Comparison of the in vitro binding characteristics of the beta-carbolines harman and norharman in rat brain and liver and in bovine adrenal medulla.

Abstract

The in vitro binding of the naturally occurring beta-carbolines harman and norharman in their tritium-labelled forms to cell membranes from the rat brain and liver and from bovine adrenal medulla was investigated. Displacement of the specific [3H]harman binding in bovine adrenal medulla and rat liver by several beta-carbolines and monoamine oxidase (MAO) inhibitors revealed the pharmacological profile of a single, high-affinity binding site (KD 4.92 +/- 0.43 nmol/l, Bmax 8.47 +/- 0.17 pmol/mg protein; adrenal medulla) which corresponded to the active site of MAO type A (MAO-A). Similar characteristics have previously been found for brain tissue from rat, marmoset and pig. In order to determine the temperature dependence of the [3H]harman binding, the KD and Bmax values for rat cerebral cortex were calculated from the results of saturation experiments at 5 temperatures (range: 0 degree C-37 degrees C). Whereas the Bmax values under all conditions were approximately 4 pmol/mg protein, the KD values, with increasing temperature, ranged from approximately 3 nmol/l to 30 nmol/l. The calculated linear van't Hoff plot (-ln KD against 1/T) suggested an enthalpy-driven binding of [3H]harman to MAO-A. At least three different [3H]norharman-binding sites were detected. In the rat forebrain, approximately 85% of the specific binding (at about 2 nmol/l of [3H]norharman) can be attributed to a MAO binding site of type B: the binding is displaceable, in nmol/l concentrations by the potent and selective MAO-B inhibitors MDL 72,974 A, R(-)-deprenyl and pargyline and, in mumol/l concentrations, by S(+)-deprenyl and the potent and selective MAO-A inhibitors clorgyline, harmine, harman, harmaline, brofaromine 5-F-alpha-methyltryptamine. After suppression of the MAO binding sites with 1 mumol/l clorgyline and 1 mumol/l R(-)-deprenyl, a second binding site was found. However, the binding at this site was biphasically displaceable by harman and norharman (Hill-slopes about 0.5 and 0.6, curvilinear Rosenthal plots) suggesting the presence of negative co-operativity or of two binding sites (states). A similar clorgyline/R(-)-deprenyl resistant single (Hill-slopes of displacement by norharman, harman and 6-hydroxy-beta-carboline about unity; linear Rosenthal plots) high affinity binding sites (KD 7.5 +/- 2 nmol/l, Bmax 130+/- 30 fmol/mg protein) was found in bovine adrenal medullary cell membranes. A third quite different clorgyline/R(-)-deprenyl resistant high-affinity (KD approximately 14 nmol/l) and high-density (Bmax 10-30 pmol/mg protein) binding site was detected in the liver.(ABSTRACT TRUNCATED AT 400 WORDS)