Temperature Dependence of Drug Interaction with the Platelet 5‐Hydroxytryptamine Transporter: A Clue to the Imipramine Selectivity Paradox

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Abstract
Although [3H]imipramine is a selective radioli‐gand for the 5‐hydroxytryptamine (5‐HT) transporter in human platelets, its affinity for binding to the 5‐HT transporter complex at 0°C (0.6 nM) is significantly higher than its potency for inhibition of [3H]5‐HT uptake at the physiological temperature of 37°C (Ki= 29 nM). As this apparent discrepancy could be related to the assay temperature, we studied the thermodynamics of drug interaction with the 5‐HT transporter at assay temperatures between 0°C and 37°C, using as radioligands [3H]imipramine (0°C and 20°C) and [3H]paroxetine (20°C and 37°C), a newly available probe for the 5‐HT transporter. At 20°C, Ki values of 14 tricyclic and nontricyclic drugs for inhibition of [3H]imipramine and [3H]paroxetine binding to human platelet membranes were highly significantly correlated (r = 0.98, p < 0.001), validating the use of these two radioligands to study the 5‐HT transporter over a temperature range larger than was previously possible with [3H]imipramine alone. The affinity of imipramine for the 5‐HT transporter is progressively enhanced with decreasing incubation temperature, thus favoring the selectivity of [3H]imipramine for the 5‐HT transporter at 0°C. At 37°C, the Ki of imipramine for inhibition of [3H]paroxetine binding is 32 nM, and equals its Ki value for inhibition of 5‐HT uptake into human platelets. With the exception of chlorimipramine, other tricyclic 5‐HT uptake inhibitors showed a temperature sensitivity in their interaction with the 5‐HT transporter similar to that of imipramine. On the other hand, the temperature sensitivity of nontricyclic 5‐HT uptake inhibitors was much less pronounced. Moreover, Ki values for drug inhibition of [3H]5‐HT uptake into human platelets were significantly (p < 0.05) better correlated with the corresponding Ki values for inhibition of [3H]paroxetine binding at 37°C (r = 0.97) than with their Ki values for inhibition of [3H]imipramine binding at 0°C (r = 0.87). Therefore, drug inhibition of [3H]paroxetine binding to the 5‐HT transporter at 37°C may be more representative of 5‐HT uptake inhibitory activity than its inhibition of [3H]imipramine binding to the 5‐HT transporter as usually measured at 0°C.