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 response s to 5-hydroxytryptamine (5HT) in Xenopus oocytes, and on the binding of [H-3]5HT to 5HT2c receptors in transfected HeLa cells. In oocytes e xpressing rat 5HT2c receptors, EEDQ inhibited the 5HT2c receptor-media ted Cl- currents; and the response did not recover more than 24 h afte r removal of the EEDQ, To see if this effect of EEDQ was on the recept or itself, the binding of 5HT to 5HT2c receptors was studied in transf ected HeLa cells, EEDQ decreased the specific binding of [H-3]5HT to 5 HT2c receptors. At approximate to 22 degrees C, incubating the membran es with 2 x 10(-4) M EEDQ for 1 h caused a 40% decrease in the B-max, without changing the K-d, At 37 degrees C, the same treatment with EED Q blocked [H-3]5HT binding completely, Half-maximal inhibition occurre d at 5 mu M EEDQ at both temperatures, and washing for 1.5 h did not r estore the binding, suggesting that the inactivation of 5HT2c receptor binding was practically irreversible. Results from both systems showe d clearly that EEDQ is an irreversible antagonist of 5HT2c receptors a nd therefore can be used for many studies of this receptor.