GABAB receptors in the rabbit uterus may mediate contractile responses

European Journal of Pharmacology, 119 (1985) 199-204

199

Elsevier

G A B A B R E C E P T O R S IN T H E R A B B I T U T E R U S M A Y M E D I A T E C O N T R A C T I L E R E S P O N S E S

MIKLOS R1ESZ and SANDOR L. ERDO * Pharmacological Research Centre, Chemical Works of Gedeon Richter, Ltd., P.O. Box 27, H-1475 Budapest, Hungary

Received 10 April 1985, revised MS received 5 August 1985, accepted 1 October 1985

M. RIESZ and S.L. ERDO, GABA 8 receptors in the rabbit uterus may mediate contractile responses, European J. Pharmacol. 119 (1985) 199-204. The effects of "r-aminobutyric acid (GABA) and related compounds on the contractility of isolated rabbit uterus were examined. GABA and baclofen (10-6-10 4 M) stimulated the spontaneous motility in a dose-dependent manner and showed cross-desensitization. The effect of baclofen was stereoselective for the (-)-enantiomer. Muscimol was ineffective, while bicuculline evoked marked contractions. Contractions elicited by submaximal electrical stimulation could be further increased by GABA and baclofen. The effects of GABA and baclofen on spontaneous contractility could not be antagonized by atropine, pbentolamine, or tetrodotoxin. These findings indicate: (1) the presence of GABA B receptors in the rabbit uterus; (2) their involvement in the modulation of uterine contractility; (3) the extraneuronal location of uterine GABA B receptors which are thus most probably on smooth muscle cells; and (4) a possible role of inhibitory GABA A receptors in the modulation of spontaneous movements of the uterus. Rabbit uterus

GABA B receptors

Spontaneous motility

1. Introduction

~,-Aminobutyric acid ( G A B A ) is a well-established inhibitory neurotransmitter in the mammalian central nervous system (Krnjevic, 1974; Curtis, 1979). There is increasing evidence that G A B A is widely distributed throughout peripheral tissues and may even have functional relevance in certain peripheral organs (recent review, see Erd6, 1985). A m o n g the peripheral tissues examined so far, the rat oviduct has been found to be the richest in G A B A (Del Rio, 1981; Erd6 et al., 1982; A p u d et al., 1984). In addition to the high G A B A concentration - over twice that in whole brain - the presence of the enzymes of G A B A metabolism, i.e. glutamate decarboxylase and GABA-2-oxoglutarate aminotransferase, in the rat oviduct has also been demonstrated (Del Rio, 1981; A p u d et al., 1984; Erd6 et al., 1984a). Moreover, specific G A B A

* To whom correspondence should be addressed. 0014-2999/85/$03.30 ¢~ 1985 Elsevier Science Publishers B.V.

Baclofen

Tetrodotoxin

Bicuculline

receptor binding sites have been identified on membranes of the mammalian oviduct (Erd6 and Lapis, 1982; Erd6 et al., 1983). Specific, G A B A - r e ceptor mediated contractile responses of isolated rabbit oviduct have been demonstrated (Erd6 et al., 1984b), and the modulation of rat oviduct contractility by G A B A has been reported (Fernhndez et al., 1984). These findings suggest that oviductal GABAergic mechanisms may be involved in the regulation of the motility of this organ. G A B A and glutamate decarboxylase, the enzyme thought to be responsible for G A B A formation, as well as specific G A B A receptor binding sites have recently been shown to be present in rat and rabbit uterus (Erd6, 1984). These findings p r o m p t e d us to examine whether this GABAergic system might be involved in the modulation of uterine contractility as is the case in the oviduct. Thus, we examined the effect of G A B A and various model c o m p o u n d s k n o w n to act on G A B A receptors on the contractility of isolated rabbit uterus.

200 2. M a t e r i a l s

and methods

2.1. Organ preparation and contractility measurement Sexually mature, virgin N e w Z e a l a n d r a b b i t s weighing 2.5-4.1 kg, were used. The a n i m a l s were caged i n d i v i d u a l l y a n d allowed free access to food a n d water. A f t e r the rabbits h a d been killed by a s h a r p blow on the head and e x s a n g u i n a t i o n , the uterus was r e m o v e d a n d l o n g i t u d i n a l strips (app r o x i m a t e l y 15 m m long and 4 m m wide) were cut from the m i d d l e p a r t of the uterine horns in o x y g e n a t e d K r e b s - H e n s e l e i t solution at r o o m temperature. A n y tissue samples not used i m m e d i a t e l y were stored in the refrigerator at 4 ° C for 8-18 h w i t h o u t o b s e r v a b l e changes in s p o n t a n e o u s motility and d r u g responsiveness d u r i n g s u b s e q u e n t e x p e r i m e n t a t i o n . The muscle strips were susp e n d e d in organ b a t h s of 10 ml volume using wire clips. The b a t h i n g m e d i u m was a m o d i f i e d KrebsHenseleit solution (Erd6 et al., 1984b), c o n t i n u ously a e r a t e d with c a r b o g e n (95% 02, 5% CO2), a n d its t e m p e r a t u r e was m a i n t a i n e d at 37°C. S p o n t a n e o u s a n d evoked m o t i l i t y of the prep a r a t i o n s was m e a s u r e d by semi-isometric transducers ( E L T R O N ) with a p r e l o a d of 1 g, a m p l i f i e d (Izometer, E L T R O N ) a n d registered on a twochannel r e c o r d e r ( K i p p and Zonen). In certain e x p e r i m e n t s we used m u s c u l o t r o p i c electrical s t i m u l a t i o n : the lower end of the muscle strip was a t t a c h e d to a p l a t i n u m hook, the o t h e r pole being a cylindrical metal p a r t of the glass, h o o d - s h a p e d electrode. S u b m a x i m a l , b i p h a s i c r e c t a n g u l a r stimuli (50 Hz, 13.3 ms width, half of the voltage eliciting m a x i m a l c o n t r a c t i o n , 20 s trains in every 2 min) were delivered b y the isolated o u t p u t of a s t i m u l a t o r ( H S E I I M Z ) . The effects of each comp o u n d a n d drug c o m b i n a t i o n were e x a m i n e d in at least 5 i n d e p e n d e n t e x p e r i m e n t s (i.e. on specimens o b t a i n e d from 5 or m o r e animals).

2.2. Chemicals used A d r e n a l i n e (Richter); a t r o p i n e s u l p h a t e ( E G I S ) ; racemic, ( + ) - a n d ( - ) - b a c l o f e n ( k i n d l y s u p p l i e d by Ciba-Geigy, Budapest): ( + )-bicuculline (Sigma); h i s t a m i n e d i h y d r o c h l o r i d e (Chinoin);

muscimol (Fluka); p h e n t o l a m i n e ( C i b a - G e i g y ) ; serotonin ( 5 - h y d r o x y t r y p t a m i n e creatinine sulphate, Serva); t e t r o d o t o x i n (Sankyo). O t h e r chemicals were of analytical grade and were p u r c h a s e d from c o m m e r c i a l sources.

3. Results Regular s p o n t a n e o u s activity of uterine strips was generally seen at the end of the a c c o m m o d a t i o n period (60 min). The motility p a t t e r n consisted of r h y t h m i c a l phasic c o n t r a c t i o n s with varying amplitude, a n d the frequency usually decreased d u r i n g the experiments. Uteri generating c o n t r a c t u r e s ( ' o u t b u r s t s ' ) s e p a r a t e d by long-lasting silent p e r i o d s of 5-10 min (3 animals) were disc a r d e d because this type of activity is u n c o m m o n in virgin r a b b i t s a n d p o i n t s to some deviation in s e x - h o r m o n a l b a c k g r o u n d . A total of 76 p r e p a r a tions o b t a i n e d from 36 a n i m a l s were evaluated. A

B

t, i

i Mus 1 0 " M

GABA 10-' M

i A

Bicu

M u 6 s °Mus

10 -sM

IO"M

5 x 10 "~M

D

o)

Bacl 10 -7 M

6

Bacl 10"6M

6

Bacl lO-SM

2 rain

Fig. I(A). GABA (10 4 M) induced frequent contractions with increased amplitude and strongly elevated basal tone in the isolated rabbit uterus. (B) Muscimol was virtually ineffective even at a high concentration. (C) On another specimen, pretreated with atropine (10 s M), bicuculline evoked a series of very frequent contractions with highly elevated basal tone that could be completely abolished by repeated administration of 5 × 10 5 M muscimol. This effect of muscimol was rarely seen so clearly. (D) Lack of effect of 10 7 M baclofen on the spontaneous contractility of isolated rabbit oviduct. (E) 10 6 M baclofen slightly increased the contractile strength with a marginal elevation of the basal tone. (F) At 10 5 M baclofen produced marked stimulation of contractions and strongly elevated the basal tone. Abbreviations used: A - atropine, Bacl - ( + ) baclofen, Bicu = ( + )bicuculline, Mus - muscimol. Records A, B, D, E and F were obtained from the same specimen, C from another preparation.

201

• GABA

I

g

• Bacl

IO"M

•

•

Bacl

GABA

each

2rain

• t Bacl wash 10" M

I

fig. 2. Cross-desensitization between GABA and baclofen on the isolated rabbit uterus. Note also the qualitatively similar contractile effects of the two drugs when applied in equimolar concentrations. For abbreviations, see legend to fig. 1.

(~le..¢l wo.~ 10.4 M

(÷)n.~ (-~'~.cl~°.,

IO"M

10"4M

2mln Fig. 3. Stereoselectivity of the baclofen effect on the spontaneous contractility of isolated rabbit uterus. See legend to fig, 1 for abbreviations.

GABA (10 6-10-4 M) exerted a dose-dependent stimulatory action on uterine preparations. This effect could be characterized by a marked

• TTX 5 x 10"lM

Bacl

10"' M

1~ 2rain e Fig. 4. Tetrodotoxin resistance of the baclofen effect on the spontaneous contractility of isolated rabbit uterus. TTX = tetrodotoxin.

increase of contraction amplitude and frequency and by a moderate elevation of basal tone. Peak effect (maximal contraction amplitude) was generally reached within 2 min (fig. 1A). These responses could also be elicited in temporarily quiescent preparations. Muscimol, a selective agonist at GABA A receptor sites (Bowery et al., 1983), was virtually ineffective in concentrations up to 10 -4 M (fig. 1B). Bicuculline (10 -4 M), a selective blocker of GABA A receptors (Bowery et al., 1983) evoked strong contractions of spontaneously active specimens, thus its interactions with G A B A and baclofen could not be tested with accuracy. This action

TABLE 1 Cumulative evaluation of the effects of GABA and various model compounds on the contractility of isolated rabbit uterus. Numbers refer to experiments on specimens from different animals. Number of specimens tested

Drug

GABA ( + )-Baclofen (-)-Baclofen ( + )-Baclofen Muscimol Tetrodotoxin" + ( _+)-Baclofen Atropine ~ + ( + )-Baclofen Phentolamine a + ( _+)-Baclofen Bicuculline

(10-6-10 -4 M) (10-6-10 -4 M) (10-%10 4 M) (10- 4 M) (10-6-10 -4 M) (5 X 10- 6 M) (10 -4 M) (10 -5 M) (10- 4 M) (2 X 10 5 M) (10- 4 M) (10 4 M)

a Pretreatment time was 5 min for all antagonists.

Total

Stimulation

No stimulation

21 32 10 11 7

17 26 9 2 1

4 6 ] 9 6

5

5

0

5

5

0

5 12

5 10

0 2

202

of bicuculline was also observed in the presence of atropine. The effect of bicuculline could be antagonized by equimolar muscimol in certain preparations (fig. 1C). The GABA B receptor agonist baclofen (10 %10 4 M) (Bowery et al., 1983) produced dose-dependent stimulation (fig. 1 D-F). The common site of action of GABA and baclofen could be demonstrated by cross-desensitization experiments (fig. 2). Preparations desensitized by these drugs, however, could be contracted again by known uterine stimulants, such as adrenaline (10 6 M), histamine (10 5 M) or serotonin (10 5 M) (data not shown); thus muscle fatigue was not responsible for the reduction of motility. The stimulant effect of baclofen was stereoselective for the ( - )-enantiomer (fig. 3). When submaximal electric stimulation was applied, the excitatory action of GABA and baclofen was manifested as an increase in contraction force, but no such augmentation could be observed in supramaximally stimulated specimens (data not shown, n = 4). Tetrodotoxin (5 × 10 ¢' M), an inhibitor of fast sodium channels, failed to antagonize the above effects of GABA and baclofen (fig. 4). Blockade of ~-adrenoceptors (phentolamine, 2 × 10 5 M) and muscarine receptors (atropine, 10 5 M) was also ineffective. The main qualitative results are summarized in table 1.

4. Discussion

The aim of the present study was to elucidate whether the GABA u receptor-mediated increase of motility was a response of isolated rabbit oviductal tissues only (Erda et al., 1984b) or whether it could be demonstrated with uterine preparations of the same species. Our findings show that a non-cholinergic non-adrenergic, tetrodotoxin-insensitive stimulatory action of GABAu receptor agonists (GABA and baclofen) could also be elicited from isolated rabbit uterus, while the GABA A receptor stimulant muscimol failed to evoke a detectable contractile response. Cross-desensitization experiments between GABA and baclofen revealed a common site of action of the two drugs. The stereoselective effect of baclofen,

with the ( )-enantiomer being active, is supportive evidence for the receptor-mediated response. The enantioselectivity of baclofen has been demonstrated on different central and peripheral neuronal elements (Bowery et al., 1981: 1983) which could be characterized by decreased neuronal transmission, but a stereoselective stimulatory action on smooth muscle tissue has been observed only in the rabbit oviduct (Riesz, unpublished) and uterus (present study). Since the involvement of neuronal release processes seems unlikely in view of the resistance to tetrodotoxin it can be supposed that GABA and baclofen exert an excitatory effect on rabbit uterine smooth muscle which results in the increase of contraction amplitude of submaximally stimulated organ strips and the occasional (although not systematically studied) decrease of the stimulation threshold. Changes in contractions elicited by supramaximal stimulation were negligible, thus the increase in contractile force in submaximally stimulated preparations is more likely to result from the enhancement of membrane depolarization, or a depolarization-coupled process, rather than direct stimulation of intracellular contractile mechanisms. The responses of isolated rabbit uteri to GABA ~ receptor agonists were similar to those observed on the oviduct of the same species. However, two important differences should be emphasized. First, the uterus was approximately 10 times more sensitive to these drugs, the maximum stimulatory action being attained at 10 4 M, while in the oviduct this was obtained at 10 3 M. Since the uterine GABA content in rabbits and rats (Erda, 1984; Fernfindez-Pardal et al., 1984) was found to be lower than that of the oviduct, it is reasonable to assume that if the GABAI~ receptor-mediated effect is physiologically relevant, the uterus would be more sensitive to the endogenous compound, GABA. GABA concentrations determined in the rabbit uterus (16.3 n m o l / g frozen tissue: Erda, 1984) seem to be adequate to exert an excitatory action under physiological conditions in this organ. The other major difference observed was that the GABA A receptor blocking agent, bicuculline (10 4 M), elicited contractions in the isolated rabbit uterus. This effect was rarely seen in rabbit

203

oviduct at this dose level, but occurred at higher doses (2-5 × 10 -4 M). This was also observed by Fern~.ndez et al. (1984) with isolated rat oviduct and the authors explained their observation by the known cholinesterase-inhibiting action of bicuculline (Breuker and Johnston, 1975). Under our conditions, however, bicuculline-induced stimulation of the rabbit oviduct and uterus could not be blocked by atropine (10 5 M), thus indirect muscarinic stimulation can be ruled out, at least in rabbits. Since the bicuculline effect could be diminished by the subsequent administration of muscimol, it seems possible that this is a specific action mediated via GABA A receptors. Taking into account the biochemical evidence for the presence of A-type GABA receptors in the female genital tract of rats (Erda) and Lapis, 1982; Erd6, 1984), rabbits (Erd6, 1984) and humans (Erd6 et al., 1983), it seems possible that GABA A receptor sites are also involved in the modulation of uterine motility. The GABA B receptor-mediated increase in the motility of the rabbit oviduct and uterus has been demonstrated in isolated organ experiments and no biochemical (receptor binding) and anatomical (autoradiographic) correlates have yet been published. Therefore, our findings do not demonstrate the exact site of action of GABA B receptor stimulants. These receptors may be located either on smooth muscle cells or on secretory cells which are able to release an endogenous compound responsible for the stimulatory action. This study did not attempt to characterize quantitatively the stimulatory action of GABA B receptor agonists on the isolated rabbit uterus. The main reason for this was the considerable difference observed in the spontaneous motility of individual organs and the decreasing contractile frequency during the experiments. It seems likely that dose-response relationships would be more easily quantified with electrically stimulated organs (using submaximal stimulus intensity) under experimental conditions minimizing spontaneous activity. In conclusion, a non-cholinergic, non-adrenergic and tetrodotoxin-insensitive stimulatory action of GABA and baclofen has been demonstrated on the contractility of isolated rabbit uterus.

This effect proved to be mediated by specific GABA B receptors on extraneuronal elements. Further investigations are needed, however, to elucidate the exact location of these receptors. Since the endogenous level of GABA in the uterine tissue and the effective concentrations of GABA and baclofen are of the same order of magnitude, an involvement of local GABAergic mechanisms in the physiological modulation of uterine contractility seems plausible.

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