NH2-terminal specificity and axonal localization of adrenocorticotropin binding sites in rat median eminence.

Abstract

Adrenocorticotropin binding sites in the rat median eminence have been localized in vivo. These binding sites occur in the basalar zone, which is rich in axonal endings. Using competitive binding and quantitative light-microscope radioautography, we found that the median-eminence binding site, in contradistinction to the adrenal receptor, binds specifically the residue 4-10 region of the adrenocorticotropin molecule. Using quantitative electron-microscope radioautography and median-eminence deafferentation, we localized the binding sites to axon terminals in this region. In time-delayed uptake studies using light-microscope radioautography, we failed to observe concentration of radiolabel in neurons of the medial basal hypothalamus after the direct injection of radioiodinated adrenocorticotropin(1-24) into the median eminence.Binding sites for blood-borne adrenocorticotropin (ACTH) have been localized to the rat median eminence by quantitative radioautography (1). The median eminence is one of the four circumventricular organs of the brain, which are targets for direct interaction with a variety of circulating peptide hormones (2). Of these four regions, the median eminence is the only brain region in which specific binding sites for blood-borne adrenocorticotropin have been demonstrated (1). In view of the various purportedly direct effects of blood-borne adrenocorticotropin and related peptides on brain function (3, 4), we have attempted to determine the cellular location and binding specificity of adrenocorticotropin binding sites in the rat median eminence by using quantitative light-and electron-microscope radioautography. In addition, we report the results of pilot radioautographic investigations of brain uptake of circulating adrenocorticotropin (5-8). METHODSSynthetic ACTH1-24 (Cortrosyn), a gift from Organon, was iodinated using lactoperoxidase under mild conditions for 30 sec, as described by McIlhinney and Schulster (8). Monoiodinated hormone prepared by this method has been shown to retain its steroidogenic properties and capacity to bind receptors isolated from rat adrenocortical cells (8). lodinated peptide was purified by the silicic acid adsorption method of Rees et al. (9) with minor modifications, as follows. Ten milligrams of silicic acid (Bio-Sil A, mesh 200-325, Bio-Rad), which had been suspended in 2 ml of 50 mM sodium phosphate buffer, pH 7.4, containing 1.5% (wt/vol) bovine serum albumin, was added to the iodination mixture to terminate the iodination reaction. After 30 min at room temperature, the pellet was washed three times with 2 ml of cold distilled water and once with 1 M HCl. The adsorbed hormone was eluted in 2 ml of acetone/water/acetic acid (40:59:1, vol/vol) and concentrated by evaporation under a stream of N2. The purified 125I-labeled ACTH1-24 (125I-ACTH1-24) had a specific activity of 124 ,uCi/,g (1 Ci = 37 GBq). After purification, >98% of the radioactivity could be adsorbed by silicic acid and >95% could be precipitated by 10% (wt/vol) trichloroacetic acid. The l...

Alpha-Melanocyte-Stimulating Hormone Abolishes IL-1- and IL-6-lnduced Corticotropin-Releasing Factor Release from the Hypothalamus in vitro

“…It has been shown previously that ether stress-induced release of ACTH can be blocked by microinjection of a-MSH and ACTH into the third ventricle of male rats [16]. Furthermore, CRF release from median eminence in vitro was inhibited by ACTH and related peptides [17], down to 1-24 residues, while a-MSH^^ was much less potent [18]; the latter is consistent with our results which show greater potency of ACTH|_24 over a-MSH j_i3.…”

Variable proopiomelanocortin expression in tanycytes of the adult rat hypothalamus and pituitary stalk

“…Hypophysiotropic axons in the rat median eminence contain large amounts of µ opioid receptors (Abbadie et al, ) that bind β‐endorphin, and tanycyte‐derived β‐endorphin would be the best positioned ligand to access these receptors. In addition, radioligand binding studies demonstrated that hypophysiotropic axons in the median eminence bind ACTH with high affinity (van Houten et al, ). This is probably due to the presence of melanocortin 4 receptors that hypophysiotropic neurons express (Tatro, ; Kishi et al, ), although the axonal presence of these receptor proteins remains to be determined.…”

Hypothalamic Control of Adrenocorticotropin Secretion: Advances since the Discovery of 41-Residue Corticotropin-Releasing Factor

“…Furthermore, unilateral transection of the lateral retrochiasmatic area (see Fig. 4), decreased the binding of radiolabeled ACTH on the side of the ME ipsilateral to the lesion (203). These data suggest that the ACTH binding sites may be located in hypophysiotrophic CRF neurons.…”

“…Interestingly, the specificity of ACTH binding sites in the ME seems to show some parallelism with the potencies of ACTH-related peptides to inhibit the release of CRF-41 (202): ACTH fragments down to residues 1-24 are fully active, while a-MSH (i.e. ACTHi_i 3 ) is much less potent (203). Furthermore, unilateral transection of the lateral retrochiasmatic area (see Fig.…”