1 1

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N a - D E P E N D E N T T R A N S P O R T O F γ - A M I N O B U T Y R I C A C I D I N S U B C E L L U L A R B R A I N P A R T I C L E S

1

S. V A R O N A N D W. W I L B R A N D T

Department of Genetics, Stanford University, Palo Alto, California, and Department of Pharmacology, University of Berne, Switzerland,

γ - A m i n o b u t y r i c acid (GABA) is of interest to the neuroscientists on a n u m b e r of grounds [ 1 1 ] . Although extensively present in lower orga- nisms and in plants, in the v e r t e b r a t e s G A B A can be found only in the central nervous system ( C N S ) . I t is formed within t h e C N S by decarboxylation of glutamic acid a n d is metabolized to succinic acid by t r a n s a m i n a t i o n (with α-ketoglutaric acid) a n d dehydrogena- tion. T h e presence of G A B A and of the three G A B A - r e l a t e d enzymes t h u s confers on t h e v e r t e b r a t e C N S t h e unique ability to b y p a s s t h e K r e b s cycle p a t h w a y for the conversion of α-ketoglutarate to suc- cinate. I t has been calculated t h a t as much as 4 0 % of t h e energetic metabolism of brain could go through this special G A B A shunt. A neurophysiological role of G A B A in t h e v e r t e b r a t e C N S is suggested by its demonstrable inhibitory effects on t h e evoked electrical a c t i v i t y of cerebral and cerebellar cortex, spinal cord, a n d other p a r t s of t h e C N S . F u r t h e r m o r e , in the crustacean peripheral nervous system, G A B A has been shown to mimic t h e stimulation of inhibitory fibers;

the finding [9] t h a t G A B A is present in such fibers, b u t n o t in motor or sensory ones, h a s raised t h e question of whether this compound acts as an inhibitory n e u r o t r a n s m i t t e r or, more generally, as an inhibitory neuromodulator. Indeed, G A B A presents [20] a striking n u m - ber of analogies, both a t the biochemical a n d t h e functional level, with the catecholamines, t h e i m p o r t a n c e of which in t h e modulation of C N S activity is well established, even though only p a r t i a l l y under- stood.

M o r e recent findings indicate t h e existence of mechanisms for intracellular t r a n s p o r t of G A B A in brain. P a r t i c u l a t e m a t t e r sedi- mented from brain homogenates retains a sizable portion of t h e 1

This work was supported in part by Grant R-178-64 from the United Cere- bral Palsy Foundation and in part by Research Grant N B 04270-03 from the National Institute for Neurological Diseases and Blindness of the National Institutes of Health.

119

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120 S. VARON AND W. WILBRANDT

t o t a l GABA content in brain, as first reported by Elliott a n d v a n Gelder [ 5 ] . Sano a n d R o b e r t s [14] showed t h a t such particles, in contrast with those from other tissues, a t 0 ° C can " b i n d " radioactive G A B A from t h e m e d i u m provided N a ions are present. Subsequent investigations, summarized in a recent symposium [22, 2 8 ] , gave evidence t h a t exogenous G A B A is actually t r a n s p o r t e d into brain p a r - ticles a t 0 ° C a n d a t higher t e m p e r a t u r e s , and t h a t in both cases N a ions are involved. This N a - d e p e n d e n c e of G A B A t r a n s p o r t parallels t h a t reported for other t r a n s p o r t systems both a t t h e cellular [2-4, 7, 16, 23-26, 29] and t h e subcellular [1] levels. T h e aim of t h e present p a p e r is a further analysis of t h e available d a t a on G A B A t r a n s p o r t

2

a n d a comparison with t h e other N a - d e p e n d e n t systems. I t will be shown t h a t t h e hypothesis [3, 4, 23-26] of an equilibrating carrier mechanism, coupling t h e t r a n s p o r t of s u b s t r a t e and N a - i o n s through a common carrier and supported by an energy-dependent extrusion of N a , a d e q u a t e l y fits t h e G A B A system a n d can in fact offer a suggestive basis for a relationship between the metabolic and n e u r o - physiological properties of G A B A in t h e v e r t e b r a t e C N S .

T H E GABA S Y S T E M A T 0°C [18-20, 27]

A 10% mouse b r a i n homogenate in 0.25 M sucrose yields, upon centrifugation between 1,500 and 15,000 g, a highly heterogeneous pellet which comprises mitochondria, microsomes, and pinched-off nerve endings ( s y n a p t o s o m e s ) . All three t y p e s of particles retain endogenous GABA, while no appreciable a m o u n t s of G A B A are found t o associate with t h e m e m b r a n o u s s t r a n d s a n d myelin m a t e r i a l of t h e pellet. T h e pellet also contains some free G A B A in t h e entrained sucrose s u p e r n a t a n t .

W h e n t h e pellet is resuspended in a buffered N a C l medium containing a small a m o u n t of

1 4

C - G A B A , incubation a t 0 ° C results in a r a p i d accumulation of r a d i o a c t i v i t y by t h e particles (reaching a m a x i - m u m within 30 minutes) which is accompanied by a relatively smaller increase of t h e p a r t i c u l a t e G A B A . P a r t of t h e acquired r a d i o a c t i v i t y remains in equilibrium with t h e m e d i u m a n d can be diluted out by adding an excess of nonradioactive free G A B A to the system. B o t h

2

These data were obtained in Dr. E . Roberts' laboratory at the City of Hope Medical Center, California, more than two years ago. They are reported and discussed elsewhere [17-22, 27, 28]. T h e additional analysis given here, and the conclusions drawn, reflect only the personal opinions of the present authors.

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T R A N S P O R T O F γ - A M I N O B U T Y R I C ACID I N B R A I N P A R T I C L E S 121

t h e r a d i o a c t i v i t y a n d t h e G A B A present in this r a p i d l y equilibrating pool are p r o m p t l y released from t h e particles after their transfer to a Na-free medium. T h e r a d i o a c t i v i t y r e m a i n i n g with t h e particles and most of their endogenous G A B A constitute a second pool, which decreases in size a n d increases in specific a c t i v i t y with prolonged incubation in N a - c o n t a i n i n g media. Such changes occur m u c h more slowly t h a n those affecting t h e first pool. T h e y become b a r e l y notice- able in Na-free media. Evidence from electron microscopy studies indicates t h a t both pools are associated with t h e same particles and are in fact present in all three t y p e s of membrane-enclosed particles occurring in t h e system. T h e following model h a s been suggested.

1. T h e m e m b r a n e s which enclose mitochondria, microsomes, a n d nerve-ending particles from brain are impermeable to free GABA.

2. These m e m b r a n e s contain sites which are capable of binding G A B A only in t h e presence of N a - i o n s . Exposure of t h e particles to a N a - c o n t a i n i n g m e d i u m t h u s results in t h e acquisition of exogenous G A B A a n d the formation of a r a p i d l y equilibrating pool.

3. T h e binding sites are mobile carriers a n d cross t h e m e m b r a n e (or t h e barriers within t h e m e m b r a n e ) a t a r a t e which, a t 0 ° C , is considerably slower t h a n t h a t a t which t h e y bind or exchange G A B A . Because of such carrier m o v e m e n t s , r a d i o a c t i v i t y enters t h e internal pool a n d internal G A B A moves out, with a n e t progressive loss of p a r t i c u l a t e G A B A owing to t h e higher G A B A concentration inside t h a n outside t h e barrier.

According to t h e model, an equilibrating carrier mechanism is r e - sponsible for t h e slow changes in t h e i n t e r n a l G A B A pool while t h e outer, r a p i d l y equilibrating, pool merely reflects the binding of free external G A B A by t h e N a - a c t i v a t e d carriers present a t a n y t i m e outside t h e barrier. T h e m e a s u r a b l e size of t h i s r a p i d l y equilibrating pool implies a relatively high n u m b e r of carrier molecules as compared with estimates for other carrier systems [ 6 ] .

T H E GABA S Y S T E M A T 29°C [17, 20, 22]

T h e same particles, suspended in buffered N a C l m e d i u m containing 1 4

C - G A B A a n d i n c u b a t e d a t 0 ° C for a brief period to allow t r a c e r accumulation, show a m a r k e d l y different behavior when further in- cubated a t 2 9 ° C u n d e r a c o n s t a n t air flow. T h r e e major processes t a k e p l a c e : massive release of p a r t i c u l a t e GABA, u p t a k e of free G A B A into some particles, and active metabolic d e g r a d a t i o n of G A B A within t h e same particles.

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1. Release of p a r t i c u l a t e G A B A occurs a t a much faster r a t e a t 29° t h a n a t 0 ° C . This can be directly d e m o n s t r a t e d for microsomes by use of pure microsomal p r e p a r a t i o n s (which, a t 0 ° C , h a v e been shown [18, 19] to behave with respect to G A B A in t h e same w a y as t h e heterogeneous suspension discussed t h u s f a r ) . I t is also clearly observable in t h e s t a n d a r d heterogeneous suspensions under such conditions as a nitrogen atmosphere, where u p t a k e and metabolism of G A B A are inhibited; the complete removal of p a r t i c u l a t e G A B A t h a t can be achieved with a sufficiently long incubation demonstrates t h a t this release process is not confined to the microsomal constituents of t h e suspension, b u t involves all its G A B A - c o n t a i n i n g particles.

U n d e r these inhibitory conditions, all the G A B A lost by the particles is recovered in t h e m e d i u m ; therefore, a plot of p a r t i c u l a t e GABA levels versus time yields a time curve for t h e release process. One of the most striking experimental findings was t h a t such a p a r t i c u l a t e G A B A t i m e curve is identical to those o b t a i n a b l e under air or pure oxygen, where both u p t a k e and metabolism of G A B A t a k e place, suggesting t h a t these two processes occur in such a w a y as to balance each other out. This is borne out by independent evidence indicating (see below) t h a t the G A B A newly t a k e n up is metabolized very r a p - idly and does not contribute significantly to the p a r t i c u l a t e GABA levels. I t has therefore been concluded t h a t essentially the same m a s - sive release occurs under both air a n d nitrogen and is in both cases depicted by t h e p a r t i c u l a t e G A B A time curve.

2. A t 0 ° C , t h e slow b u t progressive release of p a r t i c u l a t e G A B A results in a corresponding increase in t h e free G A B A levels of t h e medium. I n contrast with this p a t t e r n , a t 2 9 ° C t h e GABA content of the medium undergoes a considerable drop in the initial 30 to 60 m i n u t e s of incubation, a n d remains c o n s t a n t thereafter. T h i s d e - crease has been shown not to be due to metabolism of free GABA within t h e suspending fluid a n d can therefore only result from the u p t a k e of free G A B A by t h e particles. I n fact, u p t a k e of GABA by t h e particles m u s t occur a t a much greater extent and for a longer period t h a n indicated by t h e G A B A depletion in the medium since, as discussed in t h e preceding p a r a g r a p h , a considerable release of GABA is t a k i n g place throughout the incubation. T h e u p t a k e of GABA does not result in a n y observable increase of the p a r t i c u l a t e G A B A levels b u t is accompanied by an accumulation in t h e particles of G A B A metabolites. This finding, t h a t t h e newly t a k e n - u p G A B A is immediately m a d e available to metabolic degradation, d e m o n s t r a t e s

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t h a t the u p t a k e is not due to an increased binding c a p a c i t y of t h e particles and, furthermore, t h a t it t a k e s place in particles which h a v e the ability to metabolize G A B A (see below). T h e u p t a k e of free GABA by such particles does not occur in Na-free media and is inhibited b y cardiac glycosides, dinitrophenol, lack of oxygen, and by specific inhibitors of G A B A metabolism (such as aminooxyacetic acid) ; in the last case, addition of another energy source such as p y r u v a t e restores t h e ability of the particles to t a k e u p GABA. T h u s , G A B A u p t a k e a t 2 9 ° C is a process which is both N a - d e p e n d e n t and energy-dependent and which draws n o r m a l l y t h e required energy from the metabolic degradation of GABA itself.

3. Evidence for active G A B A metabolism is provided by the rapid and progressive decrease of the t o t a l G A B A content in t h e system.

N o G A B A metabolism has been found to occur in t h e m e d i u m (after removal of the particles) or in the microsomal particles. M i t o c h o n d r i a , on the other hand, are clearly indicated as G A B A metabolizing p a r - ticles by their reported content of G A B A - t r a n s a m i n a s e [13] a n d t h e observed involvement of K r e b s cycle steps in t h e metabolic d e g r a d a - tion of GABA t a k i n g place in t h e system [ 1 7 ] . I t is possible, although less likely [ 1 3 ] , t h a t mitochondria-containing nerve-ending particles also contribute to G A B A metabolism. Inhibition of G A B A metabolism can be achieved by incubating the particles in t h e absence of oxygen (nitrogen flow) or in t h e presence of aminooxyacetic acid, a k n o w n specific inhibitor of G A B A - t r a n s a m i n a s e . I t is also observed under all conditions (see above) where u p t a k e of free G A B A is inhibited.

This, and the i m m e d i a t e metabolic d e g r a d a t i o n undergone by t h e newly t a k e n - u p G A B A (see a b o v e ) , strongly suggest t h a t most, if not all, of the G A B A degraded in the system is m a d e available to the metabolic sites by t h e u p t a k e process.

T h e m a i n features of the 29°C G A B A system are shown d i a g r a m - m a t i c a l l y in Fig. 1. T h e particles are regarded as two functionally distinct classes (A, B) linked through the suspending fluid ( S ) . Only one class (A) is the site of active G A B A metabolism and t a k e s up free G A B A from t h e medium. T h e other class (B) r a p i d l y releases its G A B A content into t h e suspending fluid a n d t h u s m a k e s it available t o u p t a k e and metabolism by t h e A particles. Owing to t h e cyclic n a - t u r e of the sequence metabolism - » energy u p t a k e -> metabolism, interference a t a n y level will bring a b o u t the interruption of both metabolism and u p t a k e .

A better u n d e r s t a n d i n g of t h e system and an exact kinetic s t u d y

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Ouabain

Imipramine

Amino o x y a c e t a t e - - * -

Microsomes (nerve endings)

Pyruvate

FIG. 1. Schematic representation of the interrelationships between particles A and particles B.

of t h e two t y p e s of G A B A m o v e m e n t involved will obviously have to a w a i t additional d a t a on t h e separate behavior of t h e different t y p e s of particles, as well as on sodium distribution in t h e particles.

On t h e basis of t h e available evidence, however, a n u m b e r of a d d i - tional considerations can be discussed.

The Release of GABA from the Β Particles (29°C)

T h i s process is considerably faster t h a n t h e release occurring a t 0°C. I t is, on t h e other hand, only slightly, if a t all, slowed down in Na-free media. I t is n o t affected b y t h e occurrence or absence of energy-yielding metabolism in t h e A particles. I t is also unaffected

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T R A N S P O R T O F y - A M I N O B U T Y R I C ACID I N B R A I N P A R T I C L E S 125

by the concentration of free G A B A in t h e m e d i u m (which increases with time under nitrogen, decreases to a low c o n s t a n t level u n d e r a i r ) .

Based upon t h e p a r t i c u l a t e G A B A time curve, a kinetic analysis can be m a d e , as summarized in Fig. 2. I t is found t h a t a double reciprocal plot of G A B A decrement in t h e particles (p0 — p) versus time (£) yields a s t r a i g h t line, the equation for which can be written

— J — = a + β \ (1)

where a = l/p0 and the slope β characterizes t h e release a t 2 9 ° C . F r o m the previous equation, one o b t a i n s :

T h e validity of this t r e a t m e n t has been verified by i n t e g r a t i n g E q . (2) into

a n d plotting against time t h e reciprocals of t h e various experimental values for p. T h e rigorous linearity of this plot is shown in Fig. 2.

E q u a t i o n (2) states t h a t t h e r a t e a t which G A B A is released is a second-order function of t h e G A B A left in t h e particles. T h i s rules out diffusion as t h e u n d e r l y i n g mechanism for t h e release. I t is also h a r d to reconcile with t h e i n t e r p r e t a t i o n t h a t the G A B A release results from a t e m p e r a t u r e - e n h a n c e d breakdown of t h e Β particles. Possible mechanisms compatible with this second-order relationship include a carrier m e c h a n i s m operating under low s a t u r a t i o n conditions and with complexes involving two s u b s t r a t e molecules per carrier. U n d e r the same conditions, t h e above-mentioned irrelevance of t h e external G A B A concentration would be consistent with a carrier m e c h a n i s m in view of t h e large difference between the G A B A concentrations inside and outside t h e particles.

T h e r a p i d i t y with which t h e G A B A newly entering t h e particles is metabolized a n d t h e a t t r i b u t i o n to t h e releasing Β class of all G A B A - c o n t a i n i n g particles suggest t h a t u n d e r s t a n d a r d conditions

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The Uptake of GABA into the A Particles {29°C)

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126 S. VARON AND W . W I L B R A N D T

Ρ

\

I I I I I I I / 30 60 90 120 150 180 210 min

I P0^-P

0 . 5 - > ^ _ L _ = ^{+ / 3 - L}a

P0^'P

H

t

= -kp

dt

0.1 -

I I \/t 0.01 0.03

J_

Ρ

1.5-

y ir-ir

⁺

"

0.5- y

I I I I /

30 90 150 210 min

Fie. 2. Kinetic analysis of GABA release at 29°C from particles B.

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the free G A B A inside the A particles is brought down t o , a n d m a i n - tained at, negligible concentrations by its metabolic degradation. A G A B A gradient would t h u s obtain between external a n d internal free G A B A which could be t h e driving force for the G A B A u p t a k e into the A particles. I n fact, it w

T

as observed t h a t increasing G A B A concen- t r a t i o n s in t h e m e d i u m result in increasing r a t e s of disappearance from the medium. T h e N a - d e p e n d e n c e of t h e u p t a k e process, among other considerations, rules out a passive diffusion of G A B A along the metabolically m a i n t a i n e d G A B A gradient. I t is therefore t e m p t i n g to interpret t h e G A B A u p t a k e into A particles as a downhill t r a n s p o r t mediated by the same equilibrating carrier mechanism t h a t has been postulated in t h e 0 ° C system.

T h e issue is, however, complicated by other features of t h e process.

T h e sensitivity to ouabain, for instance, can h a r d l y be explained as a direct inhibition of metabolic processes by cardiac glycoside (for which no examples h a v e ever been c i t e d ) , and the possibility t h a t ouabain interferes with the N a - a c t i v a t e d binding ability of t h e carrier is ruled out by t h e demonstrable lack of such an effect a t 0 ° C . M o r e - over, there are experimental conditions under which t h e G A B A u p t a k e results in an accumulation of G A B A in t h e particles and, provided the p a r t i c u l a t e G A B A is in a free form, the system a p p e a r s to behave as a G A B A uphill t r a n s p o r t . This is the case, for example, where G A B A u p t a k e t a k e s place in spite of specifically blocked G A B A m e - tabolism (with the support of p y r u v a t e ) . W i t h o u t additional features, then, an equilibrating carrier system cannot account for all t h e observed properties of the GABA u p t a k e a t 2 9 ° C . However, the involve- m e n t of N a - i o n s and t h e sensitivity to cardiac glycosides allow for an i n t e r p r e t a t i o n of the whole process in t e r m s of a downhill equili- b r a t i n g carrier mechanism for a mixed Na-GABA carrier complex sustained by the simultanous action of a N a - p u m p . Such an inter- p r e t a t i o n is suggested by t h e comparison of t h e G A B A system with other N a - d e p e n d e n t t r a n s p o r t systems, to be discussed in the following section.

N a - D E P E N D E N T " U P H I L L " T R A N S P O R T S Y S T E M S

Since t h e 1953 report [15] t h a t cardiac glycosides inhibit sodium and potassium uphill t r a n s p o r t in red blood cells, similar observations have been m a d e in m a n y other cell t y p e s [8] ; in fact, no cell t y p e a p p e a r s to h a v e been tested t h u s far with negative results. This wide- spread inhibitory effect of cardiac glycosides on N a

+ a n d K

+

t r a n s p o r t

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is characterized b y two features: I t is m a i n l y affecting t h e uphill m o v e m e n t of t h e ions a n d it does n o t derive from interference w i t h t h e energy-yielding metabolism. I n recent y e a r s , a n u m b e r of other t r a n s p o r t systems concerning sugars a n d amino acids h a v e also been found to be inhibited b y cardiac glycosides; t h e suggestion occasion- ally h a s been m a d e t h a t cardiac glycosides h a v e a general action on p u m p i n g systems r a t h e r t h a n a specific one for t h e N a - K t r a n s p o r t . A v e r y strong point against such a suggestion is, however, t h a t in all the cases where cardiac glycoside inhibition was observed, t h e t r a n s - port was found to be N a - d e p e n d e n t . T h i s fact supports t h e a l t e r n a - tive interpretation, t h a t in these cases t h e glycoside inhibitory action is directly exerted on a sodium-potassium p u m p linked in some m a n - ner to t h e s u b s t r a t e t r a n s p o r t . If this i n t e r p r e t a t i o n is accepted, t h e question m a y be asked whether t h e relationship to t h e sodium t r a n s - port is by w a y of a direct coupling between sodium p u m p a n d s u b - s t r a t e p u m p or whether the role of the sodium p u m p is r a t h e r t o m a i n t a i n a sodium gradient. I n the case of iodide t r a n s p o r t , this question has been studied by Iff [7] by following t h e u p t a k e of radioactive iodide into t h y r o i d slices, continuously perfused directly u n d e r n e a t h a scintillation counter. I n an initial phase, t h e iodide u p t a k e was inhibited by using a lithium- instead of a sodium-containing medium.

L a t e r , ouabain was added and a sufficient time was allowed for t h e glycoside action to develop fully. T h u s , a condition was reached under which the sodium p u m p was certainly blocked, b u t a sodium accumulation h a d been p r e v e n t e d t h r o u g h t h e absence of sodium in the external medium. T h e n , lithium was replaced by sodium without remov- ing t h e glycoside. T h e restored sodium gradient r e a c t i v a t e d t h e iodide p u m p t e m p o r a r i l y even though t h e sodium p u m p still was, and re- mained, blocked. T h e answer therefore was t h a t it is n o t necessary for iodide uphill t r a n s p o r t t h a t t h e sodium p u m p be a c t u a l l y running, b u t t h a t its functioning is required for t h e m a i n t e n a n c e of a sodium gradient.

One possibility for a sodium gradient to do t r a n s p o r t work would be to h a v e the N a - t r a n s p o r t linked to a second t r a n s p o r t system by m e a n s of a common carrier. I n systems of this kind, uphill t r a n s - p o r t m a y be induced in various w a y s [ 3 0 ] . E x a m p l e s are listed in T a b l e I. Among t h e m t h e best-known case is t h a t of c o u n t e r t r a n s p o r t [ 1 2 ] , which has been observed in a n u m b e r of cell t y p e s and with different substrates. C o t r a n s p o r t in systems involving t e r n a r y complexes also h a s been reported, a n d recent i n t e r p r e t a t i o n s of amino

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TABLE I . Carrier Systems: Induced Uphill Movements of Substrate S (Si = S 2)

Induced m o v e m e n t C a r r i e r s Substrates Complexes P r i m a r y

a s y m m e t r y Induced gradients Absolute direction

Relative direction S R

S R

C S AB S

CS, CR

CS, CSS

CR, CRR CSR CRS

CS AS BS AB

R i > R2 C R ^ C R j ,

1

J

c2^{> c}1^—

R1^{> R}2

C i > C2 B ^ B ,

fr-CS^CS!

- C S R1^{> C S R}2

CR^ CR2

t

CRRi > CRR^

t

C2^{> C}1

c s2^{> c s}1 css2^>css1

^ A B ^ A B ,

t

A2^{> A !}

C S ^ C S , B S ^ B S ,

A S2^{> A S}1

Si~*S2 - C R S1^{> C R S}2^Sj—S.

S 2- S , S 2 - S , Si ~*s2

S ! - S2

$2^ Si

Counter- transport

Cotransport

Cotransport Cotransport

TRANSPORT OF γ-AMINOBUTYRIC ACID IN BRAIN PARTICLES 129

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130 S. VARON AND W. W1LBRANDT

acid t r a n s p o r t into single cells [23-26] and of glucose absorption from t h e intestine [2-4, 16] h a v e assumed mixed complexes involving one carrier molecule and different ratios of sodium ions and amino acid or sugar molecules. Of p a r t i c u l a r interest for t h e G A B A system is t h e successful analysis of glycine t r a n s p o r t into a v i a n red cells by Vidaver [ 2 3 - 2 6 ] . H e interprets t h e amino acid u p t a k e into t h e cells as a downhill m o v e m e n t of a carrier complex with 1 molecule of amino acid and 2 sodium ions. This downhill m o v e m e n t becomes in effect an uphill m o v e m e n t of the amino acid because t h e extrusion of sodium by the sodium p u m p results in the m a i n t e n a n c e of a steep gradient for t h e mixed complex. Since t h e sodium p u m p is blocked by cardiac glycosides, the uphill amino acid movement also stops in t h e presence of this inhibitor. Vidaver showed t h a t changing t h e driving force for t h e N a - m o v e m e n t either b y establishing a D o n n a n equilibrium or by reversing the sodium gradient (using t h e hemolysis method of S t r a u b ) , the m o v e m e n t of t h e amino acid could be changed a t will, and even reversed, in accordance with prediction. F r o m t h e observation t h a t a L i n e w e a v e r - B u r k plot of u p t a k e r a t e versus sodium concentration fails to give a straight line, whereas a similar plot versus the square of sodium concentration is linear, he concluded t h a t two sodium ions per molecule of amino acid are involved in t h e t r a n s p o r t complex. This was confirmed in experiments in which t h e t r a n s p o r t r a t e was v a r i e d : t r a n s p o r t increase of one molecule of glycine was accompanied by an increment of two sodium ions. A somewhat similar analysis, using short-circuit current as a measure of N a - t r a n s p o r t , was carried out in the case of glucose absorption from the intestine [ 2 , 1 6 ] .

According t o the L i n e w e a v e r - B u r k analysis, t h e effect of N a on the a p p a r e n t carrier p a r a m e t e r s appeared to be on t h e Km in Vidaver's experiments on amino acid t r a n s p o r t in pigeon red cells [ 2 3 - 2 6 ] , as well as in C r a n e ' s experiments on intestinal glucose absorption [ 3 , 4 ] . E x - periments on glucose absorption by Schultz and Zalusky [ 1 6 ] , however, indicated a n effect on Vmax. W h e t h e r , in a mixed complex system a s discussed, the effect of sodium should be expected on the a p p a r e n t Km or t h e a p p a r e n t Vmax of t h e carrier depends on t h e t y p e of binding.

T a b l e I I shows two different possibilities for 1:1 and 2 : 1 N a - s u b s t r a t e complexes with carrier ( C ) . " I n d e p e n d e n t " binding implies binding sites for N a (R) and for s u b s t r a t e (S) without interdependence (mean- ing t h a t S reacts equally well with C and with C R or C R2) . If? then, of

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all possible complexes only C R S (case 1) or C R R S (case 2) is able to move across the m e m b r a n e , the effect of R is on t h e a p p a r e n t Vmax^{. On} the other h a n d , a n effect on t h e a p p a r e n t Km emerges if S only, or preferentially, binds to C R (case 3) or C R R (case 5) r a t h e r t h a n to C (owing to spatial reasons or an allosteric effect of R on C ) . Theoretical possibilities also exist t h a t S has to bind before R (cases 4 a n d 7) or

in between the binding of t h e first a n d the second R (case 6 ) , yielding an effect of R both on Km^{and V}m&x^.

I n view of t h e successful i n t e r p r e t a t i o n of these cellular s y s t e m s in t e r m s of mixed complexes, in view f u r t h e r m o r e of t h e close chemical relationship b e t w e e n G A B A a n d α - a m i n o acids, i t a p p e a r s possible t h a t a similar m e c h a n i s m is involved in t h e N a - d e p e n d e n t , o u a b a i n - sensitive u p t a k e of G A B A i n t o t h e A particles (brain m i t o c h o n d r i a ) . As i l l u s t r a t e d b y t h e scheme given in Fig. 3, t h e p a r t i c u l a r conditions o b t a i n i n g in a mixed complex s y s t e m result in t w o c h a r a c t e r i s t i c f e a t u r e s : (a) T h e net m o v e m e n t of S a n d R (in t h i s case G A B A a n d N a ) m u s t occur in a m o l a r r a t i o fixed b y t h e t y p e of complex (in V i d a v e r ' s e x p e r i m e n t s 1:2); a n d (b) t h e unidirectional fluxes are func- tions of t h e c o n c e n t r a t i o n s for b o t h S a n d R . T h e s e functions also d e p e n d on t h e t y p e of complex involved and, for different t y p e s , are identical w i t h t h e t e r m s listed u n d e r Vs in T a b l e I I . T h e difference of t h e fluxes M i _ >2^{a n d}^M2^->i^{(the V}s t e r m s w i t h subscript 1 a n d 2 respectively) is t h e n e t r a t e in t h e general case w h e r e S2^{^ 0.}

T o t e s t the mixed complex hypothesis for t h e G A B A system, t h e analysis of a somewhat puzzling observation was found suitable. D u r - ing t h e initial 30 t o 60 minutes of 2 9 ° C incubation, t h e u p t a k e of G A B A into t h e A particles exceeds the a m o u n t s of G A B A released from particles B , so t h a t t h e free G A B A levels of t h e suspending fluid decrease considerably. After this time a constant low level is m a i n t a i n e d in t h e medium, which is interpreted a s t h e a t t a i n m e n t of a s t e a d y s t a t e between Β release and A u p t a k e . I n t h e experiment depicted in Fig. 4A [21] this s t e a d y s t a t e was disturbed, or its e s t a b - lishment prevented, by an addition of G A B A to the external m e d i u m after 5 o r 35 m i n u t e s of incubation a t 2 9 ° C . T h e resulting increase in t h e external G A B A concentration is t r a n s i e n t a n d after some time a steady s t a t e is again established. I n this new s t e a d y s t a t e , however, t h e G A B A level in t h e m e d i u m is higher t h a n in t h e control experi- m e n t s (without t h e a d d i t i o n ) . T h e difference is related t o the t o t a l a m o u n t of free G A B A in t h e m a n n e r illustrated in Fig. 4 B : T h e

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TABLE I I . Apparent Carrier Transport Parameters for Transport of Substrate S

Types of complexes:

A. Independent binding:

GS

Dicomplex

B. D e p e n d e n t binding:

Dicomplexes General conditions:

1. Equilibrium between s u b s t r a t e a n d carrier

2. Only fully s a t u r a t e d S-containing compound moves

Carrier forms Moving complex Dissociation constants

C CS CR CRS CRR CRRS CSR CRSR CSRR

1 + + + +

eg

O f

C J R | R ( § )

CR CS CR X R

ditto AR2 = • CRR

« ' = CRSR

C S R X R ditto KR& - „

NNN

CSRR

6 + + + +

eg

O f

C J R | R ( § )

d l t t

°

A S 3

" CRRS

CRS X R

d i t t

°

K

« ' = CRSR

C S R X R ditto KR& - „

NNN

CSRR

7 + + + +

eg

O f

C J R | R ( § )

d l t t

°

A S 3

" CRRS

CRS X R

d i t t

°

K

« ' = CRSR

C S R X R ditto KR& - „

NNN

CSRR

132

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Exclusively in Pluricomplexes with Carrier C and Substrate R

Types o f complexes : A. Independen t binding :

Tricomplex

B.

Dependen t binding :

(^C ^ (^J^^ ^ (^C ^

Tricomplexes

General conditions :

3 . S2^{= R} 2⁼⁰

S i = S R i = R

D = mobilit y o f th e movin g comple x containin g S

Relative concentration s ^Vs

Factors o f th e parameters

0

Fr FK

R' =

ditto R " =

Kri

R

Kr2

_S_ R' S

R' + 1 S ' + 1 CiD- R R "

CtD-

R'R" + R ' + 1 S ' + 1

y + -R ' + l R' ditto

ditto R' " =

ditto

:_S Ks2|

R"' S ' CiD — — — X -

R " ' + l R

KR3

S"' + R'R" + R ' + 1 R'R"

ditto R" " = R

Kr4

R

, , ,/

S ' Ct^{O„ ^ X}

ditto R " =R

Kr5

R " " + l 0S +„ . l/R ' + l

R ^TÏ

R

'

R

"

CtD . . . X

R' Î R' + l

I

R'R"

t

R-'R" + R ' + l | 1 + R ' |

_ ^ L Î _ L _ |

R'" + 1 J R' " +

1 1

R'R" + R ' + 1 ' " 1 S' +

i^{^ R'R' *}^{+ R * +}^{l |}

R""

t

l/R ' + 1

I

R " " + l | R " " - f l j

R'R

7

'

t

1

I

R'R" + R ' + 1

J

R'R " + R ' + 1

i

R'R" X R ' X l

° Arro w indicate s directio n o f paramete r wit h risin g R .

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134 S. VARON AND W . W I L B R A N D T

GABA addition

A B

FIG. 4. A. Change of GABA concentration in the medium (S) with time.

Full circles: spontaneous time course; open and half-open circles: time course in experiments with addition of GABA after 5 and 35 minutes, respectively.

B. Steady-state level of GABA concentration in the medium (GS) as a function of the sum of initial GABA concentration in the medium, Gif and concentration increment after the addition, Ga^.

FIG. 3. Schematic representation of the movement of GAB-4 and of sodium into and out of particles A.

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• Ξ

( in particles Β ) ( E x t e r n a l medium) ( In particles A )

Ξ

( O u t )

_b_

Fixed Fixed

J Uncoupled system

Fixed g,

r II Coupled

system

\

FIG. 5. Analysis of steady-state condition with respect to the GABA concen- tration in the medium (g2) for an uncoupled system a n d a coupled system

(GABA transport coupled to sodium transport by common carrier).

particles A a n d finally into metabolism. T h e coupled system (with mixed complexes) is compared with a n uncoupled system in which G A B A moves into particles A either b y diffusion or b y a carrier system n o t involving mixed complexes. Since t h e m o v e m e n t o u t of particles Β h a s been shown to be independent of external conditions, its r a t e a p p e a r s to control t h e s t e a d y s t a t e : both t h e m o v e m e n t into particles A a n d t h e r a t e of G A B A metabolism in t h e particles A s t e a d y - s t a t e level proved to be a linear function of t h e sum G\ -f- Ga^, these symbols indicating respectively t h e external G A B A concentration a t time 0 a n d its increment after t h e addition of G A B A .

Figure 5 shows a n analysis of t h e s t e a d y s t a t e with respect t o over-all G A B A movements from particles Β through t h e m e d i u m into

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m u s t equal this r a t e b. I n t h e case of t r a n s p o r t into particles A by a n y uncoupled system involving only G A B A itself (and possibly a c a r r i e r ) , regardless of whether this system follows linear kinetics or some t y p e of carrier kinetics, there is one a n d only one possible s t e a d y - s t a t e concentration of G A B A in the external medium. This is no longer so, however, if t h e t r a n s p o r t into A particles depends on both t h e G A B A a n d t h e sodium concentrations in t h e m a n n e r discussed, a n d if it is assumed t h a t the external concentration of N a is fixed due to the large external volume and t h a t t h e internal concentration of G A B A is also fixed due to t h e condition t h a t GABA metabolism equals b. I n this case, t h e postulate t h a t the r a t e of t r a n s - p o r t into particles A equals b can be m e t by a n y n u m b e r of pairs for t h e external concentration of G A B A and t h e internal concentration of sodium, and t h e external G A B A concentration in t h e s t e a d y state will increase if t h e internal N a - c o n c e n t r a t i o n rises. One possibility for such a rise would be t h e operation of t h e sodium p u m p a t m a x i - m u m r a t e , in which case the increased sodium u p t a k e into t h e particles during t h e t i m e after G A B A addition could n o t be compensated by increased removal t h r o u g h the p u m p . T h e r e are other possibilities for a sodium increase. Their discussion, however, would not be fruitful in view of the complete lack of additional d a t a .

D I S C U S S I O N

T h e experiments described here d e m o n s t r a t e the existence of so- d i u m - d e p e n d e n t and cardiac glycoside-inhibited t r a n s p o r t of GABA across m e m b r a n e s of subcellular particles. This finding in itself is of some interest since there are n o t m a n y examples in which t r a n s p o r t mechanisms k n o w n to operate across t h e cell m e m b r a n e in cellular systems were found in subcellular m e m b r a n e s as well. E v e n m o r p h o - logical c o m p a r a b i l i t y of cellular a n d subcellular m e m b r a n e s is not universally accepted, as current discussions on t h e s t r u c t u r e of t h e mitochondrial m e m b r a n e show. Subcellular sodium-dependent t r a n s - p o r t of amino acid has been d e m o n s t r a t e d across t h e m e m b r a n e of cell nuclei [ 1 ] , b u t t h e question of cardiac glycoside inhibition has not been tested in this case.

T h e relationship to observations in cellular systems is strengthened by t h e fact t h a t t h e hypothesis of mixed t r a n s p o r t complexes, which has been applied to cellular systems with considerable success, has also been helpful in t h e i n t e r p r e t a t i o n of t h e otherwise surprising a n d unexpected observation t h a t G A B A addition leads to higher

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s t e a d y - s t a t e G A B A levels in t h e m e d i u m . If t h e i n t e r p r e t a t i o n offered here is correct it also implies t h e existence of sodium p u m p s in s u b - cellular m e m b r a n e s , which to our knowledge, has not been described so far. T h u s t h e mechanisms o p e r a t i n g in cellular t r a n s p o r t of amino acids and in subcellular t r a n s p o r t of G A B A seem to be quite closely related.

A special feature of the subcellular system discussed here is t h a t the sodium dependence is a common element in t h e observations a t 0° and a t 2 9 ° C . I t therefore a p p e a r s possible t h a t the systems involved operate with the same carrier reacting both with sodium and GABA.

This results in an equilibrating t r a n s p o r t a t 0 ° C a n d in a potentially uphill system a t higher t e m p e r a t u r e . Actually such relationships would a p p e a r to be a n a t u r a l consequence of t h e concept of mixed complex downhill m o v e m e n t leading to an uphill transfer of one of t h e compo- nents. T h e additional feature introduced a t higher t e m p e r a t u r e , then, would be the energy-yielding, metabolic breakdown of G A B A and the utilization of this energy for the sodium p u m p .

As to t h e possible physiological bearing of t h e observations r e - viewed and the i n t e r p r e t a t i o n s offered here, it seems clear t h a t t h e experimental conditions differ widely from t h e biological s i t u a t i o n : I n the experiment particles originating from all p a r t s of t h e brain, including glia a n d nerve cells as well as nerve fibers, are mixed. T h e r e - fore, no direct analogy can be assumed with respect to biological conditions. Nevertheless, a few possibilities m a y be discussed briefly.

I n a general w a y t h e inter- or intracellular translocation of G A B A between sites of formation, storage, function, a n d removal m a y depend on the local concentrations of sodium ions. T o n a m e one specific a l - though speculative possibility, t h e e n t r y of sodium into a p r e s y n a p t i c nerve ending during excitation m i g h t trigger G A B A depletion by a l - lowing mitochondria to t a k e u p and metabolize G A B A . T h e p o t e n - tially uphill system in such a case would be used for accelerated r a t h e r t h a n for uphill m o v e m e n t . Likewise sodium m i g h t trigger ejec- tion of G A B A from an inhibitory nerve ending across t h e cell m e m - b r a n e in order to t r a n s l o c a t e it to a neighboring s t r u c t u r e for inhibi- t o r y action.

W h e r e a s in these cases sodium would be used to p r o m o t e t r a n s l o c a - tion of GABA, t h e coupling between G A B A a n d sodium m i g h t also be conceived to operate in the opposite sense, n a m e l y , by translocation of sodium induced by GABA. T h e general feature of inhibitory action, according to neurophysiological analysis, a p p e a r s to be a n increase

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in t h e ion conductance of t h e cell m e m b r a n e . W i t h respect to t h e sodium exchange across t h e m e m b r a n e , t h e mixed carrier complex formed in the presence of G A B A could act as a "sodium s h u n t . "

This might well be one of t h e m e a n s by which neurophysiological inhibition can be achieved.

S U M M A R Y

Sodium-dependent, cardiac glycoside-inhibited, uphill t r a n s p o r t systems a t t h e cellular level are discussed in t e r m s of recently suggested interpretations postulating downhill m o v e m e n t of s u b s t r a t e - sodium-carrier complexes in conjunction with t h e operation of a sodium p u m p . A subcellular t r a n s p o r t system, d e m o n s t r a t e d in brain particles for γ - a m i n o b u t y r i c acid and having in common with these systems N a - d e p e n d e n c e and cardiac glycoside sensitivity, is reviewed and discussed in t e r m s of t h e same hypothesis. I t is shown t h a t the mixed complex mechanism is compatible with all available experimental d a t a and offers interesting neurophysiological implications.

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