Alkylation of [3H]8-OH-DPAT binding sites in rat cerebral cortex and hippocampus |
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Authors: | Emmanuel K Nénonéné Fatiha Radja Martin Carli Nico M van Gelder Soheila Afkhami-Dastjerdian Tomás A Reader |
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Institution: | (1) Clinical Neurphysiology Laboratory, Department of Neurology, The Evanston Hospital, 2650 Ridge Avenue, 60201 Evanston, Illinois;(2) Centre de Recherche en Sciences Neurologiques, Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada;(3) Département de Physiologie, Faculté de Médicine, Université de Montréal, Succursale Centre-Ville, C.P. 6128, H3C 3J7 Montréal, Québec, Canada |
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Abstract: | The binding of tritiated 8-hydroxy-2-(di-n-propyl-amino)tetralin, or 3H]8-OH-DPAT, to membranes from rat cerebral cortex and hippocampus could be inhibited by serotonin (5-HT) and buspirone, and
by the 5-HT antagonists propranolol, NAN-190, pindolol, pindobind-5-HT1A, WAY100135, spiperone and ritanserin. All competition curves, except for ritanserin, best fitted a two-site model. In vitro
treatment of the membranes withN-ethylmaleimide (NEM), to alkylate sulfhydryl groups, caused dose-dependent decreases of binding; the inhibition curves were
biphasic, and the effects irreversible. Reduction of disulfide bonds withl-dithiothreitol (L-DTT) also decreased binding, but in a monophasic way; these effects were fully reversible in cortex, but only partially reversible
in hippocampus. In the latter region, but not in cerebral cortex, previous occupancy by 3H]8-OH-DPAT partially protected binding from the effects of bothL-DTT and NEM, suggesting that the thiol groups in the receptor recognition site(s) of this brain region are readily accessible.
The binding characteristics were examined with the aid of saturation curves, carried out with increasing concentrations, up
to 140 nM, of 3H]8-OH-DPAT. The saturation data were suggestive of a two-site receptor model incorporating a high-affinity site (Kh of 0.3–0.5 nM) corresponding to the 5-HT1A receptor, and a low-affinity site (Kl ofca 25 nM). After in vivo alkylations, carried out by treating rats withN-ethoxycarbonyl-2-ethoxy-1,2-dihydro-quinoline (EEDQ), the saturation curves from both control and EEDQ-treated rats were
again best fitted to a two-site model. For EEDQ-treated animals, a drastic decrease of 5-HT1A receptor activity was noted; this loss was greater in hippocampus than in cerebral cortex. Since the decrease in 5-HT1A receptors was not associated with changes in low-affinity binding, the results suggest independent regulations of the two
3H]8-OH-DPAT binding proteins. Altogether, the present data further supports the notion that 3H]8-OH-DPAT, besides labelling 5-HT1A receptors, also binds to other structures in rat cerebral cortex and hippocampus.
Special issue dedicated to Dr. Kinya Kuriyama |
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Keywords: | Serotonin1A receptors N-ethoxycarbonyl-2-ethoxy-1 2-dihydroquinoline or EEDQ 8-hydroxy-2-(di-n-propylamino)tetralin or 8-OH-DPAT l-dithiothreitol" target="_blank">l-dithiothreitol N-ethylmaleimide |
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