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Partial characterization of 3H-imipramine and 3H-paroxetine binding sites from calf striatum cerebral membranes
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Partial characterization of 3H-imipramine and 3H-paroxetine binding sites from calf striatum cerebral membranes
A. Rotondo ~, G. Giannaccini b, K. Quattrone b, D. Marazziti ~, C. Martini b, A. L u c a c c h i n i b a n d G.B. C a s s a n o ~ ~'hlstitute qf Psychiatry and I'lstituto Policattedra eli Discipline Biologiche, University q[' Pisa, 56100 Pisa. Italy Key words. 3H-Imipramine binding; 3H-Paroxetine binding; Neuronal 5-hydroxytryptamine transporter complex; Characterization; Purification Serotonergic neurons possess a reuptake system on plasma membranes of nerve terminals that provokes the termination of serotonin (5-HT) activity in the synaptic cleft. The uptake sites for 5-HT have been extensively studied in rat and human brain with several radioligands, especially 3Himipramine (3H-IMI). However, several studies have shown the existence of site heterogeneity for desipramine (DSI) sensitive 3H-IMI binding in brain membrane preparations (Habert et al., 1985; Marcusson et al., 1986; D'Amato et al., 1987). In recent years, a more selective ligand has been developed, i.e., 3H-paroxetine (-~H-PAR) (Habert el al., 1985). Therefore, our study aimed to characterize 3H-IMI and 3H-PAR binding sites in calf striatum cerebral membranes. The neuronal 5-HT transporter from cerebral membranes was solubilized with digitonin (1% w/v). 3H-IMI and 3H-PAR binding to membrane and soluble fraction was performed according to the methods described by D'Amato et al. (1987) and Habert et al. (1985), respectively. Non-specific 3H-IMI binding was estimated in the presence of 100 ,uM DSI, while 10 #M fluoxetine was used as the displacer for 3H-PAR binding. The solubilization yield of membrane-bound 3H-PAR and 3H-IMI high affinity binding sites was about 45%. Scatchard's analysis of 3H-PAR binding revealed that 3H-PAR binds with the same affinity (Kd about 0.20 nM) to a single site on both the parent membrane preparation and the solubilized 5-HT reuptake system. On the other hand, 3H-IMI binds to a high (Kd 2.08 nM) and a low (Kd 713 nM) affinity site on striatum membranes. 3H-IMI binding assay on the solubilized 5HT transporter complex showed the existence of a single binding site with Kd 24.37 nM, noticeably different from the high affinity site of parent membranes, perhaps due to the interaction of the labeled compound with a small fraction of the low affinity binding. Solubilizate gel exclusion chromatography showed that both 3H-PAR and 3H-IMI bind to an identical fraction of 205,000 MW. Nevertheless, 3H-IMI binding was also detected in the column void volume. After gel exclusion chromatography, the affinity of the purified 5HT transporter for 3H-IMI and -~H-PAR (Kd 0.15 nM and 1.25 nM, respectively) was unchanged, compared with that of the high affinity binding site on the parent membranes. Scatchard's analysis of DSI-sensitive 3H-IMI binding on the column void volume eluate revealed the presence of a fraction with low affinity binding activity (Kd not possible to estimate). Our data show that both 3H-PAR and 3H-IMI bind to the same site that, perhaps, corresponds to the neuronal 5-HT transporter: however, 3H-IMI displays additional binding to another site of a nature as yet unknown. References
D'Amato, R.J., Largent, B.L., Snowman, A.M. and Snyder, S.H. (1987) Selective labeling of serotonin re-uptake sites in rat brain by 3H-citalopram contrasted to labeling of multiple sites by 3H-imipramine. J. Pharmacol. Exp. Ther. 242, 364~371. Habert, E., Graham, D., Tahraoui, L., Claustre, Y and Langer, S.Z. (1985) Characterization of 3H-paroxetine binding to rat cortical membranes. Eur. J. Pharmacol. 118, 107-114. Marcusson, J.O., Backstrom, I.T. and Ross, S.B. (1986) Single site model of the neuronal 5-hydroxytryptamine re-uptake and imipramine binding sites. Mol. Pharmacol. 30, 121-128,
Partial characterization of 3H-imipramine and 3H-paroxetine binding sites from calf striatum cerebral membranes
A. Rotondo ~, G. Giannaccini b, K. Quattrone b, D. Marazziti ~, C. Martini b, A. L u c a c c h i n i b a n d G.B. C a s s a n o ~ ~'hlstitute qf Psychiatry and I'lstituto Policattedra eli Discipline Biologiche, University q[' Pisa, 56100 Pisa. Italy Key words. 3H-Imipramine binding; 3H-Paroxetine binding; Neuronal 5-hydroxytryptamine transporter complex; Characterization; Purification Serotonergic neurons possess a reuptake system on plasma membranes of nerve terminals that provokes the termination of serotonin (5-HT) activity in the synaptic cleft. The uptake sites for 5-HT have been extensively studied in rat and human brain with several radioligands, especially 3Himipramine (3H-IMI). However, several studies have shown the existence of site heterogeneity for desipramine (DSI) sensitive 3H-IMI binding in brain membrane preparations (Habert et al., 1985; Marcusson et al., 1986; D'Amato et al., 1987). In recent years, a more selective ligand has been developed, i.e., 3H-paroxetine (-~H-PAR) (Habert el al., 1985). Therefore, our study aimed to characterize 3H-IMI and 3H-PAR binding sites in calf striatum cerebral membranes. The neuronal 5-HT transporter from cerebral membranes was solubilized with digitonin (1% w/v). 3H-IMI and 3H-PAR binding to membrane and soluble fraction was performed according to the methods described by D'Amato et al. (1987) and Habert et al. (1985), respectively. Non-specific 3H-IMI binding was estimated in the presence of 100 ,uM DSI, while 10 #M fluoxetine was used as the displacer for 3H-PAR binding. The solubilization yield of membrane-bound 3H-PAR and 3H-IMI high affinity binding sites was about 45%. Scatchard's analysis of 3H-PAR binding revealed that 3H-PAR binds with the same affinity (Kd about 0.20 nM) to a single site on both the parent membrane preparation and the solubilized 5-HT reuptake system. On the other hand, 3H-IMI binds to a high (Kd 2.08 nM) and a low (Kd 713 nM) affinity site on striatum membranes. 3H-IMI binding assay on the solubilized 5HT transporter complex showed the existence of a single binding site with Kd 24.37 nM, noticeably different from the high affinity site of parent membranes, perhaps due to the interaction of the labeled compound with a small fraction of the low affinity binding. Solubilizate gel exclusion chromatography showed that both 3H-PAR and 3H-IMI bind to an identical fraction of 205,000 MW. Nevertheless, 3H-IMI binding was also detected in the column void volume. After gel exclusion chromatography, the affinity of the purified 5HT transporter for 3H-IMI and -~H-PAR (Kd 0.15 nM and 1.25 nM, respectively) was unchanged, compared with that of the high affinity binding site on the parent membranes. Scatchard's analysis of DSI-sensitive 3H-IMI binding on the column void volume eluate revealed the presence of a fraction with low affinity binding activity (Kd not possible to estimate). Our data show that both 3H-PAR and 3H-IMI bind to the same site that, perhaps, corresponds to the neuronal 5-HT transporter: however, 3H-IMI displays additional binding to another site of a nature as yet unknown. References
D'Amato, R.J., Largent, B.L., Snowman, A.M. and Snyder, S.H. (1987) Selective labeling of serotonin re-uptake sites in rat brain by 3H-citalopram contrasted to labeling of multiple sites by 3H-imipramine. J. Pharmacol. Exp. Ther. 242, 364~371. Habert, E., Graham, D., Tahraoui, L., Claustre, Y and Langer, S.Z. (1985) Characterization of 3H-paroxetine binding to rat cortical membranes. Eur. J. Pharmacol. 118, 107-114. Marcusson, J.O., Backstrom, I.T. and Ross, S.B. (1986) Single site model of the neuronal 5-hydroxytryptamine re-uptake and imipramine binding sites. Mol. Pharmacol. 30, 121-128,