A radioreceptor assay for catecholamines, 2

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 250 μg/tube, when 125I-CA of 50,000 c.p.m. was used. The appropriate conditions for incubation were 90 minutes at 200C in a pH 7.0 sucrose solution. 3) By this method utilizing 125I-CA, norepinephrine was detectable in a range from 500 pg to 10 ng/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 100 pg to 5 ng/tube. (author)