Linear and Cyclic α-Melanotropin [4–10]-Fragment Analogues That Exhibit Superpotency and Residual Activity

Two analogues of α-MSH (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂), Ac-[Nle⁴, Asp⁵, D-Phe⁷, Lys¹⁰]α-MSH_4–10NH₂ and Ac-[Nle⁴, Asp⁵, D-Phe⁷, Lys¹⁰] α-MSH_4–10-NH₂, were synthesized, and the melanotropic activities of the peptides were compared in several bioassays. Potencies were determined in the in vitro frog and lizard skin bioassays and in the S91 melanoma cell tyrosinase assay. Both analogues were equipotent or more potent than α-MSH in all bioassays, and the activities of the analogues were prolonged compared to α-MSH. The two analogues were very resistant to inactivation by purified proteolytic enzymes (α-chymotrypsin, trypsin, and pepsin). The two peptides could be topically applied and transdermally delivered across the skin of mice in vivo, resulting in a shift from pheomelanogenesis to eumelanogenesis within follicular melanocytes. The cyclic analogue exhibited greater potency, prolonged activity, and stability against enzyme inactivation than did the linear peptide. The significance of the findings for the further design of melanotropin analogues is discussed, as in the possible relevance of these melanotropin analogues for use in biomedical studies.     

Long-term and residual melanotropin-stimulated tyrosinase activity in S91 melanoma cells is density dependent

Summary Cell density is a factor that affects the capacity of Cloudman S91 melanoma cells to respond to melanotropins in monolayer culture. Continuous exposure of melanoma cells to α-melanotropin or its potent analog [Nle⁴,D-Phe⁷]-α-MSH, resulted in maximal stimulation of tyrosinase after 2 d of treatment, but the magnitude of stimulation decreased thereafter despite the continued presence of the melanotropins. However, when melanoma cells continually exposed to melanotropins were subcultured to an initial low cell density and maintained in contact with α-MSH or [Nle⁴,D-Phe⁷]-α-MSH (long-term culture), tyrosinase activity was rapidly restored and greatly enhanced. Also, when cells were seeded at initial densities ranging from 0.2 to 3.2×10⁶ cells/flask, and exposed for 24 h to 10⁻⁷ M α-MSH, only the cultures seeded at low densities (0.2 and 0.4×10⁶ cells/flask) exhibited maximal tyrosinase activity during the 24 h exposure to the melanotropins. Therefore, tyrosinase activity was primarily affected by cell density rather than by the duration of time the cells were in culture or by continuous exposure to melanotropin. Other flasks of various cell densities were treated with 10⁻⁷ M α-MSH or [Nle⁴,D-Phe⁷]-α-MSH for 24 h, followed byremoval of the melanotropins from the culture medium. The magnitude and duration of theresidual stimulation of melanoma tyrosinase activity by melanotropins were also found to be dependent on the initial cell density. These results reveal that there is a limited range of optimal cell densities at which melanoma cells can respond to melanotropins and express increased tyrosinase activity.     

Biological Activities of Melanotropic Peptide Fatty Acid Conjugates

Four fatty acids (FA, palmitic, myristic, decanoic, hexanoic) were individually conjugated to the N-terminus of the α-MSH fragment analog, H-Asp⁵-His⁶-D-Phe⁷-Arg⁸-Trp⁹-Lys¹⁰-NH₂. This resulted in enhanced potency of the conjugates (compared to the unconjugated melanotropin analog) as determined in the lizard skin bioassay and in the mouse melanoma cell tyrosinase bioassay. The shorter conjugates of hexanoic and decanoic acid were at least equipotent to α-MSH in the lizard skin bioassay, whereas the longer myristoyl and palmitoyl analogs were 100 times less active. The myristoyl and palmitoyl conjugates exhibited a “creeping” potency in the lizard skin bioassay—that is, potency of the peptides increased with time in contact with the skins. These observations may be related to the more lipid nature of these FA-conjugates. In the tyrosinase assay, the conjugates were 10–100 times more active than α-MSH or the unconjugated analog. Each of the FA-melanotropic peptide conjugates exhibited prolonged (residual) melanotropic activity in both the lizard skin and melanoma cell bioassays. In other words, after removal of the melanotropin conjugates from contact with the skins or cells, responses were still manifested for hours or days thereafter. As little as 1 hr of contact with melanoma cells resulted in enhanced enzyme activity as measured 48 hr later. Since the conjugates, but not H-[Ast⁵,D-Phe⁷,Lys¹⁰]α-MSH₅-₁₀-NH₂, exhibited prolonged activity, the conversion of reversible agonists to irreversible agonists was demonstrated.     

D-isomeric replacements within the 6-9 core sequence of Ac-[Nle4]-alpha-MSH4-11-NH2: a topological model for the solution conformation of alpha-melanotropin

Analogs of Ac-[Nle⁴]-α-MSH_4–11-NH₂ and Ac-[Nle⁴, D -Phe⁷]-α-MSH_4–11-NH₂ were prepared with D -isomeric replacements at the His⁶, Arg⁸, and Trp⁹ residues. The requirement for an indole moiety at position 9 also was evaluated by replacement with L -leucine in both parent fragment analogs. D -isomeric replacements at positions 6 and 8 in either series were detrimental to biological potency in frog (Rana pipiens) and lizard skin (Anolis carolinensis) in vitro melanotropic assays. However, Ac-[Nle⁴, D -Trp⁹]-α-MSH_4–11-NH₂ and Ac-[Nle⁴, D -Phe⁷, D -Trp⁹]-α-MSH_4–11-NH₂ were equipotent and 10 × more potent than Ac-[Nle⁴]-α-MSH_4–11-NH₂, respectively, in the lizard skin bioassay, and 30 and 1900 times more potent in the frog skin bioassay. Ac-[Nle⁴, D -Phe⁷, D -Trp⁹]-α-MSH_4–11-NH₂ was 3 × more potent than α-MSH in the frog skin bioassay. Proton nmr studies in aqueous solution revealed a marked preservation of the backbone conformation of these linear analogs. Chemical-shift variations due to the through-space anisotropic influence of the core aromatic amino acid residues permitted evaluation of side-chain topology. The observed topology was consistent with nonhydrogen-bonded β-like structure (? = ?139°, ψ = +135° for L -amino acids; ? = +139°, ψ = ?135° for D -amino acids) as the predominant solution conformation. The biological and conformational data suggest that high melanotropic potency requires a close spatial arrangement of the His⁶, Phe⁷, and Arg⁸ side chains.     

Biomedical Applications of Synthetic Melanotropins

Alpha-melanotropin (alpha-melanocyte stimulating hormone, alpha-MSH) is a hormone produced by the pituitary gland of most vertebrate animals. This melanotropic peptide, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2, regulates melanin pigmentation of the skin of some mammals. Although MSH may be absent from the human pituitary gland, this peptide can stimulate pigment formation in human skin. We have synthesized several analogues of alpha-MSH, which are superpotent, prolonged-acting, and resistant to inactivation by serum enzymes. One such analogue, [NLe⁴, D-Phe⁷]alpha-MSH, has proven particularly useful in a number of physiological studies. In addition, some [Nle⁴, D-Phe⁷]-substituted fragment analogues of MSH are even more active than the native hormone, alpha-MSH. For example, these analogues are 100–1,000 times more active than alpha-MSH in stimulating S-91 mouse melanoma tyrosinase activity in vitro. We have successfully labeled one such peptide to high specific activity; this melanotropin, [³H]-Ac-[Nle⁴, D-Phe⁷]alpha-MSH_4–11NH₂, has been shown by others to bind to B16 melanoma cells. We have also conjugated several ligands (fluorescein and biotin) to [Nle⁴, D-Phe⁷]alpha-MSH. These melanotropin conjugates might prove useful for melanotropin receptor studies and for the clinical localization of metastatic melanoma. We have demonstrated that [Nle⁴, D-Phe⁴]alpha-MSH can be topically applied and transdermally delivered across the skin of mice and humans in vitro, as determined by bioassay and RIA. Initial toxicologic studies indicate that the analogue is nontoxic to mice and is not mutagenic. Studies are underway to determine whether this analogue may prove useful as a “tanning hormone” for increasing the pigmentation of light-skinned individuals or possibly even for treating people with certain hypopigmentary disorders.     

The effect of N-terminal substitutions on the biological activity of MSH fragments

In order to study the role of N-terminal substitutions of peptide sequences related to the active site of α-melanotropin, [Glp⁵]α-MSH(5–10), [Glp⁵, -Phe⁷]α-MSH(5–10), [Sar⁵, -Phe⁷]α-MSH(5–10), [Nle⁴, -Phe⁷]α-MSH(4–10), [N-carbamoyl]α-MSH(5–10), and formyl and acetyl derivatives of α-MSH(5–10), [Gly⁵]α-MSH(5–10) and [Gly⁵, -Phe⁷]α-MSH(5–10), were synthesized in solution. The N-terminal acylations enhance by 2 to 10 times the melanin-dispersing activity of the unsubstituted sequences. Alkylation of the N-terminus does not change the biological activity of the parent peptide, suggesting the necessity of a carbonyl group for increasing the hormonal effect.     

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.     

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.     

Comparison of tyrosinase levels in amelanotic and melanotic melanoma cell cultures by a competitive enzyme-linked immunoadsorbent assay and by immunotitration analysis

Melanogenesis in mammalian pigment cells is regulated by changes in the activity of tyrosinase, the rate-limiting enzyme for melanin synthesis. Because recent evidence suggests that this enzyme may exist in pigment cells in both active and inactive stages, a competitive enzyme-linked immunoadsorbent assay (ELISA) was developed to compare tyrosinase levels in amelanotic and melanotic melanoma cell clones. The melanotic cell line used for this study, MEL-11A, had basal tyrosinase levels approximately 40 times that of the amelanotic cell line, AM-7. Both cell lines responded to melanocyte-stimulating hormone by demonstrating large increases in tyrosinase activity. For competitive ELISA analysis of tyrosinase levels in these two clones, microtiter plates were coated with purified tyrosinase, and trypsinized cell extracts were tested for their ability to compete with bound tyrosinase for antibody binding. Although tyrosinase activity in the amelanotic clone was 1/40 that of the melanotic clone, immunoreactive tyrosinase levels in AM-7 cells were found to be approximately one-half that present in the melanotic clone. Additional evidence for the presence of an inactive (or at least, catalytically less active) enzyme in AM-7 cells was obtained from immunotitration analysis of tyrosinase in cell extracts from both cell lines. These results suggest that at least some amelanotic melanoma cells may contain significant levels of catalytically inactive tyrosinase molecules and that the level of pigmentation in mammalian melanocytes may be regulated by a tyrosinase activation process.     

Melanotropic activity of gamma MSH peptides in melanoma cells.

We studied the pigmentary activity of the peptides gamma 1, gamma 2 and gamma 3 melanocyte stimulating hormone (MSH), which differ in the structure of their C-termini, using hamster and mouse melanoma cell lines responsive to beta-MSH by increasing tyrosinase activity. Gamma 1-MSH alone or in combination with beta-MSH had no effect on either cell line. Gamma 2-MSH alone was biologically inactive but potentiated beta-MSH stimulation of tyrosinase activity. Gamma 3-MSH at high concentration (10 microM) induced tyrosinase activity and dendrite formation in the hamster melanoma line. When added together with beta-MSH, gamma 3-MSH partially inhibited the tyrosinase activity response to beta-MSH. Thus, gamma-MSH peptides have low intrinsic melanotropic activity in mammalian melanoma cells; the specific pigmentary responses appear to be affected by the structure of the C-terminal portion.     

Relative stability of α-melanotropin and related analogues to rat brain homogenates

α-Melanotropin (α-MSH) retains less than 1% of its original activity after a 60 min incubation with 10% rat brain homogenate. [Nle⁴, D-Phe⁷]-α-MSH is nonbiodegradable in rat serum (240 min incubation) and still maintains 10% of its original activity in 10% rat brain homogenate (240 min incubation). The related fragment analogue, Ac-[Nle⁴, D-Phe⁷]-α-MSH_4–10-NH₂, retains 50% of its activity after a 240 min incubation in rat brain homogenate, whereas Ac-[Nle⁴, D-Phe⁷]-α-MSH_4–11-NH₂ is totally resistant to inactivation by rat brain homogenate. Both [Nle⁴, D-Phe⁷]-fragments are resistant to degradation by rat serum, but [Nle⁴]-α-MSH, Ac-[Nle⁴]-α-MSH_4–10-NH₂ and Ac-[Nle⁴]-α-MSH_4–11-NH₂ are rapidly inactivated under both conditions. The cyclic melanotropin, [ ]-α-MSH, is inactivated in rat brain homogenate as is the shorter Ac-[ ]-α-MSH_4–10-NH₂ analogue, but neither cyclic melanotropin is inactivated upon incubation in serum from rats. Ac-[ ]-α-MSH_4–10-NH₂ is resistant to inactivation by either rat serum or a brain homogenate. Some of these melanotropin analogues may provide useful probes for the localization and characterization of putative melanotropin receptors in both the central nervous system and peripheral tissues.     

Control of pigment production in mouse melanoma cells in vitro. Evocation and maintenance

A clonally derived amelanotic melanoma cell line repeatedly has been forced to produce pigment by the inhibitor of DNA synthesis, I-β-D-arabinofuranosylcytosine (ara-C) at sublethal levels. One ara-C-derived melanotic line has been cloned, and has continued to produce pigment for 2 years on normal medium. The inhibitor is most effective when administered to synchronized cells in four pulses on successive days at 1.8 x 10^-5 M during the S phase of the cell cycle. Colcemid at a sublethal concentration, and growth on medium solidified with agar also evoked pigment production in this line, but a large number of other inhibitors of biosynthetic processes did not, under the conditions tested. The melanotic lines are active producers of tyrosinase (DOPA oxidase), whereas the amelanotic line produces an inhibitor of tyrosinase activity. Both enzyme and inhibitor are labile at 4° C and -20° C, and decay of the inhibitor in homogenates of amelanotic cells reveals a low level of residual DOPA oxidase activity. The mean population doubling time of a cloned melanotic line is 23 hr, and that of a cloned amelanotic line 16.5 hr. A similar decrease in rate of growth is found in other melanotic lines and is believed to be a significant factor in maintaining this differentiated function. Rapid growth may be related to the production of an inhibitor by the amelanotic cells.     

Cultured Human Melanocytes Respond to MSH Peptides and ACTH

Although the administration of melanocyte-stimulating hormone (MSH) peptides results in skin darkening in man, cultured human melanocytes have been reported to be unresponsive to these peptides. This may be a consequence of the conditions under which the cells were maintained in vitro, particularly the use of phorbol esters and cholera toxin as melanocyte mitogens. By culturing the cells in the absence of these additives, we demonstrate that α-MSH and its synthetic analogue Nle⁴DPhe⁷α-MSH (NDP-MSH) induce dose-related increases in melanin content and tyrosinase activity and affect cell morphology in the majority of human melanocyte cultures. In addition, NDP-MSH induces increases in tyrosinase mRNA and tyrosinase-related protein-1 (TRP-1) mRNA. The dose-response curves for the MSH peptides are sigmoidal and the two peptides are equipotent in their effects on human melanocytes. Adrenocorticotropic hormone (ACTH) also affects morphology and stimulates melanogenesis and tyrosinase activity in human melanocytes. However, the dose-response curves for ACTH are biphasic, and the melanocytes respond to lower concentrations of ACTH than MSH peptides, similar to those normally present in human plasma. These findings may be important in understanding the role of these pro-opiomelanocortin peptides in human skin pigmentation.     

Quantitative determination of amino acid racemization in heat-alkali-treated melanotropins: Implications for peptide hormone structure-function studies

The treatment of frog skins (in vitro) and frogs (in vivo) with melanotropins that have been heated briefly in aqueous alkali resulted in prolonged skin darkening. It has been postulated that this increase in melanotropic activity is related to the partial racemization of amino acid residues of the melanotropins. Quantitative determination of the extent of racemization of eight amino acids (Val, Pro, Met, Phe, Glu, Asp, Nle, Ser) present in α-melanotropin (α-MSH), [4-norleucine]-α-MSH, β_porcine-melanotropin (β_p-MSH), and [7-norleucine]-β_p-MSH after brief heat-alkali treatment, was accomplished using a high-resolution gas chromatographic technique. Phenylalanine-7 in α-MSH and [4-norleucine]-α-MSH and phenylalanine-10 in β_p-MSH and [7-norleucine]-β_p-MSH were found to be partially racemized to a greater extent than expected. Other amino acid residues were also racemized to unexpected degrees. The subsequent synthesis of an α-MSH analog containing d-phenylalanine-7, [4-norleucine, 7-d-phenylalanine]-α-MSH, resulted in a highly potent melanotropin with ultralong biological activity, as determined by frog skin bioassay, stimulation of mouse melanoma cell tyrosinase activity, and activation of mouse melanoma adenylate cyclase.     

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.     

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     

Structural and conformational modifications of α-MSH/ACTH4–10 provide melanotropin analogues with highly potent behavioral activities

Previous studies have identified the (4–10) heptapeptide sequence as the central core of α-MSH/ACTH peptides required for mediation of important biological activities. In the present study, the structure-activity relationships of Nle⁴-substituted and -bridged cyclic α-MSH analogues, which were previously shown to exhibit a wide range of melanotropic potencies from weak agonism to super potency, were examined for grooming behavioral activity in the rat following intracerebroventricular injections. The results showed that stepwise C-terminal elongation of the linear Nle⁴-substituted Ac-α-MSH_4–10-NH₂ increased grooming potencies of the peptides in a manner similar to their actions on melanocytes. The most interesting finding was the observation that cyclization of the inactive linear “central (4–10) core” of α-MSH (Ac-α-MSH_4–10) to form Ac-[ ]-α-MSH_4–10-NH₂ resulted in a super potent agonist in the grooming assay. However, while cyclization of the (4–10) heptapeptide produced potent agonists on grooming behavior, the structure-activity relationships were different than the frog skin bioassay. These findings support the hypothesis that appropriate structural and confirmational modifications of α-MSH-related peptides can produce profound effects on the bioactivities of the peptides, and suggest that different structural-conformational requirements exist for α-MSH interactions with its various receptors.     

A Comparison Between Melanotic and Amelanotic Retinal Pigment Epithelial Cells In Vitro Concerning the Effects of L-Dopa and Oxygen on Cell Cycle

Melanin precursors and free radicals, cytotoxic substances, are produced during melanin synthesis by tyrosinase. We compared these cytotoxic effects of L-dopa and oxygen on the cell cycle of melanotic retinal pigment epithelial (RPE) cells with amelanotic RPE cells because of the differences of tyrosinase activities between melanotic and amelanotic RPE cells. Flow cytometric DNA analysis of RPE cells exposed to L-dopa (100 μM and 250 μM) were conducted at several oxygen concentrations (20%, 10%, and 5%). The dose-dependent effect of L-dopa to arrest the cell cycle (the S phase) was more pronounced in melanotic than in amelanotic RPE cells, and oxygen caused arrest in the G₁ phase.     

Membrane-active properties of α-MSH analogs: aggregation and fusion of liposomes triggered by surface-conjugated peptides

Reaction of the melanotropin hormone analogs [Nle⁴,D-Phe⁷]-α-MSH and [Nle⁴,D-Phe⁷]-α-MSH(4-10), which were extended at their N-terminus by a thiol-functionalized spacer arm, with preformed liposomes containing thiol-reactive (phospho)lipid derivatives resulted in the aggregation of the vesicles and in a partial leakage of their inner contents. This aggregation/leakage effect, which was only observed when the peptides were covalently conjugated to the surface of the liposomes, was correlated with the fusion of the vesicles as demonstrated by the observed decrease in resonance energy transfer between probes in a membrane lipid mixing assay. A limited fusion was confirmed by monitoring the mixing of the liposome inner contents (formation of 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylene bis(pyridinium bromide) complex). The membrane-active properties of the peptides could be correlated with changes in the fluorescence emission spectra of their tryptophan residue, which suggested that after their covalent binding to the outer surface of the liposomes they can partition within the core of the bilayers. A blue shift of 10 nm was observed for [Nle⁴,D-Phe⁷]-α-MSH which was correlated with an increase in fluorescence anisotropy and with changes in the accessibility of the coupled peptide as assessed by the quenching of fluorescence of its tryptophan residue by iodide (Stern-Volmer plots). These results should be related to the previously described capacity of α-MSH, and analogs, to interact with membranes and with the favored conformation of these peptides which, via a β-turn, segregate their central hydrophobic residues into a domain that could insert into membranes and, as shown here, trigger their destabilization.     

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