Differential Effects of the Alkylating Agent N‐Ethoxycarbonyl‐2‐Ethoxy‐1,2‐Dihydroquinoline on Brain α2‐Adrenoceptors and I2‐Imidazoline Sites In Vitro and In Vivo

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Abstract
Abstract— The alkylating agent N‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (EEDQ) is a peptide‐coupling agent that is being used to inactivate irreversibly α2‐adrenoceptors and other receptors. The aim of the present study was to assess the in vitro and in vivo effects of EEDQ on the newly discovered brain l2‐imidazoline sites, located mainly in mitochondria. Preincubation of rat cortical membranes with EEDQ (10−8‐10−5M) markedly decreased (20–90%) the specific binding of the selective antagonist [3H]R821002 to α2‐adrenoceptors without affecting that of [3H]idazoxan (in the presence of adrenaline) to l2‐imidazoline sites. In EEDQ‐pretreated membranes (10−5M, 30 min at 25°c), the density of l2‐imidazoline sites (Bmax= 80 ± 4 fmol/mg of protein) was not different from that determined in untreated membranes in the presence of 10−6M (‐)‐adrenaline (Bmax= 83 ± 4 fmol/mg of protein), and both densities were lower (24%, p < 0.05) than the total native density of [3H]idazoxan binding sites (Bmax= 107 ± 6 fmol/mg of protein) (l2‐imidazoline sites plus a2‐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 [3H]‐R821002 to α2‐adrenoceptors, but also the binding of [3H]idazoxan to l2‐imidazoline sites (by 44 ± 5%). Pretreatment with yohimbine (10 mg/kg, i.p.) fully protected against EEDQ‐induced α2‐adrenoceptor inactivation. In contrast, pretreatment with cirazoline (1 mg/kg, i.p.), did not protect against EEDQ‐induced inactivation of l2‐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 labeled by [3H]Ro 41–1049 or that of monoamine oxidase‐B sites labeled by [3H]Ro 19–6327 (lazabemide), two relevant mitochondrial markers. Competition experiments with cirazoline against the specific binding of [3H]idazoxan to l2‐imidazoline sites demonstrated the presence of the expected two affinity states for the drug in EEDQ‐pretreated membranes as well as in rats treated with EEDQ. The results indicate that EEDQ in vitro is a useful tool for quantitating l2‐imidazoline sites when using [3H]‐imidazoline ligands that also recognize α2‐adrenoceptors. In vivo, however, EEDQ is also able to inactivate partially brain l2‐imidazoline sites probably by an indirect mechanism. Key Words: Brain l2‐imidazoline sites—[3H]‐Idazoxan—α2‐Adrenoceptors—[3H] R821002—N ‐Ethoxycarbonyl‐2‐ethoxy‐li2‐dihydroquinoline—Monoamine oxidase‐A—[3H]Ro 41–1049—Monoamine oxidase‐B—[3H]Ro 19–6327.

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