The effect of alpha-melanocyte stimulating hormone on colonic inflammation in the rat

The effect of alpha-melanocyte stimulating hormone (alpha-MSH) on colonic inflammation in the rat. In this study, we investigated the effects of alpha-MSH administration on trinitrobenzene sulfonic acid-induced colitis and the role of nitric oxide and prostaglandins in this response. alpha-MSH treatment (25 microg/rat, intraperitoneally; twice daily for 3 days) reduced the colonic macroscopic lesions compared to untreated ones in both acute and chronic colitis groups. This effect was reversed by pretreatment with the nitric oxide donor, sodium NP (4 mg/kg, intravenously) or cyclooxygenase-1 selective antagonist indomethacin (5 mg/kg, subcutaneously) in the acute group and with the cyclooxygenase-2 selective antagonist nimesulide (3 mg/kg, subcutaneously) in the chronic group. alpha-MSH had no effect on colonic wet weight and myeloperoxidase activity compared to the untreated colitis group. However, protein oxidation was markedly elevated in the alpha-MSH-treated group compared to untreated ones. Nitroprusside and indomethacin reversed the effect of alpha-MSH on macroscopic lesions in the acute groups, whereas nimesulide showed a similar effect in the chronic group. In conclusion, the results of our study show a protective role of alpha-MSH on colonic lesions which partially involves nitric oxide and prostaglandins.     

Betulin binds to melanocortin receptors and antagonizes alpha-melanocyte stimulating hormone induced cAMP generation in mouse melanoma cells

Betulin is a principal component of birch bark and is known to possess a broad range of biological activities, including antiinflammatory, antiviral and anticancer actions. The present study was carried out in vitro to clarify the influence of betulin on melanocortin (MC) receptor-ergic signalling by using COS-7 cells transfected with corresponding human MC receptor DNA. The results showed that betulin binds to the human melanocortin MC1, three to five receptors with selectivity to the MC1 subtype (K(i) value 1.022 +/- 0.115 microM). Betulin binds to the MC receptors with the following potency order-MC > MC3 > MC5 > MC4. Betulin itself does not stimulate cAMP generation, however, it slightly antagonizes alpha-melanocyte-stimulating hormone (alpha-MSH)-induced cAMP accumulation in the mouse melanoma cell line B16-F1. As a water-insoluble substance, betulin was dissolved in DMSO therefore DMSO competition with the labelled ligand NDP-MSH for the binding to the MC receptors was tested in the identical experimental set-up. We found that DMSO competes for binding to all the MC receptor subtypes, at 20% concentration and above. Selectivity for one or another receptor subtype was not observed. We have demonstrated for the first time, the ability of the plant compound betulin to bind to the MC receptors. One may suggest MC receptor MC1 subtype as the essential target for the antimelanoma action of betulin and its structurally close molecules such as betulinic acid. Moreover, we have found a new non-peptide small molecule MC mimetic, that is betulin. Thus, we report a new chemical motif for the binding to the MC receptors that could be used as a template for the search of more selective MC mimetics.     

Analogs of alpha-melanocyte stimulating hormone with high agonist potency and selectivity at human melanocortin receptor 1b: the role of Trp(9) in molecular recognition

alpha-Melanocyte stimulating hormone (alphaMSH), Ac-Ser(1)-Tyr(2)-Ser(3)-Met(4)-Glu(5)-His(6)-Phe(7)-Arg(8)-Trp(9)-Gly(10)-Lys(11)-Pro(12)-Val(13)-NH(2), is an endogenous agonist for the melanocortin receptor 1 (MC1R), the receptor found in the skin, several types of immune cells, and other peripheral sites. Three-dimensional models of complexes of this receptor with alphaMSH and its synthetic analog NDP-alphaMSH, Ac-Ser(1)-Tyr(2)-Ser(3)-Nle(4)-Glu(5)-His(6)-D-Phe(7)-Arg(8)-Trp(9)-Gly(10)-Lys(11)-Pro(12)-Val(13)-NH(2), have been previously proposed. In those models, the 6-9 segment of the ligand was considered essential for the ligand-receptor interactions. In this study, we probed the role of Trp(9) of NDP-alphaMSH in interactions with hMC1bR. Analogs of NDP-alphaMSH with various amino acids in place of Trp(9) were synthesized and tested in vitro in receptor affinity binding and cAMP functional assays at human melanocortin receptors 1b, 3, 4, and 5 (hMC1b,3-5R). Several new compounds displayed high agonist potency at hMC1bR (EC(50) = 0.5-5 nM) and receptor subtype selectivity greater than 2000-fold versus hMC3-5R. The Trp(9) residue of NDP-alphaMSH was determined to be not essential for molecular recognition at hMC1bR.     

Neurons containing alpha-melanocyte stimulating hormone and beta-endorphin immunoreactivity in the cat hypothalamus

Immunohistochemical studies were conducted on sections of cat hypothalamus in order to determine the distribution of neurons containing alpha-melanocyte stimulating hormone and beta-endorphin immunoreactivity. A large number of neurons in the arcuate nucleus were stained after incubation of sections with antisera to either substance. Analysis of serial sections suggested that each neuron revealed with one antiserum was also revealed with the other antiserum, indicating the co-existence of alpha-melanocyte stimulating hormone and beta-endorphin immunoreactivity within these arcuate neurons. In contrast, a more diffuse group of lateral hypothalamic neurons which extended from the retrochiasmatic level to the posterior hypothalamus were stained only with the antiserum directed against alpha-melanocyte stimulating hormone. The present results largely confirm findings in the rat hypothalamus, although the lateral hypothalamic group of alpha-melanocyte stimulating hormone immunoreactive neurons appears to be more extensive in the cat.     

Prostaglandin production by melanocytic cells and the effect of alpha-melanocyte stimulating hormone

Prostaglandins are potent mediators of the inflammatory response and are also involved in cancer development. In this study, we show that human melanocytes and FM55 melanoma cells express cyclooxygenase-1 and -2 (COX-1 and -2) and thus have the capability to produce prostaglandins. The FM55 cells produced predominantly PGE2 and PGF2alpha, whereas the HaCaT keratinocyte cell line produced mainly PGE2. The anti-inflammatory peptide, alpha-melanocyte stimulating hormone (alpha-MSH), reduced prostaglandin production in FM55 and HaCaT cells and reversed the effect of the pro-inflammatory cytokine TNF-alpha in the former. These results indicate that melanocytes produce prostaglandins and that alpha-MSH, by inhibiting this response, may play an important role in regulating inflammatory responses in the skin.     

The effect of alpha-melanocyte stimulating hormone on endotoxin-induced intestinal injury.

We investigated the effect of alpha-melanocyte stimulating hormone (alpha-MSH) on endotoxin-induced intestinal inflammation and the role of nitric oxide and prostaglandins in this response. alpha-MSH treatment (25 microg/rat, intraperitoneally (i.p.); twice daily) reduced the severity of the lesions macroscopically and microscopically. This protective effect was found to be confined mainly to the distal ileum. These lesions were reversed by pretreatment with the non-selective COX inhibitor indomethacin (10 mg/kg, subcutaneously (s.c.)) but not by the selective COX-2 inhibitor nimesulide (3 mg/kg, s.c.), the NO donor sodium nitroprusside (4 mg/kg, i.v.) or the iNOS inhibitor dexamethasone (3 mg./kg, i.p.) at macroscopic level and reversed by Indo or Dex at microscopic level. Increased peroxidase activity -index of tissue neutrophil infiltration- in the distal ileum of LPS-treated rats was decreased by alpha-MSH and this effect was reversed by pretreatment with Indo. In conclusion, the neuropeptide alpha-MSH has a beneficial effect on endotoxin-induced distal intestinal lesions by a mechanism which probably involves nitric oxide and COX-1 derived prostaglandins.     

The alpha-amidation of alpha-melanocyte stimulating hormone in intermediate pituitary requires ascorbic acid

Rat intermediate pituitary cells in primary culture display a time-dependent loss of the ability to produce COOH-terminally alpha-amidated alpha MSH (Glembotski, C.C., Eipper, B.A., and Mains, R.E. (1983) J. Biol. Chem. 258, 7299-7304). Instead of des-, mono-, and diacetyl-adrenocorticotropic hormone(ACTH) (1-13)NH2, the cells produce des-, mono-, and diacetyl-ACTH(1-14)OH. Since the pituitary secretory granule-associated alpha-amidation enzyme requires copper and ascorbic acid for optimal activity (Eipper, B.A., Mains, R.E., and Glembotski, C. C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5144-5148), these cofactors were added to cultures of intermediate pituitary cells in an attempt to reverse the loss of peptide alpha-amidation ability. When the cultures were supplemented with up to 100 microM cooper (II) there was very little change in the ability to alpha-amidate alpha-melanotropin. Ascorbic acid at concentrations of up to 500 microM resulted in a dramatic increase in the ability of the cells to form the alpha-amidated peptide. Various combinations of ascorbic acid and copper additions indicated that a relatively short exposure (hours) to ascorbic acid produced the maximal response. Ascorbic acid displayed a dose-dependent effect on the alpha-amidation ability with a half-optimal concentration of about 25 microM. Pulse-chase labeling experiments demonstrated the ascorbic acid-dependent conversion of labeled ACTH(1-14)OH-related peptides to ACTH(1-13)NH2-related peptides. These results correlate with the ascorbic acid requirement of the pituitary alpha-amidation enzyme and demonstrate that the direct precursors to ACTH(1-13)NH2-related peptides are ACTH(1-14)OH-related peptides. Combined with our previous data, the present studies support the notion that a wide range of neuro- and endocrine peptides become alpha-amidated in a similar ascorbic acid-dependent manner.     

Injections of alpha-melanocyte stimulating hormone affect pineal serotonin, melatonin and N-acetyltransferase activity

To determine if exogenously administered alpha-melanocyte stimulating hormone (alpha-MSH) affects nighttime pineal N-acetyltransferase activity, pineal levels of 5-hydroxytryptophan, serotonin and melatonin, and plasma prolactin levels, adult male hamsters were injected at 1900 hr (lights out 2000-0600 hr) with two doses of the peptide and killed at 0300 hr. The low dose of alpha-MSH (200 ng) produced a significant fall in pineal serotonin, pineal NAT activity and plasma prolactin values. The high dose of the peptide (20 micrograms) increased circulating prolactin titers and pineal serotonin levels and caused a concomitant decrease in pineal melatonin levels.     

Receptor-mediated modulation of murine mast cell function by alpha-melanocyte stimulating hormone.

The proopiomelanocortin (POMC)-derived neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) is known to modulate some aspects of inflammation through direct effects on T cells, B cells, and monocytes. To determine whether alpha-MSH might similarly influence mast cell responsiveness, mast cells were examined to see if they expressed the receptor for alpha-MSH, melanocortin-1 (MC-1), and whether alpha-MSH altered mast cell function. We thus first identified MC-1 on bone marrow cultured murine mast cells (BMCMC) and a murine mast cell line (MCP-5) employing flow cytometry and through detection of specific binding. Subsequent treatment of mast cells with alpha-MSH increased the cAMP concentration in a characteristic biphasic pattern, demonstrating that alpha-MSH could affect intracellular processes. We next examined the effect of alpha-MSH on mediator release and cytokine expression. IgE/DNP-human serum albumin-stimulated histamine release from mast cells was inhibited by approximately 60% in the presence of alpha-MSH. Although activation of BMCMC induced the expression of mRNAs for the inflammatory cytokines IL-1beta, IL-4, IL-6, TNF-alpha, and the chemokine lymphotactin, mRNAs for IL-1beta, TNF-alpha, and lymphotactin were down-modulated in the presence of alpha-MSH. Finally, IL-3-dependent proliferative activity of BMCMC was slightly but significantly augmented by alpha-MSH. Taken together, these observations suggest that alpha-MSH may exert an inhibitory effect on the mast cell-dependent component of a specific inflammatory response.     

Short and long term inhibitory actions of alpha-melanocyte stimulating hormone on lordosis in rats

The effects of 200 ng of intracerebroventricularly (ICV) and 20 micrograms of subcutaneously (SC) administered alpha-melanocyte stimulating hormone (MSH) on lordosis in rats were examined. Previous research, employing crossover designs, has revealed significant effects of MSH on lordosis. The results of Experiments 1a and 1b suggest that similar designs produce significant effects even in the absence of MSH. Thus, it is not clear that previous results were due exclusively to an action of MSH. Experiment 2 employed a modification of previous procedures and indicated that MSH administered either SC or ICV inhibited receptivity in subjects displaying high levels of responding. Moreover, MSH administered SC was also found to facilitate receptivity in subjects displaying low levels of responding. However, a possible long term inhibitory action of MSH on receptivity was also revealed. Because animals were tested repeatedly, this brought into question the results of Experiment 2. Procedures were revised accordingly and the effects of MSH re-examined. The results of Experiment 3 indicated that MSH administered SC facilitated receptivity while MSH administered ICV inhibited receptivity. In addition, MSH administered ICV exerted an inhibitory effect one week after administration. Therefore, MSH appears to exert both short and long-acting effects on sexual receptivity.     

Attenuation of morphine tolerance and dependence by alpha-melanocyte stimulating hormone(alpha-MSH).

Repeated administration of morphine resulted in significant reduction of its analgesic potency. If 0.1 mg/kg α-MSH was coadministered, the tolerance development was attenuated, 1 mg/kg MIF (MSH release inhibiting factor), given simultaneously with morphine, did not affect tolerance. Injecting, however, MIF 1 hour prior to the daily opiate treatment resulted in accelerated development of tolerance supposedly by lowering the plasma α-MSH level at the time of morphine administration. Of the morphine abstinence symptoms the naloxone-induced jumping in morphine pretreated mice could not be modified either by α-MSH coadministration or by MIF pretreament, but the withdrawal body weight loss was found to be diminished by the former and increased by the latter peptide. The possible role of α-MSH in preventing the development of tolerance to the analgesic effect of endogenous opioid peptides is discussed.     

Anti-inflammatory effect of the alpha-melanocyte stimulating hormone in animal eyes undergoing extracapsular lens extraction

In this study anti-inflammatory effects of the alpha-melanocyte stimulating hormone (alpha-MSH) on ocular inflammation caused by extracapsular lens extraction (ECLE) have been investigated and the potential mechanism of an anti-inflammatory effect is discussed. Pigmented rabbit eyes after ECLE were treated locally with alpha-MSH, dexamethasone, diclofenac, or saline 4 times a day (q.i.d.) for 4 weeks. The inhibitory effect of alpha-MSH on infiltrating cells in the aqueous humor (AqH) was almost twice as good as that of dexamethasone or diclofenac for 3 days, 1 week, and 2 weeks after the operation. The eyes of Sprague-Dawley rats were treated with an intravenous injection of alpha-MSH or saline immediately after ECLE. Six hours postoperatively, the iris/ciliary body exhibited increased expression of TNF-alpha and IL-6 mRNAs, which were significantly decreased after alpha-MSH treatment. The number of activated NF-kappa B (NFkappaB)-positive cells in the iris/ciliary body was also significantly reduced by the alpha-MSH treatment. These results suggested that alpha-MSH could effectively reduce ocular inflammation after ECLE, and the potential mechanism for this is by down-regulating the expression of proinflammatory cytokines and inhibiting the NFkappaB-dependent signaling pathway.     

Modulation of regulatory T cell immunity by the neuropeptide alpha-melanocyte stimulating hormone.

Although many immunosuppressive factors have been identified in the eye, one of these factors, alpha-melanocyte stimulating hormone (alpha-MSH), both suppresses the activation of inflammatory activity by primed T cells and induces the activation of regulatory T cells (Treg cells). This neuropeptide alone at its ocular physiological concentration can account for most of the immunosuppressive activity of aqueous humor (the fluid filing the anterior chamber of the eye). Aqueous humor made devoid of alpha-MSH no longer suppresses IFN-gamma production by Th1 cells. It is alpha-MSH that mediates aqueous humor induction of regulatory T cells. What we have found is that alpha-MSH mediates the induction of C4+ CD25+ Treg cells, and that if the alpha-MSH Treg cells are specific to an autoantigen they can be used to suppress autoimmune disease. It is the objective of this review to demonstrate how we came to discover that alpha-MSH could have such an important role in the extreme regional immunity of the immune privileged eye and how this discovery could be applied to create or reestablish tolerance to prevent autoimmune disease.     

111In-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone peptide analogues for melanoma imaging

The purpose of this study was to examine the influence of the lactam bridge cyclization on melanoma targeting and biodistribution properties of the radiolabeled conjugates. Two novel lactam bridge-cyclized alpha-MSH peptide analogues, DOTA-CycMSH (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]) and DOTA-GlyGlu-CycMSH (DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]), were synthesized and radiolabeled with (111)In. The internalization and efflux of (111)In-labeled CycMSH peptides were examined in B16/F1 melanoma cells. The melanoma targeting properties, pharmacokinetics, and SPECT/CT imaging of (111)In-labeled CycMSH peptides were determined in B16/F1 melanoma-bearing C57 mice. Both (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH exhibited fast internalization and extended retention in B16/F1 cells. The tumor uptake values of (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH were 9.53+/-1.41% injected dose/gram (% ID/g) and 10.40+/-1.40% ID/g at 2 h postinjection, respectively. Flank melanoma tumors were clearly visualized with (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH by SPECT/CT images at 2 h postinjection. Whole-body clearance of the peptides was fast, with greater than 90% of the radioactivities cleared through urinary system by 2 h postinjection. There was low radioactivity (<0.8% ID/g) accumulated in blood and normal organs except kidneys at all time points investigated. Introduction of a negatively charged linker (-Gly-Glu-) into the peptide sequence decreased the renal uptake by 44% without affecting the tumor uptake at 4 h postinjection. High receptor-mediated melanoma uptakes coupled with fast whole-body clearance in B16/F1 melanoma-bearing C57 mice demonstrated the feasibility of using (111)In-labeled lactam bridge-cyclized alpha-MSH peptide analogues as a novel class of imaging probes for receptor-targeting melanoma imaging.     

Tc-99m-labeled RGD-conjugated alpha-melanocyte stimulating hormone hybrid peptides with reduced renal uptake

The purpose of this study was to examine whether the replacement of the positively-charged Lys or Arg linker with a neutral linker could reduce the renal uptake of Arg-Gly-Asp (RGD)-conjugated alpha-melanocyte stimulating hormone (α-MSH) hybrid peptide. The RGD motif {cyclic(Arg-Gly-Asp-DTyr-Asp)} was coupled to [Cys^3,4,10, D-Phe⁷, Arg¹¹]α-MSH_3-13 {(Arg¹¹)CCMSH} through the neutral βAla or Ahx {aminohexanoic acid} linker (replacing the Lys or Arg linker) to generate novel RGD-βAla-(Arg¹¹)CCMSH and RGD-Ahx-(Arg¹¹)CCMSH hybrid peptides. The receptor-binding affinity and cytotoxicity of RGD-βAla-(Arg¹¹)CCMSH and RGD-Ahx-(Arg¹¹)CCMSH were determined in B16/F1 melanoma cells. The melanoma targeting and imaging properties of ^99mTc-RGD-βAla-(Arg¹¹)CCMSH and ^99mTc-RGD-Ahx-(Arg¹¹)CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The replacement of the Lys or Arg linker with the βAla or Ahx linker retained nanomolar receptor-binding affinities and remarkable cytotoxicity of RGD-βAla-(Arg¹¹)CCMSH and RGD-Ahx-(Arg¹¹)CCMSH. The receptor-binding affinities of RGD-βAla-(Arg¹¹)CCMSH and RGD-Ahx-(Arg¹¹)CCMSH were 0.8?±?0.05 and 1.3?±?0.1 nM. Three-hour incubation with 0.1 µM of RGD-βAla-(Arg¹¹)CCMSH and RGD-Ahx-(Arg¹¹)CCMSH decreased the survival percentages of B16/F1 cells by 71 and 67 % as compared to the untreated control cells 5 days post the treatment. The replacement of the Arg linker with the βAla or Ahx linker reduced the non-specific renal uptake of ^99mTc-RGD-βAla-(Arg¹¹)CCMSH and ^99mTc-RGD-Ahx-(Arg¹¹)CCMSH by 62 and 61 % at 2 h post-injection. ^99mTc-RGD-βAla-(Arg¹¹)CCMSH displayed higher melanoma uptake than ^99mTc-RGD-Ahx-(Arg¹¹)CCMSH at 0.5, 2, 4, and 24 h post-injection. Enhanced tumor to kidney uptake ratio of ^99mTc-RGD-βAla-(Arg¹¹)CCMSH warranted the further evaluation of ¹⁸⁸Re-labeled RGD-βAla-(Arg¹¹)CCMSH as a novel MC1 receptor-targeting therapeutic peptide for melanoma treatment in the future.     

Evaluation of the human melanoma targeting properties of radiolabeled alpha-melanocyte stimulating hormone peptide analogues

The purpose of this study was to evaluate the human MC1 receptor-mediated melanoma targeting properties of two metal cyclized alpha-MSH peptide analogues, (188)Re-(Arg(11))CCMSH and (188)Re-CCMSH. Initially, the presence and density of the MC1 receptor were determined on a bank of human melanoma cell lines. All eight human melanoma cell lines tested in this study displayed the MC1 receptor at a density of 900 to 5700 receptors per cell. Receptor affinity and biodistribution properties of (188)Re-(Arg(11))CCMSH and (188)Re-CCMSH were evaluated in a cultured TXM13 human melanoma-xenografted Scid mouse model. Biodistribution results demonstrated that 3.06 +/- 0.68 %ID/g of (188)Re-(Arg(11))CCMSH accumulated in the tumors 1 h postinjection and greater than 65% of the activity at 1 h postinjection remained in the tumors at 4 h after dose administration. Whole body clearance of (188)Re-(Arg(11))CCMSH was very rapid, with approximately 82% of injected dose cleared through urinary system at 4 h postinjection. There was very little activity in blood and major organs such as liver, lung, and muscle except for the kidney. (188)Re-CCMSH exhibited similar tumor uptake and retention in TXM13 human melanoma-xenografted Scid mice as (188)Re-(Arg(11))CCMSH. However, the kidney uptake value of (188)Re-CCMSH was two times higher than that of (188)Re-(Arg(11))CCMSH. The results of this study indicate that the MC1 receptor is present on the surface of a large number of human melanoma cells, which makes the MC1 receptor a good imaging or therapeutic target. Moreover, the biodistribution properties of (188)Re-(Arg(11))CCMSH and (188)Re-CCMSH highlight their potential as therapeutic agents for human melanoma.     

Effects of alterations in the activity of tuberohypophysial dopaminergic neurons on the secretion of alpha-melanocyte stimulating hormone

Administration of gamma-butyrolactone (GBL), an anesthetic which reduces dopaminergic neuronal activity, decreased the concentration of the dopamine (DA) metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the intermediate lobe of the pituitary gland, and increased alpha-melanocyte stimulating hormone (alpha MSH) concentrations in the serum of male rats. Bilateral electrical stimulation of the rostral arcuate nucleus, which contains perikarya of tuberohypophysial DA neurons, increased DOPAC concentrations in the intermediate lobe and decreased alpha MSH concentrations in the serum of GBL-anesthetized rats. Administration of the DA antagonist haloperidol prevented the decline in serum alpha MSH levels following arcuate nucleus stimulation, but had no effect on serum alpha MSH concentrations in sham-stimulated GBL-treated rats. These results indicate that GBL-induced decreases or stimulation-induced increases in the activity of tuberohypophysial DA neurons are accompanied by corresponding changes in the metabolism of DA in the intermediate lobe of the rat pituitary gland, and by reciprocal changes in the secretion of alpha MSH.