Dopaminergic regulation of prolactin and growth hormone synthesis in rat pituitary tumor cells

The dopaminergic regulation of prolactin (PRL) and growth hormone (GH) synthesis were studied in monolayer cultures of rat pituitary tumor (GH₃) cells. PRL synthesis could not be inhibited by dopamine at concentrations ranging from 10⁻⁹ to 10⁻⁵ M. Bromocriptine inhibited PRL synthesis only at the very high concentration of 10⁻⁵ M, and this effect was not reversible by metoclopramide. GH synthesis was inhibited by 10⁻⁶ and 10⁻⁵ M dopamine but not by bromocriptine, and the dopamine effect was blocked by metoclopramide. These results suggest the existence of a defective dopaminergic regulation of PRL and GH synthesis in GH₃ cells.     

Insulin-like growth factor I: A mitogen for rat schwann cells in the presence of elevated levels of cyclic AMP

To develop effective procedures for improving the regeneration of peripheral nerves and for preventing the formation of neurofibromas, it is necessary to identify the different mitogens that stimulate the proliferation of Schwann cells. Insulinlike growth factor I (IGF-I), which is a potent autocrine growth factor in many tissues, is synthesized by proliferating Schwann cells. However, the role of IGF-I in stimulating their division is still uncertain. Here we show that nanomolar concentrations of IGF-I stimulate the growth of Schwann cells in primary culture. IGF-I alone was uneffective but in the presence of forskolin (5 μM) or dibutyryl cyclic AMP (dbcAMP, 10 μM), it became a potent mitogen. Neither IGF-II nor epidermal growth factor (EGF) were effective, even in the presence of forskolin. Insulin also stimulated Schwann cell proliferation in the presence of forskolin, but only at micromolar concentration. Receptors for IGF-I were visualized on the Schwann cell surface by indirect immunofluorescence staining using anti-human IGF-I receptor antibodies. Their presence was also assessed by binding assays using [¹²⁵I]-IGF-I as a ligand. Scatchard analysis showed a single class of high-affinity receptors (K_d = 1.5 nM). Competition studies with unlabeled IGF-I or insulin indicated a half-maximal displacement of [¹²⁵I]-IGF-I by IGF-I at about 5 nM, while insulin was about 500-fold less effective. The number of binding sites for IGF-I was increased by exposing cells for 3 days to forskolin (- forskolin: about 5,100; + forskolin: about 12,200 binding sites/cell). These results suggest that forskolin increases available receptors for IGF-I, which is consistent with the synergism between cAMP and IGF-I in stimulating Schwann cell growth. © 1993 Wiley-Liss, Inc.     

Autoradiographic Localization of Insulin-Like Growth Factor I Binding Sites in the Bovine Adrenal Gland and on Cultured Bovine Adrenal Chromaffin Cells

Previous studies have reported the presence of binding sites for insulin-like growth factor I (IGF-I) in membranes prepared from isolated bovine adrenal medullary cells, and IGF-I was found to regulate the secretory function of bovine chromaffin cells. In the present study, binding sites for IGF-I have been localized in sections of bovine adrenal gland and on cultured bovine adrenal medullary cells, using [¹²⁵ l][Thr⁵⁹]-IGF-l as the ligand in conjunction with qualitative autoradiographic techniques. Binding sites were present throughout the adrenal gland and were distributed evenly over all cortical cell layers and over both adrenaline and noradrenaline cell types in the medulla. They were also present at lower density over blood vessels and nerve bundles and over the capsule. The binding of radioligand was to a single class of sites with K_d 0.61 nM, and was completely displaced by excess unlabelled [Thr⁵⁹]-IGF-l and by insulin (Actrapid, K_d 1.04μM). Binding sites were also identified on single cells in primary monolayer cultures of bovine adrenal medullary cells. More than 96% of the cells possessed binding sites, although only 85% of such cells were chromaffin cells, as previously determined from dopamine β-hydroxylase immunohistochemical staining. The results suggest that IGF-I may affect the maturation, growth or function not only of adrenal chromaffin cells but also of many others cell types in this tissue.     

Estradiol Modulates Insulin-Like Growth Factor I Receptors and Binding Proteins in Neurons from the Hypothalamus

Trophic effects of 17β-estradiol (βE²) on in vitro developing hypothalamic cells have been reported. Insulin-like growth factor I (IGF-I) is also a potent trophic factor for cultured hypothalamic cells. An interaction between sexual steroids and insulin-like growth factors (IGFs) in modulating growth of hypothalamic cells has been suggested. Thus, we tested whether βE² modulates the levels of IGF-I, its membrane receptor and its binding proteins in rat hypothalamic culturs. Using both neuron- and glial-enriched cultures obtained from fetal rat hypothalami we found that addition of βE² elicited a significant increase in IGF-I receptor levels in neurons, without affecting its affinity. On the other hand, the three different IGF-binding proteins (IGFBPs) found in the conditioned medium of the cultures were differentially modulated by βE² in the two types of cells studied. Overall, neuronal cultures produced greater amounts of IGFBPs after treatment with βE², with IGFBP2 reaching significantly higher levels. On the contrary, treatment with βE² did not significantly alter the amounts of IGFBPs produced by glial cells. Finally, the levels of immunoreactive IGF-I found either in the medium or in cellular extracts in both neuronal and glial cultures were not modified by treatment with βE². These results strongly support previous observations of a trophic synergistic interaction between IGFs and βE² on hypothalamic cells. Thus, an increase in IGF-I receptors and/or IGFBPs after exposure to βE² may result in an enhanced response of hypothalamic neurons to IGF-I. Further, the present findings strengthen our recent observation that the effects of βE² on hypothalamic glial cells are neuronally mediated, since IGF-I receptors and IGFBPs are modulated by this sex hormone in neurons, but not in glial cells.     

Insulin-like growth factor I in the anterior pituitary of the clawed frog Xenopus laevis: immunocytochemical and autoradiographic indication for a paracrine action and corelease with prolactin

Insulin-like growth factor I (IGF-I) and its receptor are present in human and rat anterior pituitary. However, few data exist on the potential presence of IGF-I or its receptor in the non-mammalian pituitary and the cellular sites of IGF-I production have not been identified in any species. Thus, we investigated the anterior pituitary of the clawed frog Xenopus laevis which is widely used to study growth and differentiation. The study was performed with antisera against mammalian insulin-like growth factor I (IGF-I), prolactin (PRL) and growth hormone (GH) using immunohistochemical and immunocytochemical techniques. IGF-I binding was determined by in-vitro receptor autoradiography. The PRL-and GH-immunoreactive cells exhibited distinct distribution patterns. Neither at the light nor the electron microscopical level any colocalization of PRL-and GH-immunoreactivities was apparent. The PRL-immunoreactive cells exhibited round granules of medium electron density (mean diameter: 312 nm) and the GH-immunoreactive cells spherical granules of medium electron density (mean diameter: 165 nm). By the use of serial semithin sections IGF-I-immunoreactivity was exclusively located in PRL-immunoreactive cells. At the ultrastructural level, IGF-I-immunoreactivity was confined to the secretory granules in coexistence with PRL-immunoreactivity using the double labelling immunogold technique. Specific IGF-I binding sites were localized throughout the pituitary. The results provide evidence for a concomitant release of PRL and IGF-I and suggest autocrine/paracrine actions of IGF-I in the anterior pituitary.     

Identification and Localization of Insulin-Like Growth Factor Receptors in Human Infant Pituitary Gland—Similar Distribution to Somatotrophs

Insulin-like growth factors(IGF) are involved in feedback regulation of growth hormone(GH) secretion from the pituitary. Though receptors for IGF-I and IGF-II have been identified on particulate preparations of rat pituitary, their localization and relationship to GH-secreting acidophils has not been determined. We used quantitative in vitro autoradiography and immunocytochemistry to simultaneously determine the distribution of IGF receptors and GH-secreting cells in human infant pituitary gland. Frozen or fixed post-mortem human infant pituitary glands were sectioned for binding studies, or immunocytochemistry. Binding for IGF-I and IGF-II showed characteristic specificity for respectively Type I and Type II receptors. Binding sites were visualized throughout the pituitary gland, with similar density and distribution for IGF-I and IGF-II receptor sites. Receptor density was two-fold higher in anterior than posterior pituitary, with highest density in the lateral horns of the anterior pituitary. The distribution of GH-containing cells (acidophils) was similar to IGF receptor distribution. Increased density of IGF receptors in regions of GH-secreting cells may point to the mechanism whereby IGF uniquely inhibits synthesis of human GH in contrast to its promotion of synthetic processes in other cells.     

Growth hormone deficiency in Down's syndrome children

ABSTRACT. Down's syndrome (DS) children have been reported to have severe postnatal growth arrest and microcephaly. To determine if growth hormone (GH) deficiency plays a role in growth retardation in DS, 20 children were studied. The subjects (13 boys, 7 girls) were aged between 15 months and 13.9 years, had a height SDS ranging from ? 1.19 to ? 5.48, weight SDS ranging from ? 0.21 to ?4.58, head circumference SDS ranging from ? 0.40 to ? 6.6, and a skeletal age ranging from 0.9 to 4.6 SD below the mean for normal children of same age and sex. GH was evaluated by levodopa (125 mg up to 15 kg, and 250 mg between 15–30 kg), clonidine (0.15 mg m^?2) stimulation tests and hGH secretory patterns by the integrated 24 h. GH concentration (IC-GH) using a constant withdrawal pump with continuous blood collection every 30 min. The serum concentrations were: TSH, 0.7–8.0 mIU ml^?1 (0.2–5.5); T₄, 6.6–14.3 μg dl^?1 (5–12); T₃, 95–254 ng dl_?1 (85–185); LH, <2.0–8.3 mlU ml_?1 (<3); FSH, <1.3–7.2 mIU ml_?1 (<3); testosterone, <30 ng dl^?1 (5–35); estradiol, <5 ng dl^?1 (<5–25); prolactin, 35.7–2.9 (F: 5–25; m 5–15); and somatomedin-C (Sm-C), 0.14–1.98 U ml^?1 (0.08–5.90) (normal values in brackets). Peak serum GH after levodopa and clonidine was found to be below 10 ng ml^?1 for both stimulatory tests in seven out of the 20 children studied. Twelve children showed a disparity between levodopa and clonidine testing. Of the 12 children, peak serum GH after levodopa was found to be below 10 ng ml^?1 in five children; and peak serum GH after clonidine was found to be below 10 ng ml^?1 in six. One child had a clonidine peak increase in serum GH concentration exactly 10 ng ml^?1, but had a 12 h IC-GH of 1.5 ng ml^?1 (N<3.2). Two children with peak GH after clonidine above 10 ng ml^?1 had a 24 h IC-GH of 0.7 and 1.3 ng ml^?1. A fourth child who had peak GH concentrations above 10 ng ml^?1 with levodopa and clonidine had a 12 h IC-GH of 0.5 ng ml^?1. These data suggest that DS children have marked growth retardation, associated with a reduced serum GH response to levodopa and clonidine stimulation tests, or disparity in responses to the stimulatory tests and low 24 h IC-GH.     

Role of nerve growth factor in suramin neurotoxicity studied in vitro

We determined whether suramin neurotoxicity can be prevented by nerve growth factor (NGF) and if this interaction occurs at the level of the NGF receptor. Neurite outgrowth from rat dorsal root ganglia in vitro was measured serially in the presence of suramin (100–600 μM) alone or with β-NGF (50–1,000 ng/ml). Competitive NGF receptor–binding studies were done with ¹²⁵I-labeled NGF in the presence or absence of suramin. Neurite growth was inhibited in a dose-dependent manner, but at usual neurotoxic levels this inhibition could be overcome completely by increasing the concentration of NGF. Receptor-binding assays showed similar dose-dependent inhibition of ¹²⁵I-labeled NGF binding. In the presence of suramin, the dissociation constant for high-affinity binding was decreased from 1.2 × 10^?11 to 3.9 × 10^?10 and low-affinity binding from 2.7 × 10^?9 to 1.2 × 10^?8. Increasing doses of suramin inhibited ¹²⁵I-labeled NGF specific binding in a dose-dependent fashion, and doses of suramin ± 1,000 μM were able to completely inhibit ¹²⁵I-labeled NGF specific binding. Suramin-induced dorsal root ganglia damage can be ameliorated by high-dose NGF. This effect is most likely due to competition between suramin and NGF at the high-affinity NGF receptor.     

Growth Hormone Production and Action in N1E-115 Neuroblastoma Cells

Neuroblastoma cells are undifferentiated cells derived from the neural crest and are commonly used as models for studying neural function. Mouse N1E-115 neuroblastoma cells are derived from cancerous tissue and provide a model for studying the oncogenesis of neural cells. As growth hormone (GH) has been implicated as an autocrine or paracrine involved in neural regulation and in the induction or progression of cancer, the possibility that N1E-115 cells are sites of GH production and GH action was assessed. Using RT-PCR, cultured N1E-115 cells were found to express the mouse GH and GH receptor (GHR) genes. Immunocytochemistry demonstrated that both of the translated proteins (GH and its receptor) were abundantly present in the cytoplasm of these cells and their co-localization was established by confocal cytochemistry. GH action in these cells was determined in cells cultured for 72 h in the presence or absence of 10^–6 M or 10^–9 M mouse GH, which induced neurite sprouting and increased axon growth. In summary, the expression of GH and its receptor in GH responsive tumor-derived N1E-115 neuroblastoma cells suggests they provide a useful experimental model to assess GH actions in neural function or neural oncogenesis.     

Insulin-like growth factor (IGF)-I,-II and IGF-binding proteins in the cyst fluid of a patient with astrocytoma

Insulin-like growth factor (IGF)-I,-II and IGF-binding proteins (IGFBPs) were demonstrated in the cyst fluid of a patient with a hypothalamic astrocytoma. The astrocytoma cyst fluid was subjected to gel chromatography at low pH and the IGF-I and IGF-II levels were measured by specific radioimmunoassays. Immunoreactive IGF-I and IGF-II levels were 19 ng/ml and 78 ng/ml respectively. Several-fold higher IGF-II values were obtained when cyst fluid was not extracted or was extracted with acid ethanol before radioimmunoassay analysis. The immunoreactive IGFBP-1 concentration was 26 ng/ml. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent Western ligand blotting with [¹²⁵I]IGF-II revealed bands at 200, 34.5, 29.5, 24 and 21 kD as visualized by autoradiography. Binding studies demonstrated that these binding proteins bind specifically [¹²⁵I]IGF-I and [¹²⁵I]IGF-II. These observations suggest that IGFs as well as IGF-binding proteins are produced by astrocytoma cells and may act in a paracrine or autocrine fashion capable of modulating the growth of astrocytoma tumours.     

Central opiate modulation of growth hormone and insulin-like growth factor-I

The effects of central administration of morphine-sulfate (MOR:80 μg) and morphine-6-glucuronide (M6G:1 μg) on the growth hormone (GH)/insulin-like growth factor (IGF) system were assessed. MOR and M6G were injected intracerebroventricularly (ICV) in chronically catheterized 24 h fasted rats; time-matched control animals received H₂O (5 μl). MOR increased plasma GH concentrations 3-fold 2 h after ICV injection, and transiently increased the plasma concentration and liver content of IGF-I (60% and 90%, respectively) 30 min after ICV injection. M6G did not produce any significant alterations in plasma GH and IGF-I levels at the time-points measured. Both MOR and M6G increased the concentration of IGF binding protein-1 (IGFBP-1) in plasma and liver 2 h after injection. However, MOR showed 2- to 2.5-fold greater effect than M6G in stimulating plasma and liver IGFBP-1. MOR and M6G produced similar increases in plasma epinephrine (5-fold), norepinephrine (3-fold) and corticosterone (1.5-fold). Neither opiate significantly altered circulating insulin levels. These findings suggest that opiate modulation of GH and IGF may be hormone-independent and centrally modulated. We speculate that differential affinities of MOR and M6G to the different opiate receptor subtypes might be responsible for their distinct effects on GH/IGF-I system.     

Regulation of adult olfactory neurogenesis by insulin-like growth factor-I

Insulin-like growth factor-I (IGF-I) has multiple effects within the developing nervous system but its role in neurogenesis in the adult nervous system is less clear. The adult olfactory mucosa is a site of continuing neurogenesis that expresses IGF-I, its receptor and its binding proteins. The aim of the present study was to investigate the roles of IGF-I in regulating proliferation and differentiation in the olfactory mucosa. The action of IGF-I was assayed in serum-free culture combined with bromodeoxyuridine-labelling of proliferating cells and immunochemistry for specific cell types. IGF-I and its receptor were expressed by globose basal cells (the neuronal precursor) and by olfactory neurons. IGF-I reduced the numbers of proliferating neuronal precursors, induced their differentiation into neurons and promoted morphological differentiation of neurons. The evidence suggests that IGF-I is an autocrine and/or paracrine signal that induces neuronal precursors to differentiate into olfactory sensory neurons. These effects appear to be similar to the cellular effects of IGF-I in the developing nervous system.     

Localization and Characterization of Insulin-Like Growth Factor-I Receptors in Rat Brain and Pituitary Gland Using in vitro Autoradiography and Computerized Densitometry* A Distinct Distribution from Insulin Receptors

In order to identify likely sites of action of insulin-like growth factor-I (IGF-I) in rat brain and pituitary gland, we have used the technique of in vitro autoradiography and computerized densitometry to map, characterize and quantify its receptors in coronal and sagittal sections. A discrete and characteristic distribution of IGF-I receptor binding was demonstrated, with specific binding representing 85% of total binding. Displacement and specificity competition curves in the olfactory bulb were typical for authentic IGF-I receptors and computer analysis indicated a single class of binding site with a dissociation constant (K_d) of 13 nM for the choroid plexus and 5.1 nM for the olfactory cortex. IGF-I receptor density was very high in the choroid plexus in ail ventricles, but the binding in other circumventricular organs was variable, with high levels in the median eminence and the sub-fornical organ, and low levels in the organum vasculosum of the lamina terminalis. Highest binding was seen in the glomerular layer of the olfactory bulb and its associated regions the taenia tecta and anteromedial olfactory nucleus. The preoptic and septal regions showed moderate binding, while the hypothalamus, with the exception of the median eminence, showed low IGF-I binding. The pituitary gland showed very high binding density in both anterior and posterior lobes, similar to the median eminence. The thalamus had high IGF-I binding density, while it was low in basal ganglia. In the limbic system the hippocampal CA2, CAS, CA4 layers showed high binding, with little in CA1, while binding was high also in the adjacent amygdala. Binding was low in the mid and hindbrain, with the exception of the geniculate bodies, and the sensory nucleus of the trigeminal nerve. Binding was high in the primary olfactory and endopyriform cortex and in specific superficial layers. Cerebellar binding was also high in the molecular layer. Fibre layers showed no binding. Comparison with insulin receptors revealed common distribution in the choroid plexus, paraventricular nucleus, cerebellum, entorhinal cortex and amygdala, with receptor density three- to five-fold higher for IGF-I than for insulin. In contrast, in the hippocampus, insulin binding was high in the CA1 field, and low in CA2, CA3, CA4 while for IGF-I binding the converse was seen. The arcuate nucleus showed prominent insulin labelling and minimal IGF-I binding, while the median eminence showed low insulin and high IGF-I binding. The hypothalamus was more widely labelled with insulin, while in the thalamus the converse was true. Olfactory bulb laminae were labelled with differing intensity by insulin and IGF-I. In common with insulin receptor distribution was the high density of IGF-I receptors over areas of extensive dendritic arborizations which receive rich synaptic inputs, in the cerebellum, hippocampus and olfactory bulb. We conclude that IGF-I receptors are widespread throughout rat brain and pituitary gland, with concentration in regions concerned with olfaction, autonomie and sensory processing, as well as in regulation of growth hormone release, via feedback at the median eminence and pituitary gland. Many of these regions have in common high rates of metabolic and synthetic activity, which may be mediated by IGF-I and its receptors.     

Localization of [125I]IGF-I Binding on the Ovine Pars Tuberalis

In the sheep, it has been shown that the pars tuberalis of the pituitary may mediate the photoperiodic control of seasonal changes in prolactin secretion. High concentrations of melatonin receptors are present on the ovine pars tuberalis and melatonin is known to inhibit forskolin-stimulated cyclic AMP production in this tissue. Other hormonal inputs to the ovine pars tuberalis have not yet been identified. In the rat mRNA for the IGF-I receptor has been identified in the pars tuberalis using in situ hybridization. In order to define whether IGF-I may influence the function of the ovine pars tuberalis the presence of receptors for IGF-I has been investigated. Using in vitro autoradiography specific [¹²⁵I]IGF-I binding was found in high concentrations over the ovine pars tuberalis particularly associated with certain of the capillaries. Homogenate receptor assays showed saturable specific binding of [¹²⁵I]IGF-I with a mean dissociation constant (Kd) of 0.5 ± 0.1 nM (n=4). Competition studies revealed a rank order of potency of IGF-I>IGF-II> > >insulin, in displacing [¹²⁵I]IGF-I binding, indicative of a mixed population of IGF-I and IGF-II/rnannose-6-phosphate receptors and insulin-like growth factor binding proteins (IGFBPs). Cross-linking of [¹²⁵I]IGF-I to pars tuberalis membrane homogenates and analysis by SDS-PAGE under reducing conditions confirmed the presence of both IGF-I receptors and binding proteins. Autophosphorylation of a 97 kDa substrate, compatible with the β-sub-unit of the IGF-I receptor, was increased in the presence of IGF-I, indicating the existence of functional IGF-I receptors on the ovine pars tuberalis. In contrast in the rat [¹²⁵I]IGF-I binding was restricted to the median eminence region of the brain and was not detectable over the pars tuberalis.     

The insulin-like growth factor I system in the rat cerebellum: Developmental regulation and role in neuronal survival and differentiation

The developmental regulation of insulin-like growth factor I (IGF-I), its receptor, and its binding proteins (IGFBPs) was studied in the rat cerebellum. All the components of the IGF-I system were detectable in the cerebellum at least by embryonic day 19. Levels of IGF-I receptor and its mRNA were highest at perinatal ages and steadily decrease thereafter, although a partial recovery in IGF-I receptor mRNA was found in adults. Levels of IGF-I and its mRNA also peaked at early ages, although immunoreactive IGF-I showed a second peak during adulthood. Finally, levels of IGFBPs were also highest at early postnatal ages and abruptly decreased thereafter to reach lower adult levels. Since highest levels of the different components of the IGF-I system were found at periods of active cellular growth and differentiation we also examined possible trophic effects of IGF-I on developing cerebellar cells in vitro. We found a dose-dependent effect of IGF-I on neuron survival together with a specific increase of the two main neurotransmitters used by cerebellar neurons, GABA and glutamate. Analysis of cerebellar cultures by combined in vitro autoradiography and immunocytochemistry with cell-specific markers indicated that both Purkinje cells (calbindin-positive) and other neurons (neurofilament-positive) contain IGF-I binding sites. These results extend previous observations on a developmental regulation of the IGF-I system in the cerebellum and reinforce the notion of a physiologically relevant trophic role of IGF-I in cerebellar development. © 1994 Wiley-Liss, Inc.     

Heterogeneity of brain melanocortin receptors suggested by differential ligand binding in situ

The existence of multiple brain melanocortin receptor types has been postulated, based on the complex pharmacology of intracerebrally administered melanocortin (melanocyte-stimulating hormone-related) peptides. In this study, this hypothesis was tested by determining whether different brain melanocortin receptor populations can be discriminated on a pharmacologic or neuroanatomic basis. The abilities of various pharmacologically active native melanocortins and structural analogs, as well as other test substances, to compete with biologically active [¹²⁵I]Nle⁴,d-Phe⁷-α-MSH([¹²⁵I]NDP-MSH) for binding to melanocortin receptors was determined, by in vitro binding and autoradiography in frozen rat brain tissue sections. We have previously shown that native melanocortins including α-MSH, γ-MSH and ACTH_1–39 compete with [¹²⁵I]NDP-MSH for binding to brain tissue sites. In the present studies, each of the melanocortin peptides α-MSH, des-acetyl-α-MSH, β-MSH and ACTH_1–24 when present at 1 μM virtually eliminated [¹²⁵I]NDP-MSH binding in each of a series of brain structures, including medial preoptic area, caudate putamen, olfactory tubercle, bed nucleus of the stria terminalis, ventral part of the lateral septal nucleus, hypothalamic periventricular and paraventricular nuclei, dorsal anterior amygdaloid area, substantia innominata and thalamic paraventricular nucleus; as well as in extraorbital lacrimal gland, a peripheral melanocortin target. In contrast, the behaviorally and neurotrophically active melanocortin analogs Met(O₂),d-Lys,Phe⁹-α-MSH_4–9 (Org2766), ACTH_4–9 and the antipyretic peptide α-MSH_11–13 did not affect [¹²⁵I]NDP-MSH binding at concentrations up to 100 μM, implying that the receptors or receptor binding sites which mediate the actions of these analogs must comprise additional types, distinct from those which bind [¹²⁵I]NDP-MSH. Binding of [¹²⁵I]NDP-MSH was also unaffected by the nonmelanotropic peptides ACTH_1–4, ACTH_34–39 and vasoactive intestinal polypeptide (VIP) and by the antipyretic drugs acetaminophen and lysine-salicylate. Although some of the brain structures are known to express mRNA encoding a γ-MSH-preferring melanocortin receptor type known as MC3, the relative order of binding affinities of melanocortins, determined in concentration-response studies, wasNdp-MSH≥ACTH_1–24α-MSH>γ-MSH>ACTH_4–10 probably account for most of the [¹²⁵I]NDP-MSH binding detectable in the brain. Furthermore, the potency relationships between these respective peptides and the binding activity ofD-Trp⁷,D-Phe^{10-α-MSH_6–11} amide, a synthetic α-MSH antagonist, varied considerably among the brain sites and peripheral (lacrimal and melanoma) tissues studied, suggesting some degree of heterogeneity of ligand binding properties among [¹²⁵I]NDP-MSH-binding melanocortin receptor populations in different regions of the brain. Considered together with the available data on the pharmacologic actions of melanocortins and the molecular biology of melanocortin receptors, the present results provide evidence for the existence of multiple, pharmacologically distinct classes of melanocortin receptors in the brain, potentially providing a basis for the pharmacological targeting of specific populations of central melanocortin receptors.