Premium
Differential Effects of the Alkylating Agent N ‐Ethoxycarbonyl‐2‐Ethoxy‐1,2‐Dihydroquinoline on Brain α 2 ‐Adrenoceptors and I 2 ‐Imidazoline Sites In Vitro and In Vivo
Author(s) -
Miralles Antonio,
Ribas Catalina,
Olmos Gabriel,
GarcíaSevilla Jesús A.
Publication year - 1993
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1993.tb09793.x
Subject(s) - imidazoline receptor , idazoxan , yohimbine , chemistry , in vivo , antagonist , in vitro , endocrinology , pharmacology , receptor , medicine , stereochemistry , biochemistry , prazosin , biology , microbiology and biotechnology
— The alkylating agent N ‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (EEDQ) is a peptide‐coupling agent that is being used to inactivate irreversibly α 2 ‐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 l 2 ‐imidazoline sites, located mainly in mitochondria. Preincubation of rat cortical membranes with EEDQ (10 −8 ‐10 −5 M ) markedly decreased (20–90%) the specific binding of the selective antagonist [ 3 H]R821002 to α 2 ‐adrenoceptors without affecting that of [ 3 H]idazoxan (in the presence of adrenaline) to l 2 ‐imidazoline sites. In EEDQ‐pretreated membranes (10 −5 M , 30 min at 25°c), the density of l 2 ‐imidazoline sites ( B max = 80 ± 4 fmol/mg of protein) was not different from that determined in untreated membranes in the presence of 10 −6 M (‐)‐adrenaline ( B max = 83 ± 4 fmol/mg of protein), and both densities were lower (24%, p < 0.05) than the total native density of [ 3 H]idazoxan binding sites ( B max = 107 ± 6 fmol/mg of protein) (l 2 ‐imidazoline sites plus a 2 ‐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 [ 3 H]‐R821002 to α 2 ‐adrenoceptors, but also the binding of [ 3 H]idazoxan to l 2 ‐imidazoline sites (by 44 ± 5%). Pretreatment with yohimbine (10 mg/kg, i.p.) fully protected against EEDQ‐induced α 2 ‐adrenoceptor inactivation. In contrast, pretreatment with cirazoline (1 mg/kg, i.p.), did not protect against EEDQ‐induced inactivation of l 2 ‐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 [ 3 H]Ro 41–1049 or that of monoamine oxidase‐B sites labeled by [ 3 H]Ro 19–6327 (lazabemide), two relevant mitochondrial markers. Competition experiments with cirazoline against the specific binding of [ 3 H]idazoxan to l 2 ‐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 l 2 ‐imidazoline sites when using [ 3 H]‐imidazoline ligands that also recognize α 2 ‐adrenoceptors. In vivo, however, EEDQ is also able to inactivate partially brain l 2 ‐imidazoline sites probably by an indirect mechanism. Key Words: Brain l 2 ‐imidazoline sites—[ 3 H]‐Idazoxan—α 2 ‐Adrenoceptors—[ 3 H] R821002— N ‐Ethoxycarbonyl‐2‐ethoxy‐li2‐dihydroquinoline—Monoamine oxidase‐A—[ 3 H]Ro 41–1049—Monoamine oxidase‐B—[ 3 H]Ro 19–6327.