AHN 683: a fluorescent ligand for peripheral-type benzodiazepine receptors

RT McCabe, AH Newman and P Skolnick

Laboratory of Neuroscience, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland.

AHN 683 [1-(2-fluoro-5-N[1,3-dihydrol-1,1-bis(4-hydroxyphenyl)-3-oxo- 5- isobenzofurancarboxamide]-phenyl)-N-methyl-N-(1-methylpropyl)-3- isoquinoline carboxamide] is a fluorescein-derived ligand at peripheral- type benzodiazepine receptors structurally related to the isoquinoline carboxamide, PK 14105. The binding of AHN 683 to rat renal membranes measured by fluorescence techniques was saturable with a maximum number of binding sites of 2.3 +/- 0.3 pmol/mg of protein. The KD (40.4 +/- 2.2 nM) estimated by fluorescence was in good agreement with the Ki (77.4 +/- 13.5 nM) obtained in competition studies with [3H] Ro 5-4864. AHN 683 exhibited rapid and reversible binding which was significantly reduced by the histidine modifying reagent, diethylpyrocarbonate. The potencies of a pair of isoquinoline carboxamide enantiomers as well as other structurally diverse peripheral-type benzodiazepine receptor ligands estimated by inhibition of AHN 683 binding were in good agreement with values obtained using radioligand binding techniques. AHN 683 binding was unaffected by compounds that do not recognize peripheral-type benzodiazepine receptors. Moreover, a significant increase in the maximum number of binding sites of AHN 683 to rat renal membranes after chronic furosemide treatment (29.2%, P less than .02) was comparable to the increase measured using [3H]PK 11195 (35.6%, P less than .001). These findings demonstrate the feasibility of using fluorescent ligand binding techniques to quantitatively characterize peripheral-type benzodiazepine receptors.

Volume 262, Issue 2, pp. 734-740, 08/01/1992
Copyright © 1992 by American Society for Pharmacology and Experimental Therapeutics