Bicuculline-insensitive gaba receptors on peripheral autonomic nerve terminals

European Journal of Pharmacology, 71 (1981) 53--70 © Elsevier/North-Holland Biomedical Press

53

B I C U C U L L I N E - I N S E N S I T I V E G A B A R E C E P T O R S ON P E R I P H E R A L A U T O N O M I C N E R V E TERMINALS NORMAN G. BOWERY, ADAM DOBLE *,**, DAVID R. HILL, ALAN L. HUDSON, JOHN S. SHAW *, MICHAEL J. TURNBULL * and RUTH WARRINGTON * Department o f Pharmacology, St. Thomas's Hospital Medical School, London SE1 7EH, England, and * Biology Department, ICI Pharmaceuticals Ltd., Macclesfield, Cheshire, England

Received 6 October 1981, revised MS received 5 January 1981, accepted 20 January 1981

N.G. BOWERY, A. DOBLE, D.R. HILL, A.L. HUDSON, J.S. SHAW, M.J. TURNBULL and R. WARRINGTON, Bicuculline-insensitive GABA receptors on peripheral autonomic nerve terminals, European J. Pharmacol. 71 (1981) 53--70. The action of 7-aminobutyric acid (GABA) and related compounds on rat isolated atria and mouse and guineapig isolated vas deferens has been studied. GABA depressed the evoked but not basal release of [3H]noradrenaline from atria (ICs0 4 gM) and reduced the twitch responses of the vas deferens (ICs0 3 tiM) in a dose-dependent manner. These depressant effects were not prevented by recognised GABA antagonists such as bicuculline and picrotoxin. Numerous GABA analogues, in particular 3-aminopropanesulphonic acid, failed to mimic the action of GABA. However, /3-p-chlorophenyl GABA (baclofen) was stereospecifically active. Other related/~-substituted derivatives were also active but to a lesser degree than GABA. Pretreatment of the vas deferens with the neuronal GABA uptake inhibitors 2,4-diaminobutyric acid or cis-3-aminocyclohexanecarboxylic acid potentiated the action of GABA. These data suggest the presence of a bicuculline-insensitive GABA receptor on autonomic nerve terminals. Preliminary observations indicate a lack of chloride ion dependence in the action of GABA at this site. Autonomic nerve terminals Bicuculline insensitivity

Transmitter release

1. I n t r o d u c t i o n Bicuculline-sensitive receptors for GABA are n o t c o n f i n e d t o c e n t r a l n e u r o n e s in m a m m a l s b u t are also p r e s e n t o n p e r i p h e r a l n e u r o n e s ( B o w e r y a n d B r o w n , 1 9 7 4 ; S a n g i a h et al., 1 9 7 4 ; A d a m s a n d B r o w n , 1 9 7 5 ; De G r o a t , 1 9 7 0 ; K i m u r a et al., 1 9 7 7 ; F e l t z a n d Rasminsky, 1974). Activation of these peripheral receptors selectively increases the m e m b r a n e c o n d u c t a n c e t o c h l o r i d e i o n s as at c e n t r a l sites ( A d a m s a n d B r o w n , 1 9 7 5 ) . H o w e v e r , neuronal depolarization rather than hyperp o l a r i z a t i o n r e s u l t s f r o m t h i s i n c r e a s e in i o n i c ** Present address: MRC Neurochemical Pharmacology Unit, Hills Road, Cambridge, England.

GABA receptor

Baclofen

p e r m e a b i l i t y . The presence of receptors for G A B A o n n e u r o n a l cell b o d i e s o f s y m p a t h e t i c ganglia m a d e us c o n s i d e r t h e p o s s i b i l i t y t h a t t h e y m i g h t also be p r e s e n t on t h e t e r m i n a l s o f t h e s e s a m e n e u r o n e s . If so t h e n t h e d e p o larization produced on activation with GABA might reduce the evoked release of neurotransm i t t e r ( D u d e l a n d K u f f l e r , 1 9 6 1 ) . To t e s t t h i s h y p o t h e s i s we have s t u d i e d t h e e f f e c t o f G A B A on the evoked release of radiolabelled noradrenaline from rat isolated atria and on the contractile responses of the field-stimulated m o u s e a n d g u i n e a - p i g vas d e f e r e n s , a n d g u i n e a pig i l e u m p r e p a r a t i o n s . W h i l s t o u r o b s e r v a t i o n s indicate that GABA does reduce the evoked output of neurotransmitter the pharmacology of this effect differs m a r k e d l y from that

54 associated with the GABA receptors responsible for the depolarization occurring at the neuronal cell body. Brief reports of some of these results have been presented to the British Pharmacological Society (Bowery and Hudson, 1979; Bowery et al., 1979a; Bowery et al., 1981).

2. Materials and methods 2.1. Rat atria Atria (left and right treated as one preparation) were obtained from freshly killed Wistar rats. Each preparation was incubated for 40 min at 32°C in 1 ml Krebs-Henseleit solution containing 0 . 4 p M DL-[7-3H]noradrenaline (10 Ci/mmol, Radiochemical Amersham) and then in 50 ml radioactive-free solution for a further 45-60 min. The latter incubation was adopted to allow time for the basal tritium release rate to stabilise. The atria were then suspended through t w o silver ring electrodes and superfused with pre-heated (32°C)KrebsHenseleit solution at 0.45 ml/min. The upper end of the tissue was connected to a light spring. Superfusate samples were collected consecutively for 4 min periods. Scintillation fluid (PPO: dimethyl POPOP in toluene plus triton-X100) was added to each sample for determination of the tritium content. Ascorbic acid (0.1 raM) and iproniazid (0.5 mM) were present in all incubation and superfusion solutions to minimize noradrenaline catabolism. Qualitatively similar results, however, were obtained in their absence. Iproniazid (0.5 mM) did n o t affect noradrenaline transport in the atria (see also Iversen, 1967). At the end of each experiment the tissue was weighed and dissolved overnight in 1 ml Soluene (Packard) to measure the residual tritium content. The atria were stimulated either transmurally via the ring electrodes (3-5 Hz, 0.5 msec, 10 V for 1 min at 16 min intervals) or b y the addition of potassium chloride (final concentration 60 mM) to the superfusate solution.

N.G. BOWERY ET A[, Superfusate samples from three separate experiments were analysed chromatographically by the method of Graefe et al. (1973) to determine the contents of authentic radiolabelled noradrenaline (instead of just tritium). Briefly, each sample was adjusted to pH 8.2 using 0.1 N NaOH and ethylene diamine tetraacetic acid (2 mg) and sodium sulphite (2.5 mg) added. The solution was then passed through a column containing aluminium oxide (Woelm) activated by the method of Anton and Sayer (1962) and equilibrated with sodium acetate (0.2 M). Noradrenaline adsorbed to the alumina was displaced with 0.2 N acetic acid and the effluent passed through Dowex 50W resin (Sigma) equilibrated with 0.01 N HC1. Noradrenaline was eluted from this with 2 N HC1. 2.2. Vas deferens Vasa deferentia were obtained from 20 g mice or 350 g guinea pigs, and suspended in 5 ml organ baths. The tissues were bathed in magnesium-free Krebs bicarbonate buffer at 32°C. Contractions were recorded isotonically using a Palmer transducer (tension 200 mg) and displayed on a Servoscribe Is potentiometric recorder. Twitch responses of the vasa were elicited by coaxial stimulation using a Grass Stimulator 588 (trains of 50 Hz 1 msec, 60 V for 0.1 sec every 10 sec) as described by Shaw and Turnbull (1978). 2.3. Guinea-pig ileum Twitch responses of the ileum were recorded using the method described by Kosterlitz and Watt (1968). 2.4. Ganglion depolarization Depolarization of the rat superior cervical ganglia in vitro was recorded as described previously (Bowery and Brown, 1974, with a modification for superfusion, Brown and Marsh, 1975). Analogue potency was determined by comparison with GABA in each experiment.

BICUCULLINE-INSENSITIVE GABA RECEPTORS

55

2.5. Materials G A B A ( B D H a n d Sigma); 3 - a m i n o p r o p a n e s u l p h o n i c acid (Aldrich rare c h e m i c a l l i b r a r y ) ; f l - h y d r o x y G A B A , i m i d a z o l e a c e t i c acid, /3a m i n o b u t y r i c acid, g u a n i d i n o p r o p i o n i c acid, g u a n i d o a c e t i c acid, 2 , 4 ~ l i a m i n o b u t y r i c acid, 7 - h y d r o x y b u t y r i c acid (Sigma); ~-alanine, glycine, c a r b a c h o l , ascorbic acid ( B D H ) ; isop r o p y l b i c y c l o p h o s p h a t e , isoguvacine h y d r o chloride, i s o n i p e c o t i c acid h y d r o c h l o r i d e , n i p e c o t i c acid h y d r o c h l o r i d e , p i c r o t o x i n , (+)bicuculline m e t h o c h l o r i d e , (+)-bicuculline methobromide, ~-chloroGABA, muscimol and T H I P w e r e k i n d l y p r o v i d e d b y Dr. J.F. Collins, City o f L o n d o n P o l y t e c h n i c , L o n d o n a n d Dr. P. K r o g s g a a r d - L a r s e n , R o y a l Danish School of Pharmacy, Copenhagen; yohimbine h y d r o c h l o r i d e ( H o p k i n s a n d Williams); leptazol (Martindale Samoore); bemegride (Nicholas); p h e n t o l a m i n e ( C I B A - G e i g y ) ; racem i c b a c l o f e n a n d its e n a n t i o m e r s were gifts f r o m C I B A - G e i g y , H o r s h a m a n d Basle. I p r o n i azid was a gift f r o m R o c h e , Welwyn. Penicillin ( G l a x o ) . ~ - s u b s t i t u t e d G A B A derivatives ( b a c l o f e n analogues) w e r e kindly p r o v i d e d b y Dr. P.J. R o b e r t s , University o f S o u t h a m p t o n . A r e c a i d i n e (Sigma). N - m e t h y l G A B A was k i n d l y p r o v i d e d b y Dr. R. Galt, D e p t . o f C h e m i s t r y , ICI. Kojic a m i n e was a gift f r o m Dr. D. Carr, ICI. F r u s e m i d e ( H o e c h s t ) . A C H C was s y n t h e s i s e d b y Mr. C. C o o k s e y , University College L o n d o n .

3. Results

3.1. Atria -- release o f [3H] noradrenaline and the effect o f GABA An e x a m p l e o f t h e p a t t e r n o f t r i t i u m release f r o m atria p r e l o a d e d w i t h [ 3 H ] n o r a d r e n a l i n e is s h o w n in fig. 1. Washing t h e tissue p r i o r t o s u p e r f u s i o n p r o v i d e d a fairly c o n s t a n t b a c k ground efflux rate which a p p r o x i m a t e d to 0 . 0 0 2 m i n -1 tritium efflux min -1 t tissue tritium content]

9

JJ §3

'I 0

•

*

0 GABA I 0 0 ,uM ,

;

,

st/mulotion 3 H z ,5ms I0 V for Imin

,

0

Time (hours)

Fig. 1. Inhibitory effect of GABA on the evoked release of [3H] noradrenaline from rat atria. The atria from one rat were incubated, in 0.4 pM [3H]noradrenaline for 40 min followed by an incubation in radioactive-free solution for a further 45 min. The tissue was then superfused (0.45 ml/min) at 32°C with Krebs-Henseleit solution containing iproniazid (0.5 mM) and ascorbic acid (0.1 mM) and the effluent collected. The tritium content (dpm/4 min sample: ordinate) in consecutive 4 min samples is represented by the smaller dots which have been linked together and plotted against time (h: abscissa). The larger dots below correspond to 1 rain periods of rectangular stimulation (3 Hz, 0.5 msec at 10 V) of the atria via two silver ring electrodes approximately 1 cm apart. During the period indicated by the open bar at the top of the figure calcium chloride was omitted from the superfusion solution. This suppressed the evoked release of tritium. The replacement of calcium chloride (2.5 mM) produced a full recovery in the evoked release. GABA (100 pM) was added to the superfusion fluid during the period indicated by the open rectangular bar below the record (30 sec before and 1 min during stimulation). This reduced the evoked tritium release when compared with the control responses immediately before and after. The reduction produced by GABA was greater in this experiment than in two similar experiments.

T r a n s m u r a l s t i m u l a t i o n o f t h e tissue f o r p e r i o d s o f 1 m i n at 16 m i n intervals increased t h e o v e r f l o w o f t r i t i u m f r o m t h e atria. This increase was d e t e c t a b l e o n l y f o r t h e p e r i o d o f s t i m u l a t i o n , t h e t r i t i u m c o n t e n t o f t h e sub-

56

N.G. B O W E R Y ET A L

sequent collection period returning to the basal level. The evoked release of tritium was dependent on the presence of Ca 2÷ in the superfusion medium (fig. 1). Removal of Ca 2+ abolished the evoked increase in tritium release without decreasing the basal output. On replacing Ca ~+ the response to stimulation was immediately restored. The addition of GABA (0.1-300 pM) to the superfusion solution 30-60 sec before and during transmural stimulation reduced the resulting increase in tritium overflow. By contrast the application of GABA without stimulation failed to alter the basal efflux rate (fig. 1). The reduction in evoked release produced by GABA was rather small (maximum 15% at GABA 100 pM in 3 experiments)and was variable both within and between experiments (cf. fig. 1 and first part of fig. 2). The release of noradrenaline from sympa-

thetic terminals is modulated by an autoreceptor feedback mechanism (see Langer, 1977). Released noradrenaline activates a2receptors on the terminal membrane reducing further release of transmitter. In the presence of an a2 antagonist (e.g. yohimbine or phentolamine) the tritium overflow during stimulation therefore increases (Langer et al., 1977). It was conceivable that modulation of [3H]noradrenaline release in the atria might mask any further inhibition by GABA. The experiments were therefore repeated in the presence of yohimbine (2.5 pM) or phentolamine (2.5 pM). On superfusing atria with either azantagonist the evoked release of tritium was increased 2-4 fold without any change in the basal release (fig. 2). When GABA was now applied a much larger and less variable depression in the evoked release occurred as illustrated in fig. 2. In this experiment the atria

yohilh I:)i n¢ 2.S,uM

Iii

~3

\ I

• o

o 0

,L

0

•

Q

o I

I

I

I

2

• o

"

Q

•

n

i

st~re. 3 H z "3msl IOV for/m/n J GABA IOOJJM

i

3

h

Fig. 2. T h e a p p a r e n t increase b y y o h i m b i n e , t h e ~ 2 - a d r e n o c e p t o r a n t a g o n i s t , o f t h e decrease in e v o k e d [3H]n o r a d r e n a l i n e release b y G A B A in r a t atria. T h e p r o c e d u r e in t h i s e x p e r i m e n t was t h e s a m e as described in fig. 1 e x c e p t t h a t d u r i n g t h e p e r i o d i n d i c a t e d b y t h e o p e n h o r i z o n t a l b a r a b o v e t h e r e c o r d y o h i m b i n e (2.5 p M ) was a d d e d t o t h e s u p e r f u s i o n s o l u t i o n . This e n h a n c e d t h e evoked t r i t i u m release w i t h o u t altering t h e basal efflux. On a d d i n g G A B A ( 3 0 sec b e f o r e a n d d u r i n g s t i m u l a t i o n ) a m u c h greater r e d u c t i o n in e v o k e d release o c c u r r e d w h e n c o m p a r e d w i t h t h e r e d u c t i o n p r o d u c e d b e f o r e t h e a d d i t i o n of y o h i m b i n e . G A B A h a d n o effect o n basal t r i t i u m release.

BICUCULLINE-INSENSITIVE GABA RECEPTORS

were initially superfused with Krebs-Henseleit solution alone and as before GABA produced only a marginal reduction in the evoked tritium release. On adding GABA in the presence of yohimbine a larger and more consistent reduction in the evoked release occurred. This effect of GABA was dose-dependent between 0.3 pM and 300 pM (EDs0 = 4.2 +_ 1.4 #M, n = 6) producing a maximal reduction 54.1 +_ 2.34% at 100-300 pM (fig. 3). GABA was routinely applied in these experiments 30 sec before as well during stimulation. If GABA was applied for longer periods (more than 5 min) prior to stimulation this decreased its effectiveness. An example of the decrease observed with time is illustrated in fig. 4. After 30 min continuous superfusion with 100 pM GABA there was no discernible reduction in the evoked release produced by transmural stimulation. However, following an interval of > 2 0 min super-

57

fusion with GABA-free solution the reapplication of GABA (100 pM) 30 sec before stimulation produced the expected reduction in evoked release. The 'desensitisation' to GABA was therefore reversible. GABA not only reduced the tritium o u t p u t evoked by transmural stimulation but also reduced to a similar extent the increase produced by the addition of KC1 (60 mM for 2 min) to the superfusion solution (fig. 5). Superfusate samples from three experiments were analysed chromatographically for the presence of [3H]noradrenaline. Samples collected during basal periods contained only 36% of the released tritium as authentic [3H]noradrenaline. By contrast during transmural stimulation the value increased to 87%. In the presence of GABA (100 pM during stimula-

30'

60

s

1.5

.~ 20

~

©

i

-6

-5 -4 log M o l a r GABA

-3

Fig. 3. Log dose/response curve for the inhibitory action of G A B A on the evoked release of [ 3 H ] n o r adrenaline f r o m rat atria. The data derive f r o m e x p e r i m e n t s each p e r f o r m e d by the procedure described in fig. 1. The inhibitory action of different concentrations of G A B A was determined by expressing the r e d u c t i o n as a percentage o f the m e a n of the control responses obtained i m m e d i a t e l y before and after the response p r o d u c e d in the presence of GABA. Each point is the mean of at least 4 determinations in sep_parate experiments except the value obtained at log 7.5 (M) G A B A which is the m e a n of 2 determinations. Vertical bars represent S.E.M. Y o h i m b i n e (2.5 pM) was present in the superfusion fluids t h r o u g h o u t .

i

is

GABA IO0,uM

Fig. 4. The decrease with time in the inhibitory effect of G A B A on evoked [ 3 H ] n o r a d r e n a l i n e release in rat atria. The atria f r o m a single rat were incubated in [ 3 H ] n o r a d r e n a l i n e and superfused as indicated in fig. 1 except that y o h i m h i n e (2.5 p.M) was present t h r o u g h o u t the superfusion. Each histogram bar represents the increase (above basal) in tritium release ( d p m / 4 min sample: Ordinate) evoked by 1 min periods of stimulation (at 16 min intervals). G A B A (100 pM) was added 30 sec before the third period of stimulation and left in c o n t a c t with the tissue for t w o further periods of stimulation (hatched bars). After 30 min the r e d u c t i o n p r o d u c e d by G A B A was m a r k e d l y suppressed. The reapplication of G A B A ( a p p r o x i m a t e l y 60 min later) for 30 sec before stimulation p r o d u c e d the e x p e c t e d r e d u c t i o n in evoked release. Stimulation: 3 Hz, 0.5 msec, 10 V for I rain.

58 tion) the level decreased to 67%. This is not surprising since a reduction in noradrenaline release produced by GABA would allow the basal material to form a relatively larger a m o u n t of tritium unassociated with noradrenaline. This would suggest that GABA decreases the release of noradrenaline rather than metabolites. 3.2. A t r i a - - G A B A a n t a g o n i s t s

Bicuculline methobromide or methochloride (10-300 pM) affected neither the basal nor the evoked efflux of tritium from atria. More importantly these antagonists failed to modify the reduction in evoked release produced by GABA (6 e x p e r i m e n t s - example illustrated in fig. 6). In the experiment of fig. 6 phentolamine (2.5 pM) was used to antagonise a2receptors and enhance evoked release. The reduction in evoked release produced by GABA (100 gM) was clearly unaffected by the addition of bicuculline methobromide (100 pM) to the superfusion solution. A number of other recognised GABA antagonists have been tested in the same way. All of them failed to prevent the reduction in transmitter release produced by GABA. The substances tested include picrotoxin ( 170 pM), isopropylbicyclophosphate (200 pM), leptazol (100 pM), benzyl penicillin (2 mM) bemegride (1 mM) tubocurarine (1 mM) and frusemide (1 mM).

N.G. BOWERY ET AL mM. By contrast the ~ h l o r o p h e n y l derivative of GABA, baclofen, was as active as GABA in reducing the evoked release of [ 3H]noradrenaline from atria. The activity of baclofen in the atria resides in the (--)-enantiomer as shown in fig. 7. In the lower half of fig. 7 are the combined data from four experiments in which the reduction produced by various concentrations of the two baclofen enantiomers have been compared. (--)-Baclofen is clearly greater than 100 fold more active than the (+)-isomer. In the upper half of fig. 7 a comparison between the activities of GABA and the (--)-isomer is shown. The log dose/ response curves are virtually superimposed suggesting that a c o m m o n site of action is involved. 3.4. A t r i a - - c r o s s - d e s e n s i t i s a t i o n G A B A and baclofen

between

As pointed out above the depressant effects of GABA on the evoked release of [3H]noradrenaline from atria decreases with time. After 30 min continuous application GABA no longer reduced the evoked release. If baclofen (100pM) was applied during this period of 'desensitisation' it did not reduce the evoked release whereas the addition of carbachol (16.5 pM) (see Langer, 1977) still reduced the evoked release to the same extent as in the absence of GABA (fig. 8). On discontinuing the superfusion with GABA the response to baclofen returned.

3.3. A t r i a - - G A B A - r e l a ted agon ists

A variety of GABA-related substances were tested for mimetic activity in the atria. Surprisingly, many of t h e m which have been previously shown to mimic the depolarizing action of GABA in sympathetic ganglia were inactive in the atria (table 1). Even muscimol, though active, was considerably weaker (0.015) than GABA. 3-Aminopropane sulphonic acid, the sulphonic acid derivative of GABA which is at least as active as GABA at bicuculline-sensitive sites was completely inactive in the atria at concentrations up to 10

3.5. Vas d e f e r e n s - - t w i t c h r e s p o n s e to c o a x i a l stimulation : effect of GABA

The field-stimulated vas deferens has been used to assay drugs which depress noradrenaline outflow such as opiates and clonidinelike drugs. In view of the reduction in transmitter outflow from atria produced by GABA it seemed plausible that such an action would also produce a reduction in the twitch response of the vas. GABA (0.1-100 #M) did reduce the twitch response in a dose-dependent manner (fig. 9) but unlike clonidine and leucine-

59

BICUCULLINE-INSENSITIVE GABA RECEPTORS

3Hz "Sins/OV/mi~ o K + 50mM 2rain G ,, G A B A

|5

IOOA/M

3 0 s beforf • or(;

b

b

• s

3

• s

0 s

• s

0

e

0

0

e K

0

o K i

0

30

150

120

60 90 time (rain)

Fig. 5. Depression of the K+-evoked release of [3H]noradrenaline from rat atria by OABA. Atria from a single rat were ' l o a d e d ' with [3H]noradrenaline and superfused as in fig. 1. Yohimbine (2.5 p_M) was present throughout the superfusion. During the first part of the experiment the tissue was electrically stimulated fo~ 1 rain periods (as in fig. 1) at the dots ($, S). [3H]Noradrenaline release was stimulated in the latter part of the experiment by the addition of KCI (60 mM final concentration) to the superfusion fluid for 2 min periods. Each addition o f KC1 is indicated by the circles (o, K); GABA (G 100 pM) was added 30 sec before electrical or potassium stimulation and left in contact during the stimulation period. Clearly GABA reduced not only electrical but also K+-evoked release of [3H]noradrenaline.

18

~

5

¢. 12 9 6 3

•

•

•

•

•

0

•

•

• o

0 0

2

a

•

stimulation

GA BA IO0.uM h

Fig, 6. The inability o f bieucuUine methobromide to antagonise the inhibitory effect of GABA in rat atria. Experimental procedure as described in fig, 1. In this experiment phentolamine (2.5 pM) was added to the superfusion solution (during the period indicated by the horizontal bar) to antagonise a2-adrenoeeptors and enhance the electrically evoked release of [3H]-noradrenaline. Bicuculline methohromide (150 pM) was added as indicated by the lower horizontal bar. This did not prevent the reduction in evoked release produced by GABA (100 ]aM). GABA was added where indicated 30 see before and during stimulation.

60

N.G. B O W E R Y ET AL

• (-) Baclofen o OABA

6O

Fig. 7. T h e c o m p a r a t i v e i n h i b i t o r y p o t e n c i e s of t h e isomers o f b a c l o f e n a n d G A B A o n t h e evoked release of [3H] n o r a d r e n a l i n e f r o m rat atria. T h e data derive f r o m 8 s e p a r a t e e x p e r i m e n t s (4 for each graph) a n d have b e e n p l o t t e d in t h e m a n n e r described in fig. 3. Each p o i n t is t h e m e a n p e r c e n t a g e r e d u c t i o n ( o r d i n a t e ) f r o m at least 3 d e t e r m i n a t i o n s for each c o n c e n t r a t i o n or a single o b s e r v a t i o n . Vertical bars r e p r e s e n t S.E.M. In t h e l o w e r graph ( - - ) - b a c l o f e n (e) is c o m p a r e d w i t h its e n a n t i o m e r ( + ) - b a c l o f e n (c:) a n d in t h e u p p e r g r a p h ( - ) - b a c l o f e n ( e ) is c o m p a r e d w i t h G A B A (©) in t h e same e x p e r i m e n t s . Whilst G A B A a n d ( - - ) - b a c l o f e n were equiactive over t h e e n t i r e c o n c e n t r a t i o n range ( 1 0 0 / a M was m a x i m a l for b o t h ) , (+)-baclofen was m u c h less active.

]

40 20

I

10L7

I

i0-6 i0-5 Drug M

10-4

(+B~:~ 1

60-

: (-)Baclofen

T/~

4O.E "~ 20-

,o'-I Drug M

I

GABA IOO,u M

I 1

n baclofen/O0,wM

20

I

I corbachol /6"5~1MI I~ @AaA /OO~,M

16

3 0 s before st/re.

j

t 4 •

•

•

•

stim. 3Hz..5ms.

r[

O

I

O

|

I

I I

0 I

2

I

i

3

~

I

I

I

4

i o v for Imin I

h

Fig. 8. Occlusion b y G A B A of t h e i n h i b i t o r y a c t i o n of b a c l o f e n o n t h e e v o k e d release o f [ 3H ] n o r a d r e n a l i n e f r o m rat atria. Atria f r o m a single r a t were i n c u b a t e d a n d s u p e r f u s e d as i n d i c a t e d in fig. 1. Y o h i m b i n e (2.5 p M ) was p r e s e n t t h r o u g h o u t t h e s u p e r f u s i o n . G A B A (100/.aM) was a d d e d for 30 sec b e f o r e t h e s e c o n d p e r i o d o f t r a n s m u r a l s t i m u l a t i o n a n d left in c o n t a c t w i t h t h e tissue for t h e p e r i o d i n d i c a t e d b y t h e h o r i z o n t a l bar a b o v e t h e record. During this p e r i o d t h e i n h i b i t o r y a c t i o n o f G A B A o n t h e e v o k e d release decreased as e x p e c t e d (see fig. 4). Bac l o f e n ( 1 0 0 / a M ) a d d e d t o t h e s u p e r f u s i o n s o l u t i o n d u r i n g t h e p e r i o d o f p r o l o n g e d c o n t a c t w i t h G A B A failed t o decrease t h e e v o k e d release of [ 3 H ] n o r a d r e n a l i n e . By c o n t r a s t c a r b a c h o l ( 1 6 . 5 / a M ) did suppress t h e e v o k e d release. A f t e r t h e r e m o v a l of G A B A f r o m t h e s u p e r f u s i o n s o l u t i o n t h e r e a p p l i c a t i o n o f b a c l o f e n in t h e a b s e n c e o f G A B A n o w r e d u c e d t h e evoked release.

BICUCULLINE-INSENSITIVE GABA RECEPTORS

61

TABLE 1 C o m p a r a t i v e p o t e n c i e s o f G A B A a n a l o g u e s . M e a n values + S.E.M. f r o m 2 - - 1 0 d e t e r m i n a t i o n s f o r e a c h a n a l o g u e . Analogue

R a t atria: i n h i b i t i o n o f [ 3 H ] n o r a d r e n a l i n e release

M o u s e vas d e f e r e n s : twitch depression

EDs 0 (M)

Relative potency ( G A B A = 1)

EDs 0 (M)

GABA

4.2 + 1.4 x 10 -6

1

3.1 0.4

Muscimol

2.8 + 0 . 5 6 x 10 -4

0.015

~-HydroxyGABA

1.4 + 0 . 0 9 x 10 -4

Kojic a m i n e

4.2 +1.6 x 10 -3

Relative potency ( G A B A = 1)

Relative potency (GABA = 1 )

+ x 1 0 -6

1

1 ( E D s 0 -12.5 × 10 - ° )

2.6 0.4

+ x 10 -s

0.12

5.08 2

0.03

5.6 1.1

+ x 10 -s

0.055

0.27 3

0.001

1.1 0.4

+ × 10 -4

0.028

0.008

2.13 + 0.13 X 10 -4

0.014

0.005 3

N-MethylGABA Homoearnosine

7.0 + 3.0 X 10 .4

7-Hydroxybutyric acid 3-Aminopropane s u l p h o n i c acid ~ - A m i n o b u t y r i c acid Guanidnopropionic acid I m i d a z o l e a c e t i c acid I s o n i p e e o t i c acid Isoguvacine N i p e c o t i e acid THIP

nal

2,4-Diaminobutyric acid Arecaidine ~-Alanine Glycine (+)--Baclofen

na

(--)-Baclofen

4 . 2 -+

0.006

0.007

na +x 1 0 -4

0.014

na

2.2 1.1 na

na na

na na

na 0.12 3

na na na na 6.0 + 5.1 x 1 0 -4

na na na na

0 . 1 0 '* 0.011 ~ 0.23 2 na 0.05

na

0.0012 3

0.007

fla

3.4 3

na

na na 4.5 + 0 . 0 9 × 10 -6 0.8

(+)-Baclofen

Rat superior cervical ganglion : depolarization (bicueullinesensitive

0.93 1.00

x 1 0 -6

> 5 X 10 -4

<0.01

2.8 0.6

+ × 10 -6

1.11

0.01 3 na na

2.7

+

1.15

na

0.9

x 1 0 -6 <0.03

na

> 1 0 -4

1 na i n d i c a t e s p o t e n c y < 0 . 0 0 1 atria, < 0 . 0 0 3 vas d e f e r e n s , < 0 . 0 0 0 4 ganglia. 2 B o w e r y et al. ( 1 9 7 8 ) ; 3 B o w e r y a n d Brown (1974); 4 Bowery and Jones (1976).

62 enkephalin G A B A never p r o d u c e d a c o m p l e t e block of contraction. The mean m a x i m u m depression p r o d u c e d b y G A B A was similar in the m o u s e and guinea-pig p r e p a r a t i o n s (33.6 ± 6, n = 15, m o u s e ; 28 ± 2, n = 10, guinea-pig; m e a n ± S.E.M.% r e d u c t i o n in t w i t c h height). The EDs0 values for G A B A were also c o m parable 3.0 ± 0.4 pM ( m o u s e , n - - 12) and 3.3 ± 0.5 pM (guinea-pig, n = 20).

N.G. BOWERY ET AL 0.1 A

3.6. Vas d e f e r e n s - - G A B A a n t a g o n i s t s

Bicuculline m e t h o c h l o r i d e ( 3 0 0 pM) failed to p r e v e n t t h e t w i t c h r e d u c t i o n p r o d u c e d b y G A B A t h r o u g h o u t t h e effective dose range. No a n t a g o n i s m was observed in t h e m o u s e or guinea-pig preparations. As in the atria a n u m b e r o f c o m p o u n d s have been tested for possible a n t a g o n i s m o f t h e d e p r e s s a n t a c t i o n of G A B A . These included p i c r o t o x i n , strychnine, leptazol, bemegride, n a l o x o n e , a m a n t a dine and a - d i a m i n o p i m e l i c acid (all at 100 pM). No selective a n t a g o n i s m was o b t a i n e d with a n y o f these c o m p o u n d s . 3.7. Vas d e f e r e n s - - G A B A - r e l a t e d a g o n i s t s

A s u m m a r y o f the G A B A - r e l a t e d c o m p o u n d s w h i c h have been tested is s h o w n in table 1. The results are clearly c o m p a r a b l e with t h o s e o b t a i n e d in t h e atria. 3 - A m i n o p r o p a n e s u l p h o n i c acid and isoguvacine were c o m p l e t e l y inactive and m u s c i m o l was less active t h a n G A B A . Similarly b a c l o f e n was as active as G A B A the activity residing in the (--)-enantiomer. Comparative log dose/ response curves for G A B A . (±)-baclofen and m u s c i m o l in the m o u s e vas deferens are s h o w n in fig. 10a. The curves for b a c l o f e n and G A B A were s u p e r i m p o s e d and a l t h o u g h m u s c i m o l was less active the log d o s e / r e s p o n s e curve was parallel to t h a t o f G A B A . In fig. 10b are s h o w n t h e log d o s e / r e s p o n s e curves for these same agonists o n the electrically e v o k e d t w i t c h responses o f the guinea-pig ileum. G A B A , b a c l o f e n and m u s c i m o l also depressed the t w i t c h response o f this tissue. Their relative potencies were similar to t h o s e observed

Fig. 9. Inhibition by GABA of the twitch response to coaxial stimulation of the mouse vas deferens. The tissue was suspended under a tension of 100 mg in a 5 ml organ bath containing Mg2+-free Krebs solution at 37°C. Twitch responses were elicited by rectangular stimulation (1 msec pulses at 50 Hz for 100 msec at a voltage of 1.25 × maximal) every 10 sec. Cumulative doses of GABA were added at 1 min intervals as indicated by the dots above the record. The final GABA concentrations were 0.1, 0.3, 1.0, 3.0, 10 and 30 pM as indicated. GABA was finally washed out of the organ bath at the point marked by W.

in t h e vas deferens. T h e m a x i m a l inhibition p r o d u c e d in this p r e p a r a t i o n was 18.2 + 4.2% (n = 6) and t h e EDs0 for G A B A was 7.1 ± 0.5 t~M.

BICUCULLINE-INSENSITIVE GABA RECEPTORS

63

100o

GABA

•

Baciofen

~

Muscimol

100o GABA (n-12) •

Baclofen ( n - 6 )

,~ Muscirnol ( n - 6 ) 80 -t

a

"~ 60" o

_o:

.o

)

I=

.

E

'~ 40E E

20-

10-7

16-6

i0'-5

/~'--r--'~

~-4

-7

Conc. (M)

-6

-5

-4

Conc. (IOgl0M)

Fig. 10. C o m p a r a t i v e log d o s e / r e s p o n s e curves for G A B A , ( + ) - b a c l o f e n a n d m u s c i m o l o n t w i t c h r e s p o n s e s o f m o u s e vas d e f e r e n s (a) a n d guinea-pig i l e u m (b). T h e i n h i b i t o r y e f f e c t o f varying c o n c e n t r a t i o n s (log M abscissa) of G A B A (©), b a c l o f e n ( e ) or m u s c i m o l (A) is p l o t t e d in t h e ordinate as a p e r c e n t a g e of t h e m a x i m a l i n h i b i t i o n ( 1 0 0 % ) p r o d u c e d b y G A B A ( 1 0 0 p M ) w i t h i n t h e s a m e e x p e r i m e n t . In (a) t h e data derive f r o m 6 e x p e r i m e n t s for b a c l o f e n a n d m u s c i m o l a n d 12 e x p e r i m e n t s for G A B A . M e a n m a x i m a l i n h i b i t i o n was 32 + 4.9%. In (b) t h e data derive f r o m 5 e x p e r i m e n t s for each analogue. Mean m a x i m a l i n h i b i t i o n was 18.2 + 4.2%. G

BI ~

(3

Fig. 11. Failure o f b a c l o f e n ( 1 0 0 p_M) o r G A B A ( 1 0 0 p M ) t o increase t h e m a x i m a l t w i t c h i n h i b i t i o n prod u c e d b y each o t h e r in m o u s e vas deferens. T w i t c h

r e s p o n s e s were elicited as described in fig. 9. In t h e first s e c t i o n , G A B A ( G : 100 p_M) was a d d e d t o t h e b a t h i n g m e d i u m at t h e first arrow. This p r o d u c e d t h e e x p e c t e d d e p r e s s i o n in t h e t w i t c h response. B a c l o f e n (B: 1 0 0 p M ) was t h e n a d d e d b e f o r e r e m o v i n g t h e G A B A a n d n o f u r t h e r r e d u c t i o n of t h e t w i t c h r e s p o n s e o c c u r r e d . In t h e s e c o n d s e c t i o n G A B A a n d b a c l o f e n were a d d e d in reverse order. B a c l o f e n p r o d u c e d a depression comparable with that produced by GABA in t h e first s e c t i o n b u t t h e s u b s e q u e n t a d d i t i o n o f G A B A in t h e p r e s e n c e of b a c l o f e n p r o d u c e d n o f u r t h e r r e d u c t i o n . In t h e t h i r d s e c t i o n G A B A ( 1 0 0 p M ) was a d d e d at t h e first a r r o w a n d t h e n c l o n i d i n e (0.1 p M ) at t h e s e c o n d a r r o w in t h e c o n t i n u e d presence of GABA. Clonidine produced an additional decrease in t h e t w i t c h response. T h e r e c o r d i n g s were o b t a i n e d in t h e s a m e m o u s e vas deferens p r e p a r a t i o n .

64

3.8. Vas deferens -- lack o f additivity between GABA and baclofen

Additivity experiments were performed to check the possibility that baclofen (as well as the other active analogues) acts at the same site as GABA in the vas deferens. Supramaximal doses (300 pM) of GABA and baclofen which alone produced exactly the same partial reduction in twitch response were applied together. The combination did not produce a greater inhibition of the twitch height than either alone {fig. 11) whereas a combination of the same doses of either GABA or baclofen with a submaximal dose of leucine enkephalin or clonidine did produce a greater effect (fig. 11). 3.9. Vas deferens - effect o f GABA transport inhibitors

The knowledge that GABA uptake processes exist in peripheral tissues (Bowery and Brown, 1972; Brown and Galvan, 1977; Jessen et al., 1979) prompted us to consider the possibility that the action of GABA in the vas deferens may be limited by the presence of such uptake systems. Accordingly we have investigated the effect of drugs known to affect GABA uptake on the response to GABA in the guineapig vas deferens. Cumulative dose-response curves to GABA were obtained before and after the addition to the bath of 13-alanine (10 -4 M; 10 min) 2,4-diaminobutyric acid (DABA 10-%10 -s M; up to 15 min) or cis-3aminocyclohexanecarboxylic acid (ACHC 10-7-10 -5 M; 2 0 m i n ) . DABA (10 -5 M) potentiated the maximum inhibitory effect of GABA by 127 + 4% (n = 3) of the control response and reduced the dose ratio to 0.75 + 0.14 (mean +- S.E.M.) (fig. 12). This potentiation occurred irrespective of the pre-incubation time from 2 to 1 5 m i n . The same enhancement occurred following 20 min incubation in ACHC (10 -6 M). Very large enhancements (dose ratios less than 0.1 and two-fold potentiation of the maximum reduc-

N.G. BOWERY ET AI

tion by GABA) were occasionally seen in the presence of either DABA or ACHC. The simultaneous administration of DABA (10-SM) with GABA in two experiments decreased the maximum inhibitory effect of GABA (41% and 51% of normal) and increased the dose ratio (to 15 and 35 calculated at the ECs0). Thus only preincubation produced the potentiation. 13-Alanine, an inhibitor of glial GABA uptake (see Iversen and Kelly, 1975; Bowery et al., 1979b), did not affect the sensitivity of the vas to GABA. 3.10. Baclofen analogues

We have examined the activity of a number of ~-substituted GABA derivatives related to baclofen in the atria and vas deferens preparations. The compounds which were studied are listed in table 2. None of the derivatives were more active than baclofen in either the atria or vas deferens. For comparison, data obtained by Davies and Watkins (1974) on the activity of these analogues as inhibitors of cerebral neurone activity have also been included in table 2. There is a more marked similarly between these values and those obtained in the vas deferens than with those obtained in the atria. Nevertheless, the derivatives were all active in the atria whereas they were all inactive at 1 0 0 p M in displacing [3H]GABA specifically b o u n d to rat brain membrane fractions (5 nM [3H]GABA, 50 mM tris-citrate buffer pH 7.1, Bowery and Hill, unpublished observations; Galli et al., 1979) and in depolarizing sympathetic ganglion neurones (unpublished observations). Lack of activity in these latter two systems reflects an inability to act at bicuculline-sensitive GABA sites.

4. Discussion In line with our original supposition (see Introduction) GABA did reduce the evoked o u t p u t of sympathetic transmitter. However, this reduction did not appear to result from

BICUCULLINE-INSENSITIVE GABA RECEPTORS

65

16b

140

14

!20 /

120-

100 -

/

1008080-

/

/ //

6060

//

4040 20

O-

20-

-i

-4'

0

. . . .

S

-7

-6

-3

Fig. 12. Potentiation of the inhibitory action of GABA on twitch responses of the guinea-pig vas deferens by pretreatment with 2,4-diaminobutyric acid (DABA) or cis-3-aminocyclohexanecarboxylic acid (ACHC). Inhibition of twitch responses (elicited as described in fig. 9) is plotted on the ordinate as a percentage of-the maximal inhibition (100%) produced in the absence of DABA or ACHC. Abscissa: logM GABA concentration. In (a) the log dose/response curve to GABA is compared before (e) and after (~) adding DABA (10 #M) for 15 min to the organ bath in 3 experiments. In (b) comparison is made between GABA responses obtained before ($) and 20 min after (A) adding ACHC (10 pM) to the organ bath (3 experiments). Vertical bars represent S.E,M. Note the increase in maximal inhibition produced by GABA in the presence of either DABA or ACHC.

depolarization of the terminals. A presynaptic inhibitory effect of GABA on the release of transmitter from mammalian and amphibian sympathetic ganglia has been described (Brown and Higgins, 1979; Kato and Kuba, 1980). This also appeared to be unrelated to any depolarizing action and bicuculline and picrotoxin did not modify GABA's action. Although depolarization of autonomic nerve terminals occurs with GABA this effect can be blocked by bicuculline {Brown and Higgins, 1979; Galvan et al., 1981). The decrease in transmitter release observed in this present study was unaffected by high concentrations of bicuculline as well as other accepted GABA antagonists. In addition, the GABA mimetic 3-aminopropane sulphonic acid (Curtis et al.,

1961; Bowery and Brown, ]974; Zukin et al., 1974) which also mimics the depolarizing action of GABA on nerve terminals (Galvan et al., 1981) failed to mimic the depressant action of GABA in this study. By contrast, baclofen which depolarizes neither sympathetic ganglion neurones (Ault and Evans, 1978; Bowery et al., 1979a) nor preganglionic terminals (Galvan et al., 1981) and is not a GABA mimetic at bicuculline-sensitive receptors within the central nervous system (Curtis et al., 1974; Davies and Watkins, 1974; Olsen et al., 1978; Galli et al., 1979, Fox et al., 1978) reduced evoked ' [ 3H]noradrenaline output in atria and depressed twitch responses of the vas deferens. Despite the lack of a selective antagonist

66

N.G. BOWERY ET AL

TABLE 2 Activity o f ~-substituted G A B A derivatives at bicuculline-insensitive sites. Mean values f r o m at least 3 experim e n t s for each analogue. R

] NH2--C--C--C--COOH

Relative p o t e n c y ( G A B A = 1) Mouse vas deferens: twitch depression

Feline cortical n e u r o n e s 1. i n h i b i t i o n o f firing

R

Rat atria: inhibition of [ 3H ] n o r a d r e n a l i n e release

H

1

1

1

OH C1

0.03 + 0.002 < 0.01

0.05 -+ 0.01 <0.01

0.5

0.93 + 0.02

1.03 -+ 0.3

0.7

0.03-+0.02

0.5

-+0.2

0.6

0.12-+0.06

0.4

-+0.3

0.35

0.2

0.5

-+0.1

0.55

-+0.06

<0.01

Inactive

1 Data f r o m Davies and Watkins (1974).

0.2

-+0.07

Inactive

0.3

Excitatory

BICUCULLINE-INSENSITIVE GABA RECEPTORS our results suggest that baclofen and GABA act at the same site to depress transmitter release. This conclusion derives from their parallel log dose/response curves in the atria, vas deferens and ileum, their cross-desensitisation in atria and lack of additivity in vas deferens and their equal maximal responses in the three preparations. Further support derives from binding studies using radiolabelled [3H]baclofen (Hill and Bowery, 1981). Baclofen, GABA and their analogues inhibit saturable [3H] baclofen binding to rat synaptic membranes in accordance with their biological activity described in this present paper. The differences in pharmacological specificity between the GABA site on nerve terminals and sites elsewhere in the peripheral and central nervous systems suggest a separation of receptor types. On autonomic nerve terminals the receptor is not sensitive to bicuculline and the rank order of potency of agonists is baclofen = GABA > muscimol > > > 3-aminopropane sulphonic acid and isoguvacine. Whereas at 'classical sites' the rank order of agonists is muscimol > 3-aminopropane sulphonic acid = GABA > isoguvacine > > > baclofen, and sensitivity to bicuculline can be demonstrated. The division of GABA receptor types on the basis of agonist potency in mammalian systems has been noted before. For example, the GABA receptor mediating stimulation of benzodiazepines binding to rat brain membrane fractions may differ from the classical receptor in so far as 3-aminopropane sulphonic acid and isoguvacine are only partial agonists at low temperatures in vitro (Tallman et al., 1978; Braestrup et al., 1979). However, this site is still sensitive to the antagonist bicuculline. Mammalian GABA receptors which are insensitive to bicuculline have also been postulated (Johnston, 1976) on the basis of neuronal depressant action of certain GABA analogues of folded conformation which act even in the presence of bicuculline. However, what is not clear from these studies is whether GABA can mimic their depressant action in the presence of bicuculline.

67 It has been reported by Evans (1979) that baclofen and GABA do not reduce the twitch response of the mouse vas deferens. This lack of effect is difficult to reconcile with our findings and it is not clear why his results should be at variance with ours even though we have found some variation in sensitivity of the mouse vas deferens. This variation always occurred simultaneously with both GABA and baclofen and was independent of the sensitivity of the same preparation to clonidine. The guinea-pig vas deferens which has a similar sensitivity to GABA and baclofen as the mouse preparation appeared to be more consistent in its response to these agonists. We have never failed to obtain a reduction in twitch height with baclofen or GABA on over 50 consecutive guinea-pig preparations. The ability of baclofen to decrease peripheral transmitter efflux ~night lead one to expect to observe short-lasting effects on autonomic function following its administration in vivo. In support of this Fotherby et al., (1976) have reported that baclofen (1-20 mg/kg i.v.) diminished neurally evoked contractions of the rabbit and cat nictitating membrane. This decrease in response could have resulted from a reduction in transmitter release although the site of action was not defined in their study. The mechanism underlying the action of GABA (and baclofen) on nerve terminals probably differs from that associated with the depolarizing and hyperpolarizing action of GABA at peripheral and central sites. The depolarizing and hyperpolarizing actions of GABA appear to relate to a selective increase in membrane conductance to chloride ions. If chloride ions are removed from the external environment of the tissue or are injected into cells this diminishes or reverses the action of GABA (Adams and Brown, 1975; Krnjevic and Schwartz, 1966). The replacement of external chloride with impermeable sulphate anion in two atrial experiments d i d not prevent GABA depressing the evoked release even though release had been reduced by the decrease in chloride ion (unpublished observations).

68

A possible mechanism for the action of GABA may be to decrease inward Ca 2+ mobility since the release of neurotransmitters requires the influx of Ca ~+ ions during the terminal action potential (Katz and Miledi, 1967; 1969). Dunlap and Fischbach (1978) have shown that GABA (5 pM) decreases the influx of Ca :+ across the soma membrane of chick cultured dorsal root ganglion neurones in a bicuculline- and picrotoxin-insensitive manner. The decrease in influx occurs without any change in resting membrane conductance. As the authors point out if the same phenomenon occurs at nerve terminals this might effectively reduce transmitter release. The insensitivity to bicuculline and picrotoxin prompted Dunlap and Fischbach to suggest that 'another kind of GABA recep~ tor must be involved'. It is tempting to believe that the receptor envisaged by these authors is the same as that described in the present study. If so then baclofen b u t not 3-aminopropane sulphonic acid or isoguvacine should be expected to depress Ca 2÷ influx. The depressant action of baclofen on transmitter release is not confined to the periphery but extends to the central nervous system (Potashner, 1978; Bowery et al., 1980; Johnston et al., 1980). In fact it has been suggested that baclofen may exert its thera. peutic action in the treatment of spasticity by decreasing transmitter release within the central nervous system (Fox et al., 1978). Possibly this controlling effect on the release mechanism is mediated by the same GABA receptor as we describe in this present study. Whether a functional role can be ascribed to GABA at sites within the mammalian periphery remains unanswered. However, since GABA appears to be localised in peripheral neurones (Jessen et al., 1979) and neuronal uptake inhibitors can potentiate the action of applied GABA in the vas deferens this possibility must remain under consideration.

N.G. BOWERY ET AL

Acknowledgements DRH is an SRC Scholar. NGB wishes to thank CIBA Laboratories, Horsham for financial assistance We thank Mrs. J. Andrews for typing the manuscript

References Adams, P.R. and D.A. Brown, 1975, Actions of ")~aminobutyric acid on sympathetic ganglion cells, J. Physiol. London 250, 85. Anton, A.H. and D.F. Sayre, 1962, A study of th~ factors affecting the aluminum oxide-trihydroxy indole procedure for the analysis of catechol. amines, J. Pharmac. Exp. Ther. 138, 360. Ault, B. and R.H. Evans, 1978, Central depressant action of baclofen. J. Physiol. London 284,131P. Bowery, N.G. and D.A. Brown, 1972, 7-Aminobutyric acid uptake by sympathetic ganglia, Nature New Biol. 238, 89. Bowery, N.G. and D.A. Brown, 1974, Depolarizing actions of 7-aminobutyric acid and related compounds on rat superior cervical ganglia in vitro, Br. J. Pharmacol. 50, 205. Bowery, N.G., D.A. Brown, R.D. White and G. Yamini, 1979b, [aH]-7-Aminobutyric acid uptake into neuroglial cells of rat superior cervical sympathetic ganglia, J. Physiol. London 293, 51. Bowery, N.G., J.F. Collins, A.L. Hudson and M.J. Neal, 1978, Isoguvacine, isonipecotic acid, muscimol and N-methyl isoguvacine on the GABA receptor in rat sympathetic ganglia, Experientia 34, 1193. Bowery, N.G., A. Doble, D.R. Hill, A.L. Hudson, J.S. Shaw and M.J. Turnbull, 1979a, Baclofen: a selective agonist for a novel type of GABA receptor, Br. J. Pharmacol. 67,444P. Bowery, N.G., D.R. Hill, A.L. Hudson, A. Doble, D.N. Middlemiss, J. Shaw and M.J. Turnbull, 1980, (--)-Baclofen decreases neurotransmitter release in the mammalian CNS by an action at a novel GABA receptor, Nature 283, 92. Bowery, N.G. and A.L. Hudson, 1979, 7-Aminobutytic acid reduces the evoked release of [~H]-noradrenaline from sympathetic nerve terminals, Br. J. Pharmacol. 66,108P. Bowery, N.G. and G.P. Jones, 1976, A comparison of 7-aminobutyric acid and the semi-rigid analogues 4-aminotetrolic acid, 4-aminocrotonic acid and imidazole-4-acetic acid on the isolated superior cervical ganglion of the rat, Br. J. Pharmacol. 56, 323. Bowery, N.G., J.S. Shaw, M.J. Turnbull and Ruth Warrington, 1981, Inhibitors of neuronal GABA uptake potentiate the inhibitory effect of GABA on the field-stimulated guinea-pig vas deferens

BICUCULLINE-INSENSITIVE GABA RECEPTORS preparation, Br. J. Pharmacol. 72, 142P. Braestrup, C., M. Nielsen, P. Krogsgaard-Larsen and E. Falch, 1979, Partial agonists for brain GABA/ benzodiazepine receptor complex, Nature 280, 331. Brown, D.A. and M. Galvan, 1977, Influence of neuroglial transport on the action of 7-aminobutyric acid on mammalian ganglion cells, Br. J. Pharmacol. 59,373. Brown, D.A. and A.J. Higgins, 1979. Presynaptic effects of ~-aminobutyric acid in isolated rat superior cervical ganglia, Br. J. Pharmacol. 66, 108P. Brown, D.A. and S. Marsh, 1975, A very simple method for recording ganglion depolarization, J. Physiol. London 246, 24P. Curtis, D.R., C.J.A. Game, Johnston and R.M. McCulloch, 1974, Central effects of ~-(p-chlorophenyl)-~'-aminobutyric acid, Brain Res. 70, 493. Curtis, D.R., J.W. Phillis and J.C. Watkins, 1961, Actions of amino acids on the isolated hemisected spinal cord of the toad, Br. J. Pharmacol. 16, 262. Davies, J. and J.C. Watkins, 1974, The action of ~phenyl-GABA derivatives on neurones of the cat cerebral cortex, Brain Res. 70,501. De Groat, W.C., 1970, The actions of 7-aminobutyric acid and related amino acids on mammalian autonomic ganglia, J. Pharmac. Exp. Ther. 1 7 2 , 3 8 4 . Dudel, J. and S.W. Kuffler, 1961, Presynaptic inhibition at the crayfish neuromuscular junction, J. Physiol. London 155,543. Dunlap, K. and G.D. Fischbach, 1978, Neurotransmitters decrease the calcium component of sensory neurone action potentials, Nature 276, 837. Evans, R.H., 1979, Baelofen: lack of effect on neurotransmission in the mouse vas deferens, J. Pharm. Pharmacol. 31,642. Feltz, P. and M. Rasminsky, 1974, A model for the mode of action of GABA on primary afferent terminals: depolarizing effects of GABA applied iontophoretieally to neurones of mammalian dorsal root ganglia, Neuropharmacology 13,553. Fotherby, K.J., N.J. Morrish and R.W. Ryall, 1976, Is Lioresal (baclofen) an antagonist of substance P?, Brain Res. 113, 210. Fox, S., K. Krnjevic, M.E. Morris, E. Puil and R. Werman, 1978, Action of baclofen on mammalian synaptic transmission, Neuroscience 3,495. Galli, A., L. Zilletti, M. Scotton, G. Adembri and A. Giotti, 1979, Inhibition of Na÷-independent [3H]GABA binding to synaptic membranes of rat brain by f~-substituted GABA derivatives, J. Neurochem. 32, 1123. Galvan, M., P. Grafe and G. ten Bruggencate, 1981, Presynaptic actions of GABA on rat isolated sympathetic ganglia demonstrated in the presence of 4-aminopyridine, Br. J. Pharmacol. 72, 159P,

69 and in: Amino Acid Transmitters, Satellite Meeting held in France of the International Physiological Sciences Congress 1980, Proceedings (Raven Press. New York) (in press}. Graefe, K.H., F.J.E. Stefano and S.Z. Langer, 1973, Preferential metabolism of (--)-[3H]norepinephrine through the deaminated glycol in the rat vas deferens, Biochem. Pharmac. 22, 1147. Hill, D.R. and N.G. Bowery, 1981, JH-baclofen and 3H-GABA bind to bicuculline-insensitive GABA B sites in rat brain, Nature (in press}. Iversen, L.L., 1967, The Uptake and Storage of Noradrenaline in Sympathetic Nerves (Publ. Can'lbridge University Press}. Iversen, L.L. and J.S. Kelly, 1975, Uptake and metabolism of ~'-aminobutyric acid by neurones and glial cells, Biochem. Pharmacol. 24, 933. Jessen, K.R., R. Mirsky, M.E. Dennison and G. Burnstock, 1979, GABA may be a neurotransmitter in the vertebrate peripheral nervous system, Nature 281, 71. Johnston, G.A.R., 1976 Physiologic pharmacology of GABA and its antagonists in the vertebrate nervous system, in: GABA in Nervous System Function, eds. E. Roberts, T.N. Chase and D.B. Tower (Plenum Press, New York) p. 395. Johnston, G.A.R., M.H. Hailstone and C.G. Freeman, 1980, Baclofen stereospecific inhibition of excitant amino acid release, J. Pharm. Pharmacol. 32,230. Kato, E. and E. Kuba, 1980, Inhibition of transmitter release in bullfrog sympathetic ganglia induced by 7-aminobutyrie acid, J. Physiol. London 298, 271. Katz, B. and R. Miledi, 1967, Ionic requirements of synaptic transmitter release, Nature 215,651. Katz, B. and R. Miledi, 1969, Tetrodotoxin-resistant electric activity in presynaptic terminals, J. Physiol. London 203,459. Kimura, T., H. Imamura and K. Hashimoto, 1977, Facilitatory and inhibitory effects of 7-aminobutyric acid on ganglionic transmission in the sympathetic cardiac nerves of the dog, J. Pharm. Exp. Ther. 202,397. Kosterlitz, H.W. and A.J. Watt, 1968, Kinetic parameters of narcotic agonists and antagonists with particular reference to N-allylnoroxymorphone (naloxone), Br. J. Pharmacol. 33,266. Krnjevic, K. and S. Schwartz, 1966, Is 7-aminobutyric acid an inhibitory transmitter? Nature 211, 1372. Langer, S.Z., 1977, Presynaptic receptors and their role in the regulation of transmitter release, Br. J. Pharmacol. 6 0 , 4 8 1 . Langer, S.Z., E. Adler-Graschinsky and O. Giorgio, 1977, Physiological significance of 0~-adrenoreceptor-mediated negative feedback mechanism regulating noradrenaline release during nerve stimulation, Nature 265,648.

70 Olsen, R.W., D. Greenlee, P. Van Ness and M.K. Ticku, 1978, Studies on the gamma-aminobutyric acid receptor/ionophore proteins in mammalian brain, in: Amino acids as Chemical Transmitters, ed. F. F o n n u m (Plenum Press, New York). Potashner, S.J., 1979, Baclofen: effects on amino acid release and metabolism in slices of guinea-pig cerebral cortex, J. Neurochem. 32,103. Sangiah, S., J.C. Borowitz and G.K.W. Yim, 1974, Actions of GABA, picrotoxin and bicucuUine on adrenal medulla, European J. Pharmacol. 27,130.

N.G. BOWERY ET AL Shaw, J.S. and M.J. Turnbull, 1978, In vitro profile of some opioid pentapeptide analogues, European J. Pharmacol. 4 9 , 3 1 3 . Tallman, J.F., J.W. Thomas and D.W. Gallager, 1978, GABAergic modulation of benzodiazepine binding site sensitivity, Nature 274, 383. Zukin, S.R., A.B. Young and S.H. Snyder, 1974, Gamma-aminobutyric acid binding to receptor sites in rat central nervous system, Proc. Nat. Acad. Sci. U.S.A. 71, 4802.