A Radioreceptor Assay for Catecholamines

II. A Study with 4- (2-Iodoethyl) Pyrocatechol

Abstract

We have already reported on a radioreceptor assay for catecholamines utilizing the microsome fraction of bovine myocardium as a catecholamine (CA) receptor and ³H-norepinephrine as a labelled CA. In order to increase the sensitivity of the radioreceptor assay, we used 4- (2-iodoethyl) pyrocatechol (¹²⁵I-CA) as a ligand instead of ³H-norepinephrine and performed a radioreceptor assay for CA. The following results were obtained :
1) ¹²⁵I-CA was able to bind alpha-receptors prepared from bovine myocardium.
2) The optimal amount of the microsomal fraction was 250μg/tube, when ¹²⁵I-CA of 50,000 c.p.m. was used. The appropriate conditions for incubation were 90 minutes at 20°C in a pH 7.0 sucrose solution.
3) By this method utilizing ¹²⁵I-CA, norepinephrine was detectable in a range from 500pg to 10ng/tube.
4) Various compounds with a catechol nucleus showed cross-reaction in this radioreceptor assay system.
5) Whereas beta-adrenergic blocking agents did not inhibit the binding of ¹²⁵I-CA, phentholamine, a short acting type of alpha-adrenergic blocking agents, was effective in inhibiting the binding. However, dibenamine and phenoxybenzamine, long acting types of alpha adrenergic blocking agents, increased the binding of ¹²⁵I-CA to the microsomal fraction.
6) Utilizing this phenomenon, norepinephrine was detectable in the range from 100pg to 5ng/tube.