DIFFERENTIAL-EFFECTS OF THE ALKYLATING AGENT N-ETHOXYCARBONYL-2-ETHOXY-1,2-DIHYDROQUINOLINE ON BRAIN ALPHA-2-ADRENOCEPTORS AND I2-IMIDAZOLINE SITES IN-VITRO AND IN-VIVO

The alkylating agent N-ethoxycarbonyl-2-eth-oxy-1,2-dihydroquinoline ( EEDQ) is a peptide-coupling agent that is being used to inactivate irr eversibly alpha2-adrenoceptors and other receptors. The aim of the pre sent study was to assess the in vitro and in vivo effects of EEDQ on t he newly discovered brain I2-imidazoline sites, located mainly in mito chondria. Preincubation of rat cortical membranes with EEDQ (10(-8)-10 (-5) M) markedly decreased (20-90%) the specific binding of the select ive antagonist [H-3]RX821002 to alpha2-adrenoceptors without affecting that of [H-3]idazoxan (in the presence of adrenaline) to I2-imidazoli ne sites. In EEDQ-pretreated membranes (10(-5) M, 30 min at 25-degrees -C), the density of I2-imidazoline sites (B(max) = 80 +/- 4 fmol/mg of protein) was not different from that determined in untreated membrane s in the presence of 10(-6) M (-)-adrenaline (B(max) = 83 +/- 4 fmol/m g of protein), and both densities were lower (24%, p < 0.05) than the total native density of [H-3]idazoxan binding sites (B(max) = 107 +/- 6 fmol/mg of protein) (I2-imidazoline sites plus alpha2-adrenoceptors) . Treatment of rats with an optimal dose of EEDQ (1.6 mg/kg, i.p., for 2 h to 30 days) reduced maximally at 6 h (by 95 +/- 1%) the specific binding of [H-3]-RX821002 to alpha2-adrenoceptors, but also the bindin g of [H-3]idazoxan to I2-imidazoline sites (by 44 +/- 5%). Pretreatmen t with yohimbine (10 mg/kg, i.p.) fully protected against EEDQ-induced alpha2-adrenoceptor inactivation. In contrast, pretreatment with cira zoline (1 mg/kg, i.p.), did not protect against EEDQ-induced inactivat ion of I2-imidazoline sites. Treatment with EEDQ (1.6 mg/kg, i.p., for 6 h) did not alter the density of brain monoamine oxidase-A sites lab eled by [H-3]Ro 41-1 049 or that of monoamine oxidase-B sites labeled by [H-3]Ro 19-6327 (lazabemide), two relevant mitochondrial markers. C ompetition experiments with cirazoline against the specific binding of [H-3]idazoxan to I2-imidazoline sites demonstrated the presence of th e expected two affinity states for the drug in EEDQ-pretreated membran es as well as in rats treated with EEDQ. The results indicate that EED Q in vitro is a useful tool for quantitating I2-imidazoline sites when using [H-3]-imidazoline ligands that also recognize alpha2-adrenocept ors. In vivo, however, EEDQ is also able to inactivate partially brain I2-imidazoline sites probably by an indirect mechanism.