NeurophormacologyVol. 31, No. 9, pp. 895-898, 1992 Printed in Great Britain

002%3908/92$5.00 + 0.00 Pergamon Press Ltd

POLYAMINES MODULATE THE BINDING OF GABA,-BENZODIAZEPINE RECEPTOR LIGANDS IN MEMBRANES FROM THE RAT FOREBRAIN G. M. GILAD,* V. H. GILAD* and R. J. WYATT Neuropsychiatry Branch, NIMH Neuroscience Center at Saint Elizabeths Hospital, Washington, DC 20032, U.S.A. (Accepted 28 April 1992)

Summary-The effects of spermine, spennidine and putrescine on the binding of the GABA,-benzodiazepine receptor complex were examined in the hippocampus and frontal cortex membranes of the rat. The results demonstrated modulatory effects of polyamines on the binding of diazepam and flunitrazepam but not on that of GABA, muscimol and Ro 15-1788. When membranes were prepared without detergent, the polyamines enhanced the binding of diazepam. However, while the binding capacity increased after homogenization in the presence of the non-ionic detergent Triton X-100, the polyamines did not enhance the binding but inhibited the binding of diazepam and flunitrazepam at greater concentrations. Considered together with other studies, the present findings indicate that polyamines can modulate the binding characteristics of several different neurotransmitter receptor-ionophore complexes. Key words-polyamines,

diazepam, GABAJbenzodiazepine

The naturally occurring polyamines, spermidine and spermine, and their diamine precursor, putrescine, have been found to modulate the binding characteristics of several neurotransmitter receptors. Thus, they inhibit the binding of acetylcholine to synaptic vesicles from the brain of the mouse (Kuriyama, Roberts and Vos, 1968), modulate ligand binding and the function of N-methyl-D-aspartate (NMDA) receptors in brain (Ransom and Stec, 1988; Williams, Romano, Dichter and Molinoff, 1991), modulate the binding and function of A, adenosine receptors in brain (Wasserkort, Hoppe, Reddington and Schubert, 1991) and may inhibit ligand binding to sigma receptors (Paul, Kuypers, Youdim and Skolnick, 1990; but see Contreras, Bremer and Gray, 1990). While polyamines do not alter the binding of yaminobutyric acid (GABA) to synaptic vesicles (Kuriyama et al., 1968), spermidine appears to modulate GABA receptor-mediated functions, as expressed in Xenopus oocytes (Brackley, Goodnow, Nakanishi, Sudan and Usherwood, 1990). Furthermore, putrescine potentiates the binding of diazepam to cerebral cortical membranes in the rat (Morgan and Stone, 1983). The effects of polyamines on the binding of benzodiazepine receptor ligands, however, have not been investigated. In the present study, it was demonstrated that putrescine, spermidine and spermine could, under certain experimental conditions, potentiate or inhibit *Present address: Faculty of Medicine, Technion-Israel Institute of Technology, Efron Street, P.O. Box 9649, Haifa 31096, Israel.

receptor, brain, rat.

the binding of diazepam and flunitrazepam but not other ligands of the central GABA,-benzodiazepine receptor complex. METHODS Experiments were performed on 3-S-month-old male Sprague-Dawley rats. After decapitation, the brains were rapidly excised, the hippocampus and frontal cortex were removed (Gilad and Gilad, 1991) and membranes were prepared by homogenizing the tissues in 10 volumes of ice-cold 0.32 M sucrose, using a Teflon-glass homogenizer. The homogenates were centrifuged at 1OOOg for 5 min and the supernatant was centrifuged at 17,500 g for 18 min. The resulting pellet was resuspended in 10 volumes of 5 mM Tris-HCl, pH 7.4, with and without the nonionic detergent Triton X-100 (O.OS%), homogenized in a glass-glass homogenizer and centrifuged at 17,SOOg for 20 min. The pellet was washed by rehomogenizing in 10 volumes of 50 mM Tris-HCl, pH 7.4 (buffer) and re-centrifuged. This washing step was repeated twice and the resulting membranes were stored at -70°C up to 2 months for subsequent binding assays. For binding assays, the frozen membranes were homogenized and washed once more and then assayed (0.5 mg protein/ml) in duplicate in 0.5 ml of the buffer. After the addition of polyamines (in their hydrochloride form; Sigma, St Louis, Missouri), the assays were started by adding [)H]diazepam (benzodiazepine agonist, 87.6 Ci/mmol), [3H]flunitrazepam (benzodiazepine agonist, 87.0 Ci/mmol),

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G. M. GILAD et al.

896



.B’

Polyamine

Concentration







(Mf

Fig. 1. Effects of polyamines on the binding of [‘Hldiazepam (2.3 nM) to frontal cortex membranes, prepared without (A) or with (B) Triton X-100. Results: mean values of 3 separate experiments, are expressed as percentage of binding in the absence of polyamines (control, shaded area). Note the interruption of the ordinates. Standard error did not exceed 10%. Put = putrescine; Spd = spermidine; Spm = spermine.

[3H]ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4Himidazo[l,S-a][l,4]benzodiazepine-3-carboxylate (Ro 1% 1788, central-type ~nzodiaz~pine antagonist, 83.2 Ci/mmol), [3H]1-(2-chlorophenyl)-N-methyl-N(1-methylpropyl)-3-isoquinoline carboxamide (PK. I 1195, ~~pheral-ty~ ~nzodi~pine antagonist, 86.2 Ci/mmol), [3H]GABA (71 .OCi/mmol) or [-‘H]muscimol (GABA agonist, 20.0 Ci/mmol), all from New England Nuclear (Du Pont, Wilmington, Delaware). The incubations lasted for 1 hr at 23-25°C and were terminated by the addition of 1 ml ice-cold buffer, followed by a rapid vacuum filtration through Whatman GF/B filters and two additional 1 ml rinses. For the binding of flunitrazepam and PK 11195, the incubations lasted 30 min at 23-25 and 4”C, respectively. The nonspecific binding of the benzodiazepine ligands was determined in the pres-

ence of 0.1 mM unlabelled diazepam and that of GABA and muscimol in the presence of 1.0 mM unla~ll~ GABA. RESULTS

The results in Fig. l(A) demonstrate that, in membranes prepared without detergent, all polyamines enhanced the binding of diazepam, with the order of potency being spermine > spermidine > putrescine [ECSo values (M) equal: 4.5 x 1O-6 (k4.0 x lo-‘), 8.0 x IO-’ (+4.5 x 10m6) and 2.0 x 10s3 (&-1.5 x 10e4), respectively]. The enhancing effects were apparent on maximum binding (I$,,,,), but not on the affinity (I&,) values (Table 1). Since numerous binding studies have routinely used non-ionic detergents for the preparation of

Table 1.Effects of poiyamines (PA) on the E,,, fp moljmg protein) and K,, (nM) values for the binding of diazepam (l&200 nM concentration range) to frontal cortex membranes, prepared with and without Triton X-100 Ligand

PA (mM)

Bmnl

% Change

% Change

%

Without Trirm X-i00

Diazepam Diazepam 4 Put(S.0) Diaxepam + Spd(1 .O) Diazepam + Spm(O.1)

1.1 kO.1 I A f 0.2” 1.5 *0.1* 2.1 i 0.2’

Diazepam Diaxepam + Put(lO.0) Diazepam + Spd(S.0) Diazepam + Spm(5.0)

1.9kO.2 1.2fO.l* 1.4f0.1” 1.7*0.2

127 136 191

7.5 f 7.3 + 7.0 f 6.9 +

With Trim

X-100

63 74 a9

0.5 0.6 0.7 0.5

97 93 92

6.1 f 0.4 7.6 -+ 0.6

125

7.8* 0.7

128

7.1 + 0.6

126

Results, mean (+ SEM) values of 4 determinations, are also expressed as percentage of binding in the absence of PA f% change). *P < 0.05 (unpaired t-test). Put = putrescine; Spd = spermidine; Spm = spermine.

Polyamines modulate diazepam binding

I 120



I

I

I

I

I

I

897 DISCUSSION

The study showed that polyamines could potentiate the binding of diazepam by forebrain membranes in the rat in a concentration-dependent manner. After non-ionic detergent (Triton X-100) treatment of membranes, however, potentiation was no longer observed but, rather inhibition of binding occurred at large concentrations of polyamines. These effects were apparently unique to central benzodiazepine agonists, since similar changes to those noted for diazepam were observed with the binding of the flunitrazepam (results not shown) but not with the other GABA,-benzodiazepine ligands tested. Enhanced binding capacity in washed membranes, after homogenization with Triton X-100, was previously demonstrated (Sabato, Aguilar and De Robertis, 1981). A recent publication (Martijena, Salvatierra and Arce, 1992), confirmed that Triton X-100, even when present during the binding assays, could potentiate agonist binding. The present findings indicate, however, that although the receptor affinity was not changed, the treatment with detergent must have Fig. 2. Effects of polyamines on the binding of [3H]PK 11195 caused some structural changes in the receptor complex, or in its vicinity, that led to the specific (1.0 nM) to frontal cortex membranes, prepared with Triton X-100. Results: mean values of 2 separate experiments, are alterations in the effects of the polyamines. expressed as percentage of binding in the absence of The pharmacological relevance of the present polyamines (control, 0.25 f 0.02 pmol/mg protein, shaded findings is intriguing and future studies to investigate area). Put = putrescine; Spd = spermidine; Spm = spermine. the effects of polyamines on the functional response to benzodiazepines are warranted. Further studies to membranes, the effects of polyamines on the binding establish the pharmacological mechanisms of the to Triton X-loo-treated membranes was also effect of the polyamines are also of interest. The examined. In membranes that were homogenized in effects of arcaine, a polyamine analogue which bethe presence 0.05% Triton X-100, B,,,,, values for the haves as a competitive antagonist at the polyamine binding of diazepam increased by 72.7%, with no recognition site of the NMDA receptor (Reynolds, change in the KD values (Table 1). The potentiating 1990) are currently being examined. The preliminary effects of polyamines, however, were no longer obexperiments (in progress) suggest that this compound served in this membrane preparation, but fairly large competitively inhibits the effects of polyamines on concentrations of spermidine and putrescine but not the binding of diazepam. It is now clear that spermine, inhibited the binding of diazepam (Table 1 polyamines can modulate binding at a variety of and Fig. 1). The inhibitory effects of spermidine and neurotransmitter receptors, of the ligand-gated ion putrescine were evident by an apparent decrease of channels type. Polyamines may exert their effects B_ and a tendency toward increase in KD values through binding to specific receptor sites (Gilad and (Table 1). Nonspecific binding of diazepam was not Gilad, 1991; Mantione, Demirgoren and London, affected significantly by polyamines. 1990; Williams et al., 1991). Alternatively, the While the binding of diazepam was increased modulatory effects could conceivably occur through 2-fold in the presence of GABA (1 mM) or muscimol nonspecific binding of the positively charged (100 PM), as previously observed (Tallman, Thomas polyamine to negatively charged groups that may and Gallager, 1978), polyamines had no effects on the become exposed at or in the vicinity of, the recepalready enhanced binding (results not shown). In tor-ionophore complex, depending on the procedure addition, no effects of polyamines on the binding of of membrane preparation. In turn, such nonspecific GABA (in agreement with Kuriyama et al., 1968), binding of polyamines would result in alterations of muscimol or Ro 15-1788 were detected (results not specific ligand binding. shown). Preliminary studies with the peripheral-type benzodiazepine antagonist, PK 11195 indicated that REFERENCES interactions of polyamines, at this receptor, are Brackley P., Goodnow R. Jr, Nakanishi K., Sudan H. L. complex. While putrescine was observed to exert only and Usherwood P. N. R. (1990) Spermine and philanthoinhibition in detergent-treated membranes, spertoxin potentiate excitatory ammo acid responses of midine and spermine inhibited binding at small but Xenopus oocytes injected with rat and chick brain RNA. Neurosci. Left. 114: 51-57. not at larger concentrations (Fig. 2). t

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Contreras P. C., Bremer M. E. and Gray N. M. (1990) Ifenprodil and SL 82.0715 potently inhibit binding of [‘H]( + )-PPP to sigma binding sites in rat brain. Neurosci. Let?. 116: 190-193. Gilad G. M. and Gilad V. H. (1991) Polyamine uptake, binding and release in rat brain. Eur. J. Pharmac. 193: 41-45. Kuriyama K., Roberts E. and Vos J. (1968) Some characteristics of binding of gamma-aminobutyric acid and acetylcholine to a synaptic vesicle fraction from mouse brain. Brain Res. 9:-23i-252. Mantione C. R., Demirgoren S. and London E. D. (1990) Specific binding of [‘Hlspermidine to membranes of rat brain. Eur. J. Pharmac. 180: 393-395. Martijena D. I., Salvatierra N. A. and Arce A. (1992) Benzodiazepine receptor recruitment after acute stress in synaptosomal membranes from forebrain of young chicks: action of Triton X-100. J. Neural Transm. 87: 97-104. Morgan P. F. and Stone T. W. (1983) Structure-activity studies on the potentiation of benzodiazepine receptor binding by ethvlenediamine analogues and derivatives. Br. J. Pharmhc. 79: 973-978.

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Paul I. A., Kuypers G., Youdim M. B. H. and Skolnick P. (1990) Polyamines non-competitively inhibit [)H]3-

PPP binding to sigma receptors. Eur. J. Pharmac. 184: 203-205. Ransom R. W. and Stec N. L. (1988) Cooperative modulation of [3H]MK-801 binding to the N-methylD-aspartate receptor-ion channel complex by I-glutamate, glycine and polyamines. J. Neurochem. 51: 830-837. Reynolds I. J. (1990) Arcaine is a competitive antagonist of the polyamine site on the NMDA receptor. Eur. J. Pharmac. 177: 215-216.

Sabato U. C., Aguilar J. S. and De Robertis E. (1981) Benzodiazepine receptors in rat brain: action of Triton X-100 and localization in relation to the synaptic region. J. Recepr. Res. 2: 119-133. Tallman J. F., Thomas J. W. and Gallager D. W. (1978) GABA-ergic modulation of benzodiazepine binding site sensitivity. Nature 274: 383-390. Wasserkort R., Hoppe E., Reddington M. and Schubert P. (1991) Modulation of A, adenosine receptor function in rat brain by the polyamine, spermine. Neurosci. L&t. 124: 183-186. Williams K., Roman0 C., Dichter M. A. and Molinoff P. B. (1991) Modulation of the NMDA receptor by polyamines. Life Sci. 48: 469-475.

Polyamines modulate the binding of GABAA-benzodiazepine receptor ligands in membranes from the rat forebrain.

The effects of spermine, spermidine and putrescine on the binding of the GABAA-benzodiazepine receptor complex were examined in the hippocampus and fr...
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