Homologous Regulation of the MSH Receptor in Melanoma Cells

Abstract

We have examined the mechanism of homologous regulation of MSH receptor binding and receptor-mediated adenylate cyclase activation in three human and two mouse melanoma cell lines. Pretreatment with α-MSH resulted in a time- and dose-dependent up-regulation of MSH receptors in human D10 and 205 melanoma cells whereas in human HBL and in mouse B16–F1 and Cloudman S91 cells α-MSH induced receptor down-regulation. Up-regulation of receptors was maximal after a 24–h incubation period and an α-MSH concentration of 100 nM (EC₅₀ = 2.4 nM). The increase in α-MSH binding was independent of adenylate cyclase activation and protein synthesis and appeared to be caused by recruitment of spare receptors. The structural requirements of the peptide for triggering this process differed from those found in receptor-binding analyses. Receptor down-regulation was maximal after 12 h and hence more rapid than up-regulation. In B16–F1 cells, 10 nM α-MSH caused the disappearance of 85–90% of the MSH receptors, the EC₅₀ of 0.23 nM lying exactly between that for α-MSH-induced melanogenesis (0.027 nM) and the dissociation constant of receptor binding (1.31 nM). Down-regulation in B16–F1 cells appears to be the consequence of receptor internalization following MSH binding and seems to be initiated during an early step in MSH signalling, preceding the activation of adenylate cyclase and the cAMP signal. Receptor up- and down- regulation were not accompanied by an alteration in affinity to a-MSH, as demonstrated by Scatchard analysis of the binding curves.     

Radioreceptor Assay for α;-Msh Using Mouse B16 Melanoma Cells

Abstract

A radioreceptor assay for α;-MSH is described which is based on cultured mouse B16 melanoma cells and bioactive monoiodinated [Nle⁴]-α;-MSH tracer. The assay was used (1) to study the binding characteristics of α;-MSH to B16 cells, (2) to determine the relative binding activity of MSH peptides, and (3) to measure MSH in tissue extracts. The association of α;-MSH to B16 cells reached a stable plateau after 3 h at 15°C. At 25° or 37°C, the binding was transient and at 0-1°C, the association was very slow. The hormone-receptor complex was relatively stable between 0° and 15°C whereas a 50% dissociation was reached after 90 min at 25°C and after 35 min at 37°C. The mean K_D for α;-MSH of four saturation experiments was 1.3 nM and the number of receptors 9570 per cell. 1,10-Phenanthroline had a stabilizing effect in the binding assay when used at a 0.3 mM concentration. From the MSH peptides tested in the binding assay, some showed similar potencies in three bioassays (tyrosinase, melanin and Anolis skin), whereas others displayed considerably     

Interactions of α-Melanotropin and Agouti on B16 Melanoma Cells: Evidence for Inverse Agonism of Agouti

Abstract

α-Melanocyte-stimulating hormone (α-MSH, α-melanotropin) and agouti control the switch between eumelanin and pheomelanin synthesis in mammalian melanocytes. Here we investigated interactions between α-MSH, agouti protein, cAMP elevating agents and phorbol ester on mouse B16 melanoma cells. Agouti (K_d 3.7nmol/l) and α-MSH (K_d 2.3 nmol/l) had similar affinities to the MC1 melanocortin receptor. Both α-MSH and agouti induced MC1 receptor down-regulation. Agouti antagonized melanogenesis induced by α-MSH, forskolin, cholera toxin (CT), and pertussis toxin (PT). It also reduced the constitutive melanin formation of long-term cultures. Cell proliferation was inhibited by agouti (43% at 100 nM). This effect was reversed by α-MSH, forskolin, or CT. B16-G4F cells, a cell variant that lacks the MC1 receptor, did not respond to agouti. From these results we conclude that agouti shows the characteristics of an inverse agonist acting through the MC1 receptor.     

ANTAGONIST AND AGONIST ACTIVITIES OF THE MOUSE AGOUTI PROTEIN FRAGMENT (91–131) AT THE MELANOCORTIN-1 RECEPTOR

Antagonist and agonist activities of chemically synthetized mouse agouti protein fragment (91–131) (AP_91–131) at the melanocortin type-1 receptor (MC1-R) were assessed using B16-F1 mouse melanoma cells in vitro and the following assay systems: (i) receptor binding, (ii) adenylate cyclase, (iii) tyrosinase, (iv) melanin production, and (v) cell proliferation. In competition binding studies AP_91–131 was about 3-fold less potent than the natural agonist α-melanocyte-stimulating hormone (α-MSH) in displacing the radioligand [¹²⁵I]-[Nle⁴, D-Phe⁷]-α-MSH (K_i 6.5±0.8 nmol/l). α-MSH-induced tyrosinase activation and melanin production were completely inhibited by a 100-fold higher concentration of AP_91–131; the IC₅₀ values for AP_91–131 in the two assay systems were 91±22 nM and 95±15 nM respectively. Basal melanin production and adenylate cyclase activity in the absence of agonist were decreased by AP_91–131 with IC₅₀ values of 9.6±1.8 nM and 5.0±2.4 nM, respectively. This indicates inverse agonist activity of AP_91–131 similar to that of native AP. The presence of 10 nM melanin-concentrating hormone (MCH) slightly potentiated the inhibitory activity of AP_91–131 in the adenylate cyclase and melanin assays. On the other hand, AP_91–131 inhibited cell growth similar to α-MSH (IC₅₀ 11.0±2.1 nM; maximal inhibition 1.8-fold higher than that of α-MSH). Furthermore, MC1-R was down-regulated by AP_91–131 with about the same potency and time-course as with α-MSH. These results demonstrate that AP_91–131 displays both agonist and antagonist activities at the MC1-R and hence that it is the cysteine-rich region of agouti protein which inhibits and mimics the different α-MSH functions, most likely by simultaneous modulation of different intracellular signalling pathways.     

Adrenocorticotropin/α-Melanocyte-Stimulating Hormone (ACTH/MSH)-Like Peptides Modulate Adenylate Cyclase Activity in Rat Brain Slices: Evidence for an ACTH/MSH Receptor-Coupled Mechanism

Abstract: The regulation of adenylate cyclase activity by adrenocorticotropin/α-melanocyte–stimulating hormone (ACTH/MSH)-like peptides was investigated in rat brain slices using a superfusion method. Adenylate cyclase activity was concentration-dependently increased by ACTH-(1–24), α-MSH (EC₅₀ values 16 and 6 nM, respectively), and [Nle⁴,D-Phe⁷]α-MSH (EC₅₀ value 1.6 nM), in the presence of forskolin (1 μM, optimal concentration). 1-9-Dideoxy-forskolin did not augment the response of adenylate cyclase to ACTH-(1–24). Various peptide fragments were tested for their ability to enhance [³H]cyclic AMP production. [Nle⁴,D-Phe⁷]α-MSH increased [³H]cyclic AMP formation with a maximal effect of 30% and was more potent than ACTH-(1–24), ACTH-(1–16)-NH₂, α-MSH, ACTH-(1–13)-NH₂, [MetO⁴]α-MSH, [MetO₂⁴,D-Lys⁸,Phe⁹]ACTH-(4–9), ACTH-(7–16)-NH₂, ACTH-(1–10), and ACTH-(11–24), in order of potency. This structure–activity relationship resembles that found for the previously described peptide-induced display of excessive grooming. ACTH-(1–24) stimulated adenylate cyclase activity in both striatal (maximal effect, ?20%) and septal slices (maximal effect, ?40%), but not in hippocampal or cortical slices. Lesioning of the dopaminergic projections to the striatum did not result in a diminished effect of [Nle⁴,D-Phe⁷]α-MSH on [³H]cyclic AMP accumulation, which indicates that the ACTH/MSH receptor–stimulated adenylate cyclase is not located on striatal dopaminergic terminals. ACTH-(1–24) did not affect the dopamine D₁ or D₂ receptor–mediated modulation of adenylate cyclase activity. Based on the present data, we suggest that the binding of endogenous ACTH or α-MSH to a putative ACTH/MSH receptor in certain brain regions leads to the activation of a signal transduction pathway using cyclic AMP as a second messenger.     

α-MSH Receptor Autoradiography on Mouse and Human Melanoma Tissue Sections and Biopsies

Abstract

MSH receptors and their binding characteristics of [¹²⁵I]-labelled derivatives of α-MSH have been studied extensively on various mouse and human melanoma cell lines in culture. The aim of this study was to determine the binding characteristics of α-MSH radioligands to MSH receptors occurring in experimental mouse and human melanoma tumours as well as in human melanoma biopsies. For this reason, solid tumours were grown on experimental animals by inoculation of murine B16-F1 and human D10 and HBL melanoma cells. After excision and cryosectioning of the tumours, frozen tissue sections were incubated with [(¹²⁵I)Tyr²]-α-MSH or [(¹²⁵I)Tyr²,Nle⁴,D-Phe⁷]-α-MSH and specific α-MSH binding sites were visualized by subsequent autoradiography. The presence of increasing concentrations of unlabelled α-MSH during incubation with tracer led to a dose-dependent displacement of the radioligand. Quantitative analysis of the autoradiograms produced dissociation constants which were comparable with those obtained with cell binding assays: K_D = 1.87 and 1.31 nmol/l for B16 tumours and cells, respectively; 0.32 and 0.33 nmol/l for D10, and 2.24 and 1.36 nmol/l for HBL tumours and cells, respectively. This indicates similar binding properties of α-MSH radioligands to both cultured melanoma cells and tissue sections of melanoma tumours from experimental animals. Similar binding characteristics were also observed with human melanoma tissue sections originating from biopsies of melanoma patients.     

INTERACTION OF MELANIN-CONCENTRATING HORMONE (MCH), NEUROPEPTIDE E-I (NEI), NEUROPEPTIDE G-E (NGE), AND α-MSH WITH MELANOCORTIN AND MCH RECEPTORS ON MOUSE B16 MELANOMA CELLS

Melanin-concentrating hormone (MCH) and α-melanocyte-stimulating hormone (α-MSH) are known to exhibit mostly functionally antagonistic, but in some cases agonistic activities, e.g., in pigment cells and in the brain. Neuropeptide E-I (NEI) displays functional MCH-antagonist and MSH-agonist activity in different behavioral paradigms; the role of neuropeptide G-E (NGE) is not known. This study addressed the question of possible molecular interactions between α-MSH, MCH and the MCH-precursor-derived peptides NEI and NGE at the level of the pigment cell MCH receptor subtype (MCH-R_pc) and the different melanocortin (MC) receptors. Radioreceptor assays using [¹²⁵I]MCH, [¹²⁵I]α-MSH and [¹²⁵I]NEI as radioligands and bioassays were performed with MC1-R-positive and MC1-R-negative mouse B16 melanoma cells and with COS cells expressing the different MC receptors. The IC₅₀s of α-MSH and NEI or NGE for [¹²⁵I]MCH displacement from mouse MCH-R_pc were 80-fold and, respectively, > 300-fold higher than that of MCH, and the IC₅₀s for MCH and NEI or NGE for [¹²⁵I]α-MSH displacement from mouse MC1-R were 50,000-fold and > 200,000-fold higher than that of α-MSH. No high-affinity binding sites for NEI were detected on B16 melanoma cells and there was no significant displacement of [¹²⁵I]α-MSH by MCH, NEI or NGE with MC3-R, MC4-R and MC5-R expressed in COS cells. At concentrations of 100 nM to 10 μM, however, MCH, NEI and NGE induced cAMP formation and melanin synthesis which could be blocked by agouti protein or inhibitors of adenylate cyclase or protein kinase A. This shows that mammalian MCH-precursor-derived peptides may mimic MSH signalling via MC1-R activation at relatively high, but physiologically still relevant concentrations, as e.g. found in autocrine/paracrine signalling mechanisms.     

Des-acetyl MSH and γ-MSH act as partial agonists to α-MSH on the Anolis melanophore

The biological activities of α-MSH des-acetyl MSH, γ-MSH and LPH_37–58 were compared using the Anolis rate method of bioassay. Dose-response data showed LPH_37–58 to be equipotent with α-MSH, but des-acetyl MSH and γ-MSH were found to be much less active. The effect of LPH_37–58 was additive to that of α-MSH, indicating that LPH_37–58 is a full agonist of α-MSH. The lower potency peptides des-acetyl MSH and γ-MSH reduced the effect of α-MSH and are, therefore, partial agonists of α-MSH. The action of MSH peptides in vivo may be modulated by interaction with agonists.     

Desensitization of melanotropin receptors in COS-7 cells

Non-transfected COS-7 cells have been found to possess functional melanotropin receptors on their cell surface. These receptors, and the properties of the melanocyte stimulating hormone (MSH) peptides can be characterized by measuring melanotropin stimulation of cAMP accumulation in the cells. In these cells we studied the ultra-long lasting super agonist [Nle⁴-D-Phe⁷]-α-MSH (NDP-α-MSH), and compared it with the endogenous MSH peptides with respect to potency, maximal activity, duration of action, and rate of desensitization. Surprisingly, NDP- α-MSH did not act as a full agonist in COS-7 cells. In multiple experiments, it could stimulate cAMP accumulation to approximately 50% of the level of α-MSH, β-MSH and adrenocorticotropic hormone (ACTH). The MSH receptor mediating this activity is unknown. The time course of cAMP accumulation, and the duration of receptor activation was also investigated. In contrast to other systems, NDP-α-MSH did not induce prolonged activity, with respect to cAMP accumulation, in COS-7 cells. The MSH receptors present in COS-7 were found to desensitize rapidly subsequent to pretreatment by any of the MSH peptides. As expected for a partial agonist, the activity of NDP-α-MSH desensitized more rapidly than any of the full agonists. Surprisingly, desensitization induced by pretreatment with NDP-α-MSH also occurred more rapidly than desensitization induced by the other MSH analogs.     

Synthesis and Characterization of Time-resolved Fluorescence Probes for Evaluation of Competitive Binding to Melanocortin Receptors

Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining K_d values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited K_d values of 27 ± 3.9 nM and 4.2 ± 0.48 nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The K_i values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the K_i values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported.     

Dimeric DOTA-α-Melanocyte-Stimulating Hormone Analogs: Synthesis and In Vivo Characteristics of Radiopeptides with High In Vitro Activity

Dimeric analogs of α-melanocyte-stimulating hormone (α-MSH) labeled with radiometals are potential candidates for diagnosis and therapy of melanoma by receptor-mediated tumor targeting. Both melanotic and amelanotic melanomas (over-)express the melanocortin-1 receptor (MC1-R), the target for α-MSH. In the past, dimerized MSH analogs have been shown to display increased receptor affinity compared to monomeric MSH, offering the possibility of improving the ratio between specific uptake of radiolabeled α-MSH by melanoma and nonspecific uptake by the kidneys. We have designed three linear dimeric analogs containing a slightly modified MSH hexapeptide core sequence (Nle-Asp-His-d-Phe-Arg-Trp) in parallel or antiparallel orientation, a short spacer, and the DOTA chelator for incorporation of the radiometal. In vitro, all three peptides were more potent ligands of the mouse B16-F1 melanoma cell melanocortin-1 receptor (MC1-R) than DOTA-NAPamide, which served as standard. The binding activity of DOTA-diHexa(NC-NC)-amide was 1.75-fold higher, that of diHexa(NC-NC)-Gly-Lys(DOTA)-amide was 3.37-fold higher, and that of DOTA-diHexa(CN-NC)-amide was 2.34-fold higher. Using human HBL melanoma cells, the binding activity of diHexa(NC-NC)-Gly-Lys(DOTA)-amide was sixfold higher than that of DOTA-NAPamide. Uptake by cultured B16-F1 cells was rapid and almost quantitative. In vivo, however, the data were less promising: tumor-to-kidney ratios 4 hr postinjection were 0.11 for [¹¹¹In]DOTA-diHexa(NC-NC)-amide, 0.26 for diHexa(NC-NC)-Gly-Lys([¹¹¹In]DOTA)-amide, and 0.36 for [¹¹¹In]DOTA-diHexa(CN-NC)-amide, compared to 1.67 for [¹¹¹In]DOTA-NAPamide. It appears that despite the higher affinity to the MC1-R of the peptide dimers and their excellent internalization in vitro, the uptake by melanoma tumors in vivo was lower, possibly because of reduced tissue penetration. More striking, however, was the marked increase of kidney uptake of the dimers, explaining the unfavorable ratios. In conclusion, although radiolabeled α-MSH dimer peptides display excellent receptor affinity and internalization, they are no alternative to the monomeric DOTA-NAPamide for in vivo application.     

Demonstration of Type I Insulin-Like Growth Factor Receptors on Human Platelets

Abstract

Human platelets, freshly isolated from healthy human adults, express receptors for insulin-like growth factor I. The IC₅₀ for displacement of ¹²⁵I-IGF-I binding by unlabeled IGF-I was 0.2 nM, by IGF-II 32 nM and by insulin 160 nM. Scatchard analysis of IGF-I binding demonstrates dissociation constants of 0.14 ± 0.08 nM for high affinity binding site and 54 ± 18 nM for low affinty binding site. The presence of the α-subunit of type I IGF receptor, as high affinity binding site, was verified by affinity crosslinking of ¹²⁵I-IGF-I to platelet surface membranes. Under reducing con-conditions a M_r= 135,000 band was preferentially labeled. The complete type I IGF receptor complex, which revealed under nonreducing conditions, has an approximately molecular mass of M_r > 400,000. The immunoprecipitation of the ¹²⁵I-IGF-I cross-linked type I receptor with αIR-3 confirmed the results achieved by affinity crosslinking.     

Photoaffinity Labelling of Msh Receptors on Anolis Melanophores: Irradiation Technique and Msh Photolabels for Irreversible Stimulation

Abstract

Excised dorsal skin of Anolis carolinensis was exposed to high intensity UV-irradiation in the presence of different photoreactive α-MSH derivatives. The resulting covalent binding of the hormone to its receptor induced irreversible pigment dispersion. The duration of the longlasting response depended on the type and length of irradiation; it was maximal after two 5 min irradiation phases with a light intensity of ～180 mW/cm² and a spectrum from 310 to 550 nm, fresh hormone being added after the first phase. [Nα-(4-Azidophenyl-acetyl-serine¹]-α-MSH (I), [2′-(2-nitro-4-azidophenylsulphenyl)-tryptophan⁹]-α-MSH (II) and [p-azidophenylalanine¹³]-α-MSH (III) all inserted into the receptor to about the same extent, as judged from the persistence of the longlasting signal. In contrast, [D-alanine¹, p-azidophenylalanine², norvaline⁴]-α-MSH (IV) and [Nα-(4-azidophenylacetyl)-serine¹, leucine⁹]-α-MSH (V) gave much less insertion and [leucine⁹, p-azidophenylalanine¹³]-α-MSH (VI) hardly any insertion when applied in the same relative excess (5-fold the concentration inducing a maximal response). Covalent attachment of the cleavable photolabel [Nα-(4-azidophenyl)-1, 3′-dithio-propionyl-serine¹]-α-MSH (VII) and subsequent washing of the skin in buffer containing 1% β-mercaptoethanol released the peptide from the receptor. Insertion of the C-terminal photolabel [p-azidophenylalanine¹³]-α-MSH was reduced by the weak antagonist H-Phe-Ala-Trp-Gly-Gly-Pro-Val-NH₂. These experiments prove that hormone receptors can be covalently labelled in tissue with very limited light transparency.     

Synthesis and biological activity of four gamma-melanotropin peptides derived from the cryuptic region of the adrenocorticotropin/beta-lipotropin precursor.

A third melanotropin coding fragment named γ-MSH was discovered by Nakanishi et al (Nature $\text{278}$, 423–427 (1979)) in the cryptic region outside the portion coding for ACTH and β-LPH in the ACTH/β-LPH precursor mRNA isolated from the intermediate lobe of bovine pituitary. Four possible γ-MSH peptides derived from this coding fragment were synthesized by solid-phase methodology and their bioactivity determined in an $\text{in vitro}$ MSH assay as well as the anterior pituitary primary culture assay. Relative to α-MSH, the melanotropic activities of Ac-γ₁-MSH, γ₁-MSH, γ₂-MSH and γ₃-MSH are 7.3 × 10^?4, 3.3 × 10^?5, 1.4 × 10^?4 and 4.6 × 10^?7 respectively. None of these γ-MSH peptides releases LH, FSH, PRL, GH and TSH in the pituitary culture medium at a dose as high as 100 ng per dish.     

Agouti-Related Maturation and Tissue Distribution of oc-Melanocyte Stimulating Hormone in Wild-Type (AwJ/AwJ) and Mutant (Ay/a,a/a) Mice

Because of ectopic overproduction of agouti protein, yellow alleles (A^y and A^vy) of the murine agouti gene may secondarily modulate the synthesis, maturation (i.e., acetylation), and/or tissue deployment of α-Melanocyte Stimulating Hormone (MSH). We used HPLC to test the hypothesis that A^y/a mice exhibit altered concentrations of desacetyl-, monoacetyl-, and diacetyl-α-MSH in pituitaries, sera, and telogen hair bulbs when compared to black (a/a) mice. We also used RIA to measure total MSH in those same tissues of A^y/a, a/a, and white-bellied agouti (A^wJ/A^wJ) mice (Strain C57BL/6J). We found no evidence that A^y/a mice possessed an imbalance of des-, mono-, and diacetylated α-MSH species. However, radioimmunoassay (RIA) analyses of total MSH suggest that wild-type agouti mice (A^wJ/A^wJ) exhibited significantly decreased (P < 0.05) tissue levels of total α-MSH in pituitaries, sera, and regenerating hair bulbs when compared to those of mutant A^y/a and a/a mice.     

Antisense Peptides: Tools for Receptor Isolation? Lack of Antisense Msh and Acth to Interact with their Sense Peptides and to Induce Receptor-Specific Antibodies

Abstract

The use of antisense peptides for receptor isolation as proposed by Blalock and his colleages (e.g. TIBTECH 8, 140–144, 1990) was tested for human ACTH as well as α- and β-MSH. We synthesized the corresponding antisense peptides HTCA_h, HSM-α and HSM-β and analyzed them for specific interaction with the sense peptides using several types of binding assay and bioassay. Similarly HTCA_h antibodies were tested for binding to ACTH receptors and ACTH antibodies. All these experiments were negative, i.e. there was no specific interaction between sense and antisense peptides nor between the corresponding antibodies. Receptor binding of the sense peptides was not affected by the antisense peptides or HTCA_h antibodies. Unexpectedly, HTCA_h but not HSM-α or HSM-β was a weak MSH agonist acting through a site independent of the MSH receptor. A detailed analysis of the concept of antisense peptides revealed that the theoretical background of the hypothesis of the ‘molecular recognition theory’ is rather weak, explaining the failure of various attempts to obtain specific receptor antibodies.     

Hemodynamic actions and mechanisms of systemically administered α-MSH analogs in mice

α-Melanocyte-stimulating hormone (α-MSH) regulates important physiological functions including energy homeostasis and inflammation. Potent analogs of α-MSH, [Nle⁴, d-Phe⁷]-α-MSH (NDP-α-MSH) and melanotan-II (MT-II), are widely used in pharmacological studies, but the hemodynamic effects associated with their systemic administration have not been thoroughly examined. Therefore, we investigated the hemodynamic actions of these compounds in anesthetized and conscious C57Bl/6N mice using peripheral routes of administration. NDP-α-MSH and MT-II induced mild changes in blood pressure and heart rate in anesthetized mice compared to the effects observed in conscious mice, suggesting that anesthesia distorts the hemodynamic actions of α-MSH analogs. In conscious mice, NDP-α-MSH and MT-II increased blood pressure and heart rate in a dose-dependent manner, but the tachycardic effect was more prominent than the pressor effect. Pretreatment with the melanocortin (MC) 3/4 receptor antagonist SHU9119 abolished these hemodynamic effects. Furthermore, the blockade of β₁-adrenoceptors with metoprolol prevented the pressor effect and partly the tachycardic action of α-MSH analogs, while the ganglionic blocker hexamethonium abrogated completely the difference in heart rate between vehicle and α-MSH treatments. These findings suggest that the pressor effect is primarily caused by augmentation of cardiac sympathetic activity, but the tachycardic effect seems to involve withdrawal of vagal tone in addition to sympathetic activation. In conclusion, the present results indicate that systemic administration of α-MSH analogs elevates blood pressure and heart rate via activation of MC_3/4 receptor pathways. These effects and the consequent increase in cardiac workload should be taken into account when using α-MSH analogs via peripheral routes of administration.     

HOMOLOGOUS REGULATION OF MELANOCORTIN-1 RECEPTOR (MC1R) EXPRESSION IN MELANOMA TUMOR CELLS IN VIVO

ABSTRACT

As G protein-coupled receptors (GPCRs) are the target of numerous signaling molecules, including about half of the therapeutic drugs currently used, it is important to understand the consequences of homologous (ligand-induced) receptor regulation. Continuous exposure of GPCRs to agonist in vitro most frequently results in receptor down-regulation, but receptor up-regulation may occur as well. These phenomena are expected to play a role in the physiological adaptation to endogenous ligands and also in the response to repetitive administration of drugs in the clinic. However, there is little information on homologous regulation of GPCRs in vivo. Here, we report on the regulation of melanocortin-1 receptor (MC1R) expression in melanoma cells implanted into mice. Two melanoma cell lines were investigated, D10 and B16F1, which in vitro had previously been shown to undergo homologous receptor up- and down-regulation, respectively. After implantation into mice and exposure to the natural MC1R agonist α-melanocyte-stimulating hormone (α-MSH), cell-surface MC1R expression was evaluated by competition binding experiments in tumor membrane preparations. In B16F1 cells, a single injection of 50 to 500?µg α-MSH induced a rapid but moderate dose-dependent MC1R down-regulation which could be totally reverted within 16–24?h. By continuous administration of α-MSH via osmotic minipumps, MC1R down-regulation was considerably amplified and reached the level observed in vitro, demonstrating that prolonged receptor interaction was necessary to induce a maximal effect in vivo. Similar results were obtained in vitro, which demonstrates that homologous MC1R regulation in B16F1 cells is essentially independent of the physiological environment. In D10 cells, however, up-regulation could not be reproduced in vivo, suggesting that MC1R up-regulation is more dependent on the physiological environment. These results demonstrate the importance of in vivo receptor regulation studies, in particular in view of the potential use of MC1R as a target for melanoma therapy.     

Studies of conformational isomerism in α-melanocyte stimulating hormone by design of cyclic analogues

Results of energy calculations for α-MSH (α-melanocyte stimulating hormone, Ac-Ser¹-Tyr²-Ser³-Met⁴-Glu⁵-His⁶-Phe⁷-Arg⁸-Trp⁹-Gly¹⁰-Lys¹¹-Pro¹²-Val¹³-NH₂) and [D -Phe⁷]α-MSH were used for design of cyclic peptides with the general aim to stabilize different conformational isomers of the parent compound. The minimal structural modifications of the conformationally flexible Gly¹⁰ residue, as substitutions for L -Ala, D -Ala, or Aib (replacing of hydrogen atoms by methyl groups), were applied to obtain octa- and heptapeptide analogues of α-MSH(4–11) and α-MSH(5–11), which were cyclized by lactam bridges between the side chains in positions 5 and 11. Some of these analogues, namely those with substitutions of the Gly¹⁰ residue with L -Ala or Aib, showed biological activity potencies on frog skin comparable to the potency of the parent tridecapeptide hormone. Additional energy calculations for designed cyclic analogues were used for further refinement of the model for the biologically active conformations of the His-Phe-Arg-Trp “message” sequence within the sequences of α-MSH and [D -Phe⁷]α-MSH. In such conformations the aromatic moieties of the side chains of the His⁶, L/D -Phe⁷, and Trp⁹ residues form a continuous hydrophobic “surface,” presumably interacting with a complementary receptor site. This feature is characteristic for low-energy conformers of active cyclic analogues, but it is absent in the case of inactive analogues. This particular spatial arrangement of functional groups involved in the message sequence is very close for α-MSH and [D -Phe⁷]α-MSH, as well as for biologically active cyclic analogues despite differences of dihedral angle values for corresponding low-energy conformations. © 1998 John Wiley & Sons, Inc. Biopoly 46: 155–167, 1998