Identification of beta-adrenergic receptor binding sites in rat brain microvessels, using [125I]iodohydroxybenzylpindolol.

Brain microvessels were prepared from rat cerebral cortex. The purity was confirmed by phase-contrast microscopy and by the measurement of an enzymatic marker, gamma-glutamyltranspeptidase. The microvessel preparation was subjected to radioreceptor assay using a 125I-labelled beta-adrenergic antagonist, hydroxybenzylpindolol (IHYP). The binding was linear with protein concentration up to at least 80% microgram per tube. It was saturated at 200 pM IHYP concentration. The KD value calculated by Scatchard analysis was 69.4 +/- 9.9 pM. The maximum binding (Bmax) was 107 +/- 4 fmol/mg protein. The binding reached equilibrium within 30 min and was dissociated by addition of (-)-propranolol. The inhibitory effects of isomers of propranolol and isoproterenol on this binding showed that (-)-isomers were two orders of magnitude more potent than the (+)-isomers. Other neurotransmitters did not affect IHYP binding. The characteristics of the binding, saturability, high affinity, reversibility and stereospecificity, suggest tha IHYP is bound to beta-adrenergic receptor sites located on brain microvessels.