Irreversible antagonism of 5HT2c receptors by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)

To determine if N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a carboxyl group activating agent, can inactivate 5HT2c receptors, we have examined the effects of EEDQ on 5HT2c receptor-mediated responses to 5-hydroxytryptamine (5HT) in Xenopus oocytes, and on the binding of [(3)H]5HT to 5HT2c receptors in transfected HeLa cells. In oocytes expressing rat 5HT2c receptors, EEDQ inhibited the 5HT2c receptor-mediated Cl(−) currents; and the response did not recover more than 24 h after removal of the EEDQ. To see if this effect of EEDQ was on the receptor itself, the binding of 5HT to 5HT2c receptors was studied in transfected HeLa cells. EEDQ decreased the specific binding of [(3)H]5HT to 5HT2c receptors. At ≈22°C, incubating the membranes with 2 × 10(−4) M EEDQ for 1 h caused a 40% decrease in the B(max), without changing the K(d). At 37°C, the same treatment with EEDQ blocked [(3)H]5HT binding completely. Half-maximal inhibition occurred at 5 μM EEDQ at both temperatures, and washing for 1.5 h did not restore the binding, suggesting that the inactivation of 5HT2c receptor binding was practically irreversible. Results from both systems showed clearly that EEDQ is an irreversible antagonist of 5HT2c receptors and therefore can be used for many studies of this receptor.