Involvement of GABAB receptors in the regulation of the hypothalamo-pituitary-adrenocortical (HPA) axis in rats

J. Steroid Biochem. Molec. BioL Vol. 46, No. 6, pp. 767-771, 1993

0960-0760/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd

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INVOLVEMENT OF GABAB RECEPTORS IN THE REGULATION OF THE HYPOTHALAMO-PITUITARY-ADRENOCORTICAL (HPA) AXIS IN RATS A. HAUSLER,* G. MONNETand O. PETER Research Department, Pharmaceuticals Division, CIBA-GEIGY Ltd, 4002 Basel, Switzerland

(Received I June 1993; accepted 20 August 1993)

Summary--Previous experiments have shown that the GABA B receptor agonist L-baclofen given subcutaneously to male rats significantly enhanced plasma concentrations of adrenocorticotropic hormone (ACTH) and the adrenocortical hormones corticosterone and aidosterone. The goal of the present study was to investigate whether the stimulatory effects on adrenocortical steroids elicited by L-baclofen in vivo could be reversed by the selective GABAB antagonist CGP 35 348. One hour before subcutaneous administration of 3 mg/kg L-baclofen, a dose of 600 mg/kg CGP 35 348 or saline was administered intraperitoneally. The stimulatory effect of L-baclofen on ACTH, corticosterone and aldosterone was significantly reduced by 60% after pretreatment with CGP 35 348. The GABAs antagonist CGP 35 348 by itself had no effect on ACTH or the adrenocortical hormones. These results indicate that GABA B receptors are involved in the L-baciofen-induced activation of the HPA axis in rats. In vitro, however, neither L-baclofen nor CGP 35 348 had any effects on corticosterone and aldosterone release from perifused adrenal cells. These results suggest that the participation of GABAB receptors in the activation of the HPA axis induced by L-baclofen in vivo does not occur at the level of the adrenal gland, and therefore must occur at the level of the pituitary or the brain.

INTRODUCTION

The neurotransmitter y-aminobutyric acid (GABA) is widely distributed throughout the central nervous system in mammals (for a review see [1]). It is further known that GABA exerts its effects via two main receptor subtypes, namely the GABAA receptor which can be blocked by bicuculline and the GABAB receptor which is activated by L-baclofen (for a review see Ref. [2]). Recently it was shown that GABAB receptors are selectively blocked by C G P 35 348 i n rat brain after systemic administration [3]. There is growing evidence in support of an important role for GABA in the regulation of hormone secretion [4]. As for the effects on HPA function mediated by GABAB receptors, however, the information in the literature is rather scarce and controversial. On the one hand, it was shown that baclofen has an inhibitory effect in vitro on stimulated release of the rat hypothalamic corticotropin-releasing factor [5] and rat pituitary A C T H [6] and on cortisol response to insulin-induced hypoglycemia in man [7]. On

the other hand, we found a stimulatory effect of L-baclofen on plasma A C T H in rats and--with a lag-time of 1 0 m i n - - o n the adrenocortical hormones corticosterone and aldosterone [8]. A stimulatory effect of baclofen on plasma cortisol was also reported in man [9]. The goal of the present study was to investigate whether the stimulatory effects elicited by the GABAB agonist L-baclofen on A C T H and adrenocortical steroids could be reversed by the GABAB antagonist CGP 35 348 in vivo.

EXPERIMENTAL

Adult male rats [Tif:RAIf(SPF)] weighing 160-180 g had free access to food and drinking water under controlled laboratory conditions (room temperature: 2 4 + 2 ° C , lights on at 0600 h, lights off at 2000 h). The GABAa agonist L-baclofen and the GABAB antagonist CGP 35 348 [P-(3-aminopropyl)-P-diethoxymethyl-phosphinic acid; Fig. 1] were synthesized in the laboratories of C I B A - G E I G Y Ltd. CGP 35 348 shows good water solubility (45%). For in vivo experiments, the animals were

*To w h o m correspondence should be addressed. 767

768

A. H.~USLER et al.

O

II

HtN~/~/P',,l,- 0 V HO ( ~

CGP 35 348 Fig. 1. Chemicalstructure of CGP 35 348.

weighed on the day before the experiment and divided into 4-5 groups of 5 animals per group and kept in single cages. In all experiments, the animals were decapitated and trunk blood was collected in EDTA-coated Eppendorf tubes. Plasma obtained by centrifugation was stored at -20°C for hormone determination at a later date. Selected experiments were repeated and the data of the 2 experiments were pooled for statistical analysis. A f i r s t experiment was performed to study the effects of the GABAB agonist L-baclofen on plasma corticosteroids. At 0800 h five groups of rats received either 0.9% NaC1 or increasing doses of L-baclofen (0.75-6 mg/kg, s.c.). Two hours later, these animals were sacrificed and plasma was collected for hormone determinations. A second set of experiments was performed to study the antagonistic effect of CGP 35 348 on L-baclofen-induced enhancement of plasma corticosteroids. At 0800 h four groups of rats received either 0.9% NaC1 or CGP 35 348 (600 mg/kg, i.p.). L-baclofen was given 1 h later and 2 h after injection of L-baclofen the animals were sacrificed and plasma was collected for hormone determinations. In a separate experiment, plasma was collected for ACTH determination 30 min after injection of L-baclofen. For in vitro experiments, dispersed adrenal cells obtained from male rats were used in a perifusion system with a flow rate of 0.5 ml/min. The experimental design which was used represents a combined and modified version of perifusion techniques reported by Mulder et al. [10] and Goverde et al. [11]. The perifusion fluid (Earle's balanced salt solution containing 7.7mM CaC12, 5.6mM glucose and 0.5% bovine serum albumin) was collected over 5-min intervals and stored at - 2 0 ° C for the measurement of corticosterone and aldosterone at a later date. Increasing concentrations of either L-baclofen or CGP 35 348 were given to the cells in 5-min pulses ever 30 min. The viability of the cells was tested with 1 pM ACTH given to the

system for 5 min at the beginning and at the end of the experiment. Concentrations of immunoreactive corticosterone, aldosterone in plasma and perifusion fluid and of ACTH in plasma were measured by radioimmunoassays without extraction. The radioimmunoassays for corticosterone and aldosterone in plasma and for aldosterone in perifusion fluid have been described previously[12, 13]. Corticosterone in perifusion fluid was determined by using a commercial radioimmunoassay kit (Catalog No. 07-120103) from ICN Biomedicals Inc. (Costa Mesa, CA). According to the kit information, major crossreactivity with other steroids was 0.34% for desoxycorticosterone, 0.1% for testosterone, 0.05% for cortisol, 0.03% for aldosterone, 0.02% for progesterone, 0.01%0 for androstenedione and 5~-dihydrotestosterone and < 0.01% for dehydroepiandrosterone, ll-deoxycortisol and estrogens. ACTH in plasma was determined using a radioimmunoassay kit (ACTHK-PR) from CIS Bioindustries (Compagnie ORIS Industrie S.A., Gif-sur-Yvette Cedex, France). Major cross-reactions with related pituitary hormones were stated as being 0.1% for flLPH and aMSH and 0% for all other pituitary hormones tested. Statistical analysis was performed using either Dunnett's t-test [14, 15] or one-way analysis of variance (ANOVA) with differences between groups determined by Scheffe's F-test. Logtransformed data were used for samples with heterogeneous variances. Test values with P < 0.05 were considered to be statistically significant.

RESULTS In vivo experiments

The effects of the GABAB agonist L-baclofen on plasma concentrations of corticosterone and aldosterone in adult male rats are summarized in Table 1. Subcutaneous doses of 0.75-6 mg/kg L-baclofen led to a dose-dependent increase of plasma concentrations of both corticosterone and aldosterone measured 2 h after injection of L-baclofen. A submaximal dose of 3 mg/kg Lbaclofen led to an 8-fold increase of plasma corticosterone and to a 6-fold increase of plasma aldosterone. The effects of the GABAB antagonist CGP 35 348 on L-baclofen-induced increase of plasma hormones are summarized in Table 2. Pretreat-

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GABA B receptors and regulation of HPA function Table 1. Effect of L-baclofen on plasma concentrations of corticosterone and aldosterone in rats injected s.c. with either 0.9% NaCI or L-baclofen (0.7641 mg/kg) Treatment (I ml/kg, s.c.) 0.9% NaCI (Control) t-baclofen L-baclofen L-baclofen L-baclofen

Dose (mg/kg)

n

Corticosterone (ng/ml)

Aldosterone (pg/ml)

-0.75 1.5 3.0 6.0

5 5 5 5 5

29 +_ 12 97 +_ 32* 190 +_ 51"* 225 _ 32** 279 +_ 13"*

60 +_ 7 122 +_ 35 272 + 82* 387 _+ 78** 583 --6_87**

Plasma hormone concentrations (mean values + SEM) were measured 2 h after injection. *P < 0.05; **P < 0.01: Dunnett's t-test (L-baclofen vs control; log-transformed data due to unusual variability).

ment of the animals with an intraperitoneal injection of 600 mg/kg CGP 35 348 significantly antagonized the stimulatory effect of L-baclofen on corticosteroids (reduction of both hormone concentrations in the plasma by about 60%). In a separate experiment, it was shown that 3 mg/ kg L-baclofen led to an 11-fold increase of plasma ACTH measured 30min after L-baclofen administration. Again, pretreatment with 600 mg/kg CGP 35 348 reduced this effect by 60%. It was shown in all experiments that administration of CGP 35 348 without L-baclofen treatment had no significant effect on plasma ACTH and corticosteroid concentrations. In vitro experiments The effects of exposing perifused adrenal cells to increasing concentrations of L-baclofen are shown in Fig. 2. Dispersed adrenal cells obtained from male rats which were exposed in a perifusion system to 1 pM ACTH for 5 min reacted with a strong release of corticosterone (up to 200-fold above baseline) and aldosterone (up to 30-fold above baseline) into the perifusion fluid (see Fig. 2). Neither L-baclofen tested in concentrations of 0.1-100#M (Fig. 2) nor concentrations of 2-2000/~M of CGP 35 348 (data not shown) had an effect on corticosterone and aldosterone secretion of these cells.

DISCUSSION

The present results reproduce our previously reported data showing that subcutaneous administration of the GABA B agonist L-baclofen to adult male rats leads to a dose-dependent increase of plasma corticosterone and aldosterone 2 h after L-baclofen-administration [8]. This is in contrast to an inhibitory effect of baclofen found in a number of studies [5-7]. The major differences between our study and the others is that in our study the effects of L-baclofen and CGP 35 348 were studied under basal conditions whereas the other authors studied the effects of baclofen under stimulated conditions. Thus it is possible that GABAB receptor-mediated regulation of the HPA axis may respond differently to GABA8 agonists under unstimulated and stimulated conditions. L-baclofen incredsed plasma corticosteroids in a dose-range of 0.75-6 mg/kg (s.c.) in rats. This compares quite well with the dose-dependent effects found in another biological system (depression of patellar, flexor and linguomandibular reflex in cats with 0.3-3 mg/kg, i.v.)[16]. Moreover, this stimulation of hormone secretion in rats seems to result from specific activation of GABAa receptors along the HPA axis, since pretreatment with CGP

Table 2. Antagonism of L-baclofen-induced enhancement of plasma concentrations of corticosterone and aldosterone with C G P 35 348 (600 mg/kg, i.p.) given 1 h before L-baclofen (3 mg/kg, s.c.) Group A B C D

First treatment (I ml/kg, i.p.)

Second treatment (1 ml/kg, s.c.)

Corticosterone (ng/ml)

Aldosterone (pg/ml)

ACTH (ng/ml)

0.9% NaCI (Control) 0.9% NaCI (Control) C G P 35 348 (600 mg/kg) C G P 35 348 600 mg/kg)

0.9% NaCI (Control) L-baclofen (3 mg/kg) L-baclofen (3 mg/kg) 0.9% NaCI (Control)

30 _+ 18

50 _+ 16

0.16 _+ 0.05

252 + 45*

268 + 62*

1.87 + 0.78 a

94 _+ 19

107 + 22

0.61 + 0.14 b

71 _+ 19

106 _+ 30

0.12 +_0.03

Plasma concentrations of corticosterone and aldosterone (mean values _ SEM, n = 10) were measured 2 h after the second treatment. In a separate experiment, plasma A C T H concentrations (mean values +_ SEM, n = 5) were measured 30 rain after the second treatment. *Significantly different from all other groups, "significantly different from group A and D, bsignificantly different from group D (P < 0.05; one-way analysis of variance with differences between groups determined by Scheffe's F-test; log-transformed data for A C T H due to unusual variability.

770

A. H,~USLER et al.

10000

I I I 1 pM ACTH {

1000 cO

100

O o

10

r:o

i

i I I L-Baclofen (gM) i

i

I ! 1 pM ACTH l

i

10000 m

1000

100 10

o

0.1

0

30

60

90

120

150

180

, 210

~ C

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o <

0.1

Time (rain) Fig. 2. Effect of L-baclofen on corticosterone and aldosterone secretion by perifused rat adrenal cells. Adrenal cells were challenged with pulses of either 1 pM ACTH or increasing concentrations of L-baclofen of 5 rain duration. 35 348, a centrally active antagonist of G A B A B receptors [3] diminishes this L-baclofen-induced effect. Besides, these results confirm data which had been found using the same experimental design with C G P 36 742 [8], an orally active G A B A B antagonist [17]. That C G P 35 348, like C G P 36 742 [8], had no effect on H P A function in vivo could have been due to hormone concentrations being very low in the control animals. Therefore, under basal conditions, it would have been rather difficult to detect a significant decrease with a G A B A Bantagonist as one might expect from the data obtained with the G A B A B agonist. The main elements of the H P A axis include hypothalamic corticotropin-releasing factor(s) which stimulate(s) pituitary A C T H secretion which in turn stimulates the secretion of corticosteroids from the adrenal cortex. The present results do not allow to narrow down the precise location where along the H P A axis the effects of L-baclofen take place. However, the fact that the increase of A C T H in the plasma is antagonized by the G A B A 8 antagonist C G P 35 348 (present data) and that this increase precedes that of the corticosteroids [8] seems to exclude the adrenal cortex as a potential target for the stimulatory effect of L-baclofen. This assumption is further supported by the in v i t r o data showing no effect of L-baclofen on corticosteroid secretion in dispersed ACTH-responsive rat adrenal cells. Therefore, the participation of G A B A , receptors in the activation of the H P A axis must take place at the level of the pituitary or the brain. Both the pituitary and the brain have been shown to contain binding sites for G A B A [18].

Subtle discriminative investigations on the distribution of G A B A receptor subtypes revealed that GABAB receptors are detected in the brain[19] and in the anterior pituitary[20], although the receptor number is considerably lower in the anterior pituitary than in the brain [20]. These data indicate that G A B A , receptors either in the brain or in the anterior pituitary or in both are involved in the mediation of L-baclofen-induced activation of the H P A axis in rats. In conclusion, the L-baclofen-induced activation of rat H P A function which can be antagonized by GABAB antagonists points to an involvement of G A B A . receptors in the regulation of the H P A axis in these animals. These results further indicate that the effect of L-baclofen does not take place at the level of the adrenal gland, and therefore must take place at the level of the pituitary or the brain.

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