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Photoaffinity Labeling of the 5‐Hydroxytryptamine IA Receptor in Rat Hippocampus
Author(s) -
Ransom Richard W.,
Asarch Kenneth B.,
Shih Jean C.
Publication year - 1986
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1986.tb00721.x
Subject(s) - chemistry , radioligand , dissociation constant , binding site , receptor , spiperone , stereochemistry , 5 ht receptor , serotonin , biochemistry
1‐[2‐(4‐Azidophenyl)ethyl]‐4‐(3‐trifluoromethylphenyl)piperazine ( p ‐azido‐PAPP) inhibits [ 3 H]5‐hydroxytryptamine ([ 3 H]5‐HT) binding to 5‐HT IA and 5‐HT IB sites in rat brain with equilibrium dissociation constants ( K D ) of 0.9 n M and 230 n M , respectively. [ 3 H] p ‐Azido‐PAPP was synthesized and its reversible and irreversible binding properties to the hippocampal 5‐HT IA site characterized. [ 3 H] p ‐Azido‐PAPP labeled a single class of sites in rat hippocampal membranes with a K D of 1 n M and a maximal binding density of 370 fmol/mg protein. The pharmacological profile of [ 3 H] p ‐azido‐PAPP binding was consistent with the radioligand's selective interaction with the 5‐HT IA receptor. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of membranes preincubated with [ 3 H] p ‐azido‐PAPP and irradiated showed a major band of incorporation of radioactivity at approximately 55,000 daltons. This incorporation could be blocked when membranes were incubated with 1 μM of several agents that have high affinity for 5‐HT IA sites [5‐HT, 8‐hydroxy‐2‐(di‐ n ‐propylamino)tetraline, TVX Q 7821, spiperone, buspirone, d ‐lysergic acid diethylamide, metergoline]. The results indicate that on photolysis [ 3 H] p ‐azido‐PAPP irreversibly labels a polypeptide that is, or is a subunit of, the 5‐HT IA receptor in rat hippocampus.