Identification of β‐Adrenergic Receptor Binding Sites in Rat Brain Micro vessels, Using [125I]Iodohydroxybenzylpindolol

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Abstract
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-labeled .beta.-adrenergic antagonist, hydroxybenzylpindolol (IHYP). The binding was linear with protein concentration up to at least 80 .mu.g/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 2 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 that IHYP is bound to .beta.-adrenergic receptor sites located on brain microvessels.