Endogenous inhibitor of GABA binding in mammalian brain

Lifer Sciences Vol . 22, pp . 1653-1662 Printed in the U .S .A .

Pergamon Press

ENDOGENOUS INHIBITOR OF GABA BINDING IN MAMMALIAN BRAIN Donald V . Greenlee, Paul C . Van Nesa and Richard W . Olsen Department of Biochemistry, University of California, Riverside, CA

92521

(Received in final form March 20, 1978) Summary Binding of radioactive gamma-aminobutyric acid to homogenates of mammalian brain was detected by a centrifugation assay . The binding capacity of the tiaeue was maximal and stable with time only if the tiaeue was thoroughly washed to remove an endogenous inhibitor of binding . With such washed tissue, . binding to total rat or cow brain appeared to involve two populations of sites in the absence of sodium ions, the major site having a dissociation constant of 150 nM and saturating at 80 pmol/g brain, and a minor site with a R of 20 nM and saturating at 20 pmol/g wet tissue . This sodium-independent GABA binding as a whole was localised in the crude mitochondrial, microsomal, and synaptosomal membrane fractions . Many workers have attempted to measure receptor sites in brain for the major inhibitory neurotransmitter gamma-aminobutyric acid (GABA) (1-3), using radioactive ligand binding (4-10) . SNYDER, ENNA, and collaborators (7,11) have described binding sites, observed under sodium-free assay conditions, which eh owed properties consistent with identification ere postaynaptic receptor sites for GABA (11-14) . They reported one class of GABA binding sites in frozen and thawed membranes (11), but two populations of sodium-independent GABA binding sites in membranes treated with the detergent Triton R-100 (14) . We observed this sodium-independent binding of GABA to mammalian brain homogenates (15), but found difficulties in making reproducible quantitative measurements needed for further characterization of the binding . Problems apparently arise from variable amounts of an endogenous inhibitor present in the tissue eztracts which can be removed by more thorough washing . Using a method of tiaeue preparation which removes this endogenous inhibitor of GABA binding and which ie therefore more suitable for accurate binding assays, we obtained results which are in qualitative agreement with previous reports (7,11-14) . Differences were found, however, regarding the number, binding affinities, and subcellular localization of the GABA binding sites . Also, inhibition studies with drugs and analogues indicate that GABA binding measured in sodium-free buffers and using suitably prepared tissue fractions shows the chemical specificity expected of receptor sites (15-17, GREENLEE, VAN NESS 6 OLSEN, submitted) . Experimental Procedure Tieeue preparation . Mature Sprague-Dawley albino rate (60-90 days old) were sacrificed by cervical dislocation, followed by rapid decapitation . Brains, ezcluding the brain stem caudal to the cerebellum, were homogenized in a Type C Thomas homogenizer (8 passes at 400 rpm) ere a lOS solution in 0 .32 M sucrose at 0 ° . Aliquots at each stage of the preparation were stored frozen for later 0300-9653/78/0508-1653$02 .00/0 Copyright © 1978 Pergamon Press

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analysis . The homogenate was subfractionated according to WHITTARER and BARBER (18), to give fractions P 1 (crude nuclei), Pp (crude mitochondria), and P 3 (crude microsomes) . Routinely the pooled fraction P 2 + p was employed . Frac3 tion P 2 was subfractionated on a discontinuous sucrose gradient (18) to give mitoch3ndria, synaptosomes, and myelin enriched fractions . Fraction P 3 was further fractionated on a linear sucrose gradient (0 .32-1 .7 M), which was centrifuged for 5 hr at 0 ° at 50,000 z g in Beckman rotor SFT-27 . The protein was found distributed at sucrose concentrations of 0 .7-1 .4 M ; these were divided into three appro:imately equal pools, diluted with H 0, and frozen . Tissue fractions were then osnotically shocked, disrupted, frôzen and thawed according to ZURIN et al . (7), or further washed as described under Results . Binding assay . Binding of [ 3H]GABA to rat brain homogenate fractions was measured by a centrifugation assay (7), modified as in refs . 16,17, and GREEN LSS _et el ., submitted . The specifically bound ligand was calculated as the difference between the amount of radioactive ligand associated with the pelleted tissue in the (a) absence and (b) presence of excess (0 .1 mM) nonradioactive ligand (background) . Binding assays were performed with triplicate samples of resuspended membrane fractions at approzinat~ly 1 .0 mg/ml protein in plastic scintillation Mini-Vials (Beckman), using [ H]GABA (Amersham) at either 12 .6 Ci/mmol (10 nM) or 54 Ci/mmol (2 .5 nM), equivalent to 275,000 dpm/ ml . After incubating for 10 min at 0-4° , vials were then centrifuged at 20,000 rpm for 10 min (48,000 z g) in a Sorval SM24 rotor, the supernatant waa discarded, and the pellet rinsed twice lightly without disruption with 2 ml of buffer (0-4 ° ) . The samples were solubiliaed overnight at 23° in 0 .30 ml of Soluene (Packard) and counted in 5 ml of toluene containing 2,5 diphenylozazole (PPO, 5 gm/1) with a Beckman LS-100 scintillation counter ; efficiencies were typically between 32-35X . Protein was measured according to LOWRY _et _al . (19), using crystalline bovine serum albumin as the standard . Results ßstablishment of conditions suitable for assay of binding of GABA to mammalian brain particulate fractions . Dsing a centrifugation assay ae described under Me~~ binding of radioactive GABA to particulate (mitochondrial plus microeomal) fractions of rat brain homogenates could be detected in a sodiumfr~ee buffer, as first described by ZURIH et al . (7) . However, binding of [ H]GABA to rat brain fractions (synaptosomes,mitochondria, microsomee, or niztures) prepared according to that reference showed variation from preparation to preparation as well as a time-dependent decay ; this could be prevented, however, by more extensive washing of the tissue . Figure 1 (circles) illustrates that binding decreased se a function of the time during which the homogenate was incubated (without ligand) at 0-4 ° . The rate of decay of binding activity was not always linear with time and decreased after 2 hr so that less than half of the binding activity persisted after 18 hr . Other preparations ehwed less specific binding per mg protein at time zero and smaller decreases with time . In an experiment typical of numerous observations, a tissue sample of 4 .4 mg/ml was found to bind 4200 cpm initially, under the standard assay conditions . This declined to 1667 _+ 226 cpm bound following 18 hr incubation at 4 ° . When this sample was centrifuged, washed once in fresh buffer, recentrifuged, and half of the pellet resuspended in the same original supernatant fraction, it bound 2207 ± 161 cpm (protein 4 .0 mg/ml) . However, the other half of the same pellet, resuspended in fresh buffer for assay, bound 4693 + 87 cpm (protein 3 .8 mg/ml) . This suggested that the lose of binding activity with incubation time was probably due to the gradual appearance of an endogenous soluble inhibitor substance, rather than a denaturation or enzymatic degradation of the binding proteins . Different preparations exhibited quite variable binding capacity at time zero, ranging from 400-1250 cpm bound per mg

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protein under these conditions . Further washing of the membranes resulted in a highly reproducible binding of 1250 + 25 cpm/mg (average of 8) . This suggests that variable amounts of the endogenous inhibitory substance were present in different preparations . üashing the tissue by the following procedure prevented this time-dependent loss of binding capacity and alloyed further characterization of the GABA the homogenate was osmotically shocked with two sequential water binding :

i U 100 " c 0

m _0

F c

90

7w: 0

i 30

i 80

v 90

i 120

0° Incubation Time (min)

FIG . 1 Decay with time of GABA binding to um+ashed tissue and removal of decay &at brain particulate fractions (P 2 + p , 1 .8 mg/ml (inhibitor) by washing . protein) were incubated at 0° for varying times in aseaty buffer prior to aaeaying GABA binding as described in Methods . (~ ) : Tissue prepared according to ZURIH et al . (7), i .e . osmotic shock in distilled H2 0 and freeze-thawing ; ( ~) : tieeue further vaehed in both H 20 and assay buffer wYth additional freesIn both cases eodiumiag and thawing, as described in tezt, prior to assay . Points are mean of triplicates free conditions were maintained throughout . (variation roughly SX) ; specific binding :backg~ound was roughly 1 .75 :1, typical CPH being 3500/2000 . The concentration of [ A)GABA was 10 nll, 12 .6 Ci/mmol .

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washes, frozen in the second homogenate (-15° or below), thawed, and centrifuged . The pellet was resuspended in buffer (50 mM Trie-citrate, pH 7 .1 at 2 ° ), centrifuged, resuepended in the same buffer, and frozen again . On the day of the assay, the suspension was thawed, pelleted, rewashed once in buffer, and then resuspended in fresh buffer for assay . Further freezing and washing steps beyond these were not necessary, nor detrimental to binding . It was only after this extensive washing procedure that GABA binding appeared linear with protein concentration up to at least 1 .4 mg/ml (not shown) and that binding capacity was shown to be stable with respect to homogenate incubation time (Figure 1, squares) . The binding of ligand to the washed tissue was rapid (completed during the minimum time required to assay, i .e . the 10 min centrifugation), reproducible, and stable with time . Also, the radioactive GABA, which was shown by thin layer chromatography to be greater than 99X pure before assay, was not significantly metabolized (<5X) during a 15-60 min incubation at 0-2 ° with the tissue . c .m ô a o, E w ô E o.

2 .5

2.0

v c

0

a _v_

U O

.U v T t O _c_

E v E E v

c~

L0

0.5

I .0

2.0 3.0 Gamma-aminobutyric acid

4.0 (~,r.m )

5.0

FIG . 2 Variation of GABA binding with ligand concentration . ~11 pointe (average of triplicates) contained 1 .59 mg protein and 0 .125 Ci [ H]GABA in 1 ml and Tissue samples were prepared and assayed as varying nonradioactive ligand . described in Methods . Inset : same data plotted according to SCATCdAHD (described in DIXON and WEBB, 20) . The concentrations of free ligand are taken as the total ligand since a negligible quantity (<2X) of the total was bound at any point .

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Endogenous Inhibitor of GABA Binding

Under the conditions described the signal/noise ratio background was 1 .75 :1, and other preparations gave values depending upon the brain region and subcellular fraction .

of ae

1657 cam bound to high se 9 :1,

Saturation Analysis . ABA binding was measured under the standard assay conditions using 10 nl~[ [ H]GABA (12 .6 Ci/mmol) and varying amounts of nonradioactive GABA . A typical plot of bound GABA versus the concentration of free GABA is shown in Fig . 2 ; when replotted in Scatchard form, the data indicate a single class of binding sites (inset) . GABA binding isotherms from aia different experiments (rat brain mitochondrial plus microsomal fractions) yielded an

40

o CONTROL

w

W

TRITON TREATED

m

Kp(nM)

9 ma: (pmol/mq protein)

32 176

1 .17 4.51

23 146

r

0.69 3 .14

-0 .886 -0 .986 -0 .740 -0 .947

20

~o O 10

0

I

2

3

4

5

[ 3 H] GABA BOUND (pmol/mq protein)

FIG . 3 SCATCHARD plot of GABA binding to rat brain membranes ae a function of ligand concentration . Tissue was prepared ae described under Methods, and then preincubated in 50 mM Tris-citrate buffer, pH 7 .1, with or without 0 .05X (v/v) Triton %-100 at 22 ° C for 30 min, followed by centrifugation and reeuspension of the pellet in the same buffer without detergent for assay . Protein was 1 .0 ng/ml for preincubation and for assays . Triplicate samples were incubated at 0 ° for 5 min with varying aeounta of [ 3H]GABA (54 Ci/mmol, Amersh~n) for the lowest concentrations of ligand (0 .25-2 nI~S) and constant amount of [ H]GABA (4 n!i) and varying concentrations of nonradioactive ligand for the rest of the concentration range . At each point, background was estimated by triplicate samples containing the same amount of [ H]GABA and also 0 .1 mM nonradioactive GABA to displace any specific binding . The data in the figure eh w the apparent (pmoles bound/mg protein at saturation) and linear correlation coefm~: (r) cien of the linear functions shown, determined by linear regression . The high affinity functions are derived in either case from the points of GABA concentration up to 12 nli, the low affinity functions from the points over 24 n![ . Hesulte are typical of three experiments with Triton and siz without .

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Endogenous Inhibitor of GABA Binding

apparent dissociation constant value KD ~ 0 .150 y M + 0 .097, (standard deviation of the mesa) and a quantity of ligand bound at saturation (B~ z ) of 2 .90 pmol/mg protein _+ 0 .62, ae calculated from Linear regression analysis by the EadieHofetee or Scatchard method (20) . However, when higher specific activity [ 3H]GABA was employed to look for binding in the nli concentration range, a second very high affinity binding site was observed (Fig . 3), with an apparent of 24 + 6 nM (average of 3), in addition to the usual 150 nM site . The of the high affinity site KD PretréâÉment of the membranes with was 25 + 5X of the low affinity site . 0 .05X Triton R-100 resulted in virtually no change in the binding characteristics . As shown in Fig . 3, the same two binding sites of almost identical R were seen with Triton treatment ae without . The increase in the number of binding sites of both classes after detergent treatment may be explained by the solubilization of some inactive proteins in these samples ; alternatively, a slight increase in the number of binding sites detected, but not in affinity, may be occuring . Thus two populations (at least) of sodium-independent GABA binding sites are present, even when membranes are not treated with detergents . The same two binding sites have been uniformly observed in different brain regions, subcellular fractions, and species, including mouse and bovine cerebellum and cerebrum (not ehwn) . TABLE 1 . Subcellular Localization of Sodium-Independent GABA Binding Sites in Hat Brain Specific activity cpm Subfraction* bound/mg protein Supernatant l Pellet l Pellet l Pellet a Myelin Synaptoeomes Mitochondria P Pellet l Pellet Pelleta Myelin Synaptoeomee Mito~~ondria P3-1++ P 3-2++ 3-3

904 593 488 1271 240 802 534

+ + + + + + +

382 290 42 232 139 209 245

a Total activity 10 cpm bound/g brain 32 .6 5 .8 9 .7 9 .9 0 .5 4 .5 4 .3

± + _+ + _+ + +

17 .5 3 .3 3 .1 3 .9 0 .2 2 .1 2 .4

Percent of total binding recovered in fraction : + Crude homogenate P a l 23 + 13 38 + 13 39+15

6 + 2 48+22 46 + 26

35

2U

Specific b~nding was measured by centrifugation as described in Methods, *Particulate material in each fracwith 10 nM [ H]GABA at 12 .6 Ci/mmol . tion was prepared for assay as described in Methods, including osmotic shock, Protein was roughly 1 mg/ml in assays . ~reeze-thaw, and thorough washing . + standard deviation (4 ezperiments, triplicate determinations) . Average ++ One ezperiment only ; Pa_l, P3-2' and P3-a were subfractions of P g obtained from a linear sucrose gradient described in Methods, and correspond to pools of tissue banding at sucrose concentrations of <0 .8 M, 0 .8-1 .1 M, and >1 .1 M respectively .

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Fractionation of rat brain homogenates van pertormed Tissue subfractionation . as escri e in e o s" and the average results of four different ezperiments are show in Table 1 . Fractions P 2 (crude mitochondrial fraction) and P 3 (crude microsomal fraction) contained about 80X of the total binding sites in the crude homogenate, with roughly equal quantities is each . Within Fraction P2 , the binding sites per mg protein were enriched in the synaptoaomal fraction, although the mitochondrial fraction contained nearly Fraction P3 vas subfractionated on a linear gradient, is an equal amount . which 80X of the GABA binding sites in P 3 were recovered in particles eedimenting at densities of less than 1 .1 M sucrose . Discussion This paper describes further characterisation of sodium-independent GABA binding sites in mammalian brain vhich were first described by ZUKIN et al . (7) and postulated to be postaynaptic receptor sites for GABA . These bindingsites are best detected in sodium-free buffer with frozen and thawed and thoroughly disrupted tissue homogenates, procedures vhich eliminate nonreceptor uptakebindiag of GABA (7,11) . Alternatively, these receptor-like sites can be measured in membranes pretreated with low concentrations of the mild detergent, Triton 5-100 (14,21,22) . We report that brain homogenates contain an endogenous substance vhich inhibits binding unless rigorously removed . Using thoroughly washed tissue samples, two classes of GABA binding sites were found (without detergent) as yell ae greater amounts of binding and a somewhat different subcellular localization than has previously been reported . Nevertheless, these observations are consistent with previous reports (7,11,12) . The tissue and subcellular localization, ontogeny, and chemical specificity (7,1017,23,24) support the contention that the sodium-independent binding sites are related to GAHA receptors . A perfect correlation has been observed for inhibition of this binding by GABA analogues and drugs (and only those) vhich are active on GAGA synapses in intact tissue (7,11,13,16,17, GREENLEE et al . submitted) . However, those studies now appear to apply to the mixture ôf two classes of GABA binding Bites ; it seems likely that both shoe a receptor-like specificity . Ae indicated by SCATCHARD plot, the binding of GASA to rat brain detected in sodium-free buffer and with thoroughly washed tissue apparently involves two classes of sites of apparent &D ~ 150 nM and RD ~ 20 nM . It cannot be determined presently whether these two sites correspond to two types of GABA receptor, perhaps involving different cells, or to different states of a single receptor class . A similar situation has been observed with other neurotransmitter binding sites (25-27) . The high affinity binding site for GABA would appear functionally unreasonable on kinetic grounds . However, the affinity of the lover affinity binding sites for GABA is reasonably consistent with the biologically effective concentrations of the neurotransmitter substance (2,3), considering the differences in experimental conditions, e. g . the buffer, membranous homogenate ve . intact cell, multistep cooperative cellular response, etc . The quantity GABA binding sites found (80 + 15 pmol recovered/g rat brain for the low affinity site, 20 + 5 pmol/g for thé high affinity site) is a reasonable number to a :pect for transmitter receptor sites (5,17,25-28) . This quantity is 3-4 times greater than that reported by ENNA and SNYDER (11), apparently because of differences in the procedures for tissue fractionation and washing, although rat strain differences could also be involved .

of

The existence of two GABA binding sites in frozen and thawed membranes is consistent with the observation of two binding sites for GAGA in membranes treated with Triton R-100 (14,21,22, cf . discussion below), and with two

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apparent classes of binding sites for [ 3 H]muacimol (29), which preaunably correspond to the two GABA binding sites, although more evidence is needed to establish this point . The two sites may ezplain the apparent nonlinear inhibition of GI1BA binding observed with bicuculline and acme other drugs (GBEENLEE, VAN NE8S, 6 OLSEN, unpublished) . Further studies should ehw whether these correspond to pharmacologically distinct GABA receptors . An apparent increase in sodium-independent GABA binding sites of about 2-fold following freezing of the tissue was observed by ZUHLN et al . (7) . While it is possible that such a result reflects some unburyingofcryptic sites, or a conformational transition in the binding protein, our results suggest that the freeze-thaw and osmotic shock of the tissue are useful in breaking open cellular membranes to allow thorough washing out of interior contents, i .e . these procedures not only help in eliminating the capacity of the tissue to transport GABA into membrane-bound apace, and/or lowering the extent of sodium-dependent binding, but are essential for adequate removal of endogenous receptor binding ligands, including GABA itself, and perhaps other inhibitory substances, such as phoapholipide (9,30) . We did not characterize the endogenous substance responsible for inhibition of binding in our assays, but GAGA is a reasonable candidate . However, the slow appearance of inhibitor with time would seem unlikely to be due to GABA gradually entering the solution from some tieaue-bound pool . The inhibitory substance(s), when present in unknown and variable quantities, could give underestimates in binding . Washing the inhibitory substance from the tieaue with noneodium salt solutions was found valuable in obtaining reliable and optimal binding, no matter how vigorous the tieaue disruption . A similar phenomenon has also been observed with opiate receptor sites, where saline washes resulted in better removal of endogenous opioid peptides (31) and more accurate binding data . Hence, the somewhat unusual methods of tieaue preparation and binding assay are reasonably justified, and do not preclude the physiological relevance of the binding measured . Three laboratories have recently reported that sodium-independent, but not sodium-dependent GABA binding in mammalian brain was not inhibited but actually enhanced in membranes treated with a 0 .05x solution of the mild detergent Triton R-100 (14,21,22) . However, two distinct binding sites were observed in Triton-treated tieaue, is contrast to the single class of sites seen in frozen and thawed tieaue (11) . Both classes of sites appeared to be inhibited by receptor-specific drugs (13,14,21,22) . We observed the same two claeaes of binding sites without detergent treatment, using thoroughly washed tissue and high specific activity GABA for binding assays . The high affinity site ie also suggested in SCATCHARD plots of GABA binding without detergent previously reported but not discussed by other workers (11,23) . The appearance of a "new" very high affinity class of binding sites upon detergent treatment thus appears to reflect again a better removal of endogenous inhibitory ligand, rather than modification of the lower affinity state . Presumably the high affinity sites are more difficult to wash free of endogenous ligands, and preparations with variable amounts of endogenous inhibitor would allow variable detection of primarily the high affinity sites . Thus either detergent treatment or the lengthy freeze-thaw washing procedure we use here seem suitable for removing GABA uptake and endogenous ligands necessary for accurate measurement of GABA binding sites which are candidates for receptors . GABA binding sites as described here were found primarily in eynaptosomal and microsomal fractions . Although our fractionation procedure rigorously followed published procedures (18), the fractions obtained in this method are not highly purified, and the distribution of protein and binding activity between synaptosomes and the crude mitochondrial fraction was not highly

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A significant portion of the sodium-independent GABA binding, reproducible . both specific activity and total activity, was detected in the microsomal fraction, in contrast to the report by ZUKIN et al . (7), who reported no Our localizationresults, which have been binding in the microsomal fraction . reproduced numerous times, are consistent with the detection of receptor-like binding sites for several neurotransmitters (28) and for picrotoainin (17) in the light microsomal fraction . Although definitive identification of these membranes would require comparison to acceptable markers for postsynaptic membranes (which are not currently available), these membranes are probably small synaptosomes, or perhaps some free posteynaptic membranes fractionating Other with other plasma membrane fragments in the light microsomal region . membrane locations, such as endoplasmic reticulum, cannot be eacluded . Thus the subcellular distribution for GABA binding sites described here is consistent with, but does not prove, a postsynaptic membrane location . Acknowledgments : We thank Dra . W . B . LEVY, B . MEINERS, and M . R . TICKU for helpful discussion . Supported by NSF Grant BNS 73-02078 and NIH Grants NS 12422 and RCDA NS 00224 to R . W . OLSEN . R . W . OLSEN is an Alfred P . Sloan Fellow .

1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 .

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