ACTH receptors in nervous tissue. High affinity binding–sequestration of [¹²⁵I][Phe²,Nle⁴]ACTH 1–24 in homogenates and slices from rat brain

We have demonstrated specific, high affinity binding of a biologically active Tyr 23 -monoiodinated derivative of ACTH, [ 125 I][Phe 2 ,Nle 4 ]ACTH 1–24, in rat brain homogenates. Similarly, in metabolically inhibited and noninhibited rat whole brain slices there is a specific "binding–sequestration" process that is dependent on time, protein concentration, and pH. In homogenates, binding curves were best described by a two-site model and provided the following parameters: [Formula: see text], [Formula: see text]; [Formula: see text], [Formula: see text]. In metabolically viable brain slices, concentration–competition curves of [ 125 I][Phe 2 ,Nle 4 ]ACTH 1–24 binding–sequestration can be described by three components ([Formula: see text], [Formula: see text]; [Formula: see text], [Formula: see text]; [Formula: see text], [Formula: see text]). Metabolic inhibition, by removal of glucose and addition of 100 μM ouabain, abolishes the lowest affinity, highest capacity binding–sequestrian component only ([Formula: see text], [Formula: see text]; [Formula: see text], [Formula: see text]). The two binding–sequestration parameter estimates obtained from metabolically inhibited tissue slices are not significantly different from those of the two higher affinity components obtained with noninhibited tissue. Thus, metabolic inhibition permits demonstration of ACTH receptor binding only, unconfounded by sequestration or internalization of ligand: receptor complexes. Binding–sequestration of [ 125 I][Phe 2 ,Nle 4 ]ACTH 1–24 exhibited brain regional differences in site densities with the following rank order: cortex > hypothalamus > hippocampus > striatum > cerebellum > midbrain > brainstem. The specificity of [ 125 I][Phe 2 ,Nle 4 ]ACTH 1–24 binding in homogenates and binding–sequestration in tissue slices was assessed by testing a number of peptides and other compounds, including opioid peptides and opiate drugs. In homogenates, except for ACTH 1–24, the most potent competitors of [ 125 I][Phe 2 ,Nle 4 ]ACTH 1–24 binding were vasoactive intestinal polypeptide, dynorphin 1–13, and growth hormone releasing factor, all approximately equipotent to each other with a mean K i of 5.3 nM.