Effects of detergents on binding of 5-hydroxytryptamine3 receptor antagonist [3H]GR65630 to rat cortical membranes

Neurochem. Int. Vol. 22, No. 6, pp. 547-553,1993 Printedin Great Britain

0197-0186/93$6.00+ 0.00 PergamonPress Ltd

EFFECTS OF DETERGENTS ON BINDING OF 5-HYDROXYTRYPTAMINE3 RECEPTOR ANTAGONIST [3H]GR65630 TO RAT CORTICAL MEMBRANES ATSUKO INOUE, l* YOSHIMI TOMINAGA,1 HIROAKI NISHIO, 1 TOMIO S E G A W A 2 a n d YOSHIHIRO NAKATA 1Department of Pharmacology, Institute of Pharmaceutical Sciences,Hiroshima University School of Medicine, Kasumi 1-2-3,Minami-ku, Hiroshima 734, Japan 2Tsuruga Women's Junior College, Tsuruga 914, Japan (Received 5 August 1992 ; accepted 13 October 1992)

Abstract--In the absence of detergent, specific binding of [3H]GR65630, a 5-hydroxytryptamine3(5-HT3) antagonist, determined in the presence of 5 - H T 3 receptor antagonist ICS205-930, was at most 30% of the total binding. To decrease the level of nonspecific binding, the effects of detergents on [3H]GR65630 binding to rat cortical membranes were investigated. The use of a detergent (0.1% Lubrol PX or Triton X- 100)decreased nonspecificbinding, increasing the proportion of specificbindingto 70% of total binding. In the presence of0.1% Triton X-100, binding of [3H]GR65630was rapid, reversibleand saturable at 25°C. The rank order of 5-HT3receptor active drugs in inhibiting [3H]GR65630binding was quipazine > ICS205930 > 2-methyl-5-HT = 5-HT > metoclopramide, which confirmed that [3H]GR65630 efficientlylabeled 5 - H T 3 receptors in the presence of Triton X-100. Triton X-100 improved 5-HT3 receptor binding with rat brain membranes.

Receptors for the neurotransmitter 5-hydroxytryptamine (5-HT) have been classified into four major subtypes: 5-HTI, 5-HT2, 5-HT3 and 5-HT4 (Buchheit et al., 1985 ; Fozard, 1984; Richardson and Engel, 1986). 5-HT3 receptors have been found in peripheral nervous systems (Bradley et aL, 1986; Dumis et al., 1988) and cause rapid depolarization by activating ligand-gated cation channels directly (Wallis and Woodward, 1975; Wallis and North, 1978). In addition, behavioral studies have suggested that 5-HT3 receptors are present in the mammalian central nervous system. This has aroused much interest, as 5-HT3 receptor-affecting drugs may have potential in the treatment of emesis, anxiety and various psychotic illnesses (Costall et al., 1987; Hagan et aL, 1987 ; Miner et aL, 1987 ; Carboni et aL, 1988). Several radiolabelled ligands, designed to determine 5-HT3 receptor activity, have been developed, and binding studies have demonstrated 5-HT3 receptor radioligand binding sites in mammalian brain and on cultured neuronal cell lines such as [ 3H]GR65630 (Kilpatrick et al., 1987), [3H]quipazine (Milburn and Peroutka, 1989), [3H]zacopride (Barnes et al., 1988),

* To whom correspondence should be addressed.

[3H]ICS205-930 (Hoyer and Neijt, 1987) or quaternized [3H]ICS 205-930 (Watling et al., 1988). We investigated radioligand binding studies for 5HT3 receptors with rat brain membranes using 5-HT3 receptor selective antagonist [3H]GR65630. However, non-specific [3H]GR65630 binding, which was determined in the presence of 4 x 10-7M ICS205-930, a potent 5-HT3 receptor antagonist, was 70% of total [3H]GR65630 binding at 0.3 nM. Thus, specific [3H]GR65630 binding was so small that it was difficult to analyze the subsequent data. In this study, we report that certain detergents reduce the nonspecific binding of [3H]GR65630 and that a particular detergent, Triton X-100, was utilized to develop an improved radioligand binding assay for 5-HT3 receptor characterization in rat brain membranes.

EXPERIMENTAL PROCEDURES

Membrane preparation An adult Wistar rat (200-300 g) was sacrificed and brain tissue was dissected. Cerebral cortex dissected from brain was homogenized in 10 vol of 50 mM Hepes-Na buffer (pH7.4) using a Polytron homogenizer (KINEMATICA GmbH KRIENS-LUZERN Switzerland) for 10 s and then centrifuged at 49,000g for 10min.After the supematant was discarded, the pellet was resuspended in 50 mM Hepes-Na 547

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buffer (pH 7.4), incubated at 37~C tbr 15 rain, and centrifuged again at 49,000 g for 10min. The final pellet was suspended in 50 mM Hepes Na buffer (pH7.4) to a final protein concentration of 1 mg/ml and used as untreated membranes for binding assays.

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Pretreatment o[ rat brain membranes Rat cerebral cortex was homogenized with I 0 vol of 50 mM Hepes Na buffer using a Polytron homogenizer, as above, incubated with selected detergents for 30 rain at 37C and then centrifuged at 49,000 g for 10min. The pellet was washed by rehomogenization with 50mM Hepes-Na buffer using the Polytron for 10 s and centrifugation at 49,000 g for 10min. The final pellet was suspended in 50 mM Hepes-Na buffer to a final protein concentration of I mg/ml and used as pretreated membranes for binding assays.

[ ~H]GR65630 binding assay [3H]GR65630 binding was performed in a reaction mixture of 50/d of buffer or displacing drug, 50 #1 of [3H]GR65630 (3-(5-methyl-1H-imidazol-4-yl)- 1-(1-methyl[3H]-I H-indol3-yl)-l-propane, 64 Ci/mmol, final concentration equal to 0.02-2nM; DuPont-New England Nuclear, Boston, MA, U.S.A.), and 0.4 ml of tissue suspension. After incubation for 30 min at 25c'C, the reaction mixture was rapidly filtered under vacuum through GF/B glass-fiber filters (Whatman International Ltd. Maidstone, England), presoaked with 0.3 % polyethyleneimine, using a Brandel Cell Harvester, and washed 5 times with 2 ml of ice-cold 50 mM Tris-C1 buffer (pH 7.4) per wash. The filters were then placed in glass scintillation vials to which 8 ml of Creasol (Nacalai Tesque, Inc., Kyoto, Japan) had been added. Bound radioactivity on the filter was measured by liquid scintillation spectroscopy at 40% efficiency. Specific binding was defined as the excess over blanks taken in the presence of 4 x 10-7 M ICS205-930. Protein concentration was determined by the Lowry method (Lowry et al., 1951).

RESULTS

[-~H]GR65630 binding in the absence of detergents Untreated rat cortical membranes were incubated with [3H]GR65630 (0.3 nM) and varying concentrations of ICS205-930, a potent 5-HT3 receptor antagonist. Figure 1 shows that ICS205-930 inhibits [3H]GR65630 binding from 4 x 10 ' t M and maximally at 10 7M. The ICs0 value of ICS205-930 was found to be 1 . 4 × 1 0 - 9 M . Specific binding of [3H]GR65630 that was displaced by 4 x l O T M ICS205-930 represented only 30% of total binding.

Pretreatment with detergents [3H]GR65630 (0.3 riM) binding to rat cortical membranes pretreated with certain selected detergents is shown in Fig. 2. The specific binding of [3H]GR65630 was determined as the amount of bound [3H]GR65630 in excess of that in the presence of 4 x 10- 7M ICS205930. As described above, in untreated membranes the

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amount of specific binding was only 30% of total binding. In contrast, in membranes pretreated with 0.1% Triton X-100, 0.1% and 0.5% Lubrol PX or 0.1% deoxycholate (DOC) the amount of specific binding represented 50-60% of total binding, although the amount of specific binding itself did not change. In the membranes treated with 0.5% DOC, the amount of specific binding was significantly reduced. Also, in the supernatant during pretreatment with 0.1% Triton X-100 or 0.1% and 0.5% Lubrol PX, which may contain solubilized proteins and detergents, specific binding was found to represent about 60-.-70% of total binding. In the supernatant during pretreatment with 0.1 or 0.5% D O C the amount of specific binding was found to be small.

[ 3H]GR65630 binding in the presence of detergents Selected detergents were added to the untreated membrane suspension and binding studies of [3H]GR65630 (0.2 nM) were performed in the presence of such detergents (Fig.3). In the case of 0.1, 0.5% Triton-X-100, 0.05, 0.1 or 0.5% Lubrol PX, specific binding represented 6 0 - 8 0 % of total binding without affecting its amount. D O C reduced specific [3H]GR65630 binding in a dose dependent manner.

Competition experiments The association rate of [3H]GR65630 to rat cortical membranes in the presence of 0.1% Triton X-100 was rapid. It reached a steady state condition within 5 min, and remained steady for at least 60 rain at 25~C (The data are not shown.). Specific [3H]GR65630

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Fig. 3. Effects of the presence of detergents on [3H]GR65630 (0.2 nM) binding in rat cortical membranes. [] shows total[3H]GR65630 binding. • shows specific [~H]GR65630 bindingin the presence of 4 x 10-7M ICS 205-930. Each column represents the mean + SEM from 3 to 7 separate experiments with different membrane preparations.

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binding was linear with protein concentration at least I rag/tube (The data are not shown.). In all experiments, a 30 min incubation time and protein concentration of about 0.4 rag/tube were used. Competition studies of [3H]GR65630 binding to rat cortical membranes in the presence of0.1% Triton X100 were performed with metoclopramide, ICS205 930, quipazine, 5-HT or 2-methyl-5-HT. Figure 4 shows that these drugs inhibited [3H]GR65630 binding in a dose dependent manner. In the drugs tested, quipazine is the most potent competitor for the specific [3H]GR65630 binding. Competition was first observed at 3×10 '2M, and its ICs0 value was 1.9(_+0.61) × l0 ~°M with shallow inhibition curves (Hill number 0.68). ICS205-930 begins to compete at 10 '°M and competes maximally at 10 7M with a steep inhibition curve (ICs0 and Hill number are 1.8(_+0.62)× 10-gM and 0.82 respectively) Both 5HT and 2-m-5-HT, which are agonists for 5-HT~ receptors, were less potent, with ICs0 values of 6.3 (+0.78)×10 ~Mand6.1 (_+0.69)×10' SMandHill numbers of 1.7 and 1.7, respectively. Metoclopramide was found to be the weakest inhibitor, with an ICs0 value of 5.5(__+0.96) × 10 7M and a Hill number of 0.89. All drugs tested competed with as much as 70% of total [3H]GR65630 binding.

Saturation experiments Saturation studies were performed with increasing concentrations of [3H]GR65630 in rat cortical membranes in the presence of 0.1% Triton X-100 (Fig.5). Specific binding increased as a function of increasing concentrations of [3H]GR65630. Analyzed by Scatchard plot, a Kd value was 0.17(_+0.77) × 10 ')M with a B...... of 7.0(+0.73)x l0 '~ mol/mg protein (, = 5). Similar saturation studies in rat entorhinal cortical membranes showed a Kd vahtc of 0.16(+0.025) x 10 ') M and a B...... of 12.2(± 1.30)× 10 ~>tool/rag protein (n = 5). Distribution Figure 6 shows the regional distribution of specific [-~H]GR65630 binding in rat central nervous system in the presence of0.1% Triton X-100. The amount of binding is represented as a percentage of that in the neocortex. The entorhinal cortex had the highest level of specific binding (11.7 × 10 '~ tool/rag protein at 0.4 nM of [3H]GR65630). The neocortex and hippocampus had relatively high levels of binding. The striatum, brain stem containing the diencephalon and the mesencephaton and the spinal cord had relative low levels.. The cerebellum had the lowest level of specific [3H]GR65630 binding.

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DISCUSSION

Radioligand binding studies have identified 5-HT3 receptors in rat brain membranes using the radioligand [3H]GR65630. We have tried to define specific [3H]GR65630 binding in the presence of 4 x 10-7M ICS205-930 in rat brain membranes. ICS205-930 is a potent 5-HT3 receptor antagonist which has been evidenced in peripheral nervous systems (Richardson et al., 1985 ; Ireland and Tyers, 1987). In a competition study, ICS205-930 competed with the [3H]GR65630 binding with a high affinity. An IC5o value of ICS205930 was correspondent with its potency seen in peripheral tissues. Kilpatrick et al. (1989) reported specific binding of 60-85% for [3H]GR65630. However, in our present study, the maximal amount that could be displaced by ICS205-930 ( > 10-7M) was about 30% of total binding. The proportion of specific binding to total binding is so small that it is difficult to analyze the data and to determine the effects of other drugs. The proportion of specific binding was increased by pretreatment of brain membranes with the detergents Triton X-100 and Lubrol PX, but specific binding was reduced by pretreatment with DOC. At first, we

supposed that Triton X-100 or Lubrol PX might remove the endogenous 5-HT 3 receptor-masking substances (e.g. endogenous 5-HT3 receptor agonists or antagonists), similar in the case in excitatory amino acid receptors (Murphy et al., 1986) and that the supernatant during pretreatment with detergents might have inhibitory substances. Thus, we performed an [3H]GR65630 binding assay with the supernatant during pretreatment of rat brain membranes with detergents. However, contrary to our supposition, we observed considerable amounts of specific [3H]GR65630 binding in the supernatant even in the presence of 0.1% Triton X-100, 0.1 or 0.5% Lubrol PX. This suggests that 5-HT3 receptors could be solubilized by Triton X-100 or Lubrol PX. In the case of 0.1% Lubrol PX, the supernatant contained soluble protein in amounts as much as 75-85% of total protein and 70-80% of total specific binding. McKernan et al. have reported that 5-HT3 receptors can be efficiently solubilized from rat cortical and hippocampus membranes (McKernan et al., 1990b) or NCB20 cells (McKernan et al., 1990a) by DOC and Lubrol PX. Furthermore, in our study Triton X-100 and Lubrol PX at 0.1% did not significantly affect specific binding. The increased proportion of specific

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binding owes to reduced non specific binding. Thus, we performed [3H]GR65630 binding studies in rat brain membranes in the presence of detergents. In this study, DOC reduced the specific [3H]GR65630 binding. Addition of 0.5% Triton X-100 or Lubrol PX caused the reaction mixture to become so viscous that it was difficult to filter and to stop the reaction. While we have found that the effect of 0.5% detergents were as much as that of 0.1% detergents, in the presence of 0.5 % detergents, specific binding tended to be reduced. Thus, we used 0.1% Triton X-100 to define the specific [3H]GR65630 binding to rat brain membranes. Although the mechanism of these detergents remains unknown, they may mask the lipid molecules in the membranes where [3H]GR65630 adsorbs nonspecifically but not remove inhibitory substances. Competition studies in the presence of 0.1% Triton X-100 were carried out. All drugs tested inhibited [3H]-GR65630 (0.3nM) binding by 70% of total binding in the presence of 0.1% Triton X-100 with the following rank order of potency: quipazine > ICS205-930 > 5-HT = 2-m-5-HT > meto-

clopramide. The competition curve of quipazine was very shallow (Hill number 0.68.). It is possible that quipazine binds to an unknown sites, or that the 5HT3 receptor has subtypes which are distinguished by quipazine. In any case, the rank order of potency for 5-HT 3 affecting drugs was very similar to that observed at sites labelled with [3H]GR65630 in rat brain (Kilpatrick et al., 1987). A similar rank order of potency was also observed against [3H]quipazine (Milburn and Peroutka, 1989) or quaternized [3H]ICS205-930 (Watling et al., 1988) in rat brain membranes. Scatchard analysis shows the existence of an monophasic [3H]GR65630 binding site with high affinity in rat cortex. The highest specific [3H]GR65630 binding was observed in the rat entorhinal cortex, which is also consistent with previous findings (Kilpatrick e t al., 1987). It seems likely that [3H]GR65630 in this study is labelling very similar or identical 5-HT3 receptors in rat brain in the presence of Triton X-100. In summary, detergents Triton X-100 or Lubrol PX can efficiently reduce nonspecific [3H]GR65630

Effects of detergents on [~H]GR65630 binding b i n d i n g to 5-HT 3 receptors in rat b r a i n m e m b r a n e s w i t h o u t affecting specific binding. Thus, i m p r o v e d [3H]GR65630 binding in rat b r a i n m e m b r a n e s m a y be achieved in the presence o f 0.1% T r i t o n X-100. Acknowledyements--The authors greatly appreciate the excellent technical assistance provided by Mr Yuji Negishi and Miss Keiko Suzaki and the English stylistic improvement undertaken by Mr George Canning and Mr Aldric Tadashi Hama.

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