Diazepam stimulates the binding of GABA and muscimol but not thip to rat brain membranes

Diazepam stimulates the binding of GABA and muscimol but not thip to rat brain membranes

Neuroscience Letters, 38 (1983) 315-320 315 Elsevier Scientific Publishers Ireland Ltd. DIAZEPAM STIMULATE8 ~ BINDING OF GABA A N D MUSCIMOL BUT ...

631KB Sizes 0 Downloads 25 Views

Neuroscience Letters, 38 (1983) 315-320

315

Elsevier Scientific Publishers Ireland Ltd.

DIAZEPAM STIMULATE8 ~

BINDING OF GABA A N D MUSCIMOL

BUT NOT THIP TO RAT BLAIN MEMBRANES

JOHN H. SKERRITT and GRAVAM A.R. JOHNSTON*

Department of Pharmacology, University of Sydney, N.S.W., 2006 (Australia) (Received April 22nd, 1983; Accepted April 27th, 1983)

Key words: GABA receptors - multiplicity - benzodiazepine/GABA receptor complex - diazepam anticonvulsant - GABA agonists Diazepam (10-1000 nM) enhanced the binding of [-*HIGABA and of the monocyclic GABA agonist I~Hlmuscimol, but failed to alter binding of the bicydi¢ GABA agonist [~HITHIP to fresh, well washed rat brain membranes incubated at 2°C. Although stimulation of [3HIdiazepam binding by THIP was observed at higher incubation temperatures and in the presence of chloride ions, these measures did not induce a corresponding enhancement of [3HITHIP binding by diazepam. These resulss extend earlier observations of the unusual behavior of THlP as a seleclive GABA agonist, and emphasize that enhancemenl of benzodiazepin¢ binding by GABA agonisls is nol necessarily reflected in a c0n;plemenlary manner by any action of benzodiazepines on Ibe binding of GABA agonist.~.

A variety of neurochemical approaches have been applied to study the in vitro interactions between benzodiazepine and GABA receptors. Benzodiazepines slow the heat inactivation of [~Hlmuscimol binding sites, while several GABA agonists, including THlP and muscimol, reduce heal inactivation of benzodiazepine binding 17]. Many GABA analogues stimulate the binding of radiolabelled benzodiazepines to a variety of brain preparations [14, 27], and several groups have observed an enhancement of [3HIGABA binding by diazepam and other clinically useful benzodiazepines [8, 17, 23], although the latter effect may be quite labile [22l. The relatively rigid bicyclic GABA analogue, THIP, is a bicuculline-sensitive inhibitor of the firing of cat spinal interneurones of equal potency to that of GABA [15]. It is of intense interest as a potentially therapeutically useful GABAmimelic, having anticonvulsant and analgesic activity after oral administration [10, 18]. Although approximately one-fourth as potent as inhibitor of GABA binding as GABA itself, unlike GABA, THIP fails to enhance benzodiazepine binding at 4°C, and instead antagonizes GABA-induced enhancement of benzodiazepine binding [2]. However, more recent results indicate that, at higher temperatures (25-37°C), and in chloride ion-containing buffers, THIP may enhance benzodiazepine binding [26]. We have examined the effects of diazepam on the bindinq of 3 bicucullinesensitive neuronal inhibitors, namely [3HIGABA, [3Hlmuscimol and [3HITHIP, to * Author for correspondence.

.tl6

repeatedly washed fresh brain membranes. We repofl that while diazepam stimulates the binding of GABA and muscimol, THIP binding is unaltered, even under conditions whereby THIP may potently stimulate diazepam binding. Whole brains from male Sprague-Dawley rats, 12 weeks old, were homogenized in 0.32 M sucrose and synaptosomal-mitochondrial pellets formed by differential centrifugation. Pellets were osmotically shocked in distilled water and washed 8 times by centrifugation and resuspemion in buffer (50 mM Tris-citrat¢, pH 7. I at 2°C), as described earlier in detail [231. In some experiments the final two washes were in 50 mM Trig-chloride buffer, pH 7.1. Incubations were rominely performed at 2°C in Tris-citrate buffer; in some THIP binding and diazepam binding experiments incubations were at 2°C in either Tris-citrate or Tris-chloride buffer, pH 7.1. Aliquots of homogenate (0.5 mg protein) were incubated in I ml buffer for 15 min (GABA receptor ligands) or 30 min (diazcpam binding) with either 250 nM [~h]GABA (2.0 Ci/mmol after i.¢,0tope dilution), 100 nM |JHJmuscimol (I.9 Ci/mmol) or 100 nM |~HITHIP (4.9 Ci/mmol) for dose-response studies with diazcpam, or 0.67 nM [JHldiazepam (86.6 Ci/mmol) for concentration-response studies with GABA. Scatchard analysis of GABA ligand binding was performed using 12-15 ligand concentrations in the followinl~ ranges: |JH]GABA, 1.2-10,000 nM; [JH]muscimol, 0.6-10,000 nM; and [JH]THIP, 1.0-10,000 nM. Diazepam (10, I ~ or 1000 nM) or dimethylsulfoxide vehicle (0.001 e;'0v/v) was present as indicated. Non-sa;urabl¢ binding was asses~d using I mM GABA ([~HIGABA binding), 100

pM mu~imol ([JHImu~imol binding). I mM THIP ([JHITHIP binding) or 10 pM diazepam ([~Hldiaz~pam binding). Binding was terminated by rapid centrifugation at the incubation temperature used (2°C or 25°C) in the case of GABA receptor ligands for 5 min at the incubation temperature in an Eppendorf 5412 centrifuge (10.000 ,e). or rapid filtration through Whatman GF/B filters in the case of I~Hi diazepam binding, as described previously [22]. All radioligands were obtained from TABLE I KINETIC PARAMETERS FOR THE BINDING O F GABA. MUSCIMOL AND T H I P TO RAT BRAIN MEMBRANES The data represent means ~ S.E.M. of experiments performed in triplicate or quadruplicate on the number of tissue preparations shown in parentheses, using 12-15 different ligand concentrations. The kinetic parameters were calculated by computer-assisted non-linear regression analysis according to the method of Ro~nlhal [20]. ~H-Ligand

Kinetic parameters

GABA (4)

Ka; = 30.3 ± 6.0 nM, Kd: = 82". _+ 24 nM.

Bm,i = 0.174 ~ 0.005 pmol/mg B , , , : = 3.71 _, 0.13 pmoi/mg

Nlu~imol (3)

/C = 59.0 _+ 3.0 nM.

Bm,tl

T H I P (4)

Aa: = 5.5 _+. 0.8 nM. Ka: = 331 _+ 66 nM.

I]L~,~ = 0.018 _+ 0.009 pmol/mg B . , , , = 0.614 _ 0.032 pmol/mg

=

.'~. 9 . '9 ±

0.08 pmol/mg

317

New England Nuclear. Unlabelled GABA was purchased from Calbiochem and muscimol from Sigma. THIP was a gift of Dr. P. Krogsgaard-Larsen and Lundbeck AB, Copenhagen, while diazepam was donated by Roche Products, Australia. Saturable binding of [3H]GABA, [3H]muscimol and [3H]THIP was readily detectable in fresh, well washed rat brain membranes. The 3 ligands, however, differed markedly in their binding kinetics (Table 1). As demonstrated in frozen, high frequency rehomogenized or detergem-extracted homogenates [21], [3H]GABA bound to both a 'high affinity' and a "low affinity" population of sites. [3H]THIP also bound to two sites in fresh brain membranes. Despite the use of a wide range of ligand concentrations, [3H]muscimol appeared to bind to only one population of binding sites, with computer analysis of Scatchard plots [22] failing to reveal significant deviation from linearity. While the affinities of [3HITHIP for the high and low affinity binding sites are greater than those of [3HIGABA, the maximal binding capacities of the THIP sites are considerably lower, with a total density of only onesixth that of the GABA binding sites. High affinity binding of [3HITHIP was extremely low, with a Bum, of only 18 fmol/mg, varying somewhat between tissue preparations. Low concemrations of diazepam produced significant stimulation of [3HIGABA binding (Table !1). Previously published data [231 indicate that enhancement b y diazepam is concentration-dependent, with maximal enhancement (44%) occurring at 300 nM and half-maximal enhancement at 20.7 ± 3.4 nM. This effect has been shown due to a selective increase in the affinity of the lower affinity GABA binding site. Binding of 100 nM 13Hlmuscimol was also stimulated by 10-1000 nM diazepam, but to only about half the extent of stimulation of {~H]GABA binding. Matsumoto and Fukuda [16] have also shown a weak enhancement of muscimol binding by diazepam and clonazepam. TABLE II EFFECT OF DIAZEPAM ON THE BINDING OF GABA RECEPTOR L I G A N ~ IN VAEIOUS BUFFERS Data are expressed as percentages of binditqg in the absence of diazepam, means ± S.E.M. of experiments performed in pentuplicate or hextuplicate on the number of tissue preparations shown in parentheses. *P<0.01, **P¢'0.005, ***P<0.001, from controls by Student's l-test.

~H-Ligand: Tris-citrat¢ (2°C)

Tris-citrate (25°C)

Tris-chloride

(25°C) Diazepam (nM)

GABA (I 5)

(P I0 I00 IO00

I00 120 134 137

± ± ± ±

Mnscimol (5) 2 2*** 3*** 4"**

aDimethylsulfoxide vehicle.

I00 III 115 116

:t: ~ ± ±

THIP (5)

2 I00 ± 8 2* 108 ± 9 3** 91 ± 8 2** 112 ± II

GABA (5) I00 128 137 142

± ± ± ±

4 4*'* 3"** I***

THIP (6)

THIP (3)

I00 98 104 104

I00 I01 III 102

± 19 ± 6 + 9 ± 5

± 12 ± 9 + 12 ± 9

While THIP did not enhance |'Hlflunitrazqmm binding to cerehellar membranes at 0°C, at elevated temperatures (25-37°C) and in the presence of chloride ions, THIP has been found to stimulate binding |261. We observed similar results with |JHld~.epam binding t o washed synaptosomal membranes from whole brains (Table !11). However, neither at 2°C nor 25°C in the absence of chloride ions, nor at 25°C in the presence of chloride, did we observe significant alteration of [~HJTHIP binding by diazcpam (Table !1). There appears to be several basic differences hetween the actions of the 3 GABAmimetics on |~Hldiazcpam binding, and the actions of diazepam on the binding of [JH]GABA, [~HJmuscimol and [JHITHIP. While muscimol is a more potent enhancer of diazcpam binding than GABA 114], diazepam is less effective an enhancer of [JH]muscimol binding than of [~HIGABA binding under the conditions used in our studies. It should he noted, however, that the relative dqpre¢ of enhancement of [JHIGABA and [JHlmuscimol binding by diazepam may depend on the degree of saturation of each population of sites achieved with each ligand. Diazcpam also fails to alter |~H]THIP binding under conditions where THIP enhances [JHIdiazcpam binding. Other studies in our laboratory [231 show that, while diazA~pamenhancement of GABA binding is decreased or abolished by membrane treatments such as freezing, high frequency homogenization or detergent extraction, the enhancement of diazepam binding by GABA is not reduced by these modifications. These results suggest thai the subpopulation or conformation of GABA receptors to which iJHITHIP binds is insensitive to modulation by diazepam. This is in contrast to the observed enhancement of both I tHITHIP and [JHIGABA binding by pcntobarbitone [19], and together with electrophysiological [251 and other neurochemical studies [19. 28] further emphasizes thai the molecular mechanisms of enhancement of GABA-mediated inhibition by barbiturates and benzodiazcpines may be quite different. TABll~ III ENHANCEMENT OF DIAZEPAM BINDING BY T t I I P AND GABA (100 ~M) Data shown are means ~. S.E.M. of triplicate determinations ~m 3-6 membrane preparations. Contrel [~H]diazepam binding, at 0.67 nM !igand concentration, was 89 ± 5 fmol/m$ (Tris-citrate, 2°C); 2.0 _~ .~ fmol/mg (Tris-citrate. 25 ~) and 27 _+ 7 fmol/mg (Tris-chlorid¢, 25°C). 100/zM was chosen as I~..G A B A and THIP concentration sir~e at beth tempatures 100 pM G A B A provides maximal enhancement of ~lt-diazcTmm binding 122£ "P<0.0~. **P<0,001, fror,~ controls b v Student's t-test,

Buffer, teml~.'rature

Enhancer

% Control binding

Tris-citrate, 2°C

GABA ]'HIP GABA THIP GABA THIP

171.2 115.1 261.2 130.0 264.0 182.4

Tris
± _~ ± _+ +, +_

2.3r0 ** 3.7ro 5.5r0 ** 6.0~0 * 6,1% ** 15.6% **

319

Several groups have claimed iigands such as [3H]muscimol to be superior to [3H]GABA for the study of bi~uculline-sensitive GABA receptors by binding techniques, on the basis of its greater selectivity for presumed postsynaptic receptors over neuronal and glial GABA uptake carriers [1, 24]. Muscimol is, however, a substrate for the neuronal GABA uptake system i n r a t brain slices [11]. Under the sodium free conditions used in the experiments neither high nor low affinity [3HIGABA binding to washed synaptosomal membranes is displaced by GABA uptake inhibitors such as nipecotic acid [21]. Other workers [6] have suggested the presence of two sites for GABA, but only one for muscimol in membranes prepared from mouse neuronal cultures. In more extensively treated membranes, 2 or even 3 binding sites for [3Hlmuscimol may be detected II, 12, 13]. Further, differences in the displacement kinetics of GABA on [3Hlmuscimol and [3HIGABA binding have been noted [13], and the two amino acids do not have identical electrophysiological actions on lobster muscle [3]. Significantly, our kinetic results (Table !) indicate that neither [3Hlmuscimol nor [JHITHIP bind to all the sites labelled by [3H]GABA. Falch and Krogsgaard-Larsen [5] have noted dissimilar regional distributions in the brain for [3HITHIP and [3HIGABA binding, and concluded that [3H]THIP may label only a subpopulation of bicuculline-sensitive GABA receptors. Such may also be the case for [~H]muscimol, since the Bm~ of [3H]museimol binding is significantly lower than that of [~H]GABA, as noted earlier [12]. [3HlMuscimol may also bind to sites not labelled by [~HIGABA 14J. Our present studies have shown that diazepam has differing actions on the binding of the 3 GABA receptor ligands. It appears that the natural transmitter, GABA, may be the most suitable radioligand for the neurochemical study of drug action upon 'GABA receptors'. [~H]THIP and [JH]muscimol are useful for the study of the subpopulations of GABA receptors activated by these compounds, and which may be responsible for their unique pharmacological actions. Studies with a range of labelled GABA receptor ligands shotAd provide more information on the nature of the multiplicity of GABA receptor-ionophore complexes involved in neurotransmission. The authors wish to thank Ms. S. Chen Chow for technical assistance, and Dr. P. Krogsgaard-Larsen and Lundbeck AB for gifts of THIP. The study was supported by the National Health and Medical Research Council of Australia. I Beaumont D., Chillon, W.S., Yamamura, H.I. and Enna, S.J., Mu~imol binding in rat brain: association with synaplic GABA receptors, Brain Res., 148 (1978) 153-162. 2 Braeslrup Co, Nielsen, M., Krogsgaard-Larsen P. and Faith, E., Partial agonisls for brain (;ABA/benzodiaz~'pine receptor complex, Nature (Lond.), 280 (1979) 331-333. 3 Constanti, A. and Nistri A., l)iffcremial effects of sodium-free media on -v-aminobutyratc and muscimol-cvoked conductance increases recorded from lobster mu~le fibres, Neuroscience, 6 (I 981 ) 1443-1453. 4 DcFeudis, F.V., Binding studies with muscimol; relation to synapti¢ gamma-aminobutyrate reeeptors, Neuroscienc¢, 5 (1980)675-688. 5 Falch, E. and Krop£aa_rd-Larsen, P., The binding of the specific GABA agonist [JH]THIP to rat brain synaptic membranes, J. Ncurochcm., 38 (1982) 1123-1129.

J20

6 Frer¢o IILC.o Maedoemld. R.L. and Young, A.B., GABA binding and bicuculline in spinal cord and cortical membranes from adult rat and from mouse neurons in cell culture, Brain Res., 244 (19821 145-153. 7 Cta~,lt M, and Snyder S.H.. ~ e recoLmition sites on GABA receptors, Nature (Lond.). 287 (1980| 651-652. S Guidotti A., Toffano G. and Costa E., An emtdogeno~ protein modulates the affinity of GABA and ]~3zodiazepit~ rcggptor$ ill rat bcrahl, Natterg (Lond.), 275 11978) 553-555. 9 Her~qlml, M. and Baklessarini, Ig.J.. Evidcncg for two types of binding of ~H-GABA and ~Hmtt~mol in rat eerelMH cortex and eerdmlhtm, Life Sci., 24 (19791 1849-1854. 10 Hill, L C . . Mamer. R. ~ H.-H. and Roomer, R., Analgesic properties of the GABA-mimetic THIP, Eufop. J. Pharm~ol., 69 (19811 221-224. I 1 John,ton. G.A.R., Kenne~ ~. S~M.E. and Lodge. D., Mttscimol uptake, release and binding in rat brain dices. J. Neurochem.. 31 (197S) :.T,19-1523. 12 John,ton. G.A.R.. Allan. R.D.. Kenned)-, S.M.E. and Twitchin, B.. Systematic stud)" of GABA anal~ of re~ri~ed conformation. In P. Krogsgaard-Larsen. J. ScheeI-K.~uger and H. Kofod (Ed~.J. GABAoNcurotransmitter$. Munksttaard. C ~ , 1978. pp. 149-164. I J Jordan. C.C.. Matus. AA.. Pimrowski. W. and Wilkinson. D., Binding of [~HJl-aminobutyric acid and I'Hl-mu~'imol in purified rat brain synaptic p t a s m membranes and the effects of bicuculline. J. Nc~Iro~II~gn.. 39 (14]1112112~11. 14 Karobath, NI., Placheta P.. Lippitsch NI.. and KrogMgaard-Lars~'n Po, h stimulation of tg,nzodiazepine receptor binding mediated I ~ / a neroel GABA receptor++ Nature (Lond.), 2"/g (19"791 7411~749. I.~ Krogsgamrd-I ar~'n P.. Johnston (;.A~R~ Lodge D.. and Curtis. D~R~, A ne~ cla~ of GABA agonist. Nature (Lond~). 2¢M~(1977) ~3~55, 16 Mat,umolo. K, and Fukuda. It,..~timulatory and protecti~e effect~ of I~mzodiazelpines on GABA receptor~ labeled ~ith I'Hlmu~imol. IAfe ~:f~. ~)(19821 93~*~94J~ I"I .%leiner~ I]~A. and Salama A.I.. Enhancement of I~n,~odialepine and GABA bindinl~ by the no~el an~iolyti¢, tr~a/olate, l~ur{~p. J. Pharmacol.. 7t¢ (19"821 JI5 322. I I¢ .%leldrum. B. and H~rton. R.. F ffect~ of the big'clio GAFIA agoni,t, THIP, on m)~¢gloni¢and ,ei/ure re;+~sn+~++in mice and bald+on++m+ithreflex epilel~y, I:urop+ J+ PharmacoL, bl (191¢0)231 +=2J7+ 19 ()l~en R.W.. (;ABA +ben/odia,,epine.barbiturate receptor interaction,. J+ N~q,r~ohemo. 3"/(1981) 1~ol3. :, 20 Ro,~'nthal, It.E.. A graphic method for the determination and pre~ent~cio*; c, ,,inding parameters in a complex system, Analyt. Biochem., ~ (19671 525~532. 21 Skcrritt. J.It. and Johnston (;.A.R.. P ~ n a t a l development of GABA t.~rding sites and their end(~enou~ inhibitors in rat brain, Devdop Neuro~i., .,i (1~2) 189ol97. 22 Skerritt J.H., ('hen C'how S. and Johnston (;.A.R.. Differences in the interactions between GABA and benzodiazepine bi~ding sit~, Neurosci. Lett., 3J (1982) ITJ=I7S. 2.t Ske~Htt. J.II.. Willow. M. and Johnston. G.A.R.. Diazepam enhancement of low affinity GABA binding to rat brain membranes. Neuro~i. Lett...'9 (1982) 6J-66. 24 Snodgrass S.R., Use of H-musctmoi ' for (;ABA receptor studies, Nature (Lond.), 273 (1978) 392~394. 25 Study R.E. and Barker J . L . . Dia~¢pam and ( - ) p e n t o b a r b i t a l - fluctuation analysis res'¢als different mechanisms for potentiation of GABA responses in cultured central neurons, Proc. nat. Acad. Sci. U.S.A., 78 (1981) 7180-7184. 26 Su~vi|ai, P. and Karohath, M., The effect of temperature and chloride ions on the stimulation of ['Hlflunitrazepam binding by the muscimol analogues THIP and piperidine-4-sulfonic acid, Neurosci. LetL, 19 (1980) 337-J41. 27 Tallman, J., Thomas, J.W. and Gallager D., GABAergic modulation of benzodiazepine binding site ~nsitivity, Nature (Load.), 274 (1978) 383-385. 28 Willow. M. and Johnston G.A.R., Enhancement of GABA binding by pentobarbitone, Neurosci. Lett., 18 (198(t) 3,3-3,7.