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Differential effect of detergents on [3H]Ro 5-4864 and [3H]PK 11195 binding to peripheral-type benzodiazepine-binding sites
Pergamon Press
Life Sciences, Vol. 43, pp. 167-175 Printed in the U.S.A.
DIFFERENTIAL EFFECT OF DETERGENTS ON C3H]Ro 5-4864 AND C3H3PK 11195 BINDING TO PERIPHERAL-TYPE BENZODIAZEPINE-BINDING SITES M. Awad and M. Gavish Rappaport Family Institute for Research in the Medical Sciences and Department of Pharmacology, Faculty of Medicine, Technion-Israel Institute of Technology, P.O.B. 9697, Haifa 31096, Israel (Received in fiul
fm
tiy 13, 1988)
Sumnary The present study demonstsates a dffferentigl effect of various detergent treatments on C HlRo 5-4864 and C HIPK 11195 binding to peripheral benzodiazepine binding sites (PBS) Triton X-100 (0.0125%) caused a decrease of about 70% in C3HIRo 5-4864 binding to membranes from varigus peripheral tissues of rat, but had only a negligible effect on I:HlPK 11195 binding. A similar effect of Triton X-100 was observed on guinea pig and rabbit kidney membranes. The decrease in C HlRo 5-4864 binding after treatment with Triton X-100 was apparently due to a decrease in the density of PBS, since the affinity remained unaltered. The detergents 3-C(3-cholamidopropyl)-dimethylanmnoniol-l-propanesulfonate (CHAPS), Tween 20, deoxqholic acid, or digitonin (0.0125%) caused anly a minor change in I:HlRo 5-4864 and C HIPK 11195 binding to rat kidney membranes; but when concentrations were substantially incrtjased (0.1%). all detergents Saused a decrease of at least 50% in 1 HlRo 5-4864 binding, while C HIPK 11195 binding to rat kidney membranes remained unaffected by the first three detergents, with only a minor decrease (15%) after treatment with digitonin. These results may further support the assumption that Ro 5-4864 and PK 11195 are agonist and antagonist, respectively, of PBS and interact with two different conformations or domains in the peripheral-type benzodiazepine binding site molecule. Central-type benzodiazepine (BZ) receptors have been demonstrated in the central nervous system (1.2). These receptors are modul.atedby y-aminobutyric acid (GABA) and chloride ions (3-6). The binding of various BZs to the central-type BZ receptors correlates with their clinical potency as antfconvulsants and anxiolytics (7). In addition to these central-type BZ receptors located in the central nervous system, another type of binding sites for BZs has been identified in peripheral tissues such as platelets (81, mast cells (91, thymocytes (101, heart and kidney (11, 121, olfactory bulb (131, human term placenta (141, and also in the brain (15-17). Peripheral-type BZ bfnding sites (PBS) are different from central-type BZ receptors in their distribution within the brain, their lack of coupling to GABA receptors, and their specificity for ligand binding. Clonazepam binds with high affinity to the central-type BZ receptors, but has negligible affinity for PBS. The reverse is true with regard to Ro 5-4864, which bfnds selectively and with high affinity to PBS, but not to central-type BZ receptors. The isoquinoline carboxamide derivative C3HlPK 11195 has been shown to bind with high affinity to PBS in human and rat platelets (18) and to rat brain cortex (19). 0024-3205188 $3.00 + .OO Copyright (c) 1988 Pergamon Press plc
The precise physiological and pharmacological function of PBS is as yet unclear. Hypophysectomy in rats results in suppression of testosterone secretion and induces reduction in PBS in the adrenal gland and testis (20). These sites on human platelets are down-regulated by long-term neuroleptic treatment (21) and up-regulated by thyroxine administration in peripheral organs of male rats (22). Estradiol treatment of male rats results in marked reduction of PBS in the testis (23). Several studies have suggested that Ro 5-4864 and PK 11195 are agonist and antagonist, respectively, of PBS (24-28). Recently, porphyrins have been identified as a possible endogenous ligand for PBS (29).
In the preSent study, we sh?ed a differential effect of various detergent treatments on C HlRo 5-4864 and C HIPK 11195 binding to PBS. These results may further support the assumption that Ro 5-4864 and PK 11195 are agonist and antagonist, respectively, of PBS and interact with two different conformations or domains in the PBS molecule. Materials and Methods 1 mm011 and C3~I~o 5-4864 (78.9 Ci/mmol) Materials. C3~lp~ 11195 (85 Cm/ were purchased from New England Nuclear (Boston, MA). Unlabeled Ro 5-4864 was kindly supplied by Drs. H. Gutman and E. Kyburz (Hoffmann-La Roche, Basel, Switzerland). Unlabeled PK 11195 was a generous gift from Dr. G. Le Fur (Pharmuka Laboratories, Gennevilliers, France). Lumax was purchased from Lumac (Schaesberg, The Netherlands). All other compounds were purchased from commercial sources. Membrane preparation. Adult male Sprague-Dawley rats (150-200 g body weight, n=61 and adult male outbred guinea pigs (300-400 g body weight, n=3) were sacrificed by decapitation. Three-month-old female comnon European rabbits (3-3.5 kg body weight, n=2) were sacrificed by overdose of pentobarbital. Rat kidneys, heart, lungs, liver, adrenal, and testes and guinea pig and rabbit kidneys were immediately removed and frozen at -7O'C until used for detergent treatment and binding studies. Each type of tissue was homogenized in 50 volumes of 50 mM Tris-HCl buffer, pH 7.4, at 4'C with a Brinkmann Polytron (setting 10) for 10 set and centrifuged at 49,000 x g for 15 min. Each pellet was suspended in 200 volumes of Tris buffer and uSed as PBS preparation. Binding assay. Binding assays for [3~1~~ 5-4864 and C3HlPK 11195 were conducted as previously described (14,301. C HlRo 5-4864 binding was conducted in 50 mM Tris-HCl buffer, pH 7.4, at 4'C. Binding assay contained 400 ul membranes (200 ug protein) and 25 ul r3H1Ro 5-4864 in the absence (total binding) or presence (nonspecific binding) of 10 uM unlabeled PK 11195. After incubation for 60 min at 4 C, samples were filtered under vacuum over GF/B filters, pretreated with 0.3% polyethyleneimine, and washed three times with 4 ml ice-cold Tris buffer. Filters were placed in vials containing 5 ml of a 1:3 mixture of Lumax:xy ene. Samples were counted for radioactivity after 12 hr. Bindigg assay for C1HIPK 11195 was similar to the binding assay described above for C HlRo 5-4864, except that the nonspecific binding was conducted using 10 uM unlabeled Ro 5-4864 or PK 11195. Equilibrium dissociation constants (K I and the maximal number of binding siges (B 1 were cal$ulated by Scatchar3 analysis from saturation curves of r HlRo !!a$864 and I:HIPK 11195 binding. Detergent treatment. Homogenates (5 mg membranes/ml) of various for 6u min at 4'C with different concentrations (0.00625-0.2%) of various detergents prior to binding studies.
were preincubated
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Effect of Detergents on PBS
she effect various con$entrations of the Trfton X-100 [ HlRo (4 nM) C HIPK nM) binding various peripheral from rat. t a of 0.0125%, to all HlRo 5-4864 X-100 caused decrease of 70% in tissues tested, had only effect on C3ttlPK 11195 binding. At a concentration of all [ HlRo 5-4864 binding activity in ai1 peripheral tissues tested was inhibited, while only a 40-50% decrease in [ HIPK 11195 binding activity in kidney, heart, adrenal, and liver was observed, and a 75% decrease in lung and testis. After rsmoval of the detesgent following centrifugation, binding activity of [ HlRo 5-4864 and [ HIPK 11195 to PBS in tissues tested was-almost fully restored (data not shown).
Heart
Lung
Liver
Adrenal
Te8tis
150 r
c
Kidney
l-nLh._. n_, I
0.05
0.1
0.15
0.2
Triton
0.05 X - 100
0.1
0.15
0.2
0.05
0.1
0.15
0.2
Concentration(%)
FIG. 1 Effect of Triton X-100 on C3HIRo 5-4864 and C3tilPK11195 specific binding to various peripheral tissues of rat. Heart, lung, liver, kidney, adrenal, and testis membrane homogenates (5 mg membranes/ ml) were incubated with various concentrations of Triton X-100 (0.00625-0.2%) in 5 mM Tris-HCl buffer, !H 7.4, for 60 min at 4'C. !I HlRo 5-4864 (01 and [ HIPK 11195 (01 specific After incubation, I: binding was measured as described under Materials and Methods. Results are the mean of three separate experiments with less than 15% variability. Figure 2 demonstrates &he effect of variqus concentrations of the detergent Triton X-100 on CJH]Ro 5-4864 and lIJHIPK11195 binding to kidney membranes from guinea pig and rabbit. At a cgncentration of 0.006258, Triton X-100 caused a decrease of more than 50% in [ HlRo 5-4864 binding to kidney membranes from both guinea pig and rabbit, but had no significant effect on
Effect of Detergents on PBS
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[3HlPK 11195 binding. At a concentration of 0.05%, all C3HlRo 5-4864 binding to kidney membranes from guinea pig and rabbit was inhibited, but a decrease of only 40 and 65%, respectively, in C3HlPK 11195 binding was observed. 15Or
o” --
tc 0 0) P
I
Rabbit
r
Guinea
L
c
0 0
150
6
Triton
x-1
00
Pig
Concentration
(%I
FIG. 2 Effect of Triton X-100 on C3~l~o 5-4864 and C3H3PK 11195 specific binding to guinea pig and rabbit kidney membranes. Membrane homogenates (5 mg membranes/ml) were incubated with various concentrations of Triton X-100 0.00625-0.2%) in 50 mMTris-HCl buffer, pg 7.4, for 60 min at 4 b . After incubation, C3~l~o 5-4864 (01 and C HIPK 11195 (01 specific binding was measured as described under Materials and Methods. Results are the mean of three separate experiments with less than 15% variability. In order to determine whether Triton X-100 affests the affinity 05 the density of PBS in rat kidney, saturation curves of C HlRo 5-4864 and I:HIPK 11195 binding to kidney membranes, in the absence and in the presence of Triton X-100, were conducted. Scatchard analysis of such saturation curves yielded linear plots (Fig. 31, which indicates a single population of bindjng sites. At a concentration of 0.0125%, Triton X-100 caused a decrease in L:HlRo 5-4864 binding as a result of a decrease in the B value, without affecting the KB v lue. The same concentration of Triton X'pPbO did not affect the B value of 3 C HIPK 11195 binding, but caused a small increase in the K value. "fir a concentration of 0.05%, Triton X-100 caused a decrease in 13H!PK 11195 binding bq decreasing the B,, value and increasing the KB value. The B values of ["HIRo 5-4864 binding, in the absence and in the presence of O.O'f@% Triton
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Effec't of Detergents on PBS
Vol. 43, No. 2, 1988
a
TD
X-100, were 4580 + 415 and 1390 + 138 frnol/mg rotein, respective1 and the r s ectfve K values were 8.5 + U.6 and 9.2 + .8 nM, The 8 va ues of +H$'K 11195Dbindf ng, in the absence and in fhe presence of E6125% and 0.025%
Triton X-100, were 5350 + 512, 5350 + 530, and 3325 t 320 fmol/mg protein, respectively,and the reTpectiveKp Values were 1.2 r 0.1. 1.7 + 0.2. and 3.3 + 0.4 nM. L3H1PK 11195
0
2 E
r
I
1500
3000
?Hl -Ligand
4500
I
6000
Bound(fmoles/mg
I\
I
I
I\
I
1000
2000
3000
4000
5000
Protein)
FIG. 3 Scatchardanalysis of C3HlRo 5-4864 and C3HlpK 11195 saturation curves to rat kidney membranes in the absence and presence of Triton X-100. Menbrane homogenates(5 mg metiranes/ml)were incubated in 50 mM Trfs-HCl buffer, pH 7.4, in the absence (0) or presence of Triton j-100 ((I,0.0125%; l, 0.025%) fgr 60 min at 4O. After incubation,C HlRo 5-4864 (0.25-20 nM) and C HIPK 11195 (0.25-10 nM) specific binding was measured as described under Materials and Methods. Results are the mean of three separate experimentswith less than 15% variability. Figure 4 demonstratesthe effects of various concentrationsof the nfol-l-propanesu$fonate (CHAPS), detergents 3-C(3-cholamidopropylj-dfmethyla~ deoxycholicacid, Tween 20 and digitoninon C HlRo 5-4864 and I H]PK 11195 binding to rat kidney membranes. At a concentrationof O.l%, CHAPS, Tween 20 agd deoxycholfcacid caused a prease of 33, 75 and 60%, respectively,in I:HlRo 5-4864 binding, while C HIPK 11195 was no5 affected. At a concentration 05 0.1%. digitonin caused a decrease of 70% in I H]Ro 6-4864 binding, whereas 15%. At a concentrationof 0.0125%, C HIPK 11195 binding was decreasedby on digitonin caused an increase of 35% in CYHlRo 5-4864 binding, as we have previously reported (251.
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Effect of Detergents on PBS
CHAPS
r
150
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Tween
20
50 t
1
r
I
I
I
Deoxycholic
I Acid
Digitonin
150
Detergent
Concentration(%)
FIG. 4 Effect of various detergents on C3HlRo 5-4864 and C3HIPK 11195 specific binding to rat kidney membranes. Membrane homogenates (5 mg membranes/ml) were incubated with various concentrations (0.00625-0.2%) of the detergents CHAPS, Tween 20, deoxycholic a$id and digitonin in 50 mM Tris-HCl buffer, pg 7.4, for 60 min at 4 C. After incubation, C3~l~o 5-4864 (0) and C HIPK 11195 (01 specific binding was measured as described under Materials and Methods. Results are the mean of three separate experiments with less than 15% variability. Discussion PBS in peripheral rat tissues bind with high affinity the ligands Ro 5-4864 and PK 11195. Several lines of evidence suggest that Ro 5-4864 and PK 11195 are agonist and antagonist, respectively, of PBS and bind to two different conformational states or domains in the PBS molecule (24-35). Electrophysiological studies have demonstrated that Ro 5-4864 decreases the duration of intracellular action potential and contractility in guinea-pig heart preparation, and these effects are antagonized by PK 11195 (24). It has also been reported that PK 11195 antagonizes Ro 6-4864-induced convulsions in mice (25) and the increase in phospholipid methylation in hippocampal and cerebellar synaptosomes following Ro 5-4864 treatment (26). Furthermore, in DBA/2 mice, intraperitoneally administered Ro S-4864 enhances the sensitivity of the mice to audiogenic seizures (27); this effect is antagonized by PK
11195.
Recently, a differential effect of phospholipase A treatment on C3HlRo 5-4864 and C H]PK 11195 binding activity has been reporfed (28). Preincubation
Vol. 43, No. 2, 1988
Effect of Detergents on PBS
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of rat cerebral cortical, h$art, and kidney membranes with phospholipase A2 increased the KU value of I:HlRo 5-4864, with no significant effect on the 8 value of C3HIPK ill!!!: value. In contrast, phospholfpase A2 increased the 8 with no change in the KO value. It was suggested tha$Ehese results are further evidence for the assumption that Ro 5-4864 and PK 11195 are agonist and antagonist, respectively, of PBS. Thermodynamic studies also suggest that PK 11195 and Ro 5-4864 are agonist and antagonist, respectively, of PBS and may label different subclasses or conformations in PBS binding sftes (31). It has been reported that the binding affinity of C3HIRo 5-4864 is redu ed by increasing the incubation temperatures, whereas the binding affinity of C5 H!PK 11195 is not affected (31). Histidine modification of PBS decreajes the density of C HlPK 11195 blnding sites without affecting the affinity. [ HlRo 5-4864 binding is not affected by such modification, but Ro 5-4864 itself protects [ HIPK 11195 binding from histidine modification (32). These results may suggest that PK 11195 and Ro 5-4864 interact with different conformational states of PBS. Recently, it was found that arachidonate and thiol reagent treatment (33) or freezing and thaying of rat kidney membranes (34) have a differential effect on the binding of C HIPK 11195 and I:HlRo 5-4864, also suggesting that PK 11195 and Ro 5-4864 bind to two different conformational states or domains in the PBS molecule. The assumption that PK 11195 and Ro 5-4864 may interact with two different conformations or domains in the PBS molecule is further supported by our recent finding that PK 11195 binds with high affinity to both rat and calf tissues, whereas Ro 5-4864 binds with high affinity to rat tissues but with much lower affinity to calf tissues (35). PBS have been solubilired by several groups using a variety of detergents (36-39). Usually, the range of detergent concentrations used for receptor solubilization s 0.25-2%. At suEjhconcentrations the differential effect of 3H]Ro 5-4864 and I:HIPK 11195 binding is either abolished (Fig. detergents on I: 11 or less pronounced. In the present study lower concengrations of detergents Tre used, in order to achieve a differential effect on [ HI Ro 5-4864 and [ HIPK 11195 binding. The major finding in this study is that C3HlRo 5-4864 binding to rarious peripheral rat tissues is more sensitive to detergent treatment than C HIPK 11195 binding. A similar differential effect was also observed when guinea pig and rabbit kidney membranes were treated with Triton X-100. These findings may provide additional support for the assumption that Ro 5-4864 and PK 11195 interact with two different conformations or domains in the PBS molecule. They may also provide additional support for the assumption that Ro 5-4864 and PK 11195 are agonist and antagonist, respectively, of PBS, since it was previously demonstrated that agonist binding to opioid receptors is more sensitive to digitonin treatment3than antagonist binding (40). The fact that most of the [ HlRo 5-4864 and C HIPK 11195 binding activity was recovered in the pellet after removing the detergent rules out the possibility that the differential loss of binding activity is due to differential solubilization. The finding that Ro 5-4864 is more sensitive to detergent treatment than PK 11195 can be explained by the fact that detergents can either bind to the binding site on the receptor molecule or affect the protein-lipid or protein-protein interactjon near the binding s$te, resulting in conformational changes which affect [ H]Ro 5-4864 but not C HIPK 11195_binding tg PBS. It is also possible tt)atdetergents can remove cofactors required for I:HlRo 5-4864 but not for [ HIPK 11195 binding.
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Vol. 43, No. 2, 1988
Acknowledgements This study was supported by a grant from the Fund for Basic Research administered by the Israel Academy of Sciences and Humanities. We thank Miss Ruth Singer for typing the manuscript. This paper is submitted by M.A. in partial fulfillment of the requirements for the D.Sc. degree at the Faculty of Medicine of the Technion-Israel Institute of Technology.
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29. A. VERttA,J.S. NYE and S.H. SNYDER, Proc. natn. Acad. Sci. U.S.A. 84: 2256-2260 (1987). 30. M. GAVISH and F. FARES, Eur. J. Pharmacol. 107: 283-284 (1985). 31. G. LE FUR, N. VAUCHER, M.L. PERRIER, A. FLAMIER, J. BENAVIDES, C. RENAULT, M.C. DUBROEUCQ, C. GUEREM and A. UZAN, Life Sci. 33: 449-457 (1983). 32. J. BENAVIDES, F. BAGASSAT, T. PHAN, C. TUR, A. UZAN, C. RENAULT, M.C. DUBROEUCQ, C. GUEREMY and G. LE FUR, Life Sci. 35: 1249-1256 (1984). 33. R. SKOWRONSKI, K. BEAUMONT and D.D. FANESTIL, Eur. J. Pharmacol. 143: 305-314 (1987). 34. A.S. BASILE, N.L. OSTROWSKI and P. SKOLNICK, J. Pharmacol. Methods 17: 149-156 (1987). 35. M. AWAD and M. GAVISH, J. Neurochem. 49: 1407-1414 (1987). 36. C. MARTINI, G. GIANNACCINIand A. LUCACCHINI, Biochim. biophys. Acta 728: 289-292 (1983). 37. J. BENAVIDES, J. MANAGER, M.C. BURGEVIN, 0. FERRIS, A. UZAN, C.R. GUEREMY and G. LE FUR, Biochem. Pharmacol. 34: 167-170 (1985). 38. M. GAVISH and F. FARES, J. Neurosci. 5: 2889-2893 (1985). 39. R.R.H. ANHOLT, U. AEBI, P.L. PEDERSEN and S.H. SNYDER, Biochemistry 25: 2120-2125 (1986). 40. Y. ITZHAK, J.M. HILLER, T.L. GIOANNINI and E.J. SIMON, Brain Res. 291: 309-315 (1984).
Life Sciences, Vol. 43, pp. 167-175 Printed in the U.S.A.
DIFFERENTIAL EFFECT OF DETERGENTS ON C3H]Ro 5-4864 AND C3H3PK 11195 BINDING TO PERIPHERAL-TYPE BENZODIAZEPINE-BINDING SITES M. Awad and M. Gavish Rappaport Family Institute for Research in the Medical Sciences and Department of Pharmacology, Faculty of Medicine, Technion-Israel Institute of Technology, P.O.B. 9697, Haifa 31096, Israel (Received in fiul
fm
tiy 13, 1988)
Sumnary The present study demonstsates a dffferentigl effect of various detergent treatments on C HlRo 5-4864 and C HIPK 11195 binding to peripheral benzodiazepine binding sites (PBS) Triton X-100 (0.0125%) caused a decrease of about 70% in C3HIRo 5-4864 binding to membranes from varigus peripheral tissues of rat, but had only a negligible effect on I:HlPK 11195 binding. A similar effect of Triton X-100 was observed on guinea pig and rabbit kidney membranes. The decrease in C HlRo 5-4864 binding after treatment with Triton X-100 was apparently due to a decrease in the density of PBS, since the affinity remained unaltered. The detergents 3-C(3-cholamidopropyl)-dimethylanmnoniol-l-propanesulfonate (CHAPS), Tween 20, deoxqholic acid, or digitonin (0.0125%) caused anly a minor change in I:HlRo 5-4864 and C HIPK 11195 binding to rat kidney membranes; but when concentrations were substantially incrtjased (0.1%). all detergents Saused a decrease of at least 50% in 1 HlRo 5-4864 binding, while C HIPK 11195 binding to rat kidney membranes remained unaffected by the first three detergents, with only a minor decrease (15%) after treatment with digitonin. These results may further support the assumption that Ro 5-4864 and PK 11195 are agonist and antagonist, respectively, of PBS and interact with two different conformations or domains in the peripheral-type benzodiazepine binding site molecule. Central-type benzodiazepine (BZ) receptors have been demonstrated in the central nervous system (1.2). These receptors are modul.atedby y-aminobutyric acid (GABA) and chloride ions (3-6). The binding of various BZs to the central-type BZ receptors correlates with their clinical potency as antfconvulsants and anxiolytics (7). In addition to these central-type BZ receptors located in the central nervous system, another type of binding sites for BZs has been identified in peripheral tissues such as platelets (81, mast cells (91, thymocytes (101, heart and kidney (11, 121, olfactory bulb (131, human term placenta (141, and also in the brain (15-17). Peripheral-type BZ bfnding sites (PBS) are different from central-type BZ receptors in their distribution within the brain, their lack of coupling to GABA receptors, and their specificity for ligand binding. Clonazepam binds with high affinity to the central-type BZ receptors, but has negligible affinity for PBS. The reverse is true with regard to Ro 5-4864, which bfnds selectively and with high affinity to PBS, but not to central-type BZ receptors. The isoquinoline carboxamide derivative C3HlPK 11195 has been shown to bind with high affinity to PBS in human and rat platelets (18) and to rat brain cortex (19). 0024-3205188 $3.00 + .OO Copyright (c) 1988 Pergamon Press plc
The precise physiological and pharmacological function of PBS is as yet unclear. Hypophysectomy in rats results in suppression of testosterone secretion and induces reduction in PBS in the adrenal gland and testis (20). These sites on human platelets are down-regulated by long-term neuroleptic treatment (21) and up-regulated by thyroxine administration in peripheral organs of male rats (22). Estradiol treatment of male rats results in marked reduction of PBS in the testis (23). Several studies have suggested that Ro 5-4864 and PK 11195 are agonist and antagonist, respectively, of PBS (24-28). Recently, porphyrins have been identified as a possible endogenous ligand for PBS (29).
In the preSent study, we sh?ed a differential effect of various detergent treatments on C HlRo 5-4864 and C HIPK 11195 binding to PBS. These results may further support the assumption that Ro 5-4864 and PK 11195 are agonist and antagonist, respectively, of PBS and interact with two different conformations or domains in the PBS molecule. Materials and Methods 1 mm011 and C3~I~o 5-4864 (78.9 Ci/mmol) Materials. C3~lp~ 11195 (85 Cm/ were purchased from New England Nuclear (Boston, MA). Unlabeled Ro 5-4864 was kindly supplied by Drs. H. Gutman and E. Kyburz (Hoffmann-La Roche, Basel, Switzerland). Unlabeled PK 11195 was a generous gift from Dr. G. Le Fur (Pharmuka Laboratories, Gennevilliers, France). Lumax was purchased from Lumac (Schaesberg, The Netherlands). All other compounds were purchased from commercial sources. Membrane preparation. Adult male Sprague-Dawley rats (150-200 g body weight, n=61 and adult male outbred guinea pigs (300-400 g body weight, n=3) were sacrificed by decapitation. Three-month-old female comnon European rabbits (3-3.5 kg body weight, n=2) were sacrificed by overdose of pentobarbital. Rat kidneys, heart, lungs, liver, adrenal, and testes and guinea pig and rabbit kidneys were immediately removed and frozen at -7O'C until used for detergent treatment and binding studies. Each type of tissue was homogenized in 50 volumes of 50 mM Tris-HCl buffer, pH 7.4, at 4'C with a Brinkmann Polytron (setting 10) for 10 set and centrifuged at 49,000 x g for 15 min. Each pellet was suspended in 200 volumes of Tris buffer and uSed as PBS preparation. Binding assay. Binding assays for [3~1~~ 5-4864 and C3HlPK 11195 were conducted as previously described (14,301. C HlRo 5-4864 binding was conducted in 50 mM Tris-HCl buffer, pH 7.4, at 4'C. Binding assay contained 400 ul membranes (200 ug protein) and 25 ul r3H1Ro 5-4864 in the absence (total binding) or presence (nonspecific binding) of 10 uM unlabeled PK 11195. After incubation for 60 min at 4 C, samples were filtered under vacuum over GF/B filters, pretreated with 0.3% polyethyleneimine, and washed three times with 4 ml ice-cold Tris buffer. Filters were placed in vials containing 5 ml of a 1:3 mixture of Lumax:xy ene. Samples were counted for radioactivity after 12 hr. Bindigg assay for C1HIPK 11195 was similar to the binding assay described above for C HlRo 5-4864, except that the nonspecific binding was conducted using 10 uM unlabeled Ro 5-4864 or PK 11195. Equilibrium dissociation constants (K I and the maximal number of binding siges (B 1 were cal$ulated by Scatchar3 analysis from saturation curves of r HlRo !!a$864 and I:HIPK 11195 binding. Detergent treatment. Homogenates (5 mg membranes/ml) of various for 6u min at 4'C with different concentrations (0.00625-0.2%) of various detergents prior to binding studies.
were preincubated
organs
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Effect of Detergents on PBS
she effect various con$entrations of the Trfton X-100 [ HlRo (4 nM) C HIPK nM) binding various peripheral from rat. t a of 0.0125%, to all HlRo 5-4864 X-100 caused decrease of 70% in tissues tested, had only effect on C3ttlPK 11195 binding. At a concentration of all [ HlRo 5-4864 binding activity in ai1 peripheral tissues tested was inhibited, while only a 40-50% decrease in [ HIPK 11195 binding activity in kidney, heart, adrenal, and liver was observed, and a 75% decrease in lung and testis. After rsmoval of the detesgent following centrifugation, binding activity of [ HlRo 5-4864 and [ HIPK 11195 to PBS in tissues tested was-almost fully restored (data not shown).
Heart
Lung
Liver
Adrenal
Te8tis
150 r
c
Kidney
l-nLh._. n_, I
0.05
0.1
0.15
0.2
Triton
0.05 X - 100
0.1
0.15
0.2
0.05
0.1
0.15
0.2
Concentration(%)
FIG. 1 Effect of Triton X-100 on C3HIRo 5-4864 and C3tilPK11195 specific binding to various peripheral tissues of rat. Heart, lung, liver, kidney, adrenal, and testis membrane homogenates (5 mg membranes/ ml) were incubated with various concentrations of Triton X-100 (0.00625-0.2%) in 5 mM Tris-HCl buffer, !H 7.4, for 60 min at 4'C. !I HlRo 5-4864 (01 and [ HIPK 11195 (01 specific After incubation, I: binding was measured as described under Materials and Methods. Results are the mean of three separate experiments with less than 15% variability. Figure 2 demonstrates &he effect of variqus concentrations of the detergent Triton X-100 on CJH]Ro 5-4864 and lIJHIPK11195 binding to kidney membranes from guinea pig and rabbit. At a cgncentration of 0.006258, Triton X-100 caused a decrease of more than 50% in [ HlRo 5-4864 binding to kidney membranes from both guinea pig and rabbit, but had no significant effect on
Effect of Detergents on PBS
170
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[3HlPK 11195 binding. At a concentration of 0.05%, all C3HlRo 5-4864 binding to kidney membranes from guinea pig and rabbit was inhibited, but a decrease of only 40 and 65%, respectively, in C3HlPK 11195 binding was observed. 15Or
o” --
tc 0 0) P
I
Rabbit
r
Guinea
L
c
0 0
150
6
Triton
x-1
00
Pig
Concentration
(%I
FIG. 2 Effect of Triton X-100 on C3~l~o 5-4864 and C3H3PK 11195 specific binding to guinea pig and rabbit kidney membranes. Membrane homogenates (5 mg membranes/ml) were incubated with various concentrations of Triton X-100 0.00625-0.2%) in 50 mMTris-HCl buffer, pg 7.4, for 60 min at 4 b . After incubation, C3~l~o 5-4864 (01 and C HIPK 11195 (01 specific binding was measured as described under Materials and Methods. Results are the mean of three separate experiments with less than 15% variability. In order to determine whether Triton X-100 affests the affinity 05 the density of PBS in rat kidney, saturation curves of C HlRo 5-4864 and I:HIPK 11195 binding to kidney membranes, in the absence and in the presence of Triton X-100, were conducted. Scatchard analysis of such saturation curves yielded linear plots (Fig. 31, which indicates a single population of bindjng sites. At a concentration of 0.0125%, Triton X-100 caused a decrease in L:HlRo 5-4864 binding as a result of a decrease in the B value, without affecting the KB v lue. The same concentration of Triton X'pPbO did not affect the B value of 3 C HIPK 11195 binding, but caused a small increase in the K value. "fir a concentration of 0.05%, Triton X-100 caused a decrease in 13H!PK 11195 binding bq decreasing the B,, value and increasing the KB value. The B values of ["HIRo 5-4864 binding, in the absence and in the presence of O.O'f@% Triton
171
Effec't of Detergents on PBS
Vol. 43, No. 2, 1988
a
TD
X-100, were 4580 + 415 and 1390 + 138 frnol/mg rotein, respective1 and the r s ectfve K values were 8.5 + U.6 and 9.2 + .8 nM, The 8 va ues of +H$'K 11195Dbindf ng, in the absence and in fhe presence of E6125% and 0.025%
Triton X-100, were 5350 + 512, 5350 + 530, and 3325 t 320 fmol/mg protein, respectively,and the reTpectiveKp Values were 1.2 r 0.1. 1.7 + 0.2. and 3.3 + 0.4 nM. L3H1PK 11195
0
2 E
r
I
1500
3000
?Hl -Ligand
4500
I
6000
Bound(fmoles/mg
I\
I
I
I\
I
1000
2000
3000
4000
5000
Protein)
FIG. 3 Scatchardanalysis of C3HlRo 5-4864 and C3HlpK 11195 saturation curves to rat kidney membranes in the absence and presence of Triton X-100. Menbrane homogenates(5 mg metiranes/ml)were incubated in 50 mM Trfs-HCl buffer, pH 7.4, in the absence (0) or presence of Triton j-100 ((I,0.0125%; l, 0.025%) fgr 60 min at 4O. After incubation,C HlRo 5-4864 (0.25-20 nM) and C HIPK 11195 (0.25-10 nM) specific binding was measured as described under Materials and Methods. Results are the mean of three separate experimentswith less than 15% variability. Figure 4 demonstratesthe effects of various concentrationsof the nfol-l-propanesu$fonate (CHAPS), detergents 3-C(3-cholamidopropylj-dfmethyla~ deoxycholicacid, Tween 20 and digitoninon C HlRo 5-4864 and I H]PK 11195 binding to rat kidney membranes. At a concentrationof O.l%, CHAPS, Tween 20 agd deoxycholfcacid caused a prease of 33, 75 and 60%, respectively,in I:HlRo 5-4864 binding, while C HIPK 11195 was no5 affected. At a concentration 05 0.1%. digitonin caused a decrease of 70% in I H]Ro 6-4864 binding, whereas 15%. At a concentrationof 0.0125%, C HIPK 11195 binding was decreasedby on digitonin caused an increase of 35% in CYHlRo 5-4864 binding, as we have previously reported (251.
172
Effect of Detergents on PBS
CHAPS
r
150
vol. 43, No. 2, 1988
Tween
20
50 t
1
r
I
I
I
Deoxycholic
I Acid
Digitonin
150
Detergent
Concentration(%)
FIG. 4 Effect of various detergents on C3HlRo 5-4864 and C3HIPK 11195 specific binding to rat kidney membranes. Membrane homogenates (5 mg membranes/ml) were incubated with various concentrations (0.00625-0.2%) of the detergents CHAPS, Tween 20, deoxycholic a$id and digitonin in 50 mM Tris-HCl buffer, pg 7.4, for 60 min at 4 C. After incubation, C3~l~o 5-4864 (0) and C HIPK 11195 (01 specific binding was measured as described under Materials and Methods. Results are the mean of three separate experiments with less than 15% variability. Discussion PBS in peripheral rat tissues bind with high affinity the ligands Ro 5-4864 and PK 11195. Several lines of evidence suggest that Ro 5-4864 and PK 11195 are agonist and antagonist, respectively, of PBS and bind to two different conformational states or domains in the PBS molecule (24-35). Electrophysiological studies have demonstrated that Ro 5-4864 decreases the duration of intracellular action potential and contractility in guinea-pig heart preparation, and these effects are antagonized by PK 11195 (24). It has also been reported that PK 11195 antagonizes Ro 6-4864-induced convulsions in mice (25) and the increase in phospholipid methylation in hippocampal and cerebellar synaptosomes following Ro 5-4864 treatment (26). Furthermore, in DBA/2 mice, intraperitoneally administered Ro S-4864 enhances the sensitivity of the mice to audiogenic seizures (27); this effect is antagonized by PK
11195.
Recently, a differential effect of phospholipase A treatment on C3HlRo 5-4864 and C H]PK 11195 binding activity has been reporfed (28). Preincubation
Vol. 43, No. 2, 1988
Effect of Detergents on PBS
173
of rat cerebral cortical, h$art, and kidney membranes with phospholipase A2 increased the KU value of I:HlRo 5-4864, with no significant effect on the 8 value of C3HIPK ill!!!: value. In contrast, phospholfpase A2 increased the 8 with no change in the KO value. It was suggested tha$Ehese results are further evidence for the assumption that Ro 5-4864 and PK 11195 are agonist and antagonist, respectively, of PBS. Thermodynamic studies also suggest that PK 11195 and Ro 5-4864 are agonist and antagonist, respectively, of PBS and may label different subclasses or conformations in PBS binding sftes (31). It has been reported that the binding affinity of C3HIRo 5-4864 is redu ed by increasing the incubation temperatures, whereas the binding affinity of C5 H!PK 11195 is not affected (31). Histidine modification of PBS decreajes the density of C HlPK 11195 blnding sites without affecting the affinity. [ HlRo 5-4864 binding is not affected by such modification, but Ro 5-4864 itself protects [ HIPK 11195 binding from histidine modification (32). These results may suggest that PK 11195 and Ro 5-4864 interact with different conformational states of PBS. Recently, it was found that arachidonate and thiol reagent treatment (33) or freezing and thaying of rat kidney membranes (34) have a differential effect on the binding of C HIPK 11195 and I:HlRo 5-4864, also suggesting that PK 11195 and Ro 5-4864 bind to two different conformational states or domains in the PBS molecule. The assumption that PK 11195 and Ro 5-4864 may interact with two different conformations or domains in the PBS molecule is further supported by our recent finding that PK 11195 binds with high affinity to both rat and calf tissues, whereas Ro 5-4864 binds with high affinity to rat tissues but with much lower affinity to calf tissues (35). PBS have been solubilired by several groups using a variety of detergents (36-39). Usually, the range of detergent concentrations used for receptor solubilization s 0.25-2%. At suEjhconcentrations the differential effect of 3H]Ro 5-4864 and I:HIPK 11195 binding is either abolished (Fig. detergents on I: 11 or less pronounced. In the present study lower concengrations of detergents Tre used, in order to achieve a differential effect on [ HI Ro 5-4864 and [ HIPK 11195 binding. The major finding in this study is that C3HlRo 5-4864 binding to rarious peripheral rat tissues is more sensitive to detergent treatment than C HIPK 11195 binding. A similar differential effect was also observed when guinea pig and rabbit kidney membranes were treated with Triton X-100. These findings may provide additional support for the assumption that Ro 5-4864 and PK 11195 interact with two different conformations or domains in the PBS molecule. They may also provide additional support for the assumption that Ro 5-4864 and PK 11195 are agonist and antagonist, respectively, of PBS, since it was previously demonstrated that agonist binding to opioid receptors is more sensitive to digitonin treatment3than antagonist binding (40). The fact that most of the [ HlRo 5-4864 and C HIPK 11195 binding activity was recovered in the pellet after removing the detergent rules out the possibility that the differential loss of binding activity is due to differential solubilization. The finding that Ro 5-4864 is more sensitive to detergent treatment than PK 11195 can be explained by the fact that detergents can either bind to the binding site on the receptor molecule or affect the protein-lipid or protein-protein interactjon near the binding s$te, resulting in conformational changes which affect [ H]Ro 5-4864 but not C HIPK 11195_binding tg PBS. It is also possible tt)atdetergents can remove cofactors required for I:HlRo 5-4864 but not for [ HIPK 11195 binding.
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Acknowledgements This study was supported by a grant from the Fund for Basic Research administered by the Israel Academy of Sciences and Humanities. We thank Miss Ruth Singer for typing the manuscript. This paper is submitted by M.A. in partial fulfillment of the requirements for the D.Sc. degree at the Faculty of Medicine of the Technion-Israel Institute of Technology.
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