Journal of Psychopharmacology

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Freddy De Paermentier, Sandra Lowther, M. Rufus Crompton, Cornelius L.E. Katona and Roger W Horton J Psychopharmacol 1997 11: 295 DOI: 10.1177/026988119701100403 The online version of this article can be found at: http://jop.sagepub.com/content/11/4/295

-Adrenoceptors in human pineal glands are unaltered in depressed suicides

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Original Papers

β-Adrenoceptors in human pineal glands are unaltered in depressed

suicides
3 , 2 1 Lowther , 1† Freddy De Paermentier Sandra , M. Rufus Crompton Cornelius L.E. Katona and 1 Horton Roger W
Forensic 2 Departments 1 of Pharmacology and Clinical Pharmacology and Medicine, St Georges Hospital Medical School, London SW17 0RE, and 3 of Psychiatry, University College London Medical School, London WIN 8AA, UK. Department
† Deceased
UK

We have measured

β-adrenoceptor binding in pineal glands obtained at post mortem from suicides with a firm retrospective diagnosis of depression, and from age and gender-matched controls. In both antidepressant-free and antidepressant-treated suicides there were no significant differences in the number or affinity of βadrenoceptors compared to controls. Within the total group of subjects we found no variation in βadrenoceptor binding in relation to time of death or season of death. There was a significant negative correlation between the number of β-adrenoceptors and age in controls, but not in suicides. These results suggest that pineal β-adrenoceptors are not altered either in depression or as a result of antidepressant
treatment.

Key

words: suicide

antidepressant drugs; β-adrenoceptors; depression; pineal gland; post-mortem human brain;

Introduction
There has been considerable interest in the involvement of /3adrenoceptors in depression and in relation to antidepressant drug action. f3-Adrenoceptors have been extensively studied in white blood cells from depressed patients and in human brain samples obtained post mortem, mainly from suicide deaths. The results, particularly with brain samples, have been conflicting with reports of increased, decreased and unaltered fl-adrenoceptor binding compared to controls (Meyerson et al., 1982; Crow et al., 1984; Mann et al., 1986; Biegon and Israeli, 1988; Arango et al., 1990; De Paermentier et al., 1990b). These discrepant findings may be a reflection of the heterogeneity of psychiatric illness associated with suicide, and with recent antidepressant drug treatment. We have previously reported modest decreases in cortical p-adrenoceptors in suicide victims with a retrospective diagnosis of depression who had been free of antidepressants for at least 3 months (De Paermentier et al., 1990b), but found little or no evidence for an antidepressant-induced down-regulation of f3-adrenoceptors in suicides who were prescribed antidepressant treatment (De Paermentier et al., 1991). Depression is associated with abnormalities of several biological rhythms including melatonin secretion. Melatonin release from the pineal gland is mediated by sympathetic nerves acting via p-adrenoceptors and is influenced by environmental lighting. The highest secretion of melatonin occurs during darkness and there is substantial evidence that the night-time peak in melatonin secretion is reduced in depressed patients (Brown et al., 1985; Frazer et al., 1986; McIntyre et al., 1986). (3-Adrenoceptors also influence the

synthesis of melatonin and lower melatonin concentrations reported in the pineal gland of suicides may indicate a fladrenoceptor subsensitivity (Stanley and Brown, 1988). Acute and short-term administration of antidepressants enhances melatonin secretion in both healthy volunteers and depressed patients (Thompson et al., 1983; Cowen et al., 1985; Demisch et al., 1986; Franey et al., 1986; Murphy et al., 1986; Sack and Lewy, 1986; Palazidou et al., 1992; Skene et al., 1994), although the effects of long-term antidepressants appear not to
have been studied. ized in rat

Although fl-adrenoceptors have been extensively characterpineal glands (Moyer et al., 1981; Craft et al., 1985; Biegon, 1986; Wilkinson et al., 1987; Gonzalez-Brito et al., 1988), there are relatively few studies in human pineal glands (Oxenkrug et al., 1990) and none in relation to depression. In the present study we have quantitated p-adrenoceptors in
Suicides

pineal glands from suicides with a retrospective diagnosis of depression, and from age- and gender-matched controls.
were

subdivided into those who had been free of

antidepressant drugs for at least 3 months and those in whom prescription of antidepressant drugs was clearly documented. Because of the importance of the pineal gland as a biological clock we have analysed /3-adrenoceptors in respect of time of death and season of death within our study group of 70 subjects.

Materials and methods

Subject selection
Deaths recorded
as

suicide at Coroners

subjected

to

retrospective diagnosis by

inquests were psychiatrist

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296

Table 1

Drug

treatment and cause of death for the

antidepressant-treated suicides

(C.L.E.K.) using hospital and Coroners records and interviews with the subjects general practitioner. Thirty-eight subjects in whom there was sufficient documentary evidence to establish a retrospective diagnosis of depression according to the criteria of Beskow et al. (1976) were selected for study. Information on previous and current drug treatment was sought and blood samples obtained at examination post mortem were screened for the presence of psychoactive drugs. On the basis of this information depressed suicides were divided into two groups.

Tissue collection, dissection and storage Tissue collection, dissection and storage were as previously described by Cheetham et al. (1988). Briefly, brains were obtained within 78 h of death and stored at - 80 C. Eighteen hours prior to dissection, brains were transferred to -20 C. Brains were cut into coronal sections (3-mm thick) and pineal glands were removed from appropriate slices (one or two slices) and stored in airtight containers at -80C until

assayed.
Tissue preparation Membranes were prepared essentially as described by Cheetham et al. (1988). Whole pineal glands were homogenized in ice-cold 0.25 M sucrose (1:30 w/v) using a motor driven Teflon pestle (eight strokes at 120 rpm) and centrifuged (at 4 C) at 1000 g for 10 min. The supernatant was stored on ice and the pellet rehomogenized in 0.25 M sucrose (1:15 w/v) and centrifuged at 750g for lOmin. Combined supernatants were diluted (1:80 w/v) with 50 mM Tris-HCl buffer, pH 7.6 (at 25 C) and centrifuged at 35000g for 10 min. The pellet was resuspended in 50mM Tris-HCl buffer (1:40 w/v) and centrifuged at 35000 g for 10 min. The final pellet was resuspended in 50 mM Tris-HCl buffer to give a concentration of 50 mg original wet weight/ml and immediately used in the

Antidepressant-free suicides There were 21 subjects who had not been prescribed antidepressants (or other psychoactive drugs) in the 3 months prior to death, and with no psychoactive drugs detected in blood samples taken post mortem. Causes of death were hanging (n = 8), drug overdosage (n = 5), carbon monoxide poisoning (n 5), jumping from height (n 2) and self-inflicted stab wound (n =1 ). Control brains from subjects dying suddenly from causes not involving the central nervous system, and without documented evidence of mental illness, were individually matched for age and gender to these suicides, with two exceptions for whom control brains were not available. Causes of death were myocardial infarction (n = 16), accidental fall (n = 1), acute asthma (n=1) and road traffic accident (n = 1).
= =

binding assays. Antidepressant-treated suicides There were 15 subjects in whom prescription of antidepressant drugs (alone or in combination with other psychoactive drugs) was clearly documented. Details of drug treatment and causes
of death for these suicides are shown in Table 1. A second group of controls were individually age- and gender-matched to these suicides. Causes of death for controls were myocardial infarction (n =11 ), road traffic accident (n = 2), acute asthma (n=1) and accidental drowning (n=1).

Binding assays

Two hundred microlitres of freshly prepared membranes, 25 pl [3H]CGP 12177 (58 Ci/mmol, Amersham International) at six concentrations (0.03-1 nM) and 25 ~.1 water (total binding) or isoprenaline (200 uM; to define total fl-adrenoceptors) or CGP 20712A (500 nM; to define fli-adrenoceptors) were incubated for 120 min at 25 C.

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297

Table 2

Demographic details of subjects studied

Data

expressed

as

meansSEM, range shown in parentheses.

n, Number

of subjects.

Table 3

/3-adrenoceptor binding sites

in

antidepressant-free

and

antidepressant-treated suicides

and controls

Binax, fmol/mg protein; K,,, nM. Values are meansSEM. statistical significance (p < 0.05, by Mann-Whitney test).

n,

Number of subjects. None of the

comparisons between controls and suicides reached

Membrane-bound radioactivity was recovered by filtration under vacuum through Whatman GF/B glass fibre filters (presoaked with 0.5% polyethylenimine) using a Brandel cell harvester. Filters were washed with 16 ml ice cold 50 mM TrisHCl buffer, pH 7.6. Radioactivity was determined by liquid scintillation counting using a Packard scintillator 299 at an efficiency of 39-44 percent. Aliquots of membrane were stored at - 20 C for subsequent protein determination by the method of Lowry et al. (1951), using bovine serum albumin as the standard. Assays were performed on coded samples, blind to subject classification, but arranged so that suicide and controls were assayed concurrently.

significant
delay
and

correlations between age and Kd Bmax or K~ in controls or suicides.

or

post

mortem

Analysis The equilibrium dissociation constant (K~) and maximum number of binding sites (Bm~,j were determined by non-linear regression fitting to a one-site binding model. Comparisons between groups were made using the non-parametric MannWhitney test, but for ease of presentation the results are expressed as means SEM. Correlations were determined using Spearmans rank correlation.

Comparison between depressed suicides and controls There were no significant differences in the number or affinity of /3-adrenoceptors or #I-adrenoceptors between antidepressant-free or antidepressant-treated suicides and controls (Table 3). When antidepressant-free suicides were divided on the basis of violence of death, there was a trend for violent suicides to have higher binding (controls: 311 +32fmol/mg protein, n =10; suicides: 341 17 fmol/mg protein, n =13) and for non-violent suicides to have lower binding (controls: 312 19 fmol/mg protein, n = 9; suicides: 275 29 fmol/mg protein, n = 8) than controls; neither of these differences reached statistical significance. When antidepressant-treated suicides were divided according to duration of treatment (less than 4 weeks or more than 11 weeks), there were no significant differences between either suicide group and their respective
controls.

Effect of time and season of death

As fl-adrenoceptors in pineal glands of suicides and controls did not differ, we have combined all 70 subjects studied and examined the effects of time and season of death. There were no significant variations in the number of /3-adrenoceptors with respect to time (Fig. 1) or season (Fig. 2) of death.

Results
The demographic details of subjects are given in Table 2. There were no significant differences in age, gender or post mortem delay (time from death to storage of tissue at -80 C) between antidepressant-free and antidepressant-treated suicides and their respective matched controls. There was a weak but statistically significant negative correlation between age and Bmax when all control subjects were considered (r 2 = 0.13, p = 0.04, n 34), but no such relationship was apparent within the suicides (r 2 = 0.01, p = 0.85, n = 36). There were no
=

Discussion
To the best of our

knowledge this is the first study to examine p-adrenoceptor binding in pineal glands from depressed
suicides and controls. The values we obtained in controls, are comparable to those reported for total fl-adrenoceptor binding by Oxenkrug et al. (1990), and demonstrate the high

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298

Figure 1 Effect of time of death on (3-adrenoceptor numbers in pineal glands. Values are meansSEM. The number of subjects in each time slot are as follows: 0-4, n=6; 4-8, n=7; 8-12, n= 16; 12-16, n =15; 16-20, n = 20; 20-24, n = 5

Figure 2 Effect of season of death on /3-adrenoceptor numbers in pineal glands. Values are means + SEM. The number of subjects are as follows: spring (March-May), n=26; summer (June-August) n = 16; autumn (September-November), n =15; winter (December3 February), n =13

found no evidence of higher fl-adrenoceptor binding, irrespective of violence of death (De Paermentier et al., 1990b). In antidepressant-treated suicides, there were no significant differences in total fl- or p-adrenoceptor binding. We found no evidence in support of an antidepressant-mediated downregulation of J3-adrenoceptors. In contrast there was an unexpected trend for an increase in fl-adrenoceptor binding. In control subjects we observed a significant negative correlation between total fl-adrenoceptor number and age. This was weak however and was not seen in suicides. The relevance of the difference between suicides and controls is unclear, but it is unlikely to have influenced our overall interpretation as controls and suicides were individually matched for age. Various studies have measured changes in pineal gland Padrenoceptor number in relation to time of day. In experimental animals where day length can be controlled, results appear to be conflicting depending on species and light:dark ratio. In the rat highest fl-adrenoceptor binding occurs in the late light phase or early dark phase, whereas in hamster the reverse is seen (Wilkinson et al., 1987; Gonzalez-Brito et al., 1988; Pangrel et al., 1990). Oxenkrug et al. (1990) have examined p-adrenoceptor binding in human pineal glands and found a distinct peak between 18.00 and 20.00 hours. In our study with a comparable number of subjects, there was no significant variation throughout a 24-h period (Fig. I). This inconsistency may be related to a number of factors. Despite the large number of subjects we examined, the proportion of subjects within different time periods varied widely, e.g. 16.0020.00 hours, n = 20, 20.00-24.00 hours, n = 5. The peak in fladrenoceptor number found by Oxenkrug et al. (1990) occurs at a time which would be light or dark depending on the season of year. Although we found no overall seasonal effect (Fig. 2), the numbers we were able to study do not allow us to exclude an interaction between season and time. It is possible that there is a different relationship between p-adrenoceptors and time of day or season in suicides and controls. Inspection of the data did not support this, although the distribution of subjects within each time period was such that meaningful statistical comparisons were precluded.

Acknowledgement
<

We gratefully acknowledge financial support from the Sir Jules Thorn Charitable Trust.

density of {3-adrenoceptors in the pineal gland. These values are four to five times the density we have reported in human cerebral cortex (De Paermentier et al., 1990a). In the pineal gland, (3-adrenoceptors are almost exclusively of the flisubtype ( > 90 %, Table 3) and this is in agreement with the rat pineal gland (Dickinson et al., 1986). In antidepressant-free suicides, we found no differences in the number or affinity of total fl- or ~31-adrenoceptors. This is compatible with previous findings of unchanged ~-adrenoceptor binding in cortical samples from suicide victims (Meyerson
al., 1982; De Paermentier et al., 1990b; Stockmeier and Meltzer, 1991). We found a tendency for pineal gland fladrenoceptor binding to be higher in those subjects who had died by violent means and lower in those who had died by nonviolent means. In our previous study of nine brain regions we
et

Address for correspondence

Dr R.W. Horton Department of Pharmacology and Clinical Pharmacology St Georges Hospital Medical School London SW17 ORE UK Email: hortonr@sghms.ac.uk

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