5‐HT1A receptor agonist‐antagonist binding affinity difference as a measure of intrinsic activity in recombinant and native tissue systems

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Abstract
It has been reported that radiolabelled agonist : antagonist binding affinity ratios can predict functional efficacy at several different receptors. This study investigates whether this prediction is true for recombinant and native tissue 5‐HT1A receptors. Saturation studies using [3H]‐8‐OH‐DPAT and [3H]‐MPPF revealed a single, high affinity site (KD∼1 nM) in HEK293 cells expressing human 5‐HT1A receptors and rat cortex. In recombinant cells, [3H]‐MPPF labelled 3–4 fold more sites than [3H]‐8‐OH‐DPAT suggesting the presence of more than one affinity state of the receptor. [3H]‐Spiperone labelled a single, lower affinity site in HEK293 cells expressing h5‐HT1A receptors but did not bind to native tissue 5‐HT1A receptors. These data suggest that, in transfected HEK293 cells, human 5‐HT1A receptors exist in different affinity states but in native rat cortical tissue the majority of receptors appear to exist in the high agonist affinity state. Receptor agonists inhibited [3H]‐MPPF binding from recombinant 5‐HT1A receptors in a biphasic manner, whereas antagonists and partial agonists gave monophasic inhibition curves. All compounds displaced [3H]‐8‐OH‐DPAT and [3H]‐spiperone binding in a monophasic manner. In rat cortex, all compounds displaced [3H]‐MPPF and [3H]‐8‐OH‐DPAT in a monophasic manner. Functional evaluation of compounds, using [35S]‐GTPγS binding, produced a range of intrinsic activities from full agonism, displayed by 5‐HT and 5‐CT to inverse agonism displayed by spiperone. [3H]‐8‐OH‐DPAT : [3H]‐MPPF pKi difference correlated well with functional intrinsic activity (r=0.86) as did [3H]‐8‐OH‐DPAT : [3H]‐spiperone pKi difference with functional intrinsic activity (r=0.96). Thus agonist : antagonist binding affinity differences may be used to predict functional efficacy at human 5‐HT1A receptors expressed in HEK293 cells where both high and low agonist affinity states are present but not at native rat cortical 5‐HT1A receptors in which only the high agonist affinity state was detectable. British Journal of Pharmacology (2000) 130, 1108–1114; doi:10.1038/sj.bjp.0703394

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