CHRONIC TREATMENT WITH CITALOPRAM DOES NOT AFFECT THE EXPRESSION OF a

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SHORT COMMUNICATION

CHRONIC TREATMENT WITH CITALOPRAM DOES NOT AFFECT THE EXPRESSION OF a

¹

-ADRENERGIC RECEPTOR ( a

¹

-AR) SUBTYPES

Grzegorz Kreiner, Adam Bielawski, Agnieszka Zelek-Molik, Marta Kowalska, Irena Nalepa

Laboratory of Intracellular Signaling, Department of Biochemistry, Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków, Poland

Chronic treatment with citalopram does not affect the expression of a-adrenergic receptor subtypes. G. KREINER, A. BIELAWSKI, A. ZELEK- MOLIK, M. KOWALSKA, I. NALEPA. Pol. J. Pharmacol., 2004, 56, 831–836.

We previously reported that chronic treatment with imipramine and electroconvulsive shock up-regulate the density and a₎-adrenergic receptor (a₎-AR) mRNA level in the rat prefrontal cortex, while the expression of the a_* subtype was unchanged. The present study examined whether repeatedly given citalopram, a selective serotonin reuptake inhibitor, induces any changes in the expression ofa₎anda_*subtypes ofa-AR. The receptors density was assessed in the rat cerebral cortex by [^!H]prazosin binding while the expression ofa₎anda_*receptors’ mRNA was measured in the rat prefrontal cortex by Northern blot analysis or competitive reverse transcription and polymerase chain reaction (RT-PCR), respectively. We did not find any changes ina₎- anda_*-AR density or mRNA expression in the investigated rat brain structures of citalopram-treated rats. Thus, it seems that up-regulation ofa₎-AR subtype is characteristic only of those antidepressant agents in which a noradrenergic component is involved in their pharmacological mechanism of action.

Key words: citalopram, [^!H]prazosin binding, mRNA expression, brain cortex, rat

Polish Academy of Sciences Pol. J. Pharmacol., 2004, 56, 831–836

ISSN 1230-6002

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Abbreviations: AR – adrenergic receptors, b – base, bp – base pair, PCR – polymerase chain reac- tion, RT – reverse transcription

INTRODUCTION

Citalopram is a specific and potent inhibitor of serotonin reuptake, and it is believed that its antidepressant effect depends on the ability to enhance serotonergic transmission because of increasing the availability of serotonin in the vicinity of the receptor site. By comparison with other antidepressants, citalopram little affects other neurotransmitter receptors, e.g. serotonergic, dopaminergic, muscarinic, histaminic and opioid [7] and also it does not interfere directly with adrenergic receptors (AR) [14]. Al- though citalopram appears to have no direct effect on noradrenaline system, it may affect this system indi- rectly which is manifested by some adaptive changes in noradrenergic receptors induced by chronic administration of the drug [9].

a₁-ARs and their signaling system are important targets of antidepressant drugs [13–15]. Last decade revealed the existence of three subtypes of a₁-AR, namely a_1A, a_1B and a_1D, which are en- coded by separate genes and have distinct pharmacological profiles [21]. They are differently distrib- uted and their mRNA expression is not always pa- ralleled by protein expression. All of them are coupled with Gq/11 protein family and employ phospholipase C system to mediate responses induced by noradrenaline. In the rat brain, two subtypes, a_1A and a_1B, are mostly represented [20].

Both of them are equally expressed in the prefrontal cortex, while a_1A-AR dominate in the hippo- campus and a_1B-AR in the thalamus [5, 10]. The functional differences between a_1A- and a_1B-AR are not clear, but it had been postulated that both subtypes differ between themselves in their role in signal transduction [8].

Recently, we have found that chronic administration of antidepressant agents, electroconvulsive shock (ECS) and imipramine, increase specifically the expression ofa_1A-AR mRNA in the prefrontal cortex of rat without affecting thea_1Bsubtype [10].

In the current study we investigated whether citalopram, an antidepressant of different pharmacological profile than imipramine, may influence the expression ofa₁-AR subtypes.

MATERIALS and METHODS

Procedures involving animals and their care were conducted in conformity with the institutional guidelines, in compliance with national and inter- national laws and policies.

Reagents

Citalopram was a gift from Lundbeck A/S (Denmark). [³H]prazosin (spec. activity 24 Ci/mmol), Hybond N⁺membrane and RapidHyb buffer were purchased from Amersham/Pharmacia Biotech, UK.

DEPC (diethylpyrocarbonate), formaldehyde, WB4101 ([2,6-dimethoxyphenoxyethyl]aminomethyl-1,4-ben- zodioxane) were from Sigma, St. Louis, MO, USA.

Agarose I, agarose HRB, agarose SFR, AMV reverse transcriptase, EDTA (ethylenediaminetetra- acetic acid), ethidium bromide, dNTP, formamide, MOPS (morpholinopropane sulfonic acid), SDS (sodium dodecyl sulfate) were purchased from Amresco, Solon, OH, USA. Trizol reagent was from Invitrogen, Carlsbad, CA, USA. Whatman GF/C membrane – from Whatman, USA. Taq polymerase was from Finnzymes OY, Finnland. DNA molecular weight marker [PhiX174 DNA/BsuRI (HaeIII)] was supplied by MBI Fermentas, Lithua- nia. NorthernMax gel loading solution was purchased from Ambion, USA. RediPrime II labeling kit was obtained from Amersham/Pharmacia, UK.

Primers for PCR reaction were designed using OLIGO Primer Analysis Software (ver. 5.0, NBI), while their synthesis was done by TIB MolBiol, Poland.

Animals treatment, tissue and RNA isolation Male Wistar rats (weighing 200–250 g) were in- jected with citalopram (10 mg/kg, once daily, ip) or saline for 14 days. After 24 h from the last injec- tion, the animals were decapitated, their brains were excised and dissected on ice cold porcelain plate. All tissues were stored at –70°C until RNA extraction or membrane preparation.

Analysis ofa₎-anda_*-AR mRNA levels Total RNA was isolated from the prefrontal cortex and purified by the method of Chomczynski [2]

utilizing Trizol reagent and following the produ- cers’ protocol. RNA quantity and purity were determined by spectrophotometry (Pharmacia Ultrospec 2000 UV/Vis).

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The mRNA fora₎-AR subtype was analyzed using Northern blot hybridization with cDNA probes prepared as described previously [10].

Briefly, samples of total RNA were denatured (65°C for 15 min) in Northern Max gel loading solution. Each sample of total RNA was loaded in du- plicate (20 mg/slot) and electrophoresed in 1%

HRB agarose/2.2M formaldehyde denaturing gel and 1× Northern running buffer at 90V for 3.5 h.

Then, RNA was transferred to the Hybond N⁺ membrane by overnight capillary action in 10×

SSC (saline-sodium citrate) buffer. The blots were cross-linked on UV. Filters were pre-hybridized (20 min at 65°C) in RapidHyb buffer and hybridized with the a_1A-AR cDNA probe (ca. 6 × 10⁶ cpm/ml) at 65°C for 2.5 h. After completed hybridization the blots were washed once for 20 min in 2 × SSC/0.1% SDS at room temperature and twice for 15 min in 0.1× SSC/0.1% SDS at 65°C.

Blots were exposed to autoradiographic screen (overnight) and analyzed quantitatively by phos- phoimager (Fuji BAS 5000, Japan) and Science Lab 4.0 software. Ethidium bromide fluorescence

of 28S ribosomal RNA was used as an internal standard to control for loading errors [3].

For analysis ofa_*-AR mRNA level, the com- petitive RT-PCR technique was applied as described previously [5]. Primer sequences (sense primer: 5’-GTA GCC CAG CTA GAA CAC CA -3’; antisense primer: 5’-GGA AAA GAA AGC AGC CAA AAC CT-3’) were selected from the se- quence of rat mRNA for thea_1B-AR (Rattus norve- gicus, Voigt MM, 1993; GenBank accession:

X51585) to generate the 151 base pair (bp) seg- ment ofa_1BcDNA. Synthesis of cDNA from total RNA (RT reaction) was performed in a 20ml reaction mixture containing 1× AMV RT buffer, 1 mM dNTP, 1mM antisense primer, 2 mg RNA and 10 units of AMV. Reverse transcriptase (RT) reaction was performed as follows: 72°C/5 min (denaturation), 4°C/3 min (cooling – AMV reverse transcriptase added), 25°C/10 min (pre-heating), 42°C/60min (reverse transcription). Then, cDNA for the a_1B-AR was co-amplified with prepared 248 bp internal standard [5] in PCR reaction in a 25ml volume containing 1× PCR buffer, 0.625 units Taq polymerase,

0 50 100 150

200 SAL

CIT

1A- ARmRNAexpression [%ofcontrol]

28S

18S kb

SAL CIT

Fig. 1. Lack of effect of chronic treatment with citalopram (CIT) on steady-state level ofa)-AR mRNA in the rat prefrontal cortex determined by Northern blot analysis. Samples of 20mg of total RNA were loaded in duplicates and mRNA levels were analyzed as described under Materials and Methods. Re- sults are expressed as a percent of controls treated with saline (SAL) and are the means ± SEM of values from five individuals.

Lower panel shows a representative Northern blot analysis of a)-AR mRNA. Arrows show location ofa)-AR (~3000 b) and of residual 28S and 18S ribosomal RNA visualized by ethidium bromide staining

0 50 100 150

200 SAL

CIT

1B-AR

mRNAexpression [%ofcontrol]

wz cDNA

SAL CIT

Fig. 2. Lack of effect of chronic treatment with citalopram (CIT) on steady-state level ofa*-AR mRNA in the rat prefrontal cortex determined by competitive RT-PCR. Competitive RT-PCR analysis was conducted as described in Materials and Methods. Results are expressed as a percent of controls treated with saline (SAL) and are the means ± SEM of values from six individuals. Lower panel shows a competitive RT-PCR analysis of a*-AR transcript. Arrows show location of a* cDNA (151 bp) reverse transcribed from total RNA of rat cerebral cortex and internal standard (WZ, 248 bp) co-amplified in PCR

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1.5 mM dNTP, 0.4mM sense and antisense primers, 1ml of RT mix (containing a_1BcDNA) and 1ml of chosen concentration of internal standard (usually ca. 0.5 pg/ml). The PCR was performed in tripli- cates with T3 Thermocycler (Biometra, Germany) with the following cycles parameters: one denaturation cycle at 94°C/5 min; 34 cycles of 1 min at 94°C (denaturation), 1 min. annealing at 64°C, 1 min of extension at 72°C and of final elongation cycle at 72°C/7 min. The relative fluorescence of cDNA PCR product vs. internal standard fragment were measured in EtBr-stained 3% agarose SFR gel with Fluorescent Image Analyser (Fuji LAS-1000, Japan).

Receptor binding assay

The membrane preparation (P₂ fraction) was prepared from cortical tissue and the receptor den- sities and affinities in saline- and citalopram- treated rats were assessed by standard procedures as described previously [10]. Experimental details are given in Table 1. The B_maxand K_Dvalues were calculated from binding isotherm using GraphPad Prism 2.01 software.

Statistical analysis

Statistical analysis of the results was performed with Statistica 5.0 software using one-way analysis of variance.

RESULTS and DISCUSSION

Chronic treatment with antidepressant drugs may induce various adaptive changes at intracellular levels of signal transduction, including modula- tion of transcriptional activity of genes [12, 19].

Our previous studies showed that chronic administration of citalopram, unlike ECS and imipramine,

increased the reactivity of a₁- and b-AR to nora- drenaline reflected by an enhanced generation of inositol phosphates and cAMP, respectively, [14].

The augmented responsiveness of a₁-AR in rat brain cortex did not result from any changes in receptors level, since we have not observed any significant change in receptor density. However, the earlier studies could not take into account the current knowledge about the existence of subtypes of a₁-AR.

Recently, our attention has been focused on two subtypes ofa₁-AR,a_1Aanda_1B, which are mostly represented in the rat brain [20] and may differ between themselves in their role in signal transduction [8]. At least in smooth muscles,a_1Asubtype is coupled selectively to dihydropyridine-sensitive voltage-gated calcium channel, while a_1B mainly operates through phospholipase C system [4].

Though in the brain the functional differences be- tweena_1Aanda_1Breceptors are not clear, our previous data showed that, interestingly, only one of them, thea_1Asubtype, underwent adaptive changes after repeated administration of ECS and imipramine [10]. Our finding that both antidepressant agents induced the increase in a density of the binding sites anda_1Areceptor mRNA expression in the rat prefrontal cortex raised the question whether the phenomenon is characteristic also of other antidepressants. For this reason we investigated how chronic treatment with citalopram affects the a_1A and a_1B receptor mRNA expression in this brain area. In addition, since our previous results showed that ECS and imipramine increased the density of corticala_1A-AR receptor [10] and in view of autoradiographic study indicating important differences in the regulation of a₁-AR in various areas of rat cerebral cortex (at least after ECS) [1], we assessed the binding sites for both receptor subtypes as well as the total density ofa₁-AR in the whole cortex.

Table 1. Lack of effect of 14-day treatment with citalopram on [³H]prazosin binding sites in the cerebral cortex of the rat

Treatment Totala1-AR a1A-AR a1B-AR

B_max [fmol/mg prot.]

K_D [nM]

SAL (5) CIT (5)

108.0 ± 3.4 118.8 ± 3.4

0.29 ± 0.03 0.28 ± 0.03

89.8 ± 8.4 98.6 ± 6.4

0.15 ± 0.01 0.17 ± 0.02

63.9 ± 5.5 60.4 ± 4.7

0.90 ± 0.09 0.84 ± 0.07 Six concentrations of [³H]prazosin ranging from 0.086 to 3.46 nM were used. Total binding (including botha1A-AR anda1B-AR) was assessed by the difference in binding in the absence and presence of 10mM WB4101. The a1Asites were assessed from the difference in [³H]prazosin binding in the absence and presence of 2 nM WB4101. The difference between total anda1A-AR binding was assumed to bea1B-AR binding. The data are expressed as the mean ± SEM from five independent experiments; CIT – citalopram, SAL – saline

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The present results show that, in contrast to imipramine and ECS, which produce up-regulation of the a_1A-AR expression [10], chronically admini- stered citalopram does not affect the a_1A-AR mRNA level in the prefrontal cortex (Fig. 1) or the density and affinity of this receptor subtype in the whole cortex (Tab. 1). On the other hand, similarly to the above-mentioned antidepressant agents, citalopram changed neither the a_1B-AR density (Tab.

1) nor mRNA level (Fig. 2). In this study, we also did not observe any influence of 14-day treatment with 10 mg daily dose of citalopram on the density of [³H]prazosin binding sites, assessed by using of 10mM WB4101 as a displacer, which represent the total a₁-AR binding (Tab. 1). The latter finding is in agreement with our previous observation, though at that time citalopram was used at a higher dose (20 mg) and phentolamine served as a “cold”

ligand [14]. It should be noted that some investigators described a significant increase in the total a₁-AR density after chronic administration of citalopram [6, 16, 18]. The exact reasons for the dis- crepancies between their results and ours are not clear, but there are some differences that might be responsible for that. Thus, higher doses of citalopram and different route of drug administration as well as the different displacer drug were used in their experiments. However, the same investigators noted the great variability in effects of antidepressants on a₁-AR density depending on the time of the day.

Serotonergic and noradrenergic signal transmission pathways are mutually connected and serotonergic component of antidepressants may mask adaptive changes that occur in the AR system after chronic administration of these drugs. For instance, venlafaxine, a dual serotonergic and noradrenergic reuptake inhibitor, caused the cAMP downregulation only under conditions of experimentally induced absence of serotonin [11]. Since the expression of the gene coding for the a_1A-AR was shown to be positively regulated by adrenergic ago- nists [17], one can presume that the a_1A-AR up- regulation may be specifically induced by those antidepressant drugs which employ a noradrenergic component to their primary pharmacological action.

Acknowledgments. Thanks are due to H. Lundbeck A/S for the generous gift of citalopram. The gift of plasmid with cDNA for thea)-AR by Dr. J. W. Lomasney is gratefully acknowledged. This work was supported by grant No. 6

P05A 072 21 from the State Committee for Scientific Re- search, Warszawa, Poland.

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Received: September 7, 2004; in revised form: November 18, 2004.