295.fullb-Adrenoceptors in human pineal glands are unaltered in depressed suicides

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http://jop.sagepub.com/Journal of Psychopharmacology

http://jop.sagepub.com/content/11/4/295The online version of this article can be found at:

DOI: 10.1177/026988119701100403

1997 11: 295J PsychopharmacolFreddy De Paermentier, Sandra Lowther, M. Rufus Crompton, Cornelius L.E. Katona and Roger W Horton-Adrenoceptors in human pineal glands are unaltered in depressed suicides

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Original Papers

-Adrenoceptors in human pineal glands are unaltered in depressed

suicides

Freddy De Paermentier1, Sandra Lowther1, M. Rufus Crompton2, Cornelius L.E. Katona3 andRogerW Horton1

1Departmentsof Pharmacology and Clinical Pharmacologyand2Forensic Medicine, St Georges HospitalMedical School, London SW17 0RE, UK

and3Department ofPsychiatry, University College London Medical School, London WIN8AA, UK.

Deceased

We have measured -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 -

adrenoceptors comparedto controls. Within the total

groupof

subjectswe found no variation in -

adrenoceptor binding in relation to time of death or season of death. There was a significant negative

correlation between the number of -adrenoceptors and age in controls, but not in suicides. These results

suggest that pineal -adrenoceptors are not altered either in depression or as a result of antidepressant

treatment.

Key words: antidepressant drugs; -adrenoceptors; depression; pineal gland; post-mortem human brain;suicide

Introduction

There has been considerable interest in the involvement of /3-

adrenoceptorsin

depressionand in relation to

antidepressantdrug action. f3-Adrenoceptors have been extensively studied inwhite blood cells from depressed patients and in human brain

samples obtained post mortem, mainly from suicide deaths.The results, particularly with brain samples, have beenconflicting with reports of increased, decreased and unalteredfl-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 theheterogeneity of psychiatric illness associated with suicide, andwith recent antidepressant drug treatment. We have previouslyreported modest decreases in cortical p-adrenoceptors insuicide victims with a

retrospective diagnosisof

depressionwho had been free of antidepressants for at least 3 months (DePaermentier et al., 1990b), but found little or no evidence foran antidepressant-induced down-regulation of f3-adrenocep-tors in suicides who were prescribed antidepressant treatment

(De Paermentier et al., 1991).Depression is associated with abnormalities of several

biological rhythms including melatonin secretion. Melatoninrelease from the pineal gland is mediated by sympatheticnerves acting via p-adrenoceptors and is influenced byenvironmental lighting. The highest secretion of melatoninoccurs during darkness and there is substantial evidence thatthe night-time peak in melatonin secretion is reduced in

depressedpatients

(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 fl-adrenoceptor subsensitivity (Stanley and Brown, 1988).Acute

and short-term administrationof

antidepressantsenhances

melatonin secretion in both healthy volunteers and depressed

patients (Thompson et al., 1983; Cowen et al., 1985; Demischet 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.

Although fl-adrenoceptors have been extensively character-ized in rat pineal 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. Inthe present study we have quantitated p-adrenoceptors in

pineal glandsfrom suicides with a

retrospective diagnosisof

depression, and from age- and gender-matched controls.Suicides 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 biologicalclock we have analysed /3-adrenoceptors in respect of time ofdeath and season of death within our study group of 70

subjects.

Materials and methods

Subject selectionDeaths recorded as suicide at Coroners inquests were

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Table 1 Drug treatment and cause of death for the antidepressant-treated suicides

(C.L.E.K.) using hospital and Coroners records and inter-views with the subjects general practitioner. Thirty-eightsubjects in whom there was sufficient documentary evidenceto establish a retrospective diagnosis of depression accordingto 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 postmortem were

screened for the presence of psychoactive drugs.On the basis of this information depressed suicides weredivided into two groups.

Antidepressant-free suicidesThere were 21 subjects who had not been prescribed anti-depressants (or other psychoactive drugs) in the 3 months priorto 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 fromcauses not involving the central nervous system, and without

documented evidence of mental illness,were

individuallymatched for age and gender to these suicides, with twoexceptions for whom control brains were not available.Causes of death were myocardial infarction (n = 16), accidentalfall (n = 1), acute asthma (n=1) and road traffic accident (n = 1).

Antidepressant-treated suicidesThere were 15 subjects in whom prescription of antidepressantdrugs (alone or in combination with other psychoactive drugs)was clearly documented. Details of drug treatment and causesof death for these suicides are shown in Table 1.Asecond

group of controls were individually age- and gender-matchedto 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).

Tissue collection, dissection and storageTissue collection, dissection and storage were as previouslydescribed by Cheetham et al. (1988). Briefly, brains wereobtained within 78 h of death and stored at - 80 C. Eighteenhours prior to dissection, brains were transferred to -20 C.Brains were cut into coronal sections (3-mm thick) and pinealglands were removed from appropriate slices (one or two

slices) and stored in airtight containers at -80C untilassayed.

Tissue preparationMembranes were prepared essentially as described byCheetham et al. (1988). Whole pineal glands were homo-genized in ice-cold 0.25 M sucrose (1:30 w/v) using a motordriven Teflon pestle (eight strokes at 120 rpm) and centrifuged(at 4 C) at 1000 g for 10 min. The supernatant was stored onice and the pellet rehomogenized in 0.25 M sucrose (1:15 w/v)and centrifuged at 750g for lOmin. Combined supernatantswere 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 pelletwas

resuspended in 50mM Tris-HCl buffer (1:40 w/v) andcentrifuged at 35000 g for 10 min. The final pellet wasresuspended in 50mM Tris-HCl buffer to give a concentrationof 50 mg original wet weight/ml and immediately used in thebinding assays.

Binding assays ,Two hundred microlitres of freshly prepared membranes, 25 pl[3H]CGP 12177 (58 Ci/mmol,Amersham International) at sixconcentrations (0.03-1 nM) and 25 ~.1 water (total binding) orisoprenaline (200 uM; to define total fl-adrenoceptors) or CGP

20712A (500 nM; to define fli-adrenoceptors)were

incubatedfor 120 min at 25 C.



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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. n, Number of subjects. None of the comparisons betweencontrols and suicides reached

statistical significance (p < 0.05, by Mann-Whitney test).

Membrane-bound radioactivity was recovered by filtration

under vacuum through Whatman GF/B glass fibre filters

(presoaked with 0.5% polyethylenimine) using a Brandel cellharvester. Filters were washed with 16 ml ice cold 50 mM Tris-

HCl buffer, pH 7.6. Radioactivity was determined by liquidscintillation 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 themethod of Lowry et al. (1951), using bovine serum albuminas the standard.Assays were performed on coded samples,blind to subject classification, but arranged so that suicide andcontrols were assayed concurrently.

AnalysisThe 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. Comparisonsbetween groups were made using the non-parametric Mann-

Whitney test, but for ease of presentation the results are

expressed as means SEM. Correlations were determined

using Spearmans rank correlation.

Results

The demographic details of subjects are given in Table 2. Therewere 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 andtheir 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

significant correlations between age and Kd or post mortem

delay and Bmax or K~ in controls or suicides.

Comparison between depressed suicides and controls

There were no significant differences in the number or affinityof /3-adrenoceptors or #I-adrenoceptors between antidepres-sant-free or antidepressant-treated suicides and controls (Table3). When antidepressant-free suicides were divided on the basis

of violence of death, there was a trend for violent suicides tohave higher binding (controls: 311 +32fmol/mg protein,n =10; suicides: 341 17 fmol/mg protein, n =13) and fornon-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 suicideswere divided according to duration of treatment (less than 4

weeks or more than 11 weeks), there were no significantdifferences between either suicide group and their respectivecontrols.

Effect of time and seasonofdeath

As fl-adrenoceptors in pineal glands of suicides and controlsdid not differ, we have combined all 70 subjects studied andexamined the effects of time and season of death. There were

no significant variations in the number of /3-adrenoceptorswith respect to time (Fig. 1) or season (Fig. 2) of death.

Discussion

To the best of our knowledge this is the first study to examine

p-adrenoceptor binding in pineal glands from depressedsuicides 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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Figure 1 Effect of time of death on (3-adrenoceptor numbers in pinealglands. Values are meansSEM. The number of subjects in eachtime 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 inpineal glands. Values are means + SEM. The number of subjects areas follows: spring (March-May), n=26; summer (June-August)n = 16; autumn (September-November), n =15; winter (December-February), n =133

density of {3-adrenoceptors in the pineal gland. These valuesare four to five times the density we have reported in humancerebral cortex (De Paermentier et al., 1990a). In the pinealgland, (3-adrenoceptors are almost exclusively of the fli-subtype ( > 90 %, Table 3) and this is in agreement with therat pineal gland (Dickinson et al., 1986).

In antidepressant-free suicides, we found no differences inthe number or affinity of total fl- or ~31-adrenoceptors. This iscompatible with previous findings of unchanged ~-adrenocep-tor binding in cortical samples from suicide victims (Meyersonet al., 1982; De Paermentier et al., 1990b; Stockmeier andMeltzer, 1991). We found a tendency for pineal gland fl-adrenoceptor binding to be higher in those subjects who haddied by violent means and lower in those who had died by non-

violent means. In our previous study of nine brain regions we

found no evidence of higher fl-adrenoceptor binding, irrespec-tive of violence of death (De Paermentier et al., 1990b).

In antidepressant-treated suicides, there were no significantdifferences in total fl- or p-adrenoceptor binding. We foundno evidence in support of an antidepressant-mediated down-regulation of J3-adrenoceptors. In contrast there was anunexpected trend for an increase in fl-adrenoceptor binding.

In control subjects we observed a significant negativecorrelation 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 overallinterpretation as controls and suicides were individuallymatched for age.

Various studies have measured changes in pineal gland P-adrenoceptor number in relation to time of day. In experi-mental animals where day length can be controlled, resultsappear to be conflicting depending on species and light:darkratio. In the rat highest fl-adrenoceptor binding occurs in thelate light phase or early dark phase, whereas in hamster thereverse is seen (Wilkinson et al., 1987; Gonzalez-Brito et al.,1988; Pangrel et al., 1990). Oxenkrug et al. (1990) haveexamined p-adrenoceptor binding in human pineal glands andfound a distinct peak between 18.00 and 20.00 hours. In our

study with a comparable number of subjects, there was nosignificant variation throughout a 24-h period (Fig. I). Thisinconsistency may be related to a number of factors. Despitethe large number of subjects we examined, the proportion ofsubjects within different time periods varied widely, e.g. 16.00-20.00 hours, n = 20, 20.00-24.00 hours, n = 5. The peak in fl-adrenoceptor number found by Oxenkrug et al. (1990) occursat a time which would be light or dark depending on the seasonof year.Although we found no overall seasonal effect (Fig. 2),the numbers we were able to study do not allow us to excludean interaction between season and time. It is possible thatthere is a different relationship between p-adrenoceptors andtime of day or season in suicides and controls. Inspection ofthe data did not support this, although the distribution of

subjects within each time period was such that meaningfulstatistical comparisons were precluded.

Acknowledgement

295.fullb-Adrenoceptors in human pineal glands are unaltered in depressed suicides

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