Antiinflammatory influences of alpha-MSH molecules: central neurogenic and peripheral actions.

alpha-Melanocyte-stimulating hormone (alpha-MSH1-13) and its COOH-terminal tripeptide alpha-MSH11-13 (Lys Pro Val) inhibit inflammation when administered systemically. Recent evidence indicates that alpha-MSH1-13 can likewise inhibit inflammation in the skin solely via an action within the brain. Because of the potential importance of this discovery to understanding the control of inflammation and because alpha-MSH molecules might be useful for treatment of inflammation, experiments were performed to learn more about the mechanisms of action of these peptides. In tests on inflammation induced in the mouse ear by intradermal injections of recombinant human interleukin-1 beta, alpha-MSH1-1-13 administered intracerebroventricularly effectively reduced inflammation. This effect of centrally administered alpha-MSH1-13 was inhibited by systemic injection of the nonspecific beta-adrenergic receptor blocker propranolol and by administration of a specific beta 2-adrenergic receptor antagonist; the effect was not altered by blockade of cholinergic, alpha-adrenergic, or beta 1-adrenergic receptors. In mice with inflammation induced in a hind paw and with the spinal cord transected, the antiinflammatory effect of centrally administered alpha-MSH1-13 was prevented, indicating that intact descending neuronal pathways are required for the antiinflammatory influence of the central peptide. Systemic injection of alpha-MSH1-13 in animals with spinal cord transection had a smaller and later antiinflammatory effect, which suggests that the molecule also has an action, albeit lesser, in the periphery. However, alpha-MSH11-13 injected intraperitoneally had marked antiinflammatory activity in animals with spinal cord transection.(ABSTRACT TRUNCATED AT 250 WORDS)

The dipeptide Lys-Pro attenuates interleukin-1 beta-induced anorexia.

The carboxyl-terminal tripeptide of alpha-MSH(1-13), Lys-Pro-Val, antagonizes anorexia induced by interleukin-1 beta (IL-1). The present studies were undertaken to determine if the Lys-Pro dipeptide portion of this tripeptide likewise antagonizes anorexia induced by ICV administration of 0.5 pmol IL-1 in rats previously deprived of food. This dose of Lys-Pro did significantly attenuate the IL-1-induced anorexia, but only for 1 h after infusion. Simultaneous administration of a larger dose (5.0 pmol) of Lys-Pro reversed the IL-1-induced anorexia during both the 0-1-h and 2-4-h periods. In addition, both 0.5 and 5.0 pmol of the D-substitute tripeptide, Lys-D-Pro-Thr (LDPT), similarly attenuated the IL-1-induced anorexia. The ICV administration of 5.0 pmol Lys-Pro also did not have any significant effects on food consumption. These results indicate that the dipeptide Lys-Pro may have a short-term antagonistic action against the anorexia induced by IL-1, and it is possible that this action may be partially mediated by the blockade of IL-1 on its own receptor.

Effects of a melanotropic peptide on melanoma cell growth, metastasis, and invasion.

Melanocyte stimulating hormone (alpha-MSH, alpha-melanotropin),Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Ly-Pro-Va l-NH2, regulates melanogenesis within epidermal melanocytes of many animals. An MSH analogue ([Nle4,D-Phe7]alpha-MSH) that exhibits superpotency and prolonged biological activity has been synthesized, biologically characterized, and is presently in clinical trials to determine its possible clinical use in tanning of the skin. It also has potential for the diagnosis, localization, and chemotherapy of melanoma. The effects of this analogue on the growth, metastatic behavior, and invasive potential of a melanotic variant of Cloudman S-91 murine melanoma are reported here. In an intracutaneous murine model of melanoma cell tumor growth, the analogue did not increase primary tumor growth (size) after the period of administration of the peptide hormone analogue and did not affect spontaneous lung metastases. Survival times for the control and melanotropin-treated groups were similar, suggesting that overall tumor burden was not affected by treatment with the hormone analogue. Last, melanoma cell invasion through a human amniotic basement membrane in vitro was not enhanced compared to untreated cells.

Melanocortins stimulate proliferation and induce morphological changes in cultured rat astrocytes by distinct transducing mechanisms.

Melanocyte stimulating hormone (MSH), adrenocorticotropic hormone (ACTH), and several peptides derived from pro-opiomelanocortin, are present in the dorsolateral hypothalamus and arcuate nucleus of several vertebrate species. These peptides affect central nervous system (CNS) functions including behavior, memory, and foetal brain development. In this study we investigated the effects of ACTH1-24, ACTH1-17, ACTH4-10, alpha-MSH, beta-MSH, and a potent analog (Nle4,D-Phe7)-alpha-MSH (melanocortins) on immunocytochemically defined astroglial cells prepared from primary cultures of 1-2-day-old rat brains. A cyclic adenosine 3',5'-monophosphate (cAMP) response to the melanocortins was only detected in astrocytes and not in other cell types in the culture. The extent of the cAMP response was greatest on day 21, the latest time tested. On the other hand, (methyl3H)-thymidine incorporation in astrocytes was significantly stimulated (1.5-2-fold) by melanocortins only in 7 and not in 14 and 21 day cultures. This mitogenic activity of melanocortins was not mimicked by other agents such as forskolin or isoproterenol which efficiently stimulate cAMP production in astrocytes. ACTH1-17 as a melanocortin representative induced significant morphological changes in 7 and 14 day cultures which included rounding of the cell body and process extension. This response, however, resembled that induced by forskolin and hence appears to be cAMP mediated. These findings suggest that astrocytes in the CNS may serve as a target for melanocortins. These peptides appear to affect differentiation and proliferation of these cells during certain developmental periods. While the morphological effects of melanocortins seem to be cAMP mediated, induction of proliferation of the astrocytes by melanocortins appears to involve an alternative signal transduction pathway.

Synthesis and biological activities of fatty acid conjugates of a cyclic lactam alpha-melanotropin.

Four fatty acid conjugates of a cyclic lactam-bridged alpha-MSH fragment analogue were synthesized and their potencies and biological activities compared in several melanotropin bioassays. Palmitoyl, myristoyl, decanoyl, and hexanoyl conjugates of H-Asp-His-D-Phe-Arg-Trp-Lys-NH2 were prepared. In the in vitro mouse melanoma cell assay, each of the conjugates was 10-100 times more potent than alpha-MSH or the substrate peptide in elevating tyrosinase activity. The shorter conjugates of hexanoic and decanoic acid were as potent as alpha-MSH in the lizard skin bioassay, whereas the longer myristoyl and palmitoyl analogues were about 100 times less potent. The potency of the myristoyl and palmitoyl conjugates increased with time in contact with the skins. These observations may be related to the more lipid-like nature of these peptide-fatty acid conjugates. Each of the conjugates exhibited prolonged melanotropic activity in the lizard skin bioassays and in the mouse S91 melanoma tyrosinase bioassay, since the biological response continued following removal of the conjugates from the incubation media. The prolonged residual melanotropic activity resulted from conjugation of the fatty acids to the MSH fragment analogue since the analogue itself did not exhibit prolonged 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 alpha-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 alpha-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 alpha-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-[Asp5, D-Phe7, Lys10]alpha-MSH5-10-NH2, exhibited prolonged activity, the conversion of reversible agonists to irreversible agonists was demonstrated.

Effects of an ACTH/MSH(4-9) analog (HOE 427) on the sleep EEG and nocturnal hormonal secretion in humans.

The synthetic ACTH/MSH(4-9) analog HOE 427 ("ebiratide"), which is behaviorally the most potent ACTH-derived peptide but which is devoid of endocrine activity, was administered intravenously in a pulsatile mode 4 times (120 micrograms each) at 2200, 2300, 2400 and 0100 to study its effect on the sleep EEG and on concomitant hormonal secretion of cortisol and growth hormone. In comparison to placebo, the peptide produced signs of general activation associated with specific deteriorating effects on the quality of sleep. Sleep onset latency and intermittent wakefulness were increased, slow wave sleep was reduced, but only during the first 3 hours of the sleep period. The nocturnal secretory patterns of cortisol and growth hormone were unaffected by HOE 427. These effects are different from those reported in similar studies in which corticotropin-releasing hormone (CRH) was applied in humans, and they suggest that peripherally administered neuropeptides have specific nonendocrine behavioral effects.

Anti-inflammatory activity of alpha-MSH(11-13) analogs: influences of alteration in stereochemistry.

D-Amino acid substitutions in the anti-inflammatory/antipyretic Ac-alpha-MSH(11-13)-NH2 tripeptide of Ac-alpha-MSH(1-13)-NH2 were made and the altered peptides were injected in mice treated with picryl chloride. Ear swelling, measured 3 and 6 h after application of the irritant, was reduced by IP injections of Ac-alpha-MSH(11-13)-NH2, in confirmation of previous observations. Ac-[D-Lys11]alpha-MSH(11-13)-NH2 effected similar anti-inflammatory activity but Ac-[D-Pro12]alpha-MSH(11-13)-NH2 was inactive. Ac-[D-Val13]alpha-MSH(11-13)-NH2 and Ac-[D-Lys11,D-Val13]alpha-MSH(11-13)-NH2 generally had greater anti-inflammatory activity than the parent tripeptide molecule; the dose-response relations exhibited the bell-shaped characteristics seen previously with MSH peptides. The results indicate that the L-Pro12 is essential for the anti-inflammatory activity of Ac-alpha-MSH(11-13)-NH2 whereas the L-Lys11 is not. D-Val13 substitution increased anti-inflammatory activity approximately four-fold over Ac-alpha-MSH(11-13)-NH2. These results provide new structure-activity relationships of the anti-inflammatory Ac-alpha-MSH(11-13)-NH2 molecule. The data support the developing idea that alpha-MSH and its COOH-terminal fragments modulate host responses, perhaps by antagonizing the actions of cytokines.

Putative neurotrophic factors and functional recovery from peripheral nerve damage in the rat.

1. In rats, recovery of sensory-motor function following a crush lesion of the sciatic or tibial nerve was monitored by measuring foot reflex withdrawal from a local noxious stimulation of the foot sole. 2. Putative neurotrophic compounds were tested on this functional recovery model: melanocortins (peptides derived from ACTH (corticotropin) and alpha-MSH (melanotropin], gangliosides and nimodipine were effective whereas isaxonine and TRH (thyrotropin releasing hormone) were not. 3. Structure-activity studies with melanocortins revealed a similar effectiveness of alpha-MSH, [N-Leu4, D-Phe7]-alpha-MSH, desacetyl-alpha-MSH and the ACTH analogue ORG 2766, questioning the validity of the previously suggested notion that the melanotrophic properties of these peptides are responsible for their neurotrophic effect. 4. As recovery of function after peripheral nerve damage follows a similar time course in hypophysectomized (five days post operation) and sham-operated rats, effective melanocortin therapy does not mimic an endogenous peptide signal in the repair process from pituitary origin. 5. Subcutaneous treatment with ORG 2766 (7.5 micrograms kg-1 48 h-1) facilitates recovery of function following peripheral nerve damage in young (6-7 weeks old), mature (5 month old) and old (20 month old) rats. 6. In view of the diversity in structure of the effective neurotrophic factors and the complexity of nerve repair, the present data support the notion that peripheral nerve repair may be facilitated by different humoral factors likely to be active on different aspects of the recovery process.

Alpha-melanocyte-stimulating hormone reduces interleukin-1 beta effects on rat stomach preparations possibly through interference with a type I receptor.

Contractions elicited by CaCl2 on isolated rat stomach strip preparations have been reported to be potentiated by interleukin-1 beta (IL-1 beta). We have investigated whether this effect can be reduced by the putative IL-1 beta antagonist, alpha-melanocyte-stimulating hormone (alpha MSH). Additionally, the effects of alpha MSH on the specific binding of IL-1 beta to B- and T-cells have been investigated to further clarify its inhibitory activities. Both alpha MSH and its carboxyl terminal tripeptide concentration dependently reduced the potentiation of CaCl2-induced contractions caused by IL-1 beta but not those caused by leukotriene D4, the parent molecule being approximately 250 times more active. Additionally, both peptides potently and selectively reduced 125I-IL-1 beta binding to the T-cell sub-clone EL4-6.1 but not to the B-cell sub-clone 1H7. The results indicate that IL-1 beta effects on rat stomach may be mediated through a type-I (80 kDa) IL-1 beta receptor.

A chelating derivative of alpha-melanocyte stimulating hormone as a potential imaging agent for malignant melanoma.

A chelating derivative of alpha-melanocyte stimulating hormone (MSH) has been synthesised, in which two molecules of the hormone are cross-linked by diethylenetriamine pentaacetic acid (DTPA). This compound, bisMSH-DTPA, was equipotent with MSH in an in vitro tyrosinase assay with Cloudman S91 melanoma cells. When DBA/2 mice bearing the same tumour were injected with bisMSH-DTPA labelled with the gamma-emitting isotope indium-111 (111In), the radioactivity became rapidly associated with the melanoma tissue. By 24 h post-injection, radioactivity in tumour tissue was significantly higher (P less than 0.001) than in spleen, lung, brain, eye and skin. Uptake of radioactivity by the tumours was inhibited by a 200-fold molar excess of MSH, whereas uptake by liver, kidney, spleen, lung, brain, eye and skin was unaffected. We conclude that bisMSH-DTPA may offer an alternative to antibody targeting in the imaging of malignant melanoma.

Alpha-MSH peptides inhibit acute inflammation and contact sensitivity.

Alpha-melanocyte stimulating hormone [alpha-MSH(1-13)] occurs within the CNS, skin, circulation and in other body sites. This tridecapeptide and its COOH-terminal tripeptide, alpha-MSH (11-13), have antipyretic and anti-inflammatory actions. Studies of the anti-inflammatory effects of these molecules have been confined mainly to tests of inhibition of histamine and endogenous pyrogen-induced increases in capillary permeability in rabbits and acute inflammation of ear tissue in mice. The aim in the present experiments was to learn if alpha-MSH peptides also antagonize inflammation in two additional models: acute edema induced in the mouse paw and contact sensitivity. Significant anti-inflammatory effects were observed with MSH peptides in both models. These findings converge with previous results to indicate that alpha-MSH peptides modulate inflammation. Because circulating alpha-MSH increases after treatment of animals with endogenous pyrogen or endotoxin, administration of the peptides may simply mimic a naturally occurring modulation of host defense reactions.

Melanotropin structure-activity studies on melanocytes of the teleost fish, Synbranchus marmoratus.

The minimal sequence of alpha-MSH required for full agonism on fish (Synbranchus marmoratus) melanocytes was determined to be Ac-alpha-MSH5-10-NH2 since Ac-alpha-MSH6-10-NH2 and Ac-alpha-MSH6-9-NH2 were inactive. The N-terminal tripeptide sequence, Ser-Tyr-Ser, lacked any contribution to potency since the 4-13 (Ac-[Nle4]-alpha-MSH4-13-NH2) sequence was equipotent to alpha-MSH. The important potentiating amino acids were found to be Met at position 4 of the amino terminus and Val at position 13 of the carboxy terminus of the hormone, since Ac-alpha-MSH4-10-NH2 was about 100 times more potent than the Ac-alpha-MSH5-10-NH2 sequence, and Ac-[Nle4]-alpha-MSH4-13-NH2 was about 10 times more active than Ac-[Nle4]-alpha-MSH4-12-NH2. The minimal sequence for equipotency to alpha-MSH was demonstrated to be Ac-[Nle4]-alpha-MSH4-13-NH2. [Nle4, D-Phe7]-alpha-MSH was about 10 times more active than alpha-MSH. Unexpectingly, several conformationally restricted cyclic melanotropins were either partial agonists ([Cys4, Cys10]-alpha-MSH) or totally inactive (Ac[Cys4, Cys10]-alpha-MSH4-10-NH2) on fish melanocytes. These results point out some rather remarkable differences between S. marmoratus and tetrapod melanophores relative to structural requirements for MSH receptor recognition and signal transduction.

Phorbol ester impairs melanotropin receptor function and stimulates growth of cultured M2R melanoma cells.

We have examined the effects of a biologically active tumor promoting phorbol ester (phorbol 12-myristate, 13-acetate (PMA] which activates protein kinase C (PKC) on melanotropin receptor function and cell growth in the M2R mouse melanoma cell clone. Treatment of M2R cells with PMA resulted in a significant loss of beta-MSH binding. The effect was both time- and concentration-dependent. The inhibition of beta-MSH binding resulted from a decrease (greater than 85%) in active membranal receptors available on the external cell surface and not from either enhanced internalization or change in the binding affinity. Agonist-stimulated cyclic AMP accumulation was profoundly increased in a non-selective manner following short-term incubation (3 h) with PMA. This effect was completely reversed during long-term (72-96 h) incubation with the tumor promoting agent. Long-term culturing of M2R cells with PMA resulted in enhanced (+50%) proliferation of the melanoma cells. This enhancement was blocked by the addition of agents which stimulate the production of cAMP. Hence, phorbol esters are powerful growth promoters in transformed melanocytes and our findings indicate that the effects of melanotropins are selectively impaired during the process of growth promotion.