Brain Research, 521 (1990) 143-147
143
Elsevier BRES 15655
Na+-dependent GABA transport system scavenges endogenous external GABA and prevents desensitization of GABAA receptors in rat cerebrocortical synaptoneurosomes Wha Bin Im, David P. Blakeman and John P. Davis CNS Diseases Research, The Upjohn Company, Kalamazoo, MI 49001 (U.S.A.) (Accepted 9 January 1990)
Key words: y-Aminobutyric acid A receptor; 36C1- uptake; Desensitization; Endogenous external y-aminobutyric acid; Na÷-dependent 7-aminobutyric acid transport system
Muscimol-induced 36C1- uptake in rat cerebrocortical synaptoneurosomes was reduced upon exposure of the membrane sacs to low Na ÷ media. This Na ÷ requirement led us to examine the role of the Na÷-dependent y-aminobutyric acid (GABA) transport system in 36C1-uptake. Incubation of the synaptoneurosomes with nipecotic acid, a specific inhibitor of the GABA transport system, for 10 min increased the level of endogenous external G A B A from less than 10 to 150 /tM and induced the same signs of desensitization as observed with high muscimoi-treated synaptoneurosomes; a marked reduction of muscimol-induced 36C1-uptake and an appearance of a slow bicuculline-sensitive 36C1- uptake, probably due to a continuous recovery of a population of GABA A receptors from desensitization. Similar results were obtained upon dissipation of Na ÷ electrochemical gradient across the membranes by inhibition of Na ÷,K÷-ATPase with ouabain or by blocking energy metabolism with azide or N-ethylmaleimide. We propose that the Na+-dependent GABA transport system, its operation being dependent on inwardly directed Na ÷ electrochemical gradient, is responsible for scavenging endogenous GABA released from the synaptoneurosomes, and thus prevents desensitization of GABA A receptors.
MATERIALS AND METHODS
INTRODUCTION y - A m i n o b u t y r i c acid ( G A B A )
is a u b i q u i t o u s inhibi-
t o r y n e u r o t r a n s m i t t e r in the m a m m a l i a n central n e r v o u s s y s t e m , and i n d u c e s s y n a p t i c m e m b r a n e c h l o r i d e c o n d u c tance
by i n t e r a c t i o n
with
G A B A A r e c e p t o r s 1'5. T h e
G A B A - r e g u l a t e d c h l o r i d e c h a n n e l is a r e c e p t o r c o m p l e x c o n t a i n i n g s e p a r a t e binding sites for a variety o f psyc h o a c t i v e drugs, i.e. b e n z o d i a z e p i n e s , i m i d a z o p y r i d i n e s , b a r b i t u r a t e s , m e t a b o l i t e s of p r o g e s t e r o n e , t-butylbicyclop h o s p h o r o t h i o n a t e , a v e r m e c t i n B, and p i c r o t o x i n 3'4' 6,9,12,19-21 R e c e n t l y , c e r e b r o c o r t i c a l s y n a p t o n e u r o s o m e s f r o m t h e rat, m o u s e a n d g u i n e a pig h a v e b e e n s h o w n to t r a n s p o r t 36Cl- in r e s p o n s e to G A B A or G A B A m i m e t i c s 7'8'16 and h a v e b e e n widely used to study pharmacological
and
biochemical
actions
of v a r i o u s
ligands for the d i f f e r e n t allosteric sites on G A B A A r e c e p t o r c o m p l e x e s 13'14'18. In this study, w e h a v e obs e r v e d the d e p e n d e n c y o f m u s c i m o l - i n d u c e d 36C1- u p t a k e on N a ÷ g r a d i e n t in rat c e r e b r o c o r t i c a l s y n a p t o n e u r o s o m e s and p r o p o s e that o p e r a t i o n of the N a ÷ - d e p e n d e n t G A B A t r a n s p o r t system is essential in p r e v e n t i n g desensitization o f G A B A A r e c e p t o r s .
Rat cerebrocortical synaptoneurosomes were prepared following the procedures reported elsewhere 8. Briefly, cerebral cortices from 4 male Sprague-Dawley rats were suspended in 30 ml ice-cold solution containing (in mM): NaC1 118, KCi 5, MgSO 4 1.18, CaCI2 2.5, HEPES/Tris 20; pH 7.0 (buffer A), and D-glucose 10 and were homogenized in a Dounce homogenizer by 8 strokes with a loose-fitting pestle and then by 5 strokes with a tight-fitting pestle. The suspension was diluted to 160 ml with buffer A, and filtered through 3 layers of nylon cloth and 10-/~m Millipore filter (LC type). The filtrates were centrifuged at 3000 x g for 15 min. The membranous pellets were washed once and resuspended in buffer A or indicated buffers to a final protein concentration of 8 mg/ml. 36CI- uptake in the synaptoneurosomes was measured by a rapid filtration technique using Whatman GF/B glass microfiber filters TM with some modifications. Typical incubation medium contained 0.2 /aCi Na36Cl/ml (ICN), 118 mM NaC1, 5 mM KC1, 1.8 mM MgSO 4, 2.5 mM CaC12, 20 mM HEPES/Tris, pH 7.0, with or without testing drugs. To study the effect of Na + on muscimol-induced 36Eluptake, the concentration of Na + was varied from 0 to 140 mM by replacing Na + isoosmotically with N-methylglucamine while maintaining other ingredients the same. The same buffers without 36Elwere used to suspend membranes. The membrane suspensions were incubated for 5 min at 30 °C and, when indicated, metabolic inhibitors were introduced to the membrane during the preincubation period. The reaction was initiated by mixing equal volumes (125 ~1) of the membrane suspension (1 mg protein) and the isotope medium at 30 °C. After 5 s, unless indicated otherwise, uptake was terminated by adding ice-cold buffer A containing 500/~M bicucul-
Correspondence: W.B. Im, CNS Research 7251-209-1, The Upjohn Company, Kalamazoo, MI 49001, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
144 line or 1 mM of 4,4"-diisothiocyantostilbene-2,2"-disulfonic acid (DIDS) to minimize possible leakage of 3~'CI from the synaptoneurosomes during the termination processes. DIDS has been shown to block the effiux of 3~'C1 from the synaptoneurosomes 17. The mixture was filtered over a GF/B filter under vacuum, and the filters were washed 3 times with 15 ml of ice cold buffer A. The radioactivity was counted after mixing the filters with 15 ml Instagel. Bicuculline-sensitive 36C1 uptake refers to the portion inhibited by 500 jtM bicuculline methiodide during the reaction. To measure chloride uptake through G A B A A channels during desensitization or drug treatment, the membranes were exposed to 100 I~M muscimol or drugs for 2-5 min at 30 °C, and then mixed with the medium containing 3~'C1 and the usual ionic components of buffer A, with or without 500 ~uM bicuculline methiodide. The level of endogenous GABA in the medium was measured using the GABASE system for Pseudomonasfluorescence (Sigma). Briefly, the assay system contains ),-aminobutyric acid glutamic transaminase (0.02 U), succinic semialdehyde dehydrogenase (0.02 U), NADP (0.2 mM), a sample (100/d) or a known amount of G A B A , and 100 mM potassium pyrophosphate, pH 8.6, in a total volume of 0.95 ml. The reaction was initiated by adding 50/A of 20 mM a-ketoglutarate in the pyrophosphate buffer. The absorbance change at 340 nm was measured after reaction for 45 min at room temperature. The assays give a linear response up to at least 120 nmol GABA/ml assay medium with experimental variations less than 15%, at the concentration of GABA above 2 nmol/ml. The suspension of the synaptoneurosomes which were concentrated (16 mg protein/ml) twice as much as those used for 36C1 uptake (8 mg protein/ml) were treated with the Na+-free buffer (N-methylglucamine replacing Na+), nipecotic acid (5 mM), NEM (1 mM), azide (5 mM), or ouabain (1 mM) for 10 rain and centrifuged for 30 s using an Eppendorf microcentrifuge. An aliquot (100 ,ul) of the supernatant was analyzed. In the case of the NEM-treated sample, excess mercaptan (3 times) was added to neutralize residual NEM. Na 3~C1 was obtained from New England Nuclear. Bicuculline methiodide, DIDS, ouabain, azide, and N-ethylmaleimide were purchased from Sigma.
50
•=-
Muscimol =.° 40 /¢/~=
r=
/
30
GABA
E 20
10 ¢,J
0 0
i i i // , 40 80 120 200 [Muscimol or GABA]~_M
Fig. 1. Dose-response profiles for muscimol- and GABA-mediated 36C1 uptake in rat cerebrocortical synaptoneurosomes. Aliquots (125 ~1) of the synaptoneurosome suspensions were incubated at 30 °C for 5 min, and then mixed with an equal volume of the NaCI-36CI buffer containing final concentrations of G A B A or muscimol varying from 5 to 200/~M. The reaction was terminated 5 s later. The radioactivity associated with the synaptoneurosomes was determined using the rapid filtration techniques as described under Materials and Methods. The baseline 36C1-level was obtained from the same set of experiments without G A B A or muscimol and was used to compute GABA- or muscimol-mediated 36C1-uptake. The values represent the mean of triplicate measurements from a typical experiment. The experimental variations were less than 10%.
s u s p e n d e d a n d p r e i n c u b a t e d in t h e b u f f e r c o n t a i n i n g h i g h N a + ( > 1 0 0 m M ) , t h e 36C1- u p t a k e w a s n o t a f f e c t e d , e v e n though
the
final
Na + concentration
of the
reaction
m i x t u r e w a s l o w e r e d b e l o w 40 m M b y e m p l o y i n g t h e RESULTS 36C1- u p t a k e in r a t c e r e b r o c o r t i c a l s y n a p t o n e u r o s o m e s was d o s e - d e p e n d e n t l y s t i m u l a t e d b y G A B A
100
and musci-
m o l (Fig. 1) in g e n e r a l a g r e e m e n t w i t h e a r l i e r r e p o r t s 7'~8. This muscimol-induced bicuculline with an
36C1- u p t a k e was i n h i b i t e d b y
IC5o v a l u e o f 70 ktM. T h e b a s e l i n e
36C1- level (in t h e a b s e n c e o f e x o g e n o u s G A B A
mime-
tics), o n t h e o t h e r h a n d , was n o t n o t i c e a b l y a f f e c t e d b y b i c u c u l l i n e at 500 ktM, p r o b a b l y r e f l e c t i n g low e n d o g e nous
GABA
in
the
medium.
Replacing
Na +
with
N - m e t h y l g l u c a m i n e in t h e m e m b r a n e s u s p e n s i o n a n d t h e 36C1- r e a c t i o n b u f f e r s r e d u c e d t h e b i c u c u l l i n e - s e n s i t i v e muscimol-mediated sive c h a n g e s
36C1- u p t a k e . Fig. 2 s h o w s p r o g r e s -
in 36Cl
uptake
as a f u n c t i o n
80 --E ~E
of Na +
c o n c e n t r a t i o n s u n d e r a c o n s t a n t level of C1 at 140 m M a n d m u s c i m o l at 100 ~ M . T h e C1- u p t a k e was r e d u c e d by 5 0 % as t h e c o n c e n t r a t i o n of N a + was l o w e r e d f r o m 140 to 30 m M , a n d r e a c h e d 2 0 % o f t h e m a x i m u m level w h e n N a + was c o m p l e t e l y r e p l a c e d w i t h N - m e t h y i g l u c a m i n e . T h e p r e s e n c e o f h i g h N a + in t h e m e m b r a n e s u s p e n d i n g b u f f e r d u r i n g t h e p r e i n c u b a t i o n p e r i o d (5 m i n at 30 °C) a p p e a r e d t o b e t h e p a r t i c u l a r l y critical f a c t o r for 36C1 uptake. For example, when the synaptoneurosomes were
60
¢¢
40
._z ~-
20
~Z~
0 i 0
I
I
I
I
I
I
[
40 80 120 [Na÷] mM
Fig. 2. Effect of varying Na + concentrations in the membrane suspension and the 36C1-reaction buffers on muscimol-induced 36C[uptake in rat cerebrocortical synaptoneurosomes. The synaptoneurosomes were prepared in the typical NaCl-glucose buffer containing 118 mM NaCI, 5 mM KC1, 1.18 mM MgSO 4, 2.5 mM CaCI 2, 10 mM glucose, 20 mM HEPES/Tris, pH 7.0, but were resuspended in the media containing indicated concentrations of Na + varying from 0 to 140 raM. Na + was replaced isoosmotically with N-methylglucamine. Aliquots (125 /d) of the membrane suspensions were incubated for 5 min at 30 °C and were mixed with the 36C1 reaction buffer of the same composition as the membrane suspension buffer with or without muscimol (final concentration 100 ~M). The reaction was terminated 5 s later and the rest of the experimental procedure was the same as described in the legend of Fig. 1.
145
IA3o
TABLE I
Increases in endogenous external GABA level in the suspension of rat cerebrocortical synaptoneurosomes by blocking Na+-dependent GABA transport system or dissipation of membrane Na + electrochemicalgradiem The synaptoneurosomes were suspended in the NaCl buffer to a final protein concentration of 16 mg/ml. Then an aliquot (500/A) of the membrane suspensions was incubated at 30 °C with 5 mM nipecotic acid, 1 mM NEM, 5 mM azide or 1 mM ouabain. In the case of Na+-ffee medium, the synaptoneurosomes were suspended in the medium containing 118 mM N-methylglucamine in place of Na ÷ , and then were incubated as above. After incubation for 10 min, the suspensions were centrifuged using an Eppendorf microcentrifuge for 30 s. An aliquot (100/~1) of the supernatants was analyzed for G A B A level using the GABASE system as described in Materials and Methods. In the case of NEM-treated synaptoneurosomes, 2-mercaptan was added in excess (3 times). The values represent the mean of duplicate measurements and experimental variations were less than 15% when the G A B A level was greater than 20/~M in the sample.
Additions
GABA (pM)
N o addition Nipecotic acid 5 mM Na÷-free medium NEM 1 mM Azide 5 mM Ouabain 1 mM
< l0 150 72 109 75 110
N a + - f r e e 36C1- r e a c t i o n b u f f e r and the m e m b r a n e susp e n s i o n t w i c e m o r e c o n c e n t r a t e d t h a n the usual o n e . F u r t h e r m o r e , m u s c i m o l ( 1 0 0 / x M ) , u n d e r o p t i m a l chloride t r a n s p o r t
conditions,
i n d u c e d n o 22Na+ u p t a k e .
TABLE II
Effect of nipecotic acid on muscimol-stimulated 36C1~uptake in rat cerebrocortical synaptoneurosomes An aliquot (125/~i) of the synaptoneurosome suspensions in 118 mM NaCI, 5 mM KCI, 1.18 mM MgSO 4, 2.5 mM CaCI2, 10 mM D-glucose, and 20 mM HEPES/Tris, pH 7.0, was preincubated with 5 mM nipeeotic acid for 2, 4 and 10 rain at 30 °C. The reaction was initiated by adding an equal volume of 36C1- NaC1 buffer containing muscimol to give a final concentration of 100/~M, and was terminated 5 s later. The radioactivity associated with the synaptoneurosomes was determined using the rapid filtration technique as described under Materials and Methods. When nipecotic acid was added with 36C1-, the treatment was designated to be a 5-s one. The baseline 36C1- level was obtained from the same set of experiments without muscimol and was subtracted from the value obtained with muscimol to yield the agonist-induced 36C1 uptake.
Pre~eatment
No addition Nipecotic acid (5 mM) for 5s 2 min 4rain 10min
Muscimol-stimulated 36C1-uptake
B
50
Pentobarbital + Muscimol
40
30 ~~E 20
~.
.~'~
~'~
10
10 0 0
I
1//...-.3
1
2
10
0
Preincubation Time, min
1
2o
Time,min
Fig. 3. Plots showing constant rates of bicuculline-sensitive 36Eluptake by rat cerebrocortical synaptoneurosomes in the continuous presence of 100 pM muscimol and time course profiles for the 36Cluptake. A: the suspensions of the synaptoneurosomes were incubated in the presence of 100 pM muscimol for 10, 20, 30, 40, 60 s, 2 and l0 min. 0 s represents the addition of muscimol with 36C!-. An aliquot (125 pl) of the membrane suspensions was mixed with an equal volume of the NaCI-36CI- buffer with (0) or without (O) bicuculline methiodide (final concentration of 500 pM). The reaction was terminated 5 s later. B: the membrane suspensions were incubated in the presence or absence of 200/tM pentobarbital and/or 100 pM muscimol for 2 min. An aliquot of the membrane suspensions was mixed with an equal volume of the NaC1-36C1buffer with or without bicuculline (final concentration of 500 ~M). The reaction was terminated 5, 10, 20, 30, 40, 60 and 120 s later as described under Materials and Methods. The values for bicucullinesensitive 36C1 uptake were obtained from the differences in the presence or absence of bicuculline. The control values represent those obtained without exogenous muscimol, otherwise under identical conditions. The values in A represent the mean of duplicate measurements, and those in B with a bar represent the mean of triplicate measurements with experimental variations. (3, muscimol; O, muscimol + pentobarbital; Zk, the membrane suspensions were treated with 1 mM NEM.
T h e s e o b s e r v a t i o n s suggest that t h e a p p a r e n t N a + effect arises f r o m c h a n g e s in m e m b r a n e e n v i r o n m e n t s , and led us to e x a m i n e the roles of e n d o g e n o u s e x t e r n a l G A B A and N a + - d e p e n d e n t
GABA
t r a n s p o r t systems in 36Cl-
uptake. T r e a t m e n t o f t h e s y n a p t o n e u r o s o m e s with 5 m M n i p e c o t i c acid, a specific i n h i b i t o r of t h e N a + - d e p e n d e n t G A B A t r a n s p o r t system 11 for 10 rain i n c r e a s e d the l e v e l of e n d o g e n o u s G A B A to 150 p M
in the m e d i u m f r o m less t h a n 10
as m e a s u r e d
with the G A B A S E
system.
R e p l a c i n g N a + with N - m e t h y l g l u c a m i n e also l e d to an a c c u m u l a t i o n of e n d o g e n o u s G A B A in t h e m e d i u m to 72 /2M (Table I). Table II shows that n i p e c o t i c acid b l o c k e d m u s c i m o l -
nmol/mg protein/5 s
%
i n d u c e d c h l o r i d e u p t a k e as a f u n c t i o n o f its e x p o s u r e p e r i o d to m e m b r a n e s . F o r e x a m p l e , t h e drug as a d d e d
42.5 + 2.3
100
t o g e t h e r with 36C1- (5-s e x p o s u r e ) s h o w e d no n o t i c e a b l e
41.4 + 1.5 28.2 + 1.4 21.6_+ 1.1 8.1 __+_2.3
97 66 50 19
effect, but u p o n p r e i n c u b a t i o n with the s y n a p t o n e u r o s o m e s for 2, 4 and 10 rain, r e d u c e d the m u s c i m o l - i n d u c e d 36C1-- u p t a k e to 66, 50, and 1 9 % of the u n t r e a t e d , r e s p e c t i v e l y . A t the s a m e t i m e , t h e basal 36C1- level
146 TABLE III Inhibition o f muscimol-induced ~6C~ uptake in the synaptoneurosornes treated with azide, NEM, or ouabain
The suspensions of synaptoneurosomes were preincubated at 30 °C with drugs at the indicated concentration for 5 min. The reaction was initiated by adding the NaCl-36CI buffer with or without muscimol (a final concentration of 100/~M). Other experimental details were the same as described in Table I. The values represent the mean and standard deviations from at least 3 separate experiments or a single experiment of triplicate measurements. The experimental variations within the triplicate were less than 10%.
Treatment
36C1--Uptake (nmol/mg protein/5 s) Muscimol. . . . . . . . . . . . . Muscimol (l OOktM)
Control Azide 5 mM NEM1 mM Ouabain 1 mM
-
+
27.1 + 0.4 37.2+1.9 36.9±0.6 38.2 + 1.3
74.1 ± 4.9 48.7+3.6 45.7±4.1 42.1 + 5.2
induced~6 Cluptake (%)
47 (100) 11.5(25) 8.8(19) 3.9 (8)
m e a s u r e d without exogenous G A B A agonists rose from 27.1 to 34.6 nmol/mg protein/5 s upon preincubation of the s y n a p t o n e u r o s o m e s with nipecotic acid for 10 min. The increased portion of 36C1- uptake was sensitive to bicuculline (500 ~ M ) . These effects of nipecotic acid were readily reversed upon washing the treated synaptoneurosomes, and were similar to the chloride transport characteristics of G A B A A receptors under desensitizing conditions as shown in Fig. 3. Treatment of the synapt o n e u r o s o m e s with high muscimol (100 ~ M ) reduced, within 5 s, the level of bicuculline-sensitive CI- u p t a k e to 2 0 - 2 5 % of the maximal rate (undesensitized conditions),
and the residual rate r e m a i n e d unchanged during 10 min with high muscimoi (Fig. 3A). The time course profiles (Fig. 3B) indicate that the bicuculline-sensitive CI uptake under the desensitizing conditions increased linearly during the first 15 s and reached an equilibrium level of 35 nmol/mg protein with a t:~ of 20 s. It should be noted that in the absence of exogenous agonists, the bicuculline-sensitive C I uptake in the synaptoneurosomes increased from 0 to merely 4 nmol/mg protein during the same uptake period (2 rain). F u r t h e r m o r e , p e n t o b a r b i t a l (200 ~ M ) s h o r t e n e d the t,~ value for the slow bicuculline-sensitive C1- u p t a k e to 4 s without affecting the equilibrium level. The sensitivities of the slow C1- uptake to bicuculline and p e n t o b a r b i t a l indicate that G A B A A receptors, even under desensitizing conditions, m e d i a t e d 36C1-flux, albeit at a r e d u c e d rate. Similar desensitizing conditions were p r o d u c e d upon abolishing m e m b r a n e Na + electrochemical gradient by inhibiting N a + , K + - A T P a s e with ouabain, or blocking energy metabolism with azide and N E M . N E M (1 mM), azide (5 mM), and ouabain (1 raM), upon interaction with the s y n a p t o n e u r o s o m e s for 5 rain, reduced the muscimol-induced 36C1 uptake by 81, 75 and 92%, respectively, and at the same time increased the basal 36CF level from 27.1 to 36.9, 37.2 and 38.2 nmol/mg protein/5 s, respectively. The increased portions of the 36C1- uptake were blocked by bicuculline at 500 ktM, and showed a time course profile similar to that o b t a i n e d with the muscimol-desensitized receptors, as shown in Fig. 3 for the N E M - t r e a t e d and in Table IV for the ouabainand azide-treated synaptoneurosomes. A l s o , the level of endogenous G A B A in the m e d i u m increased to 109, 110 and 75 ¢tM upon treatments with N E M (1 mM), ouabain (1 raM), or azide (5 raM), respectively (Table I).
TABLE IV Increases in bicuculline-sensitive 36C1- uptake in rat cerebrocortical synaptoneurosomes upon desensitization with muscimol or treatment with NEM, azide or ouabain
The suspensions of the synaptoneurosomes were exposed to 100/~M muscimol, 1 mM NEM, 5 mM azide or 1 mM ouabain at 30 °C for 5 min. The reaction was then initiated by adding the NaCI-36CI- buffer with or without 1 mM bicueulline methiodide (final concentration 500/~M), and was terminated 30 s later. The rest of the experimental details were the same as described in Table I. The bicucullinesensitive uptake was computed from the differences in 36C1- uptake measured with and without bicueulline methiodide. The values represent the mean + S.D. of 3 experiments done in triplicate measurements. Pretreatment
Bicuculline-sensitive 36Cl- uptake (nmol/mg protein/30 s)
No addition Muscimo1100/~M NEM 1 mM Azide 5 mM Ouabain 1 mM
1.91 _-+0.4 24.3 -L-_3.2 21.7 + 1.1 16.9 + 0.7 20.5 -+ 3.4
DISCUSSION The m a j o r point of this r e p o r t is that the Na ÷d e p e n d e n t G A B A transport system scavenges endogenous external G A B A and prevents desensitization of G A B A A receptors in rat cerebrocortical synaptoneurosomes. We have found that inhibition of the Na +d e p e n d e n t G A B A transport system by a specific inhibitor, nipecotic acid, m a r k e d l y enhanced endogenous external G A B A (over 100/~M) and elicited the signs of desensitization; i.e. m a r k e d reduction of muscimolinduced 36C1 uptake, and an increase in slow, bicuculline-sensitive 36C1- uptake without exogenous G A B A mimetics. Also, similar desensitizing conditions were p r o d u c e d upon abolishing the N a ÷ electrochemical gradient by removal of N a ÷ from m e m b r a n e suspending m e d i u m , inhibition of N a + , K + - A T P a s e or interference with energy metabolism by azide and N E M . These
147 observations are consistent with the notion that dissipation of m e m b r a n e N a ÷ electrochemical gradient caused malfunction of N a ÷ - d e p e n d e n t G A B A transport system and led to an accumulation of e n d o g e n o u s external G A B A in rat cerebrocortical synaptoneurosomes. Of some interest u n d e r desensitizing conditions is the a p p e a r a n c e of a slow, bicuculline-sensitive 36C1- uptake. Several electrophysiological studies and earlier 36Clu p t a k e studies 1°'15'18 have also r e p o r t e d the decline of the agonist-induced anion m o v e m e n t s to a new low, steadystate level in the presence of high agonists. Its nature, however, has not b e e n clear. We have felt that the a p p e a r a n c e of the bicuculline-sensitive CI- uptake under desensitizing conditions is not likely due to a slow desensitization process since the rate of the C1- uptake was unchanged during the continuous presence of high
REFERENCES 1 Bormann, J., Electrophysiology of GABA A and GABA B receptor subtypes, Trends Neurosci., 11 (1988) 112-116. 2 Cash, D.J. and Subbarao, K., Desensitization of y-aminobutyric acid receptor from rat brain: two distinguishable receptors on the same membrane, Biochemistry, 26 (1987) 7556-7562. 3 Costa, E., The role of gamma-aminobutyric acid in the action of 1,4-benzodiazepines, Trends Pharmacol. Sci., 1 (1979) 41-44. 4 Costa, E. and Guidotti, A., Molecular mechanisms in the receptor action of benzodiazepines, Annu. Rev. Pharmacol., 19 (1979) 531-545. 5 Curtis, D.R,, Duggan, A.W., Felix, D. and Johnston, G.A.R., Bicuculline, an antagonist of GABA and synaptic inhibition in the spinal cord of the cat, Brain Research, 32 (1971) 69-96. 6 Haefely, W., Kyburz, E., Gerecke, M. and Mohler, H., Recent advances in the molecular pharmacology of benzodiazepine receptors and in the structure-activity relationship of their agonists and antagonists, Advanc. Drug Res., 14 (1985) 166-322. 7 Harris, R.A. and Allan, A.M., Functional coupling of yaminobutyric acid receptor in chloride channels in brain membranes, Science, 228 (1985) 1108-1109. 8 Hollingsworth, E.B., McNeal, E.T., Burton, J.L., Williams, R.J., Daly, J.W. and Creveling, C.R., Biochemical characterization of a filtered synaptoneurosome preparation from guinea pig cerebral cortex: cyclic adenosine 3",5"-monophosphate generating systems, receptor and enzymes, J. Neurosci., 5 (1985) 2240-2253. 9 Kass, I.S., Wang, C.C., Walrond, J.P. and Stretton, A.O.W., Avermectin B1A, a paralyzing anthelmintic that affects interneurons and inhibitory motor neurons in Ascaris, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 6211-6215. 10 Krnjevic, K,, Desensitization of GABA receptors. In E. Costa, G. DiChard and G.L. Gessa (Eds.), GABA and Benzodiazepine Receptors, Raven, New York, 1981, pp. 111-120. 11 Krogsgaard-Larsen, P. and Johnston, G.A.R., Inhibition of GABA uptake in rat brain slices by nipecotic acid, various isoxazole and related compounds, J. Neurochem., 25 (1975)
muscimol for 10 min, and recent kinetic studies have detected only two quick desensitization phases of G A B A A receptors with a t,~ of 32 and 533 ms in the rat cerebrocortical s y n a p t o n e u r o s o m e s 2. R a t h e r , the slow uptake m a y arise from the continuous recovery of a small population of G A B A A receptors from desensitization, which is known to be a slow process 15. We have observed that the slow bicuculline-sensitive 36C1- u p t a k e in the s y n a p t o n e u r o s o m e s t r e a t e d with ouabain, N E M , azide and nipecotic acid was largely indistinguishable from that observed in muscimol-desensitized ones. This could be interpreted to m e a n that these drugs, by themselves, have no effect on the chloride channel activity, and their inhibitory effect on muscimol-induced 36C1- u p t a k e arises solely from desensitization of G A B A A receptors by raising e n d o g e n o u s external G A B A .
797-802. 12 Majewska, M.D., Harrison, N.L., Schwartz, R.D., Barker, J.L. and Paul, S.M., Steroid hormone metabolites are barbituratelike modulators of the GABA A receptor, Science, 232 (1986) 1004-1007. 13 Morrow, A.L. and Paul, S.M., Benzodiazepine enhancement of y-aminobutyric acid-mediated chloride ion flux in rat brain synaptoneurosomes, J. Neurochem., 50 (1988) 302-306. 14 Obata, T. and Yamamura, H.I., The effect of benzodiazepines and fl-carbolines on GABA-stimulated chloride influx by membrane vesicles from the rat cerebral cortex, Biochem. Biophys. Res. Commun., 141 (1986) 1-6. 15 Sakmann, B., Patlak, J. and Neher, E., Single acetylcholineactivated channels show burst-kinetics in presence of desensitizing concentrations of agonist, Nature (Lond.), 286 (1980) 71-73. 16 Schwartz, R.D., Skolnick, P., Hollingworth, E.B. and Paul, S.M., Barbiturate and picrotoxin-sensitive chloride efflux in rat cerebral cortical synaptoneurosomes, FEBS Lett., 175 (1984) 193-196. 17 Schwartz, R.D., Jackson, J.A., Weigert, D., Skolnick, P. and Paul, S.M., Characterization of barbiturate-stimulated chloride efflux from rat brain synaptoneurosomes, J. Neurosci., 5 (1985) 2963-2970. 18 Schwartz, R.D., Suzdak, P.D. and Paul, S.M., GABA- and barbiturate-mediated 36C1- uptake in rat brain synaptoneurosomes: evidence for rapid desensitization of GABA A receptorcoupled chloride ion channel, Mol. Pharrnacol. 30 (1986) 491-426. 19 Schwartz, R.D., The GABA A receptor-gated ion channel: biochemical and pharmacological studies of structure and function, Biochem. Pharrnacol., 37 (1988) 3369-3375. 20 Squires, R.E, Casida, J.E, Richardson, M. and Saederup, E., [35S]t-Butylbicyclophosphorothionate binds with high affinity to brain-specific sites coupled to y-aminobutyric acid-A and ion recognition sites, Mol. Pharmacol., 23 (1983) 326-336. 21 Tallman, J.F., Paul, S.M., Skolnick, P. and Gallager, D.W., Receptors for the age of anxiety: pharmacology and benzodiazepines, Science, 207 (1980) 274-281.
143
Elsevier BRES 15655
Na+-dependent GABA transport system scavenges endogenous external GABA and prevents desensitization of GABAA receptors in rat cerebrocortical synaptoneurosomes Wha Bin Im, David P. Blakeman and John P. Davis CNS Diseases Research, The Upjohn Company, Kalamazoo, MI 49001 (U.S.A.) (Accepted 9 January 1990)
Key words: y-Aminobutyric acid A receptor; 36C1- uptake; Desensitization; Endogenous external y-aminobutyric acid; Na÷-dependent 7-aminobutyric acid transport system
Muscimol-induced 36C1- uptake in rat cerebrocortical synaptoneurosomes was reduced upon exposure of the membrane sacs to low Na ÷ media. This Na ÷ requirement led us to examine the role of the Na÷-dependent y-aminobutyric acid (GABA) transport system in 36C1-uptake. Incubation of the synaptoneurosomes with nipecotic acid, a specific inhibitor of the GABA transport system, for 10 min increased the level of endogenous external G A B A from less than 10 to 150 /tM and induced the same signs of desensitization as observed with high muscimoi-treated synaptoneurosomes; a marked reduction of muscimol-induced 36C1-uptake and an appearance of a slow bicuculline-sensitive 36C1- uptake, probably due to a continuous recovery of a population of GABA A receptors from desensitization. Similar results were obtained upon dissipation of Na ÷ electrochemical gradient across the membranes by inhibition of Na ÷,K÷-ATPase with ouabain or by blocking energy metabolism with azide or N-ethylmaleimide. We propose that the Na+-dependent GABA transport system, its operation being dependent on inwardly directed Na ÷ electrochemical gradient, is responsible for scavenging endogenous GABA released from the synaptoneurosomes, and thus prevents desensitization of GABA A receptors.
MATERIALS AND METHODS
INTRODUCTION y - A m i n o b u t y r i c acid ( G A B A )
is a u b i q u i t o u s inhibi-
t o r y n e u r o t r a n s m i t t e r in the m a m m a l i a n central n e r v o u s s y s t e m , and i n d u c e s s y n a p t i c m e m b r a n e c h l o r i d e c o n d u c tance
by i n t e r a c t i o n
with
G A B A A r e c e p t o r s 1'5. T h e
G A B A - r e g u l a t e d c h l o r i d e c h a n n e l is a r e c e p t o r c o m p l e x c o n t a i n i n g s e p a r a t e binding sites for a variety o f psyc h o a c t i v e drugs, i.e. b e n z o d i a z e p i n e s , i m i d a z o p y r i d i n e s , b a r b i t u r a t e s , m e t a b o l i t e s of p r o g e s t e r o n e , t-butylbicyclop h o s p h o r o t h i o n a t e , a v e r m e c t i n B, and p i c r o t o x i n 3'4' 6,9,12,19-21 R e c e n t l y , c e r e b r o c o r t i c a l s y n a p t o n e u r o s o m e s f r o m t h e rat, m o u s e a n d g u i n e a pig h a v e b e e n s h o w n to t r a n s p o r t 36Cl- in r e s p o n s e to G A B A or G A B A m i m e t i c s 7'8'16 and h a v e b e e n widely used to study pharmacological
and
biochemical
actions
of v a r i o u s
ligands for the d i f f e r e n t allosteric sites on G A B A A r e c e p t o r c o m p l e x e s 13'14'18. In this study, w e h a v e obs e r v e d the d e p e n d e n c y o f m u s c i m o l - i n d u c e d 36C1- u p t a k e on N a ÷ g r a d i e n t in rat c e r e b r o c o r t i c a l s y n a p t o n e u r o s o m e s and p r o p o s e that o p e r a t i o n of the N a ÷ - d e p e n d e n t G A B A t r a n s p o r t system is essential in p r e v e n t i n g desensitization o f G A B A A r e c e p t o r s .
Rat cerebrocortical synaptoneurosomes were prepared following the procedures reported elsewhere 8. Briefly, cerebral cortices from 4 male Sprague-Dawley rats were suspended in 30 ml ice-cold solution containing (in mM): NaC1 118, KCi 5, MgSO 4 1.18, CaCI2 2.5, HEPES/Tris 20; pH 7.0 (buffer A), and D-glucose 10 and were homogenized in a Dounce homogenizer by 8 strokes with a loose-fitting pestle and then by 5 strokes with a tight-fitting pestle. The suspension was diluted to 160 ml with buffer A, and filtered through 3 layers of nylon cloth and 10-/~m Millipore filter (LC type). The filtrates were centrifuged at 3000 x g for 15 min. The membranous pellets were washed once and resuspended in buffer A or indicated buffers to a final protein concentration of 8 mg/ml. 36CI- uptake in the synaptoneurosomes was measured by a rapid filtration technique using Whatman GF/B glass microfiber filters TM with some modifications. Typical incubation medium contained 0.2 /aCi Na36Cl/ml (ICN), 118 mM NaC1, 5 mM KC1, 1.8 mM MgSO 4, 2.5 mM CaC12, 20 mM HEPES/Tris, pH 7.0, with or without testing drugs. To study the effect of Na + on muscimol-induced 36Eluptake, the concentration of Na + was varied from 0 to 140 mM by replacing Na + isoosmotically with N-methylglucamine while maintaining other ingredients the same. The same buffers without 36Elwere used to suspend membranes. The membrane suspensions were incubated for 5 min at 30 °C and, when indicated, metabolic inhibitors were introduced to the membrane during the preincubation period. The reaction was initiated by mixing equal volumes (125 ~1) of the membrane suspension (1 mg protein) and the isotope medium at 30 °C. After 5 s, unless indicated otherwise, uptake was terminated by adding ice-cold buffer A containing 500/~M bicucul-
Correspondence: W.B. Im, CNS Research 7251-209-1, The Upjohn Company, Kalamazoo, MI 49001, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
144 line or 1 mM of 4,4"-diisothiocyantostilbene-2,2"-disulfonic acid (DIDS) to minimize possible leakage of 3~'CI from the synaptoneurosomes during the termination processes. DIDS has been shown to block the effiux of 3~'C1 from the synaptoneurosomes 17. The mixture was filtered over a GF/B filter under vacuum, and the filters were washed 3 times with 15 ml of ice cold buffer A. The radioactivity was counted after mixing the filters with 15 ml Instagel. Bicuculline-sensitive 36C1 uptake refers to the portion inhibited by 500 jtM bicuculline methiodide during the reaction. To measure chloride uptake through G A B A A channels during desensitization or drug treatment, the membranes were exposed to 100 I~M muscimol or drugs for 2-5 min at 30 °C, and then mixed with the medium containing 3~'C1 and the usual ionic components of buffer A, with or without 500 ~uM bicuculline methiodide. The level of endogenous GABA in the medium was measured using the GABASE system for Pseudomonasfluorescence (Sigma). Briefly, the assay system contains ),-aminobutyric acid glutamic transaminase (0.02 U), succinic semialdehyde dehydrogenase (0.02 U), NADP (0.2 mM), a sample (100/d) or a known amount of G A B A , and 100 mM potassium pyrophosphate, pH 8.6, in a total volume of 0.95 ml. The reaction was initiated by adding 50/A of 20 mM a-ketoglutarate in the pyrophosphate buffer. The absorbance change at 340 nm was measured after reaction for 45 min at room temperature. The assays give a linear response up to at least 120 nmol GABA/ml assay medium with experimental variations less than 15%, at the concentration of GABA above 2 nmol/ml. The suspension of the synaptoneurosomes which were concentrated (16 mg protein/ml) twice as much as those used for 36C1 uptake (8 mg protein/ml) were treated with the Na+-free buffer (N-methylglucamine replacing Na+), nipecotic acid (5 mM), NEM (1 mM), azide (5 mM), or ouabain (1 mM) for 10 rain and centrifuged for 30 s using an Eppendorf microcentrifuge. An aliquot (100 ,ul) of the supernatant was analyzed. In the case of the NEM-treated sample, excess mercaptan (3 times) was added to neutralize residual NEM. Na 3~C1 was obtained from New England Nuclear. Bicuculline methiodide, DIDS, ouabain, azide, and N-ethylmaleimide were purchased from Sigma.
50
•=-
Muscimol =.° 40 /¢/~=
r=
/
30
GABA
E 20
10 ¢,J
0 0
i i i // , 40 80 120 200 [Muscimol or GABA]~_M
Fig. 1. Dose-response profiles for muscimol- and GABA-mediated 36C1 uptake in rat cerebrocortical synaptoneurosomes. Aliquots (125 ~1) of the synaptoneurosome suspensions were incubated at 30 °C for 5 min, and then mixed with an equal volume of the NaCI-36CI buffer containing final concentrations of G A B A or muscimol varying from 5 to 200/~M. The reaction was terminated 5 s later. The radioactivity associated with the synaptoneurosomes was determined using the rapid filtration techniques as described under Materials and Methods. The baseline 36C1-level was obtained from the same set of experiments without G A B A or muscimol and was used to compute GABA- or muscimol-mediated 36C1-uptake. The values represent the mean of triplicate measurements from a typical experiment. The experimental variations were less than 10%.
s u s p e n d e d a n d p r e i n c u b a t e d in t h e b u f f e r c o n t a i n i n g h i g h N a + ( > 1 0 0 m M ) , t h e 36C1- u p t a k e w a s n o t a f f e c t e d , e v e n though
the
final
Na + concentration
of the
reaction
m i x t u r e w a s l o w e r e d b e l o w 40 m M b y e m p l o y i n g t h e RESULTS 36C1- u p t a k e in r a t c e r e b r o c o r t i c a l s y n a p t o n e u r o s o m e s was d o s e - d e p e n d e n t l y s t i m u l a t e d b y G A B A
100
and musci-
m o l (Fig. 1) in g e n e r a l a g r e e m e n t w i t h e a r l i e r r e p o r t s 7'~8. This muscimol-induced bicuculline with an
36C1- u p t a k e was i n h i b i t e d b y
IC5o v a l u e o f 70 ktM. T h e b a s e l i n e
36C1- level (in t h e a b s e n c e o f e x o g e n o u s G A B A
mime-
tics), o n t h e o t h e r h a n d , was n o t n o t i c e a b l y a f f e c t e d b y b i c u c u l l i n e at 500 ktM, p r o b a b l y r e f l e c t i n g low e n d o g e nous
GABA
in
the
medium.
Replacing
Na +
with
N - m e t h y l g l u c a m i n e in t h e m e m b r a n e s u s p e n s i o n a n d t h e 36C1- r e a c t i o n b u f f e r s r e d u c e d t h e b i c u c u l l i n e - s e n s i t i v e muscimol-mediated sive c h a n g e s
36C1- u p t a k e . Fig. 2 s h o w s p r o g r e s -
in 36Cl
uptake
as a f u n c t i o n
80 --E ~E
of Na +
c o n c e n t r a t i o n s u n d e r a c o n s t a n t level of C1 at 140 m M a n d m u s c i m o l at 100 ~ M . T h e C1- u p t a k e was r e d u c e d by 5 0 % as t h e c o n c e n t r a t i o n of N a + was l o w e r e d f r o m 140 to 30 m M , a n d r e a c h e d 2 0 % o f t h e m a x i m u m level w h e n N a + was c o m p l e t e l y r e p l a c e d w i t h N - m e t h y i g l u c a m i n e . T h e p r e s e n c e o f h i g h N a + in t h e m e m b r a n e s u s p e n d i n g b u f f e r d u r i n g t h e p r e i n c u b a t i o n p e r i o d (5 m i n at 30 °C) a p p e a r e d t o b e t h e p a r t i c u l a r l y critical f a c t o r for 36C1 uptake. For example, when the synaptoneurosomes were
60
¢¢
40
._z ~-
20
~Z~
0 i 0
I
I
I
I
I
I
[
40 80 120 [Na÷] mM
Fig. 2. Effect of varying Na + concentrations in the membrane suspension and the 36C1-reaction buffers on muscimol-induced 36C[uptake in rat cerebrocortical synaptoneurosomes. The synaptoneurosomes were prepared in the typical NaCl-glucose buffer containing 118 mM NaCI, 5 mM KC1, 1.18 mM MgSO 4, 2.5 mM CaCI 2, 10 mM glucose, 20 mM HEPES/Tris, pH 7.0, but were resuspended in the media containing indicated concentrations of Na + varying from 0 to 140 raM. Na + was replaced isoosmotically with N-methylglucamine. Aliquots (125 /d) of the membrane suspensions were incubated for 5 min at 30 °C and were mixed with the 36C1 reaction buffer of the same composition as the membrane suspension buffer with or without muscimol (final concentration 100 ~M). The reaction was terminated 5 s later and the rest of the experimental procedure was the same as described in the legend of Fig. 1.
145
IA3o
TABLE I
Increases in endogenous external GABA level in the suspension of rat cerebrocortical synaptoneurosomes by blocking Na+-dependent GABA transport system or dissipation of membrane Na + electrochemicalgradiem The synaptoneurosomes were suspended in the NaCl buffer to a final protein concentration of 16 mg/ml. Then an aliquot (500/A) of the membrane suspensions was incubated at 30 °C with 5 mM nipecotic acid, 1 mM NEM, 5 mM azide or 1 mM ouabain. In the case of Na+-ffee medium, the synaptoneurosomes were suspended in the medium containing 118 mM N-methylglucamine in place of Na ÷ , and then were incubated as above. After incubation for 10 min, the suspensions were centrifuged using an Eppendorf microcentrifuge for 30 s. An aliquot (100/~1) of the supernatants was analyzed for G A B A level using the GABASE system as described in Materials and Methods. In the case of NEM-treated synaptoneurosomes, 2-mercaptan was added in excess (3 times). The values represent the mean of duplicate measurements and experimental variations were less than 15% when the G A B A level was greater than 20/~M in the sample.
Additions
GABA (pM)
N o addition Nipecotic acid 5 mM Na÷-free medium NEM 1 mM Azide 5 mM Ouabain 1 mM
< l0 150 72 109 75 110
N a + - f r e e 36C1- r e a c t i o n b u f f e r and the m e m b r a n e susp e n s i o n t w i c e m o r e c o n c e n t r a t e d t h a n the usual o n e . F u r t h e r m o r e , m u s c i m o l ( 1 0 0 / x M ) , u n d e r o p t i m a l chloride t r a n s p o r t
conditions,
i n d u c e d n o 22Na+ u p t a k e .
TABLE II
Effect of nipecotic acid on muscimol-stimulated 36C1~uptake in rat cerebrocortical synaptoneurosomes An aliquot (125/~i) of the synaptoneurosome suspensions in 118 mM NaCI, 5 mM KCI, 1.18 mM MgSO 4, 2.5 mM CaCI2, 10 mM D-glucose, and 20 mM HEPES/Tris, pH 7.0, was preincubated with 5 mM nipeeotic acid for 2, 4 and 10 rain at 30 °C. The reaction was initiated by adding an equal volume of 36C1- NaC1 buffer containing muscimol to give a final concentration of 100/~M, and was terminated 5 s later. The radioactivity associated with the synaptoneurosomes was determined using the rapid filtration technique as described under Materials and Methods. When nipecotic acid was added with 36C1-, the treatment was designated to be a 5-s one. The baseline 36C1- level was obtained from the same set of experiments without muscimol and was subtracted from the value obtained with muscimol to yield the agonist-induced 36C1 uptake.
Pre~eatment
No addition Nipecotic acid (5 mM) for 5s 2 min 4rain 10min
Muscimol-stimulated 36C1-uptake
B
50
Pentobarbital + Muscimol
40
30 ~~E 20
~.
.~'~
~'~
10
10 0 0
I
1//...-.3
1
2
10
0
Preincubation Time, min
1
2o
Time,min
Fig. 3. Plots showing constant rates of bicuculline-sensitive 36Eluptake by rat cerebrocortical synaptoneurosomes in the continuous presence of 100 pM muscimol and time course profiles for the 36Cluptake. A: the suspensions of the synaptoneurosomes were incubated in the presence of 100 pM muscimol for 10, 20, 30, 40, 60 s, 2 and l0 min. 0 s represents the addition of muscimol with 36C!-. An aliquot (125 pl) of the membrane suspensions was mixed with an equal volume of the NaCI-36CI- buffer with (0) or without (O) bicuculline methiodide (final concentration of 500 pM). The reaction was terminated 5 s later. B: the membrane suspensions were incubated in the presence or absence of 200/tM pentobarbital and/or 100 pM muscimol for 2 min. An aliquot of the membrane suspensions was mixed with an equal volume of the NaC1-36C1buffer with or without bicuculline (final concentration of 500 ~M). The reaction was terminated 5, 10, 20, 30, 40, 60 and 120 s later as described under Materials and Methods. The values for bicucullinesensitive 36C1 uptake were obtained from the differences in the presence or absence of bicuculline. The control values represent those obtained without exogenous muscimol, otherwise under identical conditions. The values in A represent the mean of duplicate measurements, and those in B with a bar represent the mean of triplicate measurements with experimental variations. (3, muscimol; O, muscimol + pentobarbital; Zk, the membrane suspensions were treated with 1 mM NEM.
T h e s e o b s e r v a t i o n s suggest that t h e a p p a r e n t N a + effect arises f r o m c h a n g e s in m e m b r a n e e n v i r o n m e n t s , and led us to e x a m i n e the roles of e n d o g e n o u s e x t e r n a l G A B A and N a + - d e p e n d e n t
GABA
t r a n s p o r t systems in 36Cl-
uptake. T r e a t m e n t o f t h e s y n a p t o n e u r o s o m e s with 5 m M n i p e c o t i c acid, a specific i n h i b i t o r of t h e N a + - d e p e n d e n t G A B A t r a n s p o r t system 11 for 10 rain i n c r e a s e d the l e v e l of e n d o g e n o u s G A B A to 150 p M
in the m e d i u m f r o m less t h a n 10
as m e a s u r e d
with the G A B A S E
system.
R e p l a c i n g N a + with N - m e t h y l g l u c a m i n e also l e d to an a c c u m u l a t i o n of e n d o g e n o u s G A B A in t h e m e d i u m to 72 /2M (Table I). Table II shows that n i p e c o t i c acid b l o c k e d m u s c i m o l -
nmol/mg protein/5 s
%
i n d u c e d c h l o r i d e u p t a k e as a f u n c t i o n o f its e x p o s u r e p e r i o d to m e m b r a n e s . F o r e x a m p l e , t h e drug as a d d e d
42.5 + 2.3
100
t o g e t h e r with 36C1- (5-s e x p o s u r e ) s h o w e d no n o t i c e a b l e
41.4 + 1.5 28.2 + 1.4 21.6_+ 1.1 8.1 __+_2.3
97 66 50 19
effect, but u p o n p r e i n c u b a t i o n with the s y n a p t o n e u r o s o m e s for 2, 4 and 10 rain, r e d u c e d the m u s c i m o l - i n d u c e d 36C1-- u p t a k e to 66, 50, and 1 9 % of the u n t r e a t e d , r e s p e c t i v e l y . A t the s a m e t i m e , t h e basal 36C1- level
146 TABLE III Inhibition o f muscimol-induced ~6C~ uptake in the synaptoneurosornes treated with azide, NEM, or ouabain
The suspensions of synaptoneurosomes were preincubated at 30 °C with drugs at the indicated concentration for 5 min. The reaction was initiated by adding the NaCl-36CI buffer with or without muscimol (a final concentration of 100/~M). Other experimental details were the same as described in Table I. The values represent the mean and standard deviations from at least 3 separate experiments or a single experiment of triplicate measurements. The experimental variations within the triplicate were less than 10%.
Treatment
36C1--Uptake (nmol/mg protein/5 s) Muscimol. . . . . . . . . . . . . Muscimol (l OOktM)
Control Azide 5 mM NEM1 mM Ouabain 1 mM
-
+
27.1 + 0.4 37.2+1.9 36.9±0.6 38.2 + 1.3
74.1 ± 4.9 48.7+3.6 45.7±4.1 42.1 + 5.2
induced~6 Cluptake (%)
47 (100) 11.5(25) 8.8(19) 3.9 (8)
m e a s u r e d without exogenous G A B A agonists rose from 27.1 to 34.6 nmol/mg protein/5 s upon preincubation of the s y n a p t o n e u r o s o m e s with nipecotic acid for 10 min. The increased portion of 36C1- uptake was sensitive to bicuculline (500 ~ M ) . These effects of nipecotic acid were readily reversed upon washing the treated synaptoneurosomes, and were similar to the chloride transport characteristics of G A B A A receptors under desensitizing conditions as shown in Fig. 3. Treatment of the synapt o n e u r o s o m e s with high muscimol (100 ~ M ) reduced, within 5 s, the level of bicuculline-sensitive CI- u p t a k e to 2 0 - 2 5 % of the maximal rate (undesensitized conditions),
and the residual rate r e m a i n e d unchanged during 10 min with high muscimoi (Fig. 3A). The time course profiles (Fig. 3B) indicate that the bicuculline-sensitive CI uptake under the desensitizing conditions increased linearly during the first 15 s and reached an equilibrium level of 35 nmol/mg protein with a t:~ of 20 s. It should be noted that in the absence of exogenous agonists, the bicuculline-sensitive C I uptake in the synaptoneurosomes increased from 0 to merely 4 nmol/mg protein during the same uptake period (2 rain). F u r t h e r m o r e , p e n t o b a r b i t a l (200 ~ M ) s h o r t e n e d the t,~ value for the slow bicuculline-sensitive C1- u p t a k e to 4 s without affecting the equilibrium level. The sensitivities of the slow C1- uptake to bicuculline and p e n t o b a r b i t a l indicate that G A B A A receptors, even under desensitizing conditions, m e d i a t e d 36C1-flux, albeit at a r e d u c e d rate. Similar desensitizing conditions were p r o d u c e d upon abolishing m e m b r a n e Na + electrochemical gradient by inhibiting N a + , K + - A T P a s e with ouabain, or blocking energy metabolism with azide and N E M . N E M (1 mM), azide (5 mM), and ouabain (1 raM), upon interaction with the s y n a p t o n e u r o s o m e s for 5 rain, reduced the muscimol-induced 36C1 uptake by 81, 75 and 92%, respectively, and at the same time increased the basal 36CF level from 27.1 to 36.9, 37.2 and 38.2 nmol/mg protein/5 s, respectively. The increased portions of the 36C1- uptake were blocked by bicuculline at 500 ktM, and showed a time course profile similar to that o b t a i n e d with the muscimol-desensitized receptors, as shown in Fig. 3 for the N E M - t r e a t e d and in Table IV for the ouabainand azide-treated synaptoneurosomes. A l s o , the level of endogenous G A B A in the m e d i u m increased to 109, 110 and 75 ¢tM upon treatments with N E M (1 mM), ouabain (1 raM), or azide (5 raM), respectively (Table I).
TABLE IV Increases in bicuculline-sensitive 36C1- uptake in rat cerebrocortical synaptoneurosomes upon desensitization with muscimol or treatment with NEM, azide or ouabain
The suspensions of the synaptoneurosomes were exposed to 100/~M muscimol, 1 mM NEM, 5 mM azide or 1 mM ouabain at 30 °C for 5 min. The reaction was then initiated by adding the NaCI-36CI- buffer with or without 1 mM bicueulline methiodide (final concentration 500/~M), and was terminated 30 s later. The rest of the experimental details were the same as described in Table I. The bicucullinesensitive uptake was computed from the differences in 36C1- uptake measured with and without bicueulline methiodide. The values represent the mean + S.D. of 3 experiments done in triplicate measurements. Pretreatment
Bicuculline-sensitive 36Cl- uptake (nmol/mg protein/30 s)
No addition Muscimo1100/~M NEM 1 mM Azide 5 mM Ouabain 1 mM
1.91 _-+0.4 24.3 -L-_3.2 21.7 + 1.1 16.9 + 0.7 20.5 -+ 3.4
DISCUSSION The m a j o r point of this r e p o r t is that the Na ÷d e p e n d e n t G A B A transport system scavenges endogenous external G A B A and prevents desensitization of G A B A A receptors in rat cerebrocortical synaptoneurosomes. We have found that inhibition of the Na +d e p e n d e n t G A B A transport system by a specific inhibitor, nipecotic acid, m a r k e d l y enhanced endogenous external G A B A (over 100/~M) and elicited the signs of desensitization; i.e. m a r k e d reduction of muscimolinduced 36C1 uptake, and an increase in slow, bicuculline-sensitive 36C1- uptake without exogenous G A B A mimetics. Also, similar desensitizing conditions were p r o d u c e d upon abolishing the N a ÷ electrochemical gradient by removal of N a ÷ from m e m b r a n e suspending m e d i u m , inhibition of N a + , K + - A T P a s e or interference with energy metabolism by azide and N E M . These
147 observations are consistent with the notion that dissipation of m e m b r a n e N a ÷ electrochemical gradient caused malfunction of N a ÷ - d e p e n d e n t G A B A transport system and led to an accumulation of e n d o g e n o u s external G A B A in rat cerebrocortical synaptoneurosomes. Of some interest u n d e r desensitizing conditions is the a p p e a r a n c e of a slow, bicuculline-sensitive 36C1- uptake. Several electrophysiological studies and earlier 36Clu p t a k e studies 1°'15'18 have also r e p o r t e d the decline of the agonist-induced anion m o v e m e n t s to a new low, steadystate level in the presence of high agonists. Its nature, however, has not b e e n clear. We have felt that the a p p e a r a n c e of the bicuculline-sensitive CI- uptake under desensitizing conditions is not likely due to a slow desensitization process since the rate of the C1- uptake was unchanged during the continuous presence of high
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