Gαq/11, QL), 1 : 3,000 (anti-GRK2), 1 : 1,000 (anti-NF-L), and 1 : 3,000 (anti-α-tubulin). Immunoreactivity was detected using the enhanced chemiluminescence (ECL) western blot detection system (Amersham International, Little Chalfont, U.K.) followed by exposure to film (Amersham ECL Hyperfilm) for 5-30 min. Films were scanned in an image analyzer (Bio Image, Millipore, Ann Arbor, MI, U.S.A.) (Escribá et al., 1994). The antibodies used labeled bands with molecular masses of 70 and 50 kDa (mature and nonglycosylated α2A-adrenoceptor), 40 kDa (Gαi1/2), 42 kDa (Gαq/11), and 80 kDa (GRK2) (see Fig. 1) in good agreement with previous studies (Escribá et al., 1994, 1996 ; Sastre and García-Sevilla, 1994a ; Ozaita et al., 1998). For the α2A-adrenoceptor, only the 70-kDa mature receptor was quantitated (see Fig. 1). The ECL detection system allowed blots to be stripped and reprobed with different antibodies in the same brain sample of a given subject, which provided the best conditions for correlation analyses between the different target proteins in brains of suicide victims.

image

Figure 1. Immunological detection of α2A-adrenoceptors (α2AR), guanine nucleotide binding proteins (Gαi1/2), and membrane-associated GRK2 in the human brain (prefrontal cortex, Brodmann area 9). Representative immunoblots (same gel) from an antidepressant drug-free depressed suicide victim (D was a 39-year-old female ; PMD, 20 h) and a matched healthy control subject (C was a 39-year-old female ; PMD, 30 h) are shown. The amounts of total protein loaded per gel well were 42.1 μg (C) and 43.4 μg (D). The two main bands for α2AR correspond to the 70-kDa mature receptor and 50-kDa nonglycosylated receptor, and both were up-regulated in subjects with suicide/depression. Note that in the depressed suicide victim, the immunoreactivity of the three proteins was markedly increased (mature α2AR, 77% ; Gαi1/2, 96% ; GRK2, 66%).

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The procedure for the quantitation of target proteins in human brain by immunoblotting has been described in detail previously (Escribá et al., 1994). In a given experiment, the amount of a target protein in the prefrontal cortex of one or two suicide victims was compared with that of a control subject matched for sex, age (± 8 years), and PMD (± 12 h). In this way, duplicate or triplicate samples from suicide victims were evaluated using standard curves (i.e., total protein loaded vs. integrated optical density), which consisted of at least four points of different protein content (usually 10/15 to 80/120 μg of protein, resulting in linear relationships) from the same matched control subject all loaded on the same gel. Moreover, in a given gel, duplicate samples from other controls were similarly evaluated as problem samples to assess the intraassay (5-6%) and interassay (7-10%) coefficients of variation. Given a known amount of membrane protein from a problem sample loaded in the gel well (PR, the real amount), the percentage change with respect to control samples was calculated as the ratio between the amount of protein corresponding to the integrated optical density value of the problem sample interpolated in the control standard curve (PT, the theoretical amount) and PR. Thus, the percentage change is (PT/PR) × 100, where this value is 100 for a healthy control sample used as the standard. In brain specimens from suicide victims, the mean interassay coefficients of variation were 18% (α2A-adrenoceptor ; n = 51), 22% (Gαi1/2 ; n = 29), 13% (Gαq/11 ; n = 16), and 19% (GRK2 ; n = 32).

Statistics

Results are expressed as mean ± SEM values. The onesample t test (identical in its implementation to the paired t test) or Student's t test was used for the statistical evaluations. Pearson's correlation coefficients were calculated to test for possible association among variables. The level of significance was chosen at p = 0.05.

Materials

The antiserum against the human α2A-adrenoceptor (third intracellular loop, peptide 262-276, NH2-GGAEAEPLPTQLNGA-COOH) was produced and characterized in this laboratory (A. Ozaita, P. V. Escribá, and J. A. García-Sevilla, unpublished results). The specificity of this polyclonal antiserum was assessed in membranes from human platelets (normal expression of α2A-adrenoceptors) and in membranes from Sf9 (Spodoptera frugiperda) cells overexpressing the human recombinant α2A-adrenoceptor (Research Biochemicals International, Natick, MA, U.S.A.). In these membranes (Escribá et al., 1996) and in human brain, the antibody labeled a protein band of ~70 kDa and a lesser band of ~50 kDa (see Fig. 1). These molecular masses corresponded to the mature (glycosylated) and the precursor (nonglycosylated) forms of α2A-adrenoceptors, respectively (Kobilka et al., 1987). On deglycosylation (0.6 U of the enzyme N-glycosidase for 19 h at 37°C), only the 50-kDa band was observed. None of these bands (70 and 50 kDa) was observed after incubation of membranes with the preimmune serum. Moreover, dot blot and ELISA experiments demonstrated the specific recognition of the peptide used for the antibody production by the immune serum (data not shown). In membranes from Sf9 cells expressing the human recombinant α2C-adrenoceptor, the antiserum did not cross-react with this α2-adrenoceptor subtype (data not shown).

The production and specificity of the anti-GRK2/3 antiserum (a gift from Dr. F. Mayor, Jr., Madrid, Spain) have been described elsewhere (Murga et al., 1996). The higher immunoreactivity of this antibody against GRK2 and the higher abundance of GRK2 in brain (Arriza et al., 1992) suggest that, under the present experimental conditions, the GRK2 isotype was the main species detected. Thus, in human brain (Ozaita et al., 1998), the antibody labeled a single immunoreactive band of 80 kDa, corresponding to the GRK2 protein (see Fig. 1) and which comigrated with recombinant purified GRK2.

Anti-Gαi1/2 (AS/7) and anti-Gαq/11 (QL) polyclonal rabbit antisera were from Du Pont de Nemours/NEN Products, Regensdorf, Switzerland. Anti-68-kDa neurofilament (NF-L) protein monoclonal antibody was from Amersham. Anti-α-tubulin monoclonal antibody (clone DM 1A) was from Sigma Chemical (St. Louis, MO, U.S.A.). Secondary antibodies, chemiluminescence reagents, and film for autoradiography were from Amersham. Other chemicals were from Sigma.

Density of α2A-adrenoceptors, Gi, and GRK2 proteins in the prefronal cortex of suicide victims

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. Density of α2A-adrenoceptors, Gi, and GRK2 proteins in the prefronal cortex of suicide victims
  6. DISCUSSION
  7. Acknowledgements

The levels of α2A-adrenoceptors and associated regulatory proteins were increased in the prefrontal cortex of suicide victims. Thus, significant increases in the immunoreactivities of α2A-adrenoceptors (31 ± 3%, n = 51, t = 9.23, p < 0.0001), Gαi1/2 proteins (42 ± 6%, n = 29, t = 7.16, p < 0.0001), and membrane-associated GRK2 enzyme (24 ± 4%, n = 32, t = 5.77, p < 0.0001) were found in suicide victims compared with sex-, age-, and PMD-matched controls (Figs. 1 and 2). The immunoreactivity of these proteins was not associated with sex or age. In suicide victims, the increases observed in men were not different from those observed in women (data not shown) and were not age related (r = -0.12 to 0.19, n = 29-51 ; age range, 19-84 years).

image

Figure 2. Levels of α2A-adrenoceptors (α2AR, n = 51), guanine nucleotide binding proteins (Gαi1/2, n = 29), and membrane-associated GRK2 (n = 32) in the prefrontal cortex (Brodmann area 9) of suicide victims as mean ± SEM (bars) percentages of the levels in matched controls. Mean ± SEM values for healthy subjects evaluated as problem samples were 99 ± 1% (n = 31) for α2AR, 99 ± 2% (n = 18) for Gαi1/2, and 101 ± 2% (n = 18) for GRK2. *Significantly different from levels in matched controls (p < 0.0001, one-sample t test).

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The levels of α2A-adrenoceptors in the prefrontal cortex were significantly increased in the two series of suicide victims investigated (HUG series : 35 ± 4%, n = 34, t = 8.44, p < 0.0001 ; UIB series : 24 ± 6%, n = 17, t = 4.24, p < 0.001). In contrast to Gαi1/2 proteins, the level of Gαq/11 proteins in brains of suicide victims was not changed with respect to that in matched controls (control subjects : 101 ± 7% ; suicide victims : 99 ± 3% ; n = 16). Total GRK2 enzyme was modestly but significantly increased (11 ± 4%, n = 17, t = 2.64, p < 0.02). The presence of ethanol in brains of some suicide victims (blood concentration, 1.52 ± 0.63 g/L) did not alter the increased levels of α2A-adrenoceptors (ethanol : 29 ± 5%, n = 11, p < 0.001 ; ethanol-free : 38 ± 6%, n = 22, p < 0.0001), Gαi1/2 proteins (ethanol : 43 ± 9%, n = 10, p < 0.005 ; ethanol-free : 41 ± 8%, n = 19, p < 0.0001), and membrane-associated GRK2 enzyme (ethanol : 24 ± 5%, n = 10, p < 0.001 ; ethanol-free : 23 ± 5%, n = 22, p < 0.001) in the prefrontal cortex (Fig. 3).

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Figure 3. Effects of ethanol on the levels of α2A-adrenoceptors (α2AR ; ethanol-free, n = 22 ; ethanol-detected, n = 11), guanine nucleotide binding proteins (Gαi1/2 ; ethanol-free, n = 19 ; ethanol-detected, n = 10), and membrane-associated GRK2 (ethanol-free, n = 22 ; ethanol-detected, n = 10) in the prefrontal cortex (Brodmann area 9) of suicide victims as mean ± SEM (bars) percentages of the levels in matched control subjects. Ethanol in blood samples, 1.52 ± 0.63 g/L ; range, 0.03-7.19 g/L ; n = 11. Increased levels of α2AR, Gαi1/2, and GRK2 did not differ in ethanol-free and ethanol-detected suicide victims (Student's t test). *Significantly different from levels in matched control subjects (at least p < 0.005, one-sample t test).

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To evaluate the specificity of the up-regulation of α2A-adrenoceptors, Gαi1/2 proteins, and GRK2 enzyme in brains of suicide victims, the abundance of 68-kDa neurofilament (NF-L) protein, a neuron-specific marker, was assessed. The immunoreactive levels of NF-L proteins were not changed compared with those in matched controls (control subjects : 100 ± 1% ; suicide victims : 94 ± 4%, n = 17, p > 0.05). In a similar manner, the levels of α-tubulin, used as a loading negative control, also were found unchanged (control subjects : 98 ± 2% ; suicide victims : 102 ± 3% ; n = 16, p > 0.05).

Density of α2A-adrenoceptors and associated regulatory proteins in the prefrontal cortex of depressed suicide and other suicide victims

In depressed suicide victims (n = 23, HUG and UIB series), relevant increases in the levels of α2A-adrenoceptors (35 ± 5%, n = 23, t = 7.38, p < 0.0001), Gαi1/2 proteins (52 ± 8%, n = 13, t = 6.23, p < 0.0001), and membrane-associated GRK2 enzyme (23 ± 6%, n = 16, t = 3.66, p < 0.005) in the prefrontal cortex were also found compared with those in matched controls (Fig. 4). In contrast, no significant changes in the levels of Gαq/11 proteins (8 ± 5% increase, n = 6, p > 0.05) and total GRK2 enzyme (10 ± 8% increase, n = 6, p > 0.05) were observed. The brain levels of NF-L (control subjects : 98 ± 3% ; depressed suicide victims : 93 ± 3%, n = 8, p > 0.05) and α-tubulin (control subjects : 99 ± 3% ; depressed suicide victims : 98 ± 4%, n = 6, p > 0.05) also were unchanged in depressed suicide victims. Ethanol was detected in six depressed suicide victims (blood concentration, 0.83 ± 0.45 g/L), but it did not alter the levels of the target proteins (data not shown).

image

Figure 4. Levels of α2A-adrenoceptors (α2AR), guanine nucleotide binding proteins (Gαi1/2), and membrane-associated GRK2 in the prefrontal cortex (Brodmann area 9) of depressed suicide victims (α2AR, n = 23 ; Gαi1/2, n = 13 ; GRK2, n = 16) and in antidepressant drug-free (α2AR, n = 10 ; Gαi1/2, n = 9 ; GRK2, n = 9) and antidepressant drug-treated (α2AR, n = 6 ; Gαi1/2, n = 4 ; GRK2, n = 6) depressed suicide victims as mean ± SEM (bars) percentages of the levels in matched controls. Asterisks indicate levels significantly different from levels in matched control subjects (*p < 0.005 ; **p < 0.001 ; ***p < 0.0001 ; one-sample t test).

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In this group of depressed suicide victims, the effects of antemortem antidepressant treatment on the levels of α2A-adrenoceptors and associated regulatory proteins in the prefrontal cortex were also investigated (HUG series). In the subgroup (n = 10) of antidepressant drugfree depressed suicide victims (no antidepressant drugs detected in blood samples obtained at postmortem examination), increased levels of α2A-adrenoceptors (40 ± 6%, n = 10, t = 6.67, p < 0.0001), Gαi1/2 proteins (63 ± 10%, n = 9, t = 6.30, p < 0.0001), and membrane-associated GRK2 enzyme (32 ± 7%, n = 9, t = 4.57, p < 0.005) were also found compared with those in matched controls, and were slightly higher (5, 11, and 9%, respectively) than those found in the group of depressed suicide victims (Figs. 1 and 4). In contrast, in the subgroup (n = 6) of antidepressant drug-treated depressed suicide victims (detection of the prescribed antidepressant drugs in blood samples), nonsignificant increases in the levels of α2A-adrenoceptors (34 ± 14%, n = 6, t = 2.46, p > 0.05), Gαi1/2 proteins (31 ± 10%, n = 4, t = 3.08, p > 0.05), and membrane-associated GRK2 enzyme (14 ± 12%, n = 6, t = 1.17, p > 0.05) were found compared with those in matched controls (Fig. 4). Comparison between antidepressant-free and antidepressant-treated depressed suicide victims indicated that only the abundance of Gαi1/2 proteins was significantly reduced (32%, p < 0.05) after antidepressant treatment. Toxicological screening was not performed in one depressed suicide victim in whom prescription of antidepressant drugs was documented. In this subject, the levels (taken as the control value of 100%) of α2A-adrenoceptors, Gαi1/2 proteins, and membrane-associated GRK2 enzyme were 130, 119, and 98%, respectively.

In the subgroup of suicide victims without a diagnosis, increased levels of α2A-adrenoceptors (30 ± 6%, n = 18, t = 4.87, p < 0.001), Gαi1/2 proteins (36 ± 13%, n = 9, t = 2.83, p < 0.05), and membrane-associated GRK2 enzyme (29 ± 7%, n = 9, t = 4.23, p < 0.005) in the prefrontal cortex were also found compared with those in matched controls. In a similar manner also to the findings in depressed suicide victims, the levels of Gαq/11 proteins (3 ± 4% decrease, n = 9, p > 0.05) and total GRK2 enzyme (8 ± 6% increase, n = 9, p > 0.05) were found not significantly changed in suicide victims without a diagnosis.

In the subgroup of suicide victims with various diagnoses, the number of subjects in each category was very limited, which precluded any relevant statistical analysis. However, it is noteworthy that the levels of α2A-adrenoceptors and associated regulatory proteins (Gαi1/2 and GRK2) did not appear to be altered in suicide victims with alcohol dependence (n = 3) or schizophrenia (n = 3), whereas they showed a clear tendency to be increased in suicide victims with schizoaffective disorder (n = 2) or personality disorder (n = 2) (data not shown).

Correlations between target protein immunoreactivities in the prefrontal cortex of suicide victims

Because agonist (noradrenaline) occupancy of α2A-adrenoceptors leads to activation of Gi-coupling proteins and regulatory membrane-associated GRK2 enzyme, the relation between these proteins was investigated through correlation analyses of the different immunoreactivities quantitated in the same brain samples. In the prefrontal cortex of suicide victims, the levels of α2A-adrenoceptors correlated weakly with those of Gαi1/2 proteins (r = 0.32, n = 29, p = 0.08), but they showed a positive and significant correlation with those of membrane-associated GRK2 enzyme (r = 0.43, n = 32, p = 0.02). Moreover, the levels of Gαi1/2 proteins also correlated with those of GRK2 enzyme in membranes (r = 0.54, n = 29, p = 0.002) (Fig. 5). It is interesting that the levels of α2A-adrenoceptors did not correlate with those of total GRK2 enzyme (r = 0.03, n = 17). A multiple regression analysis between the levels of membrane-associated GRK2 enzyme (dependent variable) and those of α2A-adrenoceptors and Gαi1/2 proteins (independent variables) in the same brain samples of suicide victims also resulted in a positive and significant correlation (r = 0.56, n = 29, p = 0.008). Similar results were obtained in the prefrontal cortex of depressed suicide victims, where the levels of α2A-adrenoceptors clearly correlated with those of membrane-associated GRK2 enzyme (r = 0.62, n = 16, p = 0.011). A similar multiple regression analysis between the three target proteins (see above) in these depressed suicide victims also resulted in a significant correlation (r = 0.54, n = 13, p = 0.041).

image

Figure 5. Correlations between the levels of α2A-adrenoceptors and guanine nucleotide binding (Gαi1/2) proteins (r = 0.32, p = 0.08, 27 df), α2A-adrenoceptors and membrane-associated GRK2 enzyme (r = 0.43, p = 0.02, 30 df), and Gαi1/2 proteins and membrane-associated GRK2 enzyme (r = 0.54, p = 0.002, 27 df) in the same brains (prefrontal cortex, Brodmann area 9) of suicide victims. Each point represents a different subject and the solid lines represent the regression of the correlations. Both axes are expressed as percentages of the immunoreactive levels in matched control subjects.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. Density of α2A-adrenoceptors, Gi, and GRK2 proteins in the prefronal cortex of suicide victims
  6. DISCUSSION
  7. Acknowledgements

The results of this study indicate that in the prefrontal cortex of suicide victims and antidepressant drug-free depressed suicide victims, (1) the levels of immunoreactive α2A-adrenoceptors are increased (31-40%), which is in line with previous radioligand binding studies ; (2) the density of Gαi1/2 proteins is also up-regulated (42-63%), which suggests an increase in signal transduction through α2A-adrenoceptors ; (3) the abundance of plasma membrane-associated GRK2 enzyme is also increased (24-32%), probably because of the abnormal higher functioning of the α2A-adrenoceptor/Gαi1/2 complex ; (4) there is a significant correlation between the levels of GRK2 enzyme (dependent variable) and those of α2A-adrenoceptors and Gαi1/2 proteins (independent variables) in the same brain samples (r = 0.56), which further suggests the existence of supersensitive α2A-adrenoceptors in subjects with suicide and depression ; and (5) antemortem antidepressant drug treatment was associated with a significant reduction in the levels of Gαi1/2 proteins (32%) but with modest decreases in the levels of the α2A-adrenoceptor protein (6%) and GRK2 enzyme (18%) in brains of depressed suicide victims.

To our knowledge, this is the first report on the immunodetection and quantitation of α2A-adrenoceptors in the human brain (prefrontal cortex), using a subtype-specific polyclonal antibody. In a similar manner, various immunohistochemical studies have revealed prominent α2A-adrenoceptor-like immunoreactivity in the prefrontal cortex of monkeys (Aoki et al., 1994 ; Wang and Lidow, 1997) and in various brain regions of the rat brain (Rosin et al., 1993 ; Aoki et al., 1994 ; Venkatesan et al., 1996). Immunoreactive α2A-adrenoceptors were localized in cortical (pyramidal) neurons, dendrites, and axonal processes (catecholaminergic and noncatecholaminergic terminals), suggesting that these inhibitory receptors may operate as autoreceptors as well as heteroreceptors (Rosin et al., 1993 ; Aoki et al., 1994). Immunoelectron microscopy further revealed the presence of α2A-adrenoceptors in presynaptic terminals, postsynaptic membranes, and glia (Aoki et al., 1994 ; Venkatesan et al., 1996). In the current study, the levels of immunoreactive α2A-adrenoceptors were increase in the prefrontal cortex of suicide and depressed suicide victims, as well as in suicide victims without a recorded diagnosis. This latter group of suicide victims most probably included undiagnosed depressed subjects, because major depression is one of the most important risk factors for suicide (Lester, 1993 ; Cheng, 1995). The results agree well with previous radioligand binding studies demonstrating an increased density of α2-adrenoceptor agonist (Meana and García-Sevilla, 1987 ; Meana et al., 1992a ; González et al., 1994 ; Ordway et al., 1994 ; Callado et al., 1998) and antagonist (De Paermentier et al., 1997) binding sites in various brain regions of suicide and depressed suicide victims. Specifically, major depression appears to be associated with increased density of α2A-adrenoceptor agonist binding sites (Callado et al., 1998) and levels of the receptor protein (present results). However, one limitation of the present and previous studies is the impossibility to specifically quantitate presynaptic α2A-adrenoceptors and to ascertain whether the observed up-regulation of these receptors in depression is related to α2A-autoreceptors/heteroreceptors only or to α2A-adrenoceptors on pre- and postsynaptic membranes as well as on glial cells (Aoki et al., 1994 ; Venkatesan et al., 1996).

The density of Gαi1/2 proteins was also increased in the prefrontal cortex of the same suicide, depressed suicide, and suicide victims without a diagnosis. Because the high-affinity conformation of the α2A-adrenoceptor is most probably related to the complex with Gαi2 proteins (Simonds et al., 1989 ; Sastre and García-Sevilla, 1994a), which in turn induces the inhibition of adenylyl cyclase, an enhanced expression of Gαi1/2 proteins linked to a higher abundance of brain α2A-adrenoceptors was also expected. In contrast, the levels of Gαq/11 proteins, linked to the activation of protein kinase C, were found unchanged in brains of suicide and depressed suicide victims, in agreement with a previous study (Pacheco et al., 1996). These findings suggest that signal transduction through the α2A-adrenoceptor/Gαi2 complex is increased in suicide victims and subjects with major depression. A similar increase in the abundance of Gαi2 proteins has been observed in platelets of drug-free depressed patients (García-Sevilla et al., 1997c). In contrast to these results, other studies have reported normal (Ozawa et al., 1993 ; Cowburn et al., 1994) or reduced (Pacheco et al., 1996) levels of brain Gαi1/2 proteins in depressed suicide victims and in depressed patients ; however, photoaffinity GTP labeling of Gαi proteins was found increased in postmortem brains of depressed patients (Ozawa et al., 1993). It is noteworthy that reduced adenylyl cyclase activity has been reported in brains of depressed suicide victims (Cowburn et al., 1994 ; Lowther et al., 1996), and recovery from depression, spontaneous or after antidepressant treatment, was associated with increased enzyme activity in platelets of depressed patients (García-Sevilla et al., 1990). This could be related, respectively, to the increased levels of inhibitory Gαi2 proteins found in brains of depressed suicide victims (present results) and in platelets of depressed patients (García-Sevilla et al., 1997c), and to the down-regulation of Gαi2 proteins induced by antidepressant drugs in brains of depressed suicide victims (present results) and in platelets of depressed patients (García-Sevilla et al., 1997c). Thus, a greater or lesser tonic inhibition of this transduction system through Gαi2 proteins would result, as observed, in a reduced or enhanced adenylyl cyclase activity in brain/platelet of depressed patients. In any case, the greater abundance of α2A-adrenoceptors and Gαi1/2 proteins, and the clear trend for a positive correlation between the two proteins in the same brain samples, strongly suggest an increase in signal transduction through these receptors in suicide victims and subjects with major depression.

Recent in vitro studies have demonstrated that the activated state of a G protein-coupled receptor (e.g., the activated α2A-adrenoceptor/Gαi2 complex in brains of suicide victims) is regulated by specific GRK enzymes that promote the phosphorylation/desensitization of the agonist-occupied receptor (Premont et al., 1995). This process is facilitated by the increased Gβγ complex availability that binds, activates, and targets the cytoplasmic GRKs to the plasma membrane (Lohse et al., 1996). For the α2A-adrenoceptor, the specific GRKs involved in receptor phosphorylation are the GRK2/3 isotypes (Jewell-Motz and Liggett, 1996). However, very little is known on neurotransmitter receptor regulation by GRK enzymes in the human brain. Recently, the level of membrane-associated GRK2 was found to be increased in the prefrontal cortex of human heroin addicts (Ozaita et al., 1998), suggesting a role for this kinase in the in vivo regulation of μ-opioid receptors. The present results also demonstrate for the first time that the abundance of the plasma membrane-associated GRK2 is increased in the prefrontal cortex of suicide, depressed suicide, and suicide victims without a diagnosis. The increase in GRK2 is probably related to the abnormal higher functioning of the α2A-adrenoceptor/Gαi2 complex observed in the brains of these suicide victims. However, and because GRK2 phosphorylates other neurotransmitter receptors (Chuang et al., 1996) whose densities are increased in brains of suicide and depressed suicide victims (e.g., α1- and β-adrenoceptors, serotonin receptors, dopamine receptors, μ-opioid receptors) (Mann et al., 1986 ; Arango et al., 1993 ; Gabilondo et al., 1995 ; Bowden et al., 1997), the observed increase in GRK2 also may be related to the regulation of these receptors. In spite of this lack of GRK2/receptor selectivity, the positive and significant correlations found between the levels of membrane-associated (but not total) GRK2 enzyme and those of α2A-adrenoceptors or Gαi1/2 proteins (and Gβγ availability) strongly suggest that this kinase plays a crucial role in the regulation of the α2A-adrenoceptor in the human brain. Furthermore, in the context of receptor regulation in subjects with depression, the significant correlations (multiple regression analyses) observed between the levels of plasma membrane-associated GRK2 (dependent variable) and those of α2A-adrenoceptors and Gαi1/2 proteins (independent variables) in the same brain samples of suicide or depressed suicide victims was a relevant finding. That these proteins increase in concert and are correlated with one another clearly suggests the existence of supersensitive α2A-adrenoceptors in suicide victims and subjects with depression.

Antemortem antidepressant drug treatment in depressed subjects resulted in reduced levels of brain α2A-adrenoceptors (6%), Gαi1/2 proteins (32%), and GRK2 enzyme (18%) at postmortem examination, compared with those levels in antidepressant-free depressed suicide victims. However, only the reduction in the abundance of Gαi1/2 proteins reached statistical significance, in agreement with a previous ex vivo study in depressed patients (García-Sevilla et al., 1997c) ; the level of GRK2 showed a clear tendency for a reduction after antidepressant treatment. In any case, the limited number of cases investigated does not allow a clear comparison between treated and drug-free depressed suicide victims. Chronic antidepressant treatments have been repeatedly shown to induce down-regulation of α2-adrenoceptors in rat brain (Smith et al., 1981 ; Menargues et al., 1990 ; Kovachich et al., 1993) and platelets of depressed patients (García-Sevilla et al., 1986, 1990). Recently, antidepressant drug treatment also has been shown to reduce the density of α2A-adrenoceptors (and other α2-adrenoceptor subtypes) in various brain regions of depressed suicide victims (De Paermentier et al., 1997). These results appear to suggest that the number of α2A-adrenoceptor agonist/antagonist binding sites (Smith et al., 1981 ; Kovachich et al., 1993 ; De Paermentier et al., 1997) but not the abundance of the receptor protein (present results) is readily down-regulated by antidepressant treatment in the human brain. Further studies are needed to assess the effects of anti-depressant drugs on the levels of immunoreactive α2A-adrenoceptors and associated regulatory proteins in brains of depressed suicide victims.

To evaluate the specificity of the present findings in brains of suicide and depressed suicide victims, the levels of NF-L proteins, a neuron-specific marker, and α-tubulin, a cytoskeletal protein used as a loading control, were determined in the same brain samples. The negative results obtained for these proteins discounted possible effects of unspecific variables or nonspecific effects of drug treatment on brain α2A-adrenoceptors and associated regulatory proteins. Moreover, the levels of immunoreactive α2A-adrenoceptors, Gαi1/2 proteins, and GRK2 enzyme were significantly up-regulated in brains of suicide and depressed suicide victims, regardless of the presence of ethanol in blood samples. These results agree well with previous studies demonstrating a lack of effects of ethanol on the density of α2A-adrenoceptors (Meana et al., 1992b), Gαil/2 proteins (Escribá et al., 1994), and GRK2 enzyme (Ozaita et al., 1998) in the human brain.

Together, the present findings indicate that further understanding of the hyperactivated brain α2A-adrenoceptors and associated regulatory mechanisms in subjects with suicide/depression is likely to contribute to the knowledge of the biochemical basis and treatment of depressive disorders. In this context, the novel antidepressant mirtazapine is an antagonist at α2-adrenoceptors that enhances the release and synthesis of noradrenaline and serotonin in brain (Nutt and Pinder, 1996).

Acknowledgements

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. Density of α2A-adrenoceptors, Gi, and GRK2 proteins in the prefronal cortex of suicide victims
  6. DISCUSSION
  7. Acknowledgements

This study was supported by FNSRS grant 3100-052242.97 (J.G.), Bern, Switzerland, and by DGICYT grant PB94-0002-Mod C (J.A.G.-S.), Madrid, Spain. A.O. was supported by a fellowship from the Basque Government, Vitoria/Gasteiz, Spain. We thank Dr. Ch. Staub of the Unité de Toxicologie, Université de Genève, who supplied the toxicological data of the HUG series and Dr. A. Moreno, Servicio de Psiquiatría, Hospital “Son Dureta,” who supplied the clinical data of the UIB series. J.A.G.-S. is a member of the Institut d'Estudis Catalans.

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