Studies of the biogenic amine transporters. VI. Characterization of a novel cocaine binding site, identified with [125I]RTI-55, in membranes prepared from whole rat brain minus caudate
| Autor: | R B, Rothman, M L, Silverthorn, M H, Baumann, C B, Goodman, J L, Cadet, D, Matecka, K C, Rice, F I, Carroll, J B, Wang, G R, Uhl |
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| Rok vydání: | 1995 |
| Předmět: |
Male
Serotonin Plasma Membrane Transport Proteins Dopamine Plasma Membrane Transport Proteins Serotonin Binding Sites Membrane Glycoproteins Dopamine Brain Membrane Transport Proteins Nerve Tissue Proteins In Vitro Techniques Tritium Rats Iodine Radioisotopes Rats Sprague-Dawley Radioligand Assay Cocaine Animals Caudate Nucleus Carrier Proteins |
| Zdroj: | The Journal of pharmacology and experimental therapeutics. 274(1) |
| ISSN: | 0022-3565 |
| Popis: | Previous studies showed that the cocaine analog [125I]RTI-55 labels dopamine and serotonergic (5-HT) biogenic amine transporters (BATs) with high affinity. Here we characterized [125I]RTI-55 binding to membranes prepared from whole rat brain minus the caudate nuclei. Paroxetine (50 nM) was used to block [125I]RTI-55 binding to 5-HT transporter sites. Initial experiments identified drugs that displaced [125I]RTI-55 binding with moderately low slope factors. Binding surface analysis of the interaction of 3 beta-(4-chlorophenyl)tropan-2 beta-carboxylic acid phenyl ester hydrochloride (RTI-113) and 3 beta-(4-iodophenyl)tropan-2 beta-carboxylic acid phenyl ester hydrochloride (RTI-122) with [125I]RTI-55 binding sites readily resolved two binding sites for [125I]RTI-55 with Kd values of 0.44 nM and 17 nM and Bmax values of 31 and 245 fmol/mg protein. Potent 5-HT and noradrenergic uptake inhibitors had low affinity for both sites. Whereas cocaine, CFT and WIN35,065-2 were 6.0-, 25- and 14-fold selective for the first site, benztropine, PCP and the novel pyrrole, (+-)-(2RS,3aSR,8bRS)-1,2,3,3a,4,8b-hexahydro- 2-benzyl-1-methylindeno-[1,2-b]pyrrole resorcylate [(+-)-HBMP, formerly called (+-)-RTI-4793-14], were moderately selective for the second site. A single binding site with the characteristics of site 1 was resolved using COS cells transiently expressing the cloned rat dopamine transporter. Lesion studies with 6-hydroxydopamine and 5,7-dihydroxytryptamine were conducted to test the hypothesis that site 1 and site 2 are physically distinct. The data showed that these neurotoxins differentially decreased [125I]RTI-55 binding to sites 1 and 2. The differential distribution of sites 1 and 2 in rat brain provides further support for this hypothesis. Viewed collectively, these data show that [125I]RTI-55 labels a novel binding site in rat brain membranes, termed DATsite2, which is not associated with the classic dopamine, serotonin or norepinephrine transporters. |
| Databáze: | OpenAIRE |
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