Differential inactivation and G protein reconstitution of subtypes of [3H]5-hydroxytryptamine binding sites in brain
The sulfhydryl reagents p-chloromercuribenzoate and N-ethylmaleimide (NEM) inactivate high affinity [3H]serotonin [( 3H]5-HT) binding to bovine and rat brain membranes in a concentration-dependent manner. In both species, 15-25% of total specific high affinity [3H]5-HT binding is relatively insensit...
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Published in | Molecular pharmacology Vol. 34; no. 4; pp. 527 - 536 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
Bethesda, MD
American Society for Pharmacology and Experimental Therapeutics
01.10.1988
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Subjects | |
Online Access | Get full text |
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Summary: | The sulfhydryl reagents p-chloromercuribenzoate and N-ethylmaleimide (NEM) inactivate high affinity [3H]serotonin [( 3H]5-HT)
binding to bovine and rat brain membranes in a concentration-dependent manner. In both species, 15-25% of total specific high
affinity [3H]5-HT binding is relatively insensitive to NEM. This study examines the NEM sensitivity of the various high affinity
[3H]5-HT binding subtypes, using selective ligands, tissues, and pharmacological masks to study each subtype. Reconstitution
of NEM-inactivated binding by addition of GTP-binding proteins (G proteins, Gi and Go) is also described. Agonist binding
to 5-HT1A, 5-HT1B, and 5-HT1D sites in rat brain and to 5-HT1A and 5-HT1D sites in bovine brain is sensitive to NEM. Binding
of [3H]dihydroergotamine and [125I]iodocyanopindolol, both of which are weak partial agonists to 5-HT1B sites is relatively
insensitive to NEM. Binding of [3H]5-HT to 5-HT1C sites in rat and bovine brain and choroid plexus is relatively insensitive
to NEM. In the presence of spiperone to mask binding of 5-HT2 sites, binding of antagonist [( 3H]mesulergine) to 5-HT1C sites
is also insensitive to NEM. Likewise, binding of the agonist [3H]4-bromo-2,5-dimethoxyphenylisopropylamine and of the antagonist
[3H]ketanserin to 5-HT2 sites is not inhibited by NEM treatment of membranes. These findings suggest that agonist binding
to 5-HT1A, 5-HT1B, and 5-HT1D sites is sensitive to NEM alkylation. Binding of neither agonist nor antagonist to 5-HT1C and
5-HT2 sites is sensitive to NEM. Inability of high concentrations of a variety of ligands to protect the sensitive binding
sites against NEM inactivation indicates that the critical sulfhydryl group(s) are not located in the ligand binding domain
of the NEM-sensitive binding sites. When membranes are treated with NEM, displacement of [125I]iodocyanopindolol by 5-HT is
no longer sensitive to 5'-guanylyl imidodiphosphate (Gpp(NH)p). Gpp(NH)p sensitivity of agonist displacement of 5-HT1B binding
to NEM-treated membranes is restored by addition of purified guanine nucleotide binding proteins (Gi plus Go). In addition,
NEM-inactivated binding to 5-HT1A and 5-HT1D sites can be restored by addition of Gi plus Go. These data suggest that NEM
exerts its effects on 5-HT1A, 5-HT1B, and 5-HT1D binding sites by inactivating the G protein(s) associated with the 5-HT receptor
subtypes. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0026-895X 1521-0111 |