Effect of paroxetine, a selective 5-hydroxytryptamine uptake inhibitor, on β-adrenoceptors in rat brain: Autoradiographic and functional studies

NeuropharmacologyVol. 30, No. 6, pp.607-616,1991 Printed in Great Britain

0028-3908/91$3.00+ 0.04 Pergamon Press plc

EFFECT OF PAROXETINE, A SELECTIVE 5HYDROXYTRYPTAMINE UPTAKE INHIBITOR, ON j?-ADRENOCEPTORS IN RAT BRAIN: AUTORADIOGRAPHIC AND FUNCTIONAL STUDIES D. R. NELSON,’ G. D. PRATT,* K. J. PALMER,’ A. M. JOHNSON’and N. G. BOWERY’ ‘SmithKline Beecham Pharmaceuticals, Research and Development, The Pinnacles, Coldharbour Road, Harlow, Essex CM19 SAD and zThe School of Pharmacy, Department of Pharmacology, University of London, Brunswick Square, London WClN IAX, U.K. (Accepted 20 December 1990) Summary-Quantitative receptor autoradiography was used to investigate the effects of paroxetine (8.3 mg/kg), amitriptyline (26 mg/kg) and desipramine (17 mg/kg), administered daily in the drinking water for 21 days, on the number of /I,- and /?,-adrenoceptors in the cortex of the rat. In addition, the effect of these drugs on the function of /I-adrenoceptors was examined by measuring noradrenaline- and isoprenaline-stimulated production of cyclic AMP in slices of cortex. Paroxetine did not alter the number of cortical /I, or /I,-adrenoceptors nor did it induce any functional changes in j?-adrenoceptor-linked adenylyl cyclase. In contrast, desipramine caused a significant reduction in the density of /I,-adrenoceptors and in the sensitivity of both noradrenaline and isoprenaline-stimulated adenylyl cyclase. Although amitriptyline significantly reduced the number of &adrenoce.ptors in cortical membranes, no such changes could be detected by autoradiography. It is apparent from these and other studies, that the ability of antidepressants to down-regulate central j?-adrenoceptors is not a property shared by all antidepressants. In particular, the more potent and selective inhibitors of the uptake of S-HT, such as paroxetine, appear to be devoid of effects on this receptor system. Key words-paroxetine, cyclase.

antidepressants,

5-HT uptake, beta-adrenoceptors,

Chronic administration of the majority of antidepressants or electroconvulsive shock causes a reduction of B-adrenoceptor-formation of stimulated cyclic AMP (CAMP) and/or number of /?-adrenoceptors in the brain of the rat. Although these effects are thought to be linked to the therapeutic action of antidepressants (Vetulani, Stawarz, Dingell and Sulser, 1976; Banergee, Kung, Riggi and Chanda, 1977; Wolfe, Harden, Spot-n and Molinoff, 1978), a number of antidepressants fail to reduce the number of /I-adrenoceptors in membrane preparations from whole cortex, e.g. maprotiline (Barbaccia, Ravizzia and Costa, 1986), citalopram (Hyttel, Overo and Arnt, 1984) and fluoxetine (Wang, Reid, Bymaster and Threlkeld, 1985). However, studies using the technique of receptor autoradiography have demonstrated that the specific inhibitors of the uptake of 5-hydroxytryptamine (5HT) fluoxetine (Byerley, McConnell, McCabe, Dawson, Grasser and Wamsley, 1988) and sertraline (Byerley, McConnell, McCabe, Dawson, Grosser and 1987) decreased the binding of Wamsley, [3H]dihydroalprenolo1 (DHA) to /I-adrenoceptors in specific laminae in the frontal cortex of the rat. Paroxetine, an effective antidepressant (Lund, Laursen, Mikkelsen, Rasmussen and le Fevre Honore, 1985; Fabre, 1988; Feighner and Boyer,

autoradiography,

adenylyl

1989), is a potent and selective inhibitor of the uptake of 5-HT in vitro and in vivo (Thomas, Nelson and Johnson, 1987). Previous studies have shown that repeated intraperitoneal (Nelson, Thomas and Johnson, 1989) and oral (Nelson, Palmer and Johnson, 1990) administration of paroxetine did not alter the total number or affinity of fl, or /3*adrenoceptors in membrane preparations of whole cortex in the rat. The aim of this investigation was to determine whether or not repeatedly-administered paroxetine could produce numerical or functional changes in /3-adrenoceptors in cortex of the rat. Receptor autoradiography was employed to investigate the effect of paroxetine on fi,- or fl,-adrenoceptors, within individual lamina of the frontal cortex. The sensitivity of noradrenaline- and isoprenaline- stimulated adenylyl cyclase (CAMP) in cortical tissue was investigated, to provide a functional measure of P-adrenoceptors. The effect of the selective p-adrenoceptor agonist, isoprenaline, on production of CAMP was also measured, because the stimulation of production of CAMP by noradrenaline is not entirely mediated by fl-adrenoceptors but also, in part, by a-adrenoceptors (Mobley and Sulser, 1979). Doses of paroxetine were used, which have previously been shown not to reduce the number of fi,-adrenoceptors 607

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D. R. NELSONet

but which persistently inhibit uptake of 5-HT and reduce numbers of 5-HT, receptors in cortical preparations of the rat (Nelson et al., 1990). The tricyclic antidepressants, desipramine and amitriptyline, were also included in this study. In this and previous studies (Nelson et al., 1990), the effect of chronic administration of desipramine, amitriptyline and paroxetine was investigated on the binding of p,- and &adrenoceptors to cortical membrane preparations of the rat, using the specific ligand [3H] CGP 12177, (4-3(-tert. butyl-amino-2-hydroxypropoxy)-benzimidazole-2-on hydrochloride) (De Paermentier, Cheetham, Crompton and Horton, 1989). However, for the autoradiographical studies, [“‘I] iodopindolol, which possesses a greater specific activity, was used to aid a more rapid visualization of /I-adrenoceptors (Ordway, Gambarana and Frazer, 1988). METHODS

Treatment with drug

Male CFY rats (Interfauna, weight 140-180 g at the beginning of the study), housed 6 to a cage, were acclimatized for 1 week with food and water ad Zibitum. After acclimatization, the test drugs were added to the drinking water, with the aim of achieving a daily intake of 10 mg/kg for paroxetine, 20 mg/kg for desipramine and 30 mg/kg for amitriptyline (doses. calculated as base). Fluid intakes and body weights were monitored daily and doses were adjusted accordingly. The actual mean daily doses f SEM, received by the rats, were 8.3 f 0.1 mg/kg for paroxetine, 17 f 0.3 mg/kg for desipramine and 26 f 0.5 mg/kg for amitriptyline. On day 21, the solutions of drug were replaced by water and 24 hr later the animals killed, cerebral cortices removed rapidly and stored on ice. Autoradiography of fi-adrenoceptors

For the quantitative autoradiographic studies of receptors, after 24 hr drug-free period, the rats were anaesthetised with sodium pentobarbitone (40 mg/kg i.p.) and perfused-fixed with 250 ml 0.1% paraformaldehyde in 0.1 M phosphate-buffered saline (pH 7.4) by intracardiac administration. Brains were then frozen in iso-pentane, cooled to -40°C in liquid nitrogen and stored at -80°C until use. Parasagittal and coronal sections (10 /*m) were cut at -20°C and thaw-mounted onto glass slides. Sections were thawed and incubated for 60 min at room temperature, in a Tris/saline buffer (20 mM Tris HCl, 135 mM NaCl; pH 7.4), containing ( - )-[1251]iodopindolol, with 5 concentrations between 37.5-600 pM. Non-specific binding was defined using ( - )-isoprenaline (200 p M), whilst the selective labelling of /I,-adrenoceptors was achieved in the presence of the /I,-adrenoceptor antagonist, ICI 118551 (erythro-Dr_-l-(7-methylindan-4-yloxy)-3-isopropyl-aminobutan-2-01) (50 nM), as described by

al.

Ordway et al., 1988; Beer, Richardson, Poat, Iversen and Stahl, 1988 and De Paermentier et al., 1989. Binding to /I,-adrenoceptors was taken as the difference between total and /I,-adrenoceptor binding. After incubation, the sections were washed for 2 x 5 min periods in buffer at 4°C followed by a 3 second rinse in ice-cold distilled water, to remove buffer salts and then quickly dried in a stream of cold air. Sections and calibrated brain paste standards were then apposed to LKB tritium-sensitive ‘Ultrofilm’ to generate autoradiograms. Optical densities of ( - )-[‘251] iodopindolol binding to the individual laminae of the frontal cortex were measured in nCi/mg tissue, using a Quantimet 970 image analysis system (Cambridge Instruments) and converted to fmol/mg tissue. The kinetic parameters Kd and B,,, of the binding were derived by Scatchard analysis and statistical comparisons between control and drug-treated animals made using Student’s ‘t ‘-test (2-tailed). For the purpose of this study, autoradiographic density measurements were restricted to the frontal cortex, which was taken to be represented by the anterior third of the cerebral cortex. Assay of cyclic AMP

Individual whole cortices were cross-chopped (250 x 250 pm), using a McIlwain tissue chopper and dispersed gently in 10ml of oxygenated Krebs’Ringer bicarbonate (KRB) buffer, containing NaCl, 118 mM; KCl, 4.7 mM; CaCl,, 2.5 mM; MgSO,, 1.2 mM; KH2P0,, 1.2 mM; NaHCO,, 25 mM; glucose, 11 mM; ascorbic acid, 0.28 mM and theophylline (a phosphodiesterase inhibitor), 2.0 mM. The mini slices were washed for 30 and 60 min in 2 x 100 ml KRB at 37°C gassed with 02/C02 (95: 5%). After 90 min the KRB was aspirated off and 50 ~1 aliquots (containing _ 1 mg protein) of the resultant tissue suspension added to flat-bottomed plastic tubes, containing fresh KRB. The tubes were preincubated for 5 min at 37°C and immediately gassed to mix the contents. The reaction was initiated with either noradrenaline (final concentration 100 PM) or isoprenaline (10 PM) in final volume of 500~1. After lOmin, the reaction was terminated in a boiling water-bath for 3 min. Fifty microlitre aliquots of the supernatants were assayed for CAMP, using the radioimmunoassay method based essentially on Brown, Albano, Ekins, Sgherzi and Tampion, (1972). Concentrations of protein were determined using the Bradford assay with bovine serum albumin as the standard (Bradford, 1976). Membrane binding assay

The binding of /I-adrenoceptors in membrane preparations from the cortex of the rat was quantified (Ko and B,,,,, values) by the method of De Paermentier et al. (1989) using [3H] CGP 12177 as the specific ligand (see Nelson et al., 1990). Briefly, non-specific binding for /II-plus fi,-adrenoceptors was

Fig. 1. Autoradiograms of j-adrenoceptor-binding in brain of rat. (a) and (b) Total (8, and #?J binding of ( - )-[‘251]iodopindolol (150 PM). (c) and (d) Non-specific binding defined by ( - )-isoprenaline (2OOpM). (e) and (f) /I,-Adrenoceptor binding (defined in the presence of ICI 118551, 50 nM). (g) and (h) fi,-Adrenoceptor binding (defined in the presence of CGP 20712A, 100 nM). Note the absence of binding in the combined presence of ICI 118551 and CGP 20712A (i) and (i). The parasagittal and coronal sections are approximately represented by plates 81 and 30, respectively (Paxinos and Watson, 1986).

609

Fig. 2. Autoradiograms of total (8, and &) adrenoceptor binding (a, c, e, g) and resolved &-adrenoceptor binding (b, d, f, h) to parasagittal sections of brain from control (a, b) and desipramine- (c, d), amitriptyline- (e, f) or paroxetine- (g, h) treated rats.

610

Paroxetine: effect on j?-adrenoceptors

611

both the paroxetineand amitriptyline-treated animals were not significantly different to the control rats. In contrast, the weight of rats receiving desipramine was significantly smaller than the control rats from day 5, but the rate of gain in weight was similar after week 1. The binding of [i2rI] iodopindolol to total, 8, or p,-adrenoceptors, as well as non-specific sites, is shown in parasagittal (a, c, e, g, i) and coronal (b, d, f, h, j) sections of brain from control animals (Fig. 1). &Adrenoceptors were visualised in the presence of ICI 118551 (50 nM), a specific a-adrenoceptor antagonist, whilst b,-adrenoceptors were visualised in the presence of CGP 20712A (100 nM), a fl,-adrenoceptor antagonist. This figure demonstrates that the binding of /I-adrenoceptors was clearly laminated into 3 bands, which have been delineated as lamina I, II-III and V-VI (Zilles and Wree, 1985). /I,Adrenoceptors (Figs le and f) predominated in the cerebral cortex, hippocampus (CA1 region), caudate putamen (CPU), ventral posteromedial thalamic nucleus (VPM) and the gelatinosus thalamic nucleus (g). /I,-Adrenoceptors (Figs lg and h) predominated in the molecular layer of the cerebellum (mol), the nucleus of the optic tract (OT), the optic tract (OPT), lamina IV of the parietal cortex (IV), olfactory tubercle (Tu), central medial thalamic nucleus (CM) and the globus pallidus (GP). The effects of paroxetine, desipramine and amitriptyline on total and &adrenoceptor binding were visualised in the autoradiograms shown in Figure 2. Treatment with desipramine caused a marked reduction of both total and &adrenoceptor binding, in all laminae of the frontal cortex, whereas

defined with isoprenaline (200pM) and for /I,adrenoceptors with CGP 20712 (2-hydroxy-5-(2-((2hydroxy-3-)4-((l-methyl I-4-trifluromethyl)lH-imidazol-2yl)-phenoxy)propyl)-amino)ethoxy-benzamide monoethane-sulphonate; 50 nM. Specific &binding was obtained by subtraction [8r = (/I, + fir) - /.I,].The appropriate control and desipramine-treated membranes were analysed together (present investigation) whilst the appropriate control and either paroxetine or amitryptiline-treated membranes analysed in a second or third series of experiments (Nelson et al., 1990). Materials

Desipramine HCI and amitriptyline HCl were obtained from Courtin and Warner and paroxetine HCI was supplied by SmithKline Beecham Pharmaceuticals. [3H] Cyclic AMP (specific activity 33 Ci/mmol), [12’1] iodopindolol (specific activity 2000 Ci/mmol) and [‘HI CGP 12177 (specific activity 49-53 Ci/mmol) were obtained from Amersham, U.K. The CGP 20712A and ICI 118551 were kindly supplied by CibaGeigy and ICI. All other chemicals were obtained from Sigma Chemical Co. The CAMP binding protein was prepared from bovine adrenal glands. RESULTS

The mean volume of water consumed/day/rat in these studies was reduced significantly by 19, 31 and 55% in paroxetine-, amitriptyline- and desipraminetreated animals, respectively. Although the intake of water was reduced, the increases in body weight of

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Fig. 3. Scatchard plots showing (A) total (/?, and &) adrenoceptor binding, (B) resolved fi,-adrenoceptor binding in lamina I of the frontal cortex of control (0) and desipramine- (O), amitriptyline- (A) or paroxetine- (A) treated rats. Each Scatchard point was derived by triplicate determinations from 5 rats, for which standard errors were < 10% of the mean.

D.R. NELSON et al.

612

of /I,-adrenoceptors (20% P < O.OS), without altering /I,-adrenoceptors, while chronic treatment with paroxetine failed to alter either sub-type. Figure 5(a) shows the effect of repeatedly administered paroxetine, desipramine and amitriptyline on noradrenaline-stimulated production of CAMP in slices of whole cortex of the rat. Neither paroxetine nor amitriptyline altered noradrenaline (NA)-stimulated production of CAMP, whereas desipramine reduced production of CAMP by 72% (P < 0.001). Figure 5(b) also shows the effect of paroxetine, desipramine and amitriptyline on isoprenaline-stimulated p-adrenergic production of CAMP in slices of cortex. Neither paroxetine nor amitriptyline altered isoprenaline-stimulated production of CAMP, whereas desipramine caused a 57% reduction (P < 0.05) in the sensitivity of adenylyl cyclase.

no obvious reductions were observed treatment with amitriptyline or paroxetine. A typical Scatchard plot for the saturation analysis of the binding of [‘*‘I] iodopindolol in lamina I of slices of frontal cortex from control rats, for total specific binding (defined by 200 PM isoprenaline) revealed a single high affinity site (K, 114 PM) with a B,,, corresponding to 2.5 fmol/mg tissue. In the presence of the &-antagonist (ICI 118551), binding to /I,-adrenoceptors accounted for 73% of the total b-adrenoceptors. Scatchard analysis of the binding of [‘*‘I]iodopindolol to both total and /?,-adrenoceptors in laminae I, II-III and V-VI of the frontal cortex was performed for each group of drugs, in order to and K,values. Figure 3 shows the determine the B,,, Scatchard plots for the binding of [“‘I] iodopindolol to lamina I of the frontal cortex of the rat for both total fl and /3,-adrenoceptors. Desipramine caused a 28% (P < 0.01) down-regulation in the number of b,-adrenoceptors, whilst paroxetine and amitriptyline were without significant effects. and Kdvalues in lamina I, II-III and The mean B,,,,, V-VI for total and pi-adrenoceptors, for each treatment with drug, are shown in Table 1. Repeated treatment with desipramine reduced the B,,,,, values for total P-adrenoceptors in all laminae. Although the binding of P,-adrenoceptors appeared to be down-regulated in all laminae, the changes only reached statistical significance in laminae V-VI. No changes in the B,,,values were observed in any laminae after treatment with paroxetine or amitriptyline and none of the treatments with drugs altered the Kd values for either of the sites studied. The effects of repeated orally-administered desipramine on the binding of [‘HI CGP 12177 to /I, and &-adrenoceptors in whole cortical membranes of the rat is shown in Figure 4. Desipramine reduced the number (B,,,)of /I,-adrenoceptors by 41% (P < O.OOl), without altering the number of p2adrenoceptors or the dissociation constants (KJ.For comparison, the effects of amitriptyline and paroxetine on the numbers of PI- and fi,-adrenoceptors are also included in Figure 4 (results previously reported by Nelson et al., 1990). Repeated treatment with amitriptyline significantly reduced the number Table

I. The effects of desipramine,

DISCUSSION

This study has investigated the effect of chronically-administered paroxetine, amitriptyline or desipramine on the number of /I-adrenoceptors (visualised by receptor autoradiography and radioligand binding) and function (through changes in activity of adenylyl cyclase. as mediated by the production of the second messenger, CAMP). Concurrent biochemical studies have validated the method of administration of antidepressants to rats through the drinking water (Nelson et al., 1990). For example, it was demonstrated that paroxetine, administered in the drinking water for 21 days, inhibited the uptake of [3H]5-HT into cortical synaptosomes (ED,, = 3.0 mg/kg), when examined 2 hr after withdrawal of drinking water to which drugs had been added. Furthermore, paroxetine, at a dose of 8.6 mg/kg for 21 days, caused a down-regulation of cortical 5-HT2 receptors (21% P < 0.01) 24 hr after withdrawal of drug. In addition, repeatedly administered amitriptyline (9.2 and 27 mg/kg) caused a significant reduction (23 and 34%, respectively) in the maximum number of cortical 5-HT, receptors under the same conditions. Results from autoradiographic analysis have demonstrated that desipramine significantly and

amitriptyline and paroxetine on the binding of ( -)-[‘251] iodopindolol B,-adrenoceators in frontal cortex of the rat Frontal

I

II-III

Treatment Control Amitriptyline Desipramine Paroxetine

Control Amitriptyline Desipramine Paroxetine

2.61 2.90 1.97 2.96

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1345 17 126 f IO 119*9 130112

The kinetic parameters K, and B,,,,, were derived **P < 0.01; *P < 0.05 (Student’s ‘I’-test).

cortex-lamina

V-VI B,,, (fmol/mg 2.69 2.56 1.91 2.56

i f f f

I tissue) f SEM

0.11 0.09 0.048’ 0.10

K, (PM) 128 * 17 I21 *II 117* 14 109 f 8 by Scatchard

to p,- and

analysis

1.83 + 0.26 2.17+0.12 1.38 f 0.09 2.16iO.14

146? 172k 178f 161 f

15 I5 II I4

of 5 rats/treatment

II-III 2.16kO.26 2.50 * 0.14 1.82 * 0.10 2.46 k 0.11

134 163 163 148

k I6 f 18 k 13 &8

(triplicate

V-VI i.68+0.18 1.79 + 0.09 1.29 + 0.09* 1.70 f 0.10

151 * 15 I81 k 19 l82&9 149+ 11 determinations).

Paroxetine: effect on @-adrenoceptors

613

BETA-1 ADRENOCEP’IQR BINDING

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Fig. 4. The effect of repeated administration of paroxetine, amit~pty~ine or desipramine on binding of [-‘HICOP 12177 to fl-adrenoceptors in cortical membranes in the rat. Rats were given paroxetine (8.9 mg/kg/day), amitriptyline (27 mg/kg/day) or desipramine (19 mg/kglday), in the drinking water, for 21 days and 24 hr after the last dose the dissociation constant (I&) and the maximum number of binding sites (B,,,) determined, using [3H] CGP 12177. The data presented represents the mean of 4 (amitriptyline) or 5 (paroxetine and desipramine) separate experiments, using individ~l brains for each dete~ination. Binding to /?,- plus &adrenoceptors was defined with isoprenaline and the binding to j?,-adrenoceptors was defined with CGP 20712. *P ~0.05, **P ~0.001 (Student’s ‘t’ test, 2 tailed), vs control. Data for experiments with amitriptyline and paroxetine have been presented previously (Nelson et al., 1990). The mean kinetic data & SEM from the control animals (n = 14) was: KD = 60 F 5 pM (8, + A) and 38 + 3 pM Go,); B,,,,, = 3.9 f 0.22 fmol/mg tissue (/I, + &), 2.67 f 0.20 fmol/ mg tissue (p,) and 1.28 k 0.55 fmol/mg tissue @). selectively reduced the numbers of /3,-adrenoceptors in the deeper Iaminae of the frontal cortex, i.e. V to VI, although there may have been an apparent reduction in the outer laminae of the cortex. These data are in agreement with Ordway et al. (1988), who also measured the effect of desipramine on J,- and &adrenoceptors, autoradio~aphi~ally and demonstrated

8 I

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01 CONTROL

‘AROX.

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DNI

Fig. 5. The effect of chronic tre atment with al idepressant on isoprenaline- and noradrenaline-stimulated production of CAMP in slices of cortex of rat. Paroxetine (8.3 mg/kg), amitriptyline (26 mg/kg) and desipramine (17 mg/kg) were administered to the drinking water of rats for 21 days. Twenty-four hours after the last administration of drug the rats were killed and slices of cortex prepared and incubated, as described in the Methods. The response shows the difference of the level in CAMP between the preparation exposed to noradrenaline or isoprenaline and that of nonexposed preparations (basal level) expressed as a percentage of- the control value. The basal level, in pm01 CAMP/ 10 min/mg protein k SEM was 0.325 k 0.048 (5), whilst noradrenaline (100 PM) produced a 315% increase and isoprenaiine (10,~M) a 196% increase in production of CAMP. Samples from each animal were determined in triplicate, *P < 0.05 and ***P < 0.001. Student’s ‘t’-test (2 tailed).

a selective reduction in ~,-adrenoceptors in laminae I-III, IV and V-VI of the somatosensory cortex. This selective reduction may implicate synaptic receptors, as it has been suggested by Grassby and Broadley (1986) that /3,-adrenoceptors are located within the noradrenergic synapse and are under the direct influence of endogenously released noradrenaline. The present investigation has also demonstrated that desipramine selectively don-regulated &-

614

D. R. NELSON et al.

adrenoceptors, in membrane preparations from the cortex of the rat, without altering /$-adrenoceptors. These results agree with Minneman, Dibner, Wolfe and Molinoff (1979) who demonstrated that chronically-administered desipramine selectively reduced /I,-adrenoceptors in cortical membranes of the rat. Desipramine also reduced the sensitivity of the noradrenaline-receptor coupled adenylyl cyclase, as measured by a significant reduction of noradrenalinestimulated production of CAMP in slices of cortex of the rat. This is in agreement with earlier studies of Newman, Lipot and Lerer (1987) and Sulser and Mobley (1981). Since the stimulation of production of CAMP by noradrenaline may also involve activation of cc-adrenoceptors, the p-adrenoceptor agonist, isoprenaline, was also studied. A similar result was obtained with isoprenaline as with noradrenaline, i.e. desipramine reduced production of CAMP. Autoradiographic analysis demonstrated that amitriptyline did not alter significantly the number of b-adrenoceptors in any laminae of the frontal cortex. No other studies appear to have examined the effect of chronic administration of amitriptyline on central quantitative b-adrenoceptors, using receptor autoradiography. It has been shown previously (Nelson et al., 1990) that amitriptyline, when administered at the same dose and under the same conditions as in the present study, selectively reduced, by 20%, the maximum number of P,-adrenoceptors in membrane preparations of whole cortex in the rat. These data compare with the findings of Heal, Butler, Hurst and Buckett (1989) who demonstrated a 28% decrease in /I,-adrenoceptors in cortical membranes, with amitriptyline (10 mg/kg p.o. for 10 days). The reasons, therefore, for the discrepancy between the autoradiographic and the membrane binding studies are unclear. However, not all investigators have found amitriptyline to be effective, for example Tang, Seeman and Kwan (1981) demonstrated that amitriptyline (10 mg/kg i.p. for 21 days) did not alter the total number of cortical /3-adrenoceptors in the brain of the rat. The functional studies also failed to show any reduction in isoprenaline or noradrenalinestimulated production of CAMP in whole slices of cerebral cortex from rats receiving amitriptyline, compared to vehicle. This latter observation contrasts with the findings of Kopanski, Turck and Schultz (1983) who demonstrated that oral treatment with amitriptyline (30 mg/kg for 9 days) reduced the response to noradrenaline. However, Wise and Halliday (1985) confirmed the findings of SellingerBarnette, Mendels and Frazer (1980) by showing that repeatedly administered desipramine was consistently more effective than amitriptyline in causing a reduction in numbers of P-adrenoceptors in cortical membranes in the rat. These authors concluded that the weaker effects of amitriptyline were most likely to be due to the failure to achieve sufficiently high levels in brain of amitriptyline or nortriptyline to inhibit

uptake of noradrenaline (as shown by Baumann, Galliard, Jonzier-Perey, Gerber and Bouras, 1984). The present data also demonstrated that amitriptyline was much less potent than desipramine in down-regulating the number and function of central /I-adrenoceptors. Repeated administration of paroxetine, at a dose known to cause persistent inhibition of the uptake of 5-HT (Nelson et al., 1990) failed to alter the number of 8, or /I,-adrenoceptors in any of the laminae of the frontal cortex, as determined by receptor autoradiography. This confirms earlier findings, using membrane preparations, that chronic treatment with paroxetine failed to have any effect (Nelson et al., 1990). In line with the autoradiographic studies, paroxetine failed to alter either noradrenaline- or isoprenaline-stimulated production of CAMP in slices of cortex. Reports of the effects of the selective inhibitors of the uptake of 5-HT on the noradrenaline-stimulated adenylyl cyclase system are somewhat inconsistent. For example, fluvoxamine has been shown either to down-regulate (Brunello, Riva, Volterra and Racagni, 1986; Claasen, 1983) or have no effect (Kopanski et al., 1983; Benfield and Ward, 1986) on the number and function of /I-adrenoceptors. Repeated administration of fluoxetine (Schmidt and Thornberry, 1977; Mishra, Janowsky and Sulser, 1979) or citalopram (Garcha, Smokum, Stephenson and Waeramanthri, 1985) did not decrease the sensitivity of fl-adrenoceptors, whilst zimeldine (Mishra, Janowsky and Sulser, 1980) and sertraline (Koe, Weissman, Welch and Browne, 1983) were both able to reduce the response of B-adrenoceptorstimulated adenylyl cyclase but only one of four investigators reported the same for fluoxetine (see Table 2 for references). However, recent autoradiographic findings indicate that the specific inhibitors of the uptake of 5-HT, fluoxetine (Byerley et al., 1988) and sertraline (Byerley et al., 1987), both decreased the binding of the /3_adrenoceptor ligand, [‘HI dihydroalprenolol, in specific laminae of slices of frontal cortex of the rat. Therefore, the present results demonstrate that repeated treatment with paroxetine to rats did not affect numbers of cortical jI-adrenoceptors and function. However, Asakura, Tsukamoto, Kubota, Imafuku, Ino, Nishizaki, Sato, Shinbo and Hasegawa (1987) demonstrated that the number of cortical /I-adrenoceptors could be reduced by treatment with either mianserin or maprotiline 6 hr, but not 24 hr, after the last dose. This indicated that the time chosen for preparing the membranes after the last dose could be important in highlighting down-regulation of padrenoceptors. It is interesting that the most potent and selective inhibitors of the uptake of 5-HT, compared to noradrenaline and dopamine, e.g. paroxetine and citalopram, did not apparently downregulate cortical /?-adrenoceptors, whereas desipramine, a potent and selective noradrenaline uptake

Paroxetine: effect on /I-adrenoceptors

615

Table 2. Comparative effects of antidepressants on inhibition of uptake of 5-HT in brain of rat and number and function of ,!l-adrenoceptor Selectivity of uptake inhibition in vitro 5-HT/NA (ratio K, values) 320 1500 180 20 190 51 0.91 0.0086

Paroxetine Citalopram Fluvoxamine Fluoxetine Sertraline Zimeldine Amitryptyline Desipramine

Chronic effects on fl-adrenergic system Membrane bindina (B,,X change) 0’ 0% 061’ @,10.“.‘2 16 1 18 19 o~&L~ 12.4.‘2.2013

Reantor autoradiography 02 nt nt i:: $ 1.H 1

Activity of adenvlvl cycG 0s 04.5 051s r-J5.‘0.‘4J’5 16 olJL ;:l;:,

0 and 1 indicate absence of or significant reductions, respectively, following chronic treatment with antidepressant. nt = not tested. I. Nelson et al. (1990); 2. Present study; 3. Hyttel et al. (1984); 4. Garcha et al. (1985); 5. Kopanski et al. (1983); 6. Benfield and Ward (1986); 7. Brunello et al. (1986); 8. Claassen (1983); 9. Wong et al. (1985); 10. Mishra et al. (1979); 1I. Maggi, U’Pritchard and Enna (1980); 12. Peroutka and Snyder (1980); 13. Byerley et al. (1988); 14. Schmidt and Thornberry (1977); 15. Baron, Ogden, Siegel, Stegeman, Ursillo and Dudley (1988); 16. Koe et al. (1983); 17. Byerley et al. (1987); 18. Mishra et al. (1980); 19. Ross, Hall, Renyi and Westerlund (1981); 20. Sulser (1982); 21. Schoffelmeer, Horneman, Sminia and Mulder (1984); 22. Tang et al. (1981); 23. Sellineer-Bamette ef al. (1980): 24. Ordwav et al. (1988). For uptake data see Thomas et al. (1987) plus unpublished findings. ”

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