BIOLOGICAL PSYCHIATRY - ORIGINAL ARTICLE

Differential modulation of alpha-1 adrenoceptor subtypesby antidepressants in the rat brain

D. Ramakrishna • M. N. Subhash

Received: 10 June 2010 / Accepted: 31 October 2010 / Published online: 7 December 2010

� Springer-Verlag 2010

Abstract The aim of the present study was to examine the

effect of chronic antidepressants treatment on the density of

a1-adrenoceptor (AR) subtypes in rat brain. Density of total

a1 and a1A- and a1B-ARs was measured in cortex and cere-

bellum of rats treated with amitriptyline (AMI), desipramine

(DMI) and fluoxetine (FLX), (10 mg/kg body wt), for

30 days, using [3H]prazosin in presence and absence of WB-

4101. The density of cortical total a1-ARs was significantly

decreased with AMI (54%) and DMI (25%) treatment,

without altering the affinity of the receptor. Fluoxetine did

not alter the density of cortical a1-ARs. The density of cor-

tical a1A-ARs was also significantly decreased with AMI

(85%) and DMI (50%) treatment, without affecting the

affinity. The density of cerebellar total a1-ARs was signifi-

cantly decreased with AMI (37%), DMI (50%) and FLX

(70%) treatment, without affecting the affinity for [3H]pra-

zosin. The density of a1A-ARs was significantly decreased

with AMI (67%), DMI (59%) and FLX (92%) treatment. a1B-

AR density was decreased only with FLX (47%) and DMI

(47%) treatment. Correspondingly the basal IP3 and NE

(10 lM) stimulated IP3 levels were significantly decreased

in AMI (47%), DMI (22%) and FLX (48%) treated rat cortex.

The results suggest that chronic antidepressant (AD) treat-

ment down-regulates the cortical and cerebellar total a1-ARs

in rat brain. However, a1A subtype is predominantly down-

regulated by AMI and DMI, where as FLX affects cerebellar

a1A-ARs. The region-specific and subtype specific down-

regulation of a1-ARs density, which occurs after prolonged

AD treatment, may underline the therapeutic mechanism of

action.

Keywords Alpha1-adrenoceptors � Antidepressants �Rat brain � [3H]Prazosin � a1A-ARs � a1B-ARs � IP3

Introduction

The noradrenergic system is involved in the regulation of

many physiological and psychological processes, including

the modulation of mood. The a-adrenergic receptors (a-ARs)

modulate norepinephrine release, as well as the release of

serotonin and other neurotransmitters, and are therefore

potential targets for antidepressant and anxiolytic drug

development. Norepinephrine (NE) at physiological con-

centration primarily binds to a-adrenoceptors, whereas epi-

nephrine binds to and activates both a- and b-adrenoceptors.

Adrenoceptors are members of the family of seven

transmembrane domain (7TM) guanine nucleotide (G) pro-

tein-coupled receptors (GPCRs) and are a significant phar-

macological target in clinical medicine. The adrenoceptors

were first classified into a and b subtypes (Ahlquist 1948) and

later into a1- and a2-adrenoceptors (Langer 1974) and b1-

and b2-adrenoceptors (Lands et al. 1967). a-ARs have been

classified into several specific subtypes (a1A, a1B, a1D, a2A,

a2B, a2C) on the basis of pharmacological distinctions. a1-

ARs are GPCRs that bind catecholamines. Sixteen distinct

human a1A-AR isoforms have been identified from human

tissues. a1-ARs couple predominantly to the pertussis-toxin-

insensitive G-protein, Gq, resulting in the hydrolysis of

membrane phospholipids; subsequent activation of phos-

pholipase C-b (PLC-b) generates the major second

D. Ramakrishna (&)

Kamineni Institute of Medical Sciences, Sreepuram,

Narketpally, Nalgonda 508254, Andhra Pradesh, India

e-mail: drramakrishna@klsindia.com

M. N. Subhash

National Institute of Mental Health and Neurosciences,

Hosur Road, P.B.No.2900, Bangalore 560029, India

e-mail: drramakrishna@klsindia.com

123

J Neural Transm (2010) 117:1423–1430

DOI 10.1007/s00702-010-0522-4

messengers inositol(1,4,5)-trisphosphate [IP3] and diacyl

glycerol (DAG) (Michelotti et al. 2000). IP3 binding to its

receptor on intracellular storage sites results in the mobili-

sation of intracellular Ca??, which ultimately leads to

smooth muscle contraction (Graham et al. 1996). The exact

magnitude of activation of PLC differs between a1-AR

subtypes (Chen et al. 1996) as does the ability of a1-AR

subtypes to stimulate IP3 formation (Schwinn et al. 1995;

Theroux et al. 1996; Zhong et al. 2001). All full-length

(7TM) a1A-AR isoforms described to date display similar

pharmacology in terms of ligand binding and noradrenaline

induced [Ca2?]i responses (Coge et al. 1999; Hirasawa et al.

1995). Because a1A- and a1B-ARs have been shown to

dimerize (Stanasila et al. 2003), this mechanism might prove

clinically relevant in some diseases in spite of lack of evi-

dence to date for such an effect.

As some of these receptors interact with one another

(Pilc and Enna 1985) and few drugs are absolutely specific

for a given class, it is often difficult to determine the degree

to which any one of these receptors contributes to a par-

ticular noradrenergic response.

Central noradrenergic system is implicated in the hypoth-

esis on the aetiology of depression and the known therapeutic

action of antidepressants (ADs) is ascribed to their inhibitory

action at pre-synaptic transporters of 5-hydroxytryptamine (5-

HT) and NE (Hauger et al. 2009). Substantiating this

hypothesis, alterations in noradrenergic indices have been

found in the brain of suicide victims (Callado et al. 1998;

Garcia-Sevilla et al. 1999), major depression (Fu et al. 2001)

and schizophrenia (Klimek et al. 1999).

Data from basic and clinical studies suggest that the

therapeutic mechanism of action of ADs may involve

alterations in the sensitivity of serotonin and adrenergic

receptor activity. Down-regulation of central adrenoceptor

system as the principle action of AD treatment has been

suggested. Acute treatment with tricyclic antidepressants

(TCAs) results in the activation of inhibitory a2C-adrener-

gic autoreceptors. a2A-adrenergic heteroreceptors and/or

5-HT1A autoreceptors regulate the synthesis of NE or 5-

HT, whereas chronic treatment with TCAs is known to

desensitise these pre-synaptic receptors (Esteban et al.

1999). Significant reduction in the density and activity of

a2-ARs in rat brain (Invernizzi et al. 2001) and also in

platelets of depressed patients (Garcia-Sevilla et al. 1990)

have been reported with chronic treatment of ADs, thus

suggesting that a2-AR antagonists are good antidepressant

agents. The structural stability of a2-ARs appears to control

its drug-induced up- and down-regulation and this under-

standing of regulation for the a1B-AR subtype provides a

model for studies of the differential regulation of the other

a1-AR subtypes and may lead to identification of new

molecular targets for therapeutic intervention in a variety

of disease states (Toews et al. 2003).

The TCAs such as desipramine (DMI), clomipramine

(CMI) and amitriptyline (AMI) are chemically closely

related, but have distinct pharmacokinetic and pharmaco-

dynamic properties. Fluoxetine and AMI are the prototypical

drugs of the class as mixed NE and 5-HT uptake inhibitors.

Apart from depression, ADs have also been effective in

treatment of panic disorders, pain syndrome and anxiety

states. DMI and AMI have been demonstrated to be effective

in treating long-term depression (Wroblewski et al. 1996).

Adaptive phenomena such as desensitisation of autorecep-

tors are considered an important factor in the achievement of

therapeutic efficacy of AD drugs after chronic treatment

(Sacchetti et al. 2001). Long-term AD treatment has been

found to reduce b-adrenergic sensitivity, while enhancing

responses to serotonergic and a-adrenergic stimulation. This

suggests that modulation of receptor sensitivity may be a

mechanism of action common to ADs, atypical ADs,

monoamine oxidase inhibitors and electro convulsive ther-

apy (Charney et al. 1981). Given the established roles of a1-

ARs in mood disorders and other neuropsychiatric disorders,

elucidation of the biological significance of the signalling

diversity and potential pharmacological roles of a1A-ARs are

important areas of future research.

Classical ADs are thought to act by raising monoamine

(5-HT and NE) levels in the brain. A time lag of 3–6 weeks

has been demonstrated before robust clinical efficacy is

observed. This delay may reflect inhibitory actions of NE

at pre-synaptic a2A-adrenergic autoreceptors or heterore-

ceptors which gradually down-regulate upon prolonged

exposure (Cordi et al. 2001). The study of long-term AD

treatment provides an insight into the mechanism of their

actions and will also help in understanding the diverse

action of ADs in different mood disorders. Therefore, the

objective of the present work was to study the effect of

chronic (30 days) administration of AMI, DMI, and FLX

on a1-AR subtypes in regions of rat brain.

Materials and methods

[3H]prazosin (s.a. 27 Ci/mmol) and [3H]myoinositol (Ci/

mmol) were obtained from Amersham Int. (UK) and New

England Nuclear (USA), respectively. Phentolamine, WB

4101, HEPES, FLX, DMI, CMI, and AMI were obtained

from Sigma Chemicals (USA); 96-well microplates with

glass fibre (GF/B) filters were obtained from M/s. Milli-

pore, India. Other chemicals, of analytical grade, were

obtained from local chemical suppliers.

Animals and administration of drugs

Adult male Sprague–Dawley rats weighing 200–250 g,

were used for all the experiments. Animals (10 rats for

1424 D. Ramakrishna, M. N. Subhash

123

each drug- a total of 30 rats), procured from Central

Animal Research Facility (CARF), NIMHANS, were

housed in cages (four rats per cage) and exposed to

regular day/night period with food and water ad libitum.

DMI (10 mg/kg body wt), AMI (10 mg/kg body wt) and

Fluoxetine (10 mg/kg body wt), were injected intraperi-

toneally, once daily, for a period of 30 days. As the

clinical efficacy of ADs has been observed over

3–6 weeks of administration, their effects on a1-AR

subtypes were studied after chronic administration for

30 days. Control rats received 0.5 ml of saline, by same

route for the same period. All the animals were killed by

decapitation under ether anaesthesia, 24 h after the last

injection. Brains were removed on ice-cold Petri dish,

cerebral cortex and cerebellum dissected out and used

for membrane preparation. Tissues obtained from three

rats were pooled for receptor binding assay.

Membrane preparation

Crude membrane pellet was obtained from brain tissue,

homogenised in 20 volumes of Tris–HCl buffer (50 mM,

pH 7.4) containing 0.32 M sucrose, following the proce-

dure described by Creese and Snyder (Creese and Snyder

1978) and as described earlier (Subhash et al. 1998). The

pellet was resuspended in 50 mM sodium–potassium

phosphate buffer (pH 7.4) for [3H]prazosin binding studies.

Protein concentration was estimated by Lowry’s method

(Lowry et al. 1951) and made to 1 mg/ml using respective

buffers.

a1-adrenoceptor binding

Membranes obtained from cortex and cerebellum of both

experimental and control rat brain were used for binding

studies. The density of a1-ARs was estimated by using

[3H]prazosin following essentially the procedure described

by Hyttel et al. (1992). In brief: an aliquot of membrane

(100 lg protein) was incubated with 6–8 different con-

centrations of [3H]prazosin (0.05–0.60 nM) in 50 mM

sodium–potassium phosphate buffer (pH 7.4) in a 96 well

microplate with GF/B filters, for 30 min at 37�C. Non-

specific binding was defined by using phentolamine

(10 lM). For a1B-ARs the binding experiments were done

in presence and absence of WB-4101 (2.0 nM) to block a1A

receptors. The reaction was stopped by the addition of ice-

cold Na–K phosphate buffer (pH 7.4) and then the reaction

mixture was rapidly filtered under vacuum. The filters were

punched by punching manifold, directly into scintillation

vials containing 5 ml of scintillation fluid and allowed to

equilibrate overnight. Radioactivity was measured using

b-counter (Packard, USA) with 67% efficiency.

IP3 estimation

The measurement of [3H]IP3 levels was done according to

the method of Chuang (1989) and as reported earlier

(Devaki et al. 2006; Subhash and Jagadeesh 1997). Briefly;

cross-chopped slices (300 lM) from rat brain samples

(0.5 g) were prepared and incubated in oxygenated KRB

buffer (pH 7.4) with 0.5 lCi of [3H]myoinositol (s.a.

17.1 Ci/mmol) for 1 h at 37�C. Tissues were washed with

the same buffer and incubated with LiCl2 (10 mM). Assay

was terminated by the addition of 10% TCA. Samples were

kept on ice for 20 min and homogenised. Then the tissues

were sedimented by centrifugation (3,000 g). Supernatant

was washed 4–5 times with water-saturated diethylether.

Diethylether washed TCA extract, containing the inositol

phosphates, was then neutralised with NaHCO3 (5 mM).

[3H]IP3 formed was separated by anion-exchange chro-

matography (Dowex-formate form). Inositol phosphates

were separated by gradient elution with ammonium for-

mate and formic acid (Subhash and Jagadeesh 1997). The

bound [3H]IP3 was eluted from the column using 0.8 M

formate and 0.1 M formic acid. The eluent was mixed with

10 ml of scintillation cocktail and radioactivity was mea-

sured in liquid scintillation counter after overnight equili-

bration. The amount of [3H]IP3 formed was expressed as

DPM/g tissue.

Data analysis

The data from the binding experiments was analysed using

‘LIGAND’ software programme (McPherson 1983) to

obtain the equilibrium dissociation constant (Kd), the den-

sity of receptors (Bmax) and the Hill coefficients. The Bmax

and Kd values were expressed in fmol/mg protein and nM,

respectively. All the data are expressed as mean ± SD. The

statistical analysis was done by the Student’s t test. Differ-

ences were considered to be significant at p \ 0.05.

Results

In vivo effect of ADs on a1-adrenoceptors

The density (Bmax) of a1-ARs was estimated in cerebral

cortex and cerebellum of both control and experimental

rats. The density of a1-ARs was almost same both in cortex

(73.2 ± 3.3 fmol/mg protein) and in cerebellum

(67.2 ± 1.6 fmol/mg protein) of control rats. The affinity

of [3H]prazosin to a1-ARs was also similar in both the

regions (Table 1). After AD treatment it was observed that

in cortex the density of total a1-ARs was significantly

decreased in AMI (54%; 33.7 ± 2.0 fmol/mg protein;

p \ 0.0001) and DMI (25%; 55.2 ± 1.0 fmol/mg protein;

Differential modulation of a1-AR subtypes by antidepressants in the rat brain 1425

123

p \ 0.0001) treated rats. However, FLX treatment did not

show any significant decrease in the density of a1-ARs

(6%; 68.5 ± 4.9 fmol/mg protein). The affinity of

[3H]prazosin to a1-ARs was unaltered after AD treatment

(Fig. 1).

When differentiated it is observed that the cortical a1A-

ARs were predominantly affected by AMI treatment,

showing nearly 85% decrease (3.8 ± 1.8 fmoles/mg pro-

tein, p \ 0.0001) and DMI treatment, where nearly 57%

decrease was seen (11.0 ± 1.2 fmoles/mg protein,

p \ 0.0001). However, the decrease in a1B-ARs was not so

significant with AMI (35%; 30.0 ± 1.1 fmoles/mg protein,

p \ 0.0001) and DMI (10%; 42.4 ± 2.9 fmoles/mg pro-

tein, p \ 0.01) treatment. With FLX treatment no change

was seen in both subtypes.

In cerebellum, however, the density of total a1-ARs was

significantly decreased in FLX (70%; 33.4 ± 0.6 fmoles/

mg protein, p \ 0.0001), DMI (50%; 19.4 ± 1.2 fmoles/

mg protein, p \ 0.0001) and AMI (37%; 42.6 ± 4.3 fmo-

les/mg protein, p \ 0.0001) treatment, without any signif-

icant change in Kd values (Table 2; Fig 2). The Hill co-

efficient values for [3H]prazosin binding sites in both the

regions were near to unity.

The effect of ADs on subtypes in cerebellum was similar

to cortex. The decrease in total a1-ARs was predominantly

contributed by a1A-ARs. The density of a1A-ARs was sig-

nificantly decreased with FLX (92%; 2.6 ± 0.6 fmoles/mg

protein, p \ 0.0001), AMI (67%; 11.9 ± 3.2 fmoles/mg

protein, p \ 0.0001) and DMI (59%; 14.6 ± 1.3 fmoles/mg

Table 1 Effect of ADs on a1-adrenoceptors in cortex of rat brain

Total a1-adrenoceptors Bmax (fmoles/mg protein) Kd (nM)

Control 73.2 ± 3.3 0.10 ± 0.02

AMI 33.7 ± 2.0* 0.07 ± 0.01

DMI 55.2 ± 1.0* 0.11 ± 0.01

Fluoxetine 68.5 ± 4.9 0.13 ± 0.02

a1A-adrenoceptors

Control 25.4 ± 2.5 0.10 ± 0.01

AMI 3.8 ± 1.8* 0.07 ± 0.01

DMI 11.0 ± 1.2* 0.13 ± 0.02

Fluoxetine 21.7 ± 3.7 0.12 ± 0.02

a1B-adrenoceptors

Control 47.7 ± 3.5 0.10 ± 0.01

AMI 30.0 ± 1.1* 0.07 ± 0.01

DMI 42.4 ± 2.9 0.12 ± 0.02

Fluoxetine 47.2 ± 3.0 0.12 ± 0.02

The density of total a1-ARs and a1A-and a1B-ARs was estimated using

[3H]prazosin, as described, in cortex of rats exposed to various anti-

depressants for 30 days. Values are mean and SD of three experi-

ments, each assayed in duplicate

*p \ 0.0001

Consolidated graph for binding of [ 3H]prazosin to alpha 1 adrenoceptors-cortex

Bound [3H]prazosin (1 x 10-11M)

0 1 2 3 4 5 6

Bo

un

d/F

ree

0.0

0.5

1.0

1.5

2.0

[3H]prazosin (nM)

0.15 0.30 0.45

Bo

un

d

1

2

3

4

5ControlDMIAMIFLX

Fig. 1 Scatchard analysis of [3H]prazosin binding to a1-adrenocep-

tors in cortex of rats treated with DMI (10 mg/kg body wt.), AMI

(10 mg/kg body wt.) and FLX (10 mg/kg body wt.), I. P., for 30 days.

Binding experiments were done using [3H]prazosin (0.05–0.60 nM),

in cortical membranes, as described. Data points are mean of three

experiments, each assayed in duplicate

Table 2 Effect of ADs on a1-adrenoceptors in cerebellum of rat

brain

Bmax (fmoles/mg protein) Kd (nM)

Total a1-adrenoceptors

Control 67.2 ± 1.6 0.11 ± 0.01

AMI 42.6 ± 4.3* 0.10 ± 0.02

DMI 19.4 ± 1.2 0.07 ± 0.01*

Fluoxetine 33.4 ± 0.6* 0.11 ± 0.01

a1A-adrenoceptors

Control 35.8 ± 3.5 0.11 ± 0.01

AMI 11.9 ± 3.2* 0.10 ± 0.02

DMI 14.6 ± 1.3* 0.07 ± 0.01*

Fluoxetine 2.6 ± 0.6* 0.13 ± 0.01

a1B-adrenoceptors

Control 31.9 ± 2.5 0.11 ± 0.01

AMI 31.9 ± 1.9 0.09 ± 0.01

DMI 18.0 ± 1.0* 0.08 ± 0.01**

Fluoxetine 16.8 ± 0.7* 0.15 ± 0.01*

The density of total a1-ARs and a1A-and a1B-ARs was estimated using

[3H]prazosin, as described, in cerebellum of rats exposed to various

antidepressants for 30 days. Values are mean and SD of three

experiments, each assayed in duplicate

*p \ 0.0001, **p \ 0.001

1426 D. Ramakrishna, M. N. Subhash

123

protein, p \ 0.0001). The density of a1B-ARs was decreased

(45%) only with FLX (16.8 ± 0.7 fmoles/mg protein,

p \ 0.0001) and DMI (18.0 ± 1.0 fmoles/mg protein,

p \ 0.0001) treatment, when compared to control values

(Table 2; Fig. 2).

Effect of ADs on IP3 formation

Both basal and NE-stimulated IP3 formation was studied in

cortical slices of control and experimental animals. In

control animals the basal IP3 (1,518 DPM/g tissue) was

stimulated by nearly 175% (4,152 DPM/g tissue) by

10 lM NE. The basal IP3 levels were significantly

decreased in cortical slices of AMI (47%; 810 DPM/g

tissue; p \ 0.001), DMI (22%; 1,180 DPM/g tissue;

p \ 0.001) and FLX (48%; p \ 0.0001) treatment. Com-

pared to this and corresponding to the down-regulation of

a1-ARs the NE-stimulated IP3 formation, which corre-

sponds to the activity of positively linked PLC to a1-ARs,

was also significantly decreased in AMI (65%;

1,460 DPM/g tissue; p \ 0.0001), DMI (50%; 2,100 DPM/

g tissue; p \ 0.0001) and FLX (47%; 2,185 DPM/g tissue;

p \ 0.0001) treated rat cortex. The percentage of stimula-

tion of basal IP3 by NE was nearly 80% in AMI and 77%

in DMI-treated rat cortex, when compared to 175% in

control rats. Only in FLX-treated rats the percentage of

stimulation was same as that of control rat brain (Table 3).

Discussion

Although our understanding of clinical aspects of depres-

sion has advanced, the precise underlying neurobiological

basis of this disorder remains to be elucidated. Distur-

bances in pre- and post-synaptic proteins in depressed

suicides have been reported. A number of studies have

found differences in serotonergic and adrenergic receptors

in the prefrontal cortex of depressed suicide victims

(Arango et al. 2002). It is likely that the pathobiology of

depression cannot be attributed to dysfunction in a single

neurotransmitter pathway. Therefore, the search for other

neurochemical abnormalities associated with depression

and pharmacological agents is continuing.

Noradrenergic and serotonergic neurotransmission dys-

function has received much attention in the aetiology of

depression (Dinan 1996; Mann 1999; Ressler and Nemer-

off 1999). However, the precise biological abnormality of

noradrenergic neurons in depression has not been eluci-

dated. A reduced number of noradrenergic neurons

(Arango et al. 1996) associated with reduced NE trans-

porter density (Klimek et al. 1997) and upregulation of

a2A-adrenoceptors (Ordway et al. 1994) have been found in

the (LC) of depressed subjects.

Various drugs acting on serotonergic and noradrenergic

transmissions have been shown to have antidepressant

activity. ADs are known to exert their effect by elevating

5-HT and/or NE content in the synaptic cleft. Most TCAs

studied so far were found to inhibit the firing rate of LC-NE

neurons when administered acutely (VanderMaelen and

Braselton 1990). Central post-synaptic a1- and a2-ARs are

also implicated in the action of ADs (Cordi et al. 2001).

However, not much is known about the alterations in

subtypes of these receptors.

Consolidated graph for binding of [3H]prazosin to alpha 1 adrenoceptors-cerebellum

Bound [3H]prazosin (1 x 10-11 M)0 1 2 3 4 5 6

Bou

nd/F

ree

0.0

0.2

0.4

0.6

[3H]prazosin (nM)

0.00 0.15 0.30 0.45

Bo

un

d

0

1

2

3

4ControlFLX

AMIDMI

Fig. 2 Scatchard analysis of [3H]prazosin binding to a1-adrenocep-

tors in cerebellum of rats treated with FLX (10 mg/kg body wt.), AMI

(10 mg/kg body wt.) and DMI (10 mg/kg body wt.), I. P., for 30 days.

Binding experiments were done using [3H]prazosin (0.05–0.60 nM),

in cerebral membranes, as described. Data points are mean of three

experiments, each assayed in duplicate

Table 3 Effect of ADs on basal and NE-stimulated [3H]IP3 forma-

tion in cortical slices of rat brain

Cortex Basal (dpm/g tissue) NE treated Stimulation (%)

Control 1,518 ± 212 4,152 ± 286 174

AMI 810 ± 76 1,460 ± 102* 80

DMI 1,188 ± 112 2,100 ± 145* 77

Fluoxetine 794 ± 68 2,185 ± 185* 175

Both basal and NE (10 lM) stimulated IP3 formation from

[3H]myoinositol was measured in cortical slices of both control and

AD treated rat brain, as described. Values are mean and SD of three

experiments, each assayed in duplicate

*p \ 0.0001

Differential modulation of a1-AR subtypes by antidepressants in the rat brain 1427

123

In this study, effects of chronic administration of DMI,

AMI and FLX on the density of cortical and cerebellar total

a1-ARs and its subtype were studied. Several clinical studies

have demonstrated the necessity of long-term AD adminis-

tration for the prophylactic effect on recurrent depressive

symptoms. Although the blockade of monoamine uptake

process takes place within a few hours of administration of

ADs, they do not exert therapeutic effect until 3–6 weeks

after the start of treatment. This delay could be due to acti-

vation of inhibitory autoreceptors by the increase in the

synaptic monoamine levels, thereby leading to reduced

neuronal firing and decreased terminal release. Therefore, in

this study ADs were administered chronically for 30 days to

understand their mechanism of action through a1-ARs. The

results demonstrate that chronic administration of these ADs

has differential effect on a1-ARs in cortex and cerebellum. A

significant down-regulation of only cortical a1-ARs was

observed with AMI and DMI. Interestingly, FLX treatment

did not produced any significant decrease in the density of a1-

ARs in cortex but there is marked decrease in FLX (SSRI)

induced basal and stimulated IP3 formation, this could be the

result of interaction between serotonergic and noradrenergic

neuronal systems at signal transduction level. Long-term

administration of serotonin reuptake inhibitors has been

reported to alter the signal transduction cascade (Brunello

et al. 1987; Mongeau et al. 1994). Such altered signal cas-

cade, presumably resulting in a decrease in basal IP3 for-

mation, which cannot be ruled out. In contrast to our findings,

increased binding of [3H]prazosin to a1-ARs in cerebral

cortex and other brain structures after repeated administra-

tion of ADs has been reported (Nowak and Przegalinski

1988; Vetulani et al. 1983). However, the lack of increase in

the density of a1-ARs after repeated administration of ADs

was also demonstrated (Hyttel et al. 1992; Stockmeier et al.

1987). In contrast, it was shown that the binding of [3H]WB-

4101 to a1-ARs of mouse hippocampal membrane was

increased by long-term AMI treatment (Rehavi et al. 1980).

The apparent discrepancies between the studies might be due

to the use of different doses of ADs and duration of treatment

and also the type and concentration of radioligand used.

These findings suggest that the modulation of a1-ARs fol-

lowing AD treatment is not a general phenomenon and the

effect may be dose and duration dependent.

The density of cerebellar a1-ARs was altered signifi-

cantly after the chronic treatment with AMI, DMI and

FLX. The decrease, in contrast to cortex, was highly sig-

nificant with FLX treatment. Furthermore, NE-stimulated

inositol phospholipid breakdown via a1-ARs was also

found to be significantly decreased after long-term DMI,

AMI and FLX treatments.

In a recent study, both IMI and ECT treatments have

been shown elevate the a1A-AR mRNA and the expression

of receptor protein in cortex. The expression of a1B-AR

mRNA remained unaffected. In contrast, in the hippo-

campus, the AD treatment augmented the density of a1A-

AR protein without changing the levels of its mRNA

expression. These results suggest that the a1A-adrenoceptor

subtype is specifically involved in the mechanism of action

of classical antidepressant treatments (Nalepa et al. 2002).

Significant down-regulation of a2-ARs only in LC has been

reported with chronic treatment with DMI (Mateo et al.

2001).

In this study chronic treatment with ADs such as AMI,

DMI and FLX resulted in a significant down-regulation of

cortical and cerebellar a1-ARs. However, the effect was

more predominant with a1A-AR subtype. Adaptive phe-

nomena such as desensitisation of autoreceptors are con-

sidered as an important factor in the achievement of

therapeutic efficacy of AD drugs with chronic treatment.

Long-term but not acute administration of ADs has been

shown to significantly decrease the sensitivity of a-ARs

(Vila et al. 1990). Earlier reports documented that the acute

in vivo inhibition of NE neuronal uptake by ADs leads to

the activation (through endogenous NE) of pre-synaptic

inhibitory a -ARs, and prolonged in vivo inhibition of NE

reuptake is followed by a slow desensitisation process of

the same receptors (Garcia-Sevilla and Zubieta 1986). It

has been reported that G-protein function may contribute to

the complex neuroadaptive mechanisms involved in the

clinical actions of ADs.

The results of this study suggest that chronic treatment

with ADs, such as AMI, DMI and FLX, significantly down-

regulates cortical and cerebellar a1-ARs. Several hypoth-

eses propose the involvement of one or few neurotrans-

mitter receptors in the mechanism of AD action. However,

recent studies have shown several distinct receptor mech-

anisms triggering different but converging intracellular

signal cascades that activate transcription factors, which, in

turn, promote the expression of genes encoding for proteins

that play a crucial role in restoring the neuronal functions

that are involved in mood regulation (Vetulani et al. 1983).

Studies have also shown a region-specific alteration of

G-protein induced activation of the phosphoinositide signal

transduction system and G-protein a-subunits that are

involved in cAMP formation, in the prefrontal cortex of

suicide victims with major depression (Pacheco et al.

1996). Therefore, the differential and region-specific effect

of ADs on a1-ARs and its subtypes may be understood well

by studying the Ga-proteins and linked second messenger

system.

Hypersensitivity of 5-HT receptor mediated responses

has been reported in certain neuropsychiatric disorders

(Subhash et al. 2000; Subhash and Jagadeesh 1997).

However, an attempt made to see the affect FLX (SSRI) on

a1-ARs density and its mediated PLC-IP3 formation. The

observed alteration in a1A- and a1B-receptor densities are

1428 D. Ramakrishna, M. N. Subhash

123

likely to be a part of adaptive neuronal changes that occur

after chronic administration of AMI, DMI and FLX and

may be related to antidepressant effect of drug. This study

suggests that these ADs especially AMI and DMI at a1-

ARs and subtypes sites, acts by modulating a1-ARs med-

iated PLC-IP3 signal pathway.

In conclusion, the region-specific and subtype specific

down-regulation of a1-ARs density and mediated IP3 for-

mation, which occurs after prolonged AD treatment, may

underline the therapeutic mechanism of action.

Acknowledgments This study was supported by the Indian Council

of Medical Research, New Delhi (Project No. 9800140). We thank

Kamineni Institute of Medical Sciences for constant encouragement

and support to publish this work and Dr. Pragna Rao for reviewing the

draft article.

Conflict of interest The authors declare that they have no com-

peting interests.

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