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Binding of the 5-HT7 receptor antagonist radioligand [3H]-SB-269970 to human 5-HT7(a) receptors expressed in HEK293 cell membranes (h5-HT7(a)/293) and to guinea-pig cerebral cortex membranes, was characterized and compared with [3H]-5-CT binding. [3H]-SB-269970 (1 nM) showed full association with h5-HT7(a)/293 membranes after 40 min. Specific binding at equilibrium represented >90% of total binding and was fully reversible by methiothepin (10 μM), full dissociation occurring by 100 min. The association (k+1) and dissociation (k−1) rate constants were 0.05 nM−1min−1 and 0.05 min−1 respectively, giving a KD (k−1/k+1) of 1.0 nM. [3H]-SB-269970 bound saturably and apparently monophasically to both h5-HT7(a)/293 and guinea-pig cortex membranes, with KD values of 1.25±0.05 and 1.7±0.3 nM respectively. The Bmax for [3H]-SB-269970 to both h5-HT7(a)/293 and guinea-pig cortex membranes (5780±380 and 125±8.2 fmoles mg protein−1 respectively) was similar to that for [3H]-5-CT (6190±940 and 143±19 fmoles mg protein−1 respectively). These data suggest that, in each tissue, both radioligands labelled the same population of receptors, which appear to be present in an agonist high affinity state. The profile of compound inhibition of [3H]-SB-269970 binding to h5-HT7(a)/293 and guineapig cortex membranes correlated well (corr. coeff. 0.98) with those for [3H]-5-CT binding and were consistent with the profiles reported previously for the human 5-HT7(a) and guinea-pig cortex 5-HT7 receptors using [3H]-5-CT. Hill slopes for inhibition of [3H]-SB-269970 and [3H]-5-CT binding were close to 1, consistent with binding to a single receptor population in both tissues. [3H]-SB-269970 represents the first selective 5-HT7 antagonist radioligand, which should aid further characterization of 5-HT7 receptors in recombinant and native tissues and help establish their role in brain function. British Journal of Pharmacology (2000) 130, 409–417; doi:10.1038/sj.bjp.0703318 |
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