Effect of metyrapone on the fluoxetine-induced change in extracellular dopamine, serotonin and their metabolites in the rat frontal cortex

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Effect of metyrapone on the fluoxetine-induced change in extracellular dopamine, serotonin and their metabolites in the rat frontal cortex

Zofia Rogó¿, Krystyna Go³embiowska

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

Correspondence:Zofia Rogó¿, e-mail: rogoz@if-pan.krakow.pl

Abstract:

Major depression is frequently associated with the hyperactivity of the hypothalamic-pituitary-adrenocortical axis, and glucocorticoid synthesis inhibitors have been shown to exert antidepressant action. Metyrapone (an inhibitor of the enzyme 11-b-hydroxylase) has been found to be effective as an adjunctive therapy in combination with other antidepressants (ADs) in both treatment-resistant depression and animal models. To understand the mechanism of the clinical efficacy of a combination of an AD and metyrapone in treatment-resistant depression, the present study was aimed at determining the influence of fluoxetine (FLU; a selective serotonin reuptake inhibitor) and metyrapone, given separately or jointly, on the extracellular level of dopamine (DA), serotonin (5-HT) and their metabolites in rat frontal cortex of freely moving rats using microdialysis and high performance liquid chromatography (HPLC) with electrochemical detection. FLU (10 mg/kg) given alone increased the extracellular level of DA and 5-HT in the rat frontal cortex. Metyrapone (100 mg/kg) alone did not change the level of monoamines. A combination of FLU and metyrapone produced the same change in the efflux of both DA and 5-HT as did FLU alone. However, the latter combination (FLU and metyrapone) produced significantly bigger increases in the levels of extracellullar DA metabolites (3,4-dihydroxyphenylacetic acid, homovanillic acid) and a 5-HT metabolite (5-hydroxyindoleacetic acid) than did FLU alone. The above findings suggest that – among other mechanisms – increases in the levels of extracellullar DA and 5-HT metabolites may play a role in the enhancement of FLU efficacy by metyrapone, and may be of crucial importance to the pharmacotherapy of drug-resistant depression.

Key words:

fluoxetine, metyrapone, monoamines, microdialysis, rats

Introduction

Major depression is frequently associated with the hyperactivity of the hypothalamic-pituitary-adrenocortical (HPA) axis [14, 18, 25, 28–30]. A large number of data indicate that such hyperactivity may be induced by a decreased inhibitory feedback mechanism [11, 33]. In fact, the synthetic glucocorticoid dexamethasone is less potent in lowering cortisol level (basal

and that induced by CRH) in the blood of depressed patients than of healthy subjects [11, 12]. The dys- function of the HPA axis is corrected during a clini- cally effective therapy with antidepressant drugs, while the persistence of dexamethasone non-suppres- sion is often associated with the risk of relapse or the lack of improvement [11, 13]. The currently used antidepressant drugs (ADs) given as a monotherapy show therapeutic efficacy in around 60–70% of depressive

Pharmacological Reports 2010, 62, 10151022 ISSN 1734-1140

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and a substance that can enhance its effect is used in the clinic [e.g., 2, 7, 26, 49]. Among agents that are expected to potentiate the efficacy of ADs are inhibitors of glucocorticoid synthesis. In fact, they have already been shown to have antidepressant-like properties in some animal models of depression [1, 10]. Also clinical studies have demonstrated some antidepressant effects of metyrapone, aminoglutethimide and keto- conazole; however, the latter drugs are used mainly in drug-resistant depression [28, 38, 39]. To date, in the clinic, glucocorticoid synthesis inhibitors or antagonists of glucocorticoid receptors have been administered alone in relatively high doses, so their side- effects are occasionally observed [38]. A combination of a glucocorticoid synthesis inhibitor and an AD should help to reduce their doses and, in consequence, also their side-effects. Of the glucocorticoid synthesis inhibitors, metyrapone (an inhibitor of the enzyme 11-b-hydroxylase) has the weakest effect on gonadal hormone levels [39]. We found previously that combined treatment with ADs and metyrapone produced a more potent antidepressant-like effect than did either of the drugs given alone to rats in the forced swimming test [40–43]. Additionally, other studies indi- cated that joint administration of ADs and metyrapone led to clinical improvement [17, 44]. To understand the mechanism of clinical efficacy of an AD and metyrapone combination therapy in treatment- resistant depression, in the present study we assessed the effect of fluoxetine (FLU), a selective serotonin reuptake inhibitor (SSRI), and metyrapone – given separately or jointly – on the extracellular level of dopamine (DA), serotonin (5-HT) and their metabolites in the frontal cortex of freely moving rats using a microdialysis.

Materials and Methods

Animals

Microdialysis studies were conducted on male Wistar rats (270–300 g) (Charles River Laboratories, Ger- many). The rats were housed in temperature- and humidity-controlled rooms on a 12-hour light/dark

procedures and housing conditions were in strict ac- cordance with the Polish state regulations concerning experiments on animals (DZ. U. 05.33.289). All the experimental protocols were approved by the Local Bioethics Commission for Animal Experiments at the Institute of Pharmacology, Polish Academy of Sci- ences in Kraków, Poland.

Microdialysis study

The rats were anesthetized with ketamine (75 mg/kg, im) and xylazine (10 mg/kg, im) and placed in stereo- taxic apparatus (David Kopf Instruments, CA, USA).

Their skulls were exposed and small holes were drilled therein to insert microdialysis probes in the frontal cortex using the following coordinates: 2.9 mm anterior from the bregma, 0.8 mm lateral from the sagittal suture, –4.6 mm ventral from the dural sur- face. For cortical recording, microdialysis probes were constructed by inserting two fused silica tubes (30 and 35 mm long, 150 µm outside diameter (o.d.) (Polymicro Technologies Inc., Phoenix, AZ, USA) into a microdialysis fibre (220 µm o.d.; AN69, Ho- spal, Bologna, Italy). The tube assembly was placed in a peak cannula (0.3 mm o.d., 6 mm in long) form- ing a haft of the probe. Portions of inlet and outlet tubes were individually placed inside a polyethylene PE-10 tubing and glued. The free end of a dialysis fibre was sealed, and 3 mm of its exposed length were used for a dialysis in the frontal cortex. All the probes were connected to a syringe pump (BAS, IN, USA) which delivered an artificial cerebrospinal fluid composed of [mM]: NaCl 145, KCl 2.7, MgCl 1.0, CaCl 1.2; pH = 7.4 at a flow rate of 2 µl/min. Baseline samples were collected every 30 min after the washout period to obtain a stable extracellular neurotransmitter level. The appropriate drugs (FLU, 10 mg/kg and metyrapone, 100 mg/kg) dissolved in distilled water, were then administered intraperitoneally (ip) in a vol- ume of 2 ml/kg separately or jointly, and dialyzate fractions were collected every 30 min for 4 h. At the end of the experiment, the rats were sacrificed and their brains were histologically examined to validate the probe placement.

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Neurotransmitter analysis

DA, 5-HT and their metabolites (3,4-dihydroxyphenylacetic acid, DOPAC; homovanillic acid, HVA and 5-hydroxyindoleacetic acid, 5-HIAA) were analyzed by HPLC with coulochemical detection. Chro- matography was performed using the Ultimate 3000 System (Dionex, USA), coulochemical detector Cou- lochem III (model 5300, ESA, USA) with a 5020 guard cell, a 5014B microdialysis cell and a Hypersil Gold-C_& analytical column (3 × 100 mm). The mo- bile phase was composed of 0.05 M potassium phos- phate buffer adjusted to pH = 3.9, 0.5 mM EDTA, 13 mg/l 1-octanesulfonic acid sodium salt, a 3.1% methanol and a 0.9% acetonitrile. The flow rate during analysis was 0.7 ml/min. The applied potential of a guard cell was +600 mV, while that of a microdialysis cell was E1 = –50 mV, E2 = +300 mV; sensitivity was set at 50 nA/V.

Chromatography data were processed by Chromeleon v. 6.80 (Dionex, USA) software run on a PC com- puter. The values were not corrected for an in vitro probe recovery which was approximately 15%.

Drugs

Fluoxetine hydrochloride (FLU, Pliva, Kraków, Poland) and 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone, Aldrich, USA) were used for the present study.

Data analysis

An average concentration of three stable samples be- fore drug administration was regarded as a control value and considered to be 100%. Statistical significance was calculated using a repeated-measures ANOVA, followed by Tukey’spost-hoc test. The comparisons between experimental groups were analyzed by a two- way ANOVA, followed by Tukey’spost-hoc test. The results were considered statistically significant when p < 0.05.

Results

Effect of FLU and metyrapone on DA, 5-HT, DOPAC, HVA and 5-HIAA extracellular levels in rat prefrontal cortex

The basal extracellular concentrations of biogenic amines (DA, 5-HT and their metabolites) in dialyzates

from the frontal cortex did not significantly differ between groups and were as follows (in pg/10 µl ± SEM):

0.98 ± 0.18 (DA), 414 ± 32 (DOPAC), 388 ± 26 (HVA), 1.8 ± 0.9 (5-HT) and 419 ± 15 (5-HIAA).

The repeated measures analysis of variance showed a significant effect of the treatment, i.e., F(3,21) = 12.93; p < 0.00001, time F(7,147) = 4.05; p < 0.0004, and the interaction of both factors F(21,147) = 2.56;

p < 0.0006 in the extracellular level of DA (Fig. 1A).

Co-administration of fluoxetine with metyrapone and neurochemical effects

Zofia Rogó¿ and Krystyna Go³embiowska

0 50 100 150 200 250 300

-60 -30 0 30 60 90 120 150 180 210 240 min

DA(%ofbaseline)

FLU MET FLU+MET CON

A

0 20 40 60 80 100 120 140 160 180 200

-60 -30 0 30 60 90 120 150 180 210 240 min

DOPAC(%ofbaseline)

FLU MET FLU+MET CON

B

0 20 40 60 80 100 120 140 160 180 200

-60 -30 0 30 60 90 120 150 180 210 240 min

HVA(%ofbaseline)

FLU MET FLU+MET CON

C

Fig. 1.The effects of fluoxetine (FLU, 10 mg/kg,ip) and metyrapone (MET, 100 mg/kg,ip), administered separately or jointly, on the extra- cellular levels of DA (A), DOPAC (B) and HVA (C) in rat frontal cortex, shown as a time-course. FLU and metyrapone were given alone or jointly at the time-point 0. Microdialysis samples were collected every 30 min for 4 h. The results represent the mean ± SEM of 610 rats

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A significant increase in DA extracellular level was found after treatement with FLU, 10 mg/kg (p < 0.0004), and FLU, 10 mg/kg, plus metyrapone, 100 mg/kg (p <

0.004) compared to the control (Tukey’s post-hoc HSD test). A significant difference was also observed between groups treated with FLU plus metyrapone and metyrapone alone (p < 0.01), but not FLU alone (p < 0.15).

The extracellular level of DOPAC was changed by the treatment; namely F(3,21) = 9.08; p < 0.0005);

there was also a difference in time F(7,147) = 7.28;

p < 0.00001 and a significant interaction between both factors F(21,147) = 2.68; p < 0.0003 (Fig. 1B).

Tukey’s post-hoc HSD test showed a significant in- crease in DOPAC level after FLU, 10 mg/kg, plus metyrapone, 100 mg/kg, treatment compared to the control (p < 0.005). A significant difference was also observed after combined administration of FLU plus metyrapone, FLU alone (p < 0.002), and metyrapone alone (p < 0.01).

ver, there was an interaction between both factors F(21,147) = 1.92; p < 0.01 (Fig. 1C). Tukey’s post- hoc HSD test showed a significant increase in HVA level after FLU, 10 mg/kg plus metyrapone, 100 mg/kg, treatment compared to the control (p < 0.006);

on the other hand, no significant difference was in HVA level between groups after treatment with FLU or metyrapone.

The extracellular level of 5-HT was changed by the treatment; namely F(3,21) = 5.78; p < 0.005;

however, there was no significant change in time F(7,147) = 1.36; p < 0.23 and no interaction between both factors F(21,147) = 0.81; p < 0.70 (Fig. 2A).

Combined treatment with FLU, 10 mg/kg, and metyrapone, 100 mg/kg, significantly changed 5-HT extracellular level compared to the control (p < 0.003);

however, no significant difference was found compared to the group treated with FLU or metyrapone alone.

There was a significant effect of the treatment on the extracellular level of 5-HIAA; F(3,21) = 8.99; p <

0.0005, but no significant effect on time F(7,147) = 1.97; p < 0.06, and no interaction between both factors F(21,147) = 1.53; p < 0.08 (Fig. 2B). A significant decrease was shown in the extracellular level of

0 50 100 150 200

-60 -30 0 30 60 90 120 150 180 210 240 min

5HT(%ofbaseline) _CON

0 20 40 60 80 100 120 140

-60 -30 0 30 60 90 120 150 180 210 240 min

5-HIAA(%ofbaseline)

FLU MET FLU+MET CON

B

Fig. 2.The effects of fluoxetine (FLU, 10 mg/kg,ip) and metyrapone (MET, 100 mg/kg,ip), administered separately or jointly, on the extra- cellular levels of 5-HT (A) and 5-HIAA (B) in rat frontal cortex, shown as a time-course. FLU and metyrapone were given alone or jointly at the time-point 0. Microdialysis samples were collected every 30 min for 4 h. The results represent the mean ± SEM of 610 rats

**

*

** **

++

**

**+

**^^++

**^^

0 200 400 600 800 1000 1200 1400 1600 1800

DA DOPAC HVA 5HT 5HIAA

AUC(%ofbasallevel)

CON FLU MET FLU+MET

Fig. 3.The cumulative effect of fluoxetine (FLU, 10 mg/kg,ip) and metyrapone (MET, 100 mg/kg,ip), administered separately or jointly, on the extracellular levels of DA, DOPAC, HVA, 5-HT and 5-HIAA in rat frontal cortex, shown as an area under the curve (AUC, the mean

± SEM)). Microdialysis samples were collected every 30 min for 4 h.

Comparisons between experimental groups were analyzed by a two-way ANOVA, followed by Tukeyspost-hoc test. * p < 0.05,

** p < 0.01 compared with the control group (CON);⁺p < 0.05,⁺⁺p <

0.01 compared with FLU; ^^ p < 0.01 compared with metyrapone.

The results represent the mean ± SEM of 610 rats

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5-HIAA compared to the control (p < 0.002) or FLU, 10 mg/kg, plus metyrapone, 100 mg/kg (p < 0.006) after administration of FLU, 10 mg/kg, alone.

The cumulative effect of FLU, 10 mg/kg, ip, and metyrapone, 100 mg/kg, ip, administered separately or jointly, on the extracellular levels of DA, DOPAC, HVA, 5-HT and 5-HIAA in rat frontal cortex, is shown as an area under the curve (AUC) in Figure 3. FLU, 10 mg/kg, induced a significant elevation in DA and 5-HT extracellular levels, while metyrapone, 100 mg/kg, was effective only in rising 5-HT concentration. FLU, 10 mg/kg, increased the level of HVA and decreased that of 5-HIAA, while metyrapone, 100 mg/kg, increased both HVA and 5-HIAA levels. Combined administration of the drugs markedly increased DOPAC level compared to FLU or metyrapone alone, and significantly elevated HVA level in comparison with the group treated with FLU alone.

Discussion

In the present study we investigated the effect of FLU, belonging to the class of SSRI, and metyrapone, given separately or jointly, on the extracellular levels of DA, 5-HT and their metabolites in rat frontal cortex. FLU significantly increased DA release without changing DOPAC and HVA extracellular levels in rat frontal cortex. We also found that FLU increased extracellular 5-HT level in rat frontal cortex. However, the effect of FLU was not statistically significant when the whole 5-HT curve was compared to the control group, that indicates that the effect of FLU on 5-HT release in rat frontal cortex is rather short-lasting and limited to the first hour of drug action. On the other hand, FLU significantly and systematically decreased 5-HIAA extracellular level in response to 5-HT reuptake inhibition, which caused disturbances in the in- traneuronal metabolism of 5-HT.

A number of earlier studies demonstrated an elevation in extracellular DA and 5-HT concentrations in such brain regions as the frontal cortex, striatum and hypothalamus following FLU administration [8, 34, 50]. Moreover, it was shown that FLU treatment induced an increase in the extracellular level of noradrenaline (NA) in the frontal cortex [23, 35, 55]. Like citalopram (SSRI), FLU elevated 5-HT concentration [3, 15, 22, 54], while citalopram – unlike FLU [19,

51] – did not increase the levels of extracellular DA and NA in rat prefrontal cortex. The most selective SSRI, citalopram [46], produced a bigger increase in 5-HT efflux than did FLU [3, 9]; moreover, its local infusion into the frontal cortex markedly increased extracellular 5-HT at 0.1–10 µM, with little or no effect on DA efflux. Thus, in spite of the fact that some data indicate an increase in DA efflux in rat frontal cortex by elevating endogenous 5-HT efflux [16, 27], the substantial elevation of 5-HT level by SSRIs in the frontal cortex shows no concomitant changes in extracellular DA [37]. This observation suggests that the increased DA and NA efflux induced by FLU alone is unlikely to be secondary to its effect on 5-HT efflux. The obtained evidence indicates that inhibition of NA uptake in the frontal cortex enhances the extracellular concentrations of both DA and NA [4, 36], which may set in motion a possible mechanism of FLU effect enhancing DA efflux [37].

Our present results show that metyrapone slightly lowered extracellular DA concentration in rat frontal cortex, had no effect on DOPAC, and elevated HVA level. The level of 5-HT was significantly elevated, while that of 5-HIAA was only slightly increased (above the basal value) by metyrapone.

A combination of FLU and metyrapone did not produce any further increase in DA release, but – in contrast – markedly increased the extracellular levels of DOPAC and HVA compared to groups treated with FLU or metyrapone alone. The mechanism of that effect is difficult to explain on the basis of the present results. However, it is possible that metyrapone may change the metabolism of monoamines by influencing the enzymatic activity of MAO, the latter being en- gaged in utilization of both these neurotransmitters, i.e., DA and 5-HT. Moreover, the pharmacokinetics of a combination of both these drugs in still unknown, but seems to be an important factor causing prolonga- tion of the drug action in the brain. It has been shown that acute administration of metyrapone is associated with cytochrome P-450 enzyme inhibition [47, 53];

moreover, the inhibitory effect of metyrapone or FLU on cytochrome P-450 (on izoenzyme CYP2D6) was already demonstrated in some earlier studies [e.g., 6, 21, 32]. In addition, metyrapone is an inhibitor of the cytochrome P450 isoenzyme (CYP11B1) which cata- lyzes glucocorticoid synthesis, it also inhibits a number of other isoenzymes participating in the metabolism of ADs [52], that may be of significance consider- ing application of a relatively high dose of metyra-

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(imipramine, fluvoxamine and nefazodone) was not changed during their joint administration with metyrapone, that indicates the lack of a pharmacokinetic interaction [17, 44].

The present biochemical data are in line with our behavioral observations in which have been shown that metyrapone potentiates the antidepressant-like activity of FLU, tianeptine, imipramine, desipramine and reboxetine in the forced swimming test in rats, and that among other mechanisms, 5-HT_1Areceptors may play a role in this effect [41–43]. Furthermore, some earlier behavioral studies demonstrated a decrease in the reactivity of the 5-HT_1Asystem after repeated treatment with metyrapone, desipramine and sertraline (an attenuated hypothermic response to an acute challenge with 8-OH-DPAT) [10, 20], moreover, they showed that 5-HT_1Areceptor function was mediated by glucocorticoids. The latter concept is consistent with the observation that adrenalectomy can alter the characteristics of receptor binding, and can increase 5-HT_1Areceptor mRNA expression [5], both these effects being reversed by corticosterone supplementation. The above findings point to some similarities between metyrapone, desipramine and sertraline despite their different biochemical properties; moreover, they suggest that the serotonin system may be involved in the mechanism underlying their antidepressant action.

Interestingly, metyrapone has been shown to exert beneficial effects in depressed patients [28, 31, 45, 49]. Furthermore, some preliminary studies indicate that joint treatment with imipramine and metyrapone of drug-resistant depressed patients shows significant therapeutic activity [44]. Similar results were reported by Jahn et al. [17] who showed in a double-blind, ran- domized, placebo-controlled trial that metyrapone was an effective adjunct in the treatment of major depression by accelerating the onset of AD (fluvoxamine and nefazodone) action. The latter authors also observed a sustained antidepressive effect. The above- cited studies have demonstrated that the plasma levels of all ADs do not change during their joint administration with metyrapone, that indicates a lack of pharmacokinetic interaction [17, 44].

In conclusion, the results described in the present paper indicate that a combination of FLU and metyrapone produced the same change in the efflux of both

DA metabolites (DOPAC or HVA) and a 5-HT metabolite (5-HIAA) than did FLU alone. The above finding suggest that – among other mechanisms – increases in the levels of extracellullar DA and 5-HT metabolites may play a role in the enhancement of FLU efficacy by metyrapone, and may be of crucial importance to the pharmacotherapy of drug-resistant depression.

Aknowledgments:

The authors wish to thankPliva, Kraków (Poland) for their generous gift of fluoxetine. We would also like to thank Ms.

Katarzyna Kamiñska for her skilful technical assistance, and would like to thank Ms. E. Smolak, M.A., for her linguistic supervision of the paper.

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Received:

May 28, 2010; in revised form: August 28, 2010.