Action Of Melanocyte Stimulating Hormone Research Articles

Experimental metastasis and differentiation of murine melanoma cells: actions and interactions of factors affecting different intracellular signalling pathways.

Various factors that modulate the differentiation of malignant cells are known to affect their experimental metastatic potential (EMP), or lung colonization after intravenous injection into syngeneic animals. However, some results and conclusions on the relation between cell differentiation and metastasis have appeared to conflict. We have reanalysed this by measurement of EMP of B16 melanoma sublines after culture with agents or conditions that acted on differentiation through various intracellular pathways. All tested agents did affect the EMP. EMP was usually positively correlated with differentiation under diverse conditions, but exceptions showed that there is no direct causal connection. Nor could all findings be explained in terms of cell proliferation or expression of major histocompatibility antigens. Some data helped to explain disparities between previous reports. Specific novel findings included the following. The stimulation of EMP by melanocyte-stimulating hormone (MSH) as well as all other tested effects of MSH were prevented by extended exposure to 12-O-tetradecanoyl phorbol acetate (TPA), suggesting a requirement for protein kinase C activity as well as G-protein coupling in MSH action. Cells grown with cholera toxin were always more differentiated than untreated cells, but the EMP could be either markedly increased or markedly decreased by cholera toxin under different conditions. The basic culture medium apparently determined this striking reversal. The EMP was also significantly affected by the extracellular pH.

The melanocortin receptor in the rat lacrimal gland: a model system for the study of MSH (melanocyte stimulating hormone) as a potential neurotransmitter

The melanocortin receptors in intraorbital and extraorbital rat lacrimal glands were studied with [ 125I][Nle 4,D-Phe 7]αMSH as radioligand and with several unlabeled melanocortin peptides. The pharmacological properties of the melanocortin receptor in both tissues appeared to be essentially identical. Receptor binding was studied in a membrane fraction sedimented at 12 000–100 000 × g, establishing for [ 125I][Nle 4,D-Phe 7]αMSH a K D of 0.76 and 2.2 nM for the intra- and extraorbital glands, respectively. Binding of the radioligand was competitively inhibited by αMSH (α-melanocyte stimulating hormone) and ACTH-(1–24) with IC 50 values in the submicromolar range. MSH binding in both tissues was abolished by EGTA and was increased dose dependently with elevation of free Ca 2+ ion concentration. The half-maximal effect on MSH binding was obtained around 200 μM Ca 2+ and maximal binding was reached at nearly 2 mM free Ca 2+ in membrane preparations from both tissues. The calmodulin-binding peptides, melittin, mastoparan and M5, the latter being the 18-amino acid synthetic analogue of the C-terminal calmodulin-binding domain of myosin light chain kinase, inhibited MSH binding in the concentration range of 1–20 μM. Macroscopic autoradiographic analysis of cryosections prepared from either lacrimal gland to which [ 125I][Nle 4,D-Phe 7]αMSH was subsequently bound, showed the melanocortin receptor to be uniformly distributed within the acinar lobes. At the microscopic level, MSH was found to be associated with the acinar cells, primarily at the basal perinuclear region. Peroxidase secretion from extraorbital lacrimal slices was stimulated by MSH, ephinephrine and carbamylcholine to a similar extent. The response of the tissue to stimulation by MSH was however not blocked by α/β-adrenoceptor blockers or by atropine, suggesting that MSH acts as a primary secretagogue in this tissue. Thus, this system seems to be uniquely suited to serve as a model for the study of both the molecular and pharmalogical details of the action of MSH and other melanocortins in a non-melanogenic tissue.

Application of percent labeled mitoses (PLM) analysis to the investigation of melanoma cell responsiveness to MSH stimulation throughout the cell cycle

Using a modification of the percent labeled mitoses (PLM) method of cell cycle analysis, the approximate lengths of the cell cycle phases of Cloudman S91 mouse melanoma cells were determined to be: G1, 10.0 h; S, 8.0 h; G2, 2.0 h; and M, 4.0 h. Studies were then carried out to investigate melanoma cell responsiveness to MSH (melanocyte stimulating hormone) throughout the cell cycle. In contrast to previous reports of a G2-specific cell response to MSH, melanoma cells demonstrated increased tyrosinase activity when exposed to MSH for the time periods: 0–8 h, 8–16 h, or 16–24 h. These times correspond to late M-G1; G1-S; and late S-G2-M, respectively. Cells exposed to cycloheximide (10 −6 M) during any of these time periods were inhibited from responding to MSH, suggesting that continued protein synthesis is required throughout the cell cycle for MSH action. These results demonstrate that MSH responsiveness is not necessarily expressed exclusively during the G2 phase of the cell cycle in Cloudman S91 mouse melanoma cells in culture, as reported by others.

The role of RNA and protein synthesis in mediating the action of MSH on mouse melanoma cells

Exposure of mouse melanoma cells in culture to MSH (melanocyte stimulating hormone) results in a marked increase in tyrosinase (O-diphenyl: O 2 oxidoreductase) activity following a lag period of 6–9 hours. Within 20 minutes after exposure of cells to MSH, the intracellular levels of cyclic AMP rise to levels which are ten times those of controls but fall to concentrations twice control values by 60 minutes. Transient increases in both protein and RNA synthetic rates also occur following MSH administration correlating in time with the dramatic but rapidly decaying increase in cellular cyclic AMP. The increase in tyrosinase activity observed in response to either MSH, dibutyryl cAMP, or theophylline, is completely suppressed by the addition of either cycloheximide (0.28 μg/ml) or actinomycin D (0.05 μg/ml) as is the basal activity of the enzyme. Results from 14C/ 3H leucine studies suggest that MSH may cause increased de novo synthesis of tyrosinase.

The Effect of The Divalent Cation Ionophore A23187 on Amphibian Melanophores and Iridophores

Calcium ions are required in the medium for the full darkening action of melanocyte-stimulating hormone, but not for its second messenger, cyclic adenosine monophosphate, on isolated frog skin. Thus, the possible effect of the divalent cation ionophore A23187 was studied, using the skin of Rana pipiens. It is a potent darkening agent over the range of 1-2 micrometer, as measured by reflectance change and microscopic observation. During the darkening response of A23187, melanosome dispersion takes place in both dermal and epidermal melanophores, as well as aggregation of iridophore reflecting platelets. The darkening is slowly reversible in Ringer's solution, thus it is not due to toxicity. The darkening is partially dependent on the presence of calcium in the medium under certain conditions, suggesting that the response involves both the uptake and release of calcium ions. Cytochalasin B reversibly inhibits the darkening produced by A23187. Since this drug is known to bring about the breakdown of microfilaments, its inhibitory action is in accord with the concept that the ionophore is stimulating microfilament function. A23187 administered to tissue cultured embryonic salamander (Ambystoma maculatum) melanophores produced an irreversible rounding up of the cells, suggesting a toxic effect. The results with frog skin are interpreted as supporting the concept that the action of melanocyte-stimulating hormone involves the production of pigment granule movements as a result of the interaction of calcium ions with intracellular microfilaments and possible also the breakdown of microtubules. The mobilization of calcium ions could be brought about by cyclic adenosine monophosphate.

Passive and active avoidance responses at two levels of shock after administration of melanocyte-stimulating hormone

Rats receiving either melanocyte-stimulating hormone (MSH) or control solution were tested in a combined active and passive avoidance task involving different visual cues and two levels of shock. The group tested at 0.8 mA had a higher ( P < 0.05) index of discrimination than the rats tested at the barely perceptible 0.4 mA. In this complex task, no effect of MSH was noted on behavior during acquisition or extinction, nor was there an effect upon adrenal or body weight. The lack of an observable action of MSH on activity as tested in the shuttle box, general activity meter, and open-field maze suggests that the previously reported influence of MSH on behavior cannot be adequately explained as a simple effect upon activity.

The Influence of Hormones on Melanogenesis

This paper reviews the action of hormones on the epidermal melanin unit. Slight attention is paid to the hormonal regulation of the melanophore. Melanin serves 2 main functions in the epidermis: 1) The pigment protects underlying structures from the harmful effects of sunlight; and 2) It serves to influence the color of the epidermis. 1 mechanism by which hormones can influence the amount of melanin involves changes in the activity to the enzyme tryosinase in the melanoblasts which synthesize the pigment. A second mechanism may involve changes in the activity of keratinocytes which engulf the melanin discharged from melanoblasts. Hormones can also influence the dispersion of melanin. Interstitial cell-stimulating hormone, estrogens, melanocyte-stimulating hormone and adrenocorticotrophin appear to increase epidermal melanin by enhancing the activity of tyrosinase. The action of interstitialcell-stimulating hormone (ICSH) upon melanogenesis has been studied in weaver birds. It has been shown that estrogens are capable of accelerating the synthesis of melanin, and that the action is a direct effect of the hormone itself, because the response occurs locally when the hormone is applied directly to the skin. It has been observed that skin color varies with the menstrual cycle. Such variations may result from the synergistic action of estrogens and progesterone. A similar mechanism accounts for the pigmentation of pregnancy. Studies have shown that estrogens also influence the color of feathers in certain birds. Melanocyte-stimulating hormone (MSH) has been shown to increase the melanin content of the epidermis of the guinea pig, and promotes the dispersion of melanin into the dendritic processes of the melanocyte and into adjacent keratinocytes. Adrenocorticotropic hormone (ACTH) prossesses some MSH activity, although it is much less potent than MSH itself. Presumably the hormone acts like MSH, directly on the melanocyte. These 2 hormones darken the skin in man, and 1 or both is responsible for the well known pigmentation which follows bilateral adrenalectomy when replacement therapy is inadequate. The influence of thyroxine upon epidermal melanin is complex and varies from species to species. The action of androgens on melanogenesis is both complex and ill-understood. Adrenoline and noradrenaline inhibit the action of MSH on frog skin but there is no direct evidence that these hormones influence the mammalian melanocyte. Further investigations are needed.

Role of cyclic AMP in mediating the effects of MSH, norepinephrine, and melatonin on frog skin color.

Evidence has previously been published indicating that melanocyte stimulating hormone (MSH) acts on the frog skin to increase intracellular concentrations of adenosine 3′,5′- monophosphate (cyclic AMP) and that this nucleotide in turn mediates the action of MSH in causing dispersion of melanin granules with consequent darkening of the frog skin. In the present study both norepinephrine and melatonin were found to inhibit the increase in cyclic AMP levels in response to MSH, and both agents prevented or reversed MSH-induced darkening of frog skin. Neither agent had any effect on frog skin darkening induced by exogenous cyclic AMP or on the in vitro enzymatic degradation of cyclic AMP. The inhibitory effects of norepinephrine on the actions of MSH were prevented by the α-adrenergic blocking agents dihydroergotamine and phentolamine, but these drugs were ineffective against melatonin. In contrast, the β-adrenergic agent isoproterenol had no significant inhibitory effect on MSH-induced cyclic AMP accumulation, and the marked inhibitory effect of norepinephrine could not be prevented by α-adrenergic blocking agents. It is concluded that norepinephrine and melatonin both inhibit the action of MSH by preventing the formation of cyclic AMP in response to this hormone. Since the inhibitory effect of norepinephrine can be prevented by α-adrenergic blocking agents while the effect of melatonin cannot, it is inferred that the action of norepinephrine is mediated by an α-adrenergic receptor while that of melatonin might be mediated by a different type of receptor. Both types of receptors, nevertheless, seem to be closely related to adenyl cyclase. These data lend support to the hypothesis that α-adrenergic responses in general might be mediated by decreases in intracellular concentrations of cyclic AMP. (Endocrinology85: 674, 1969)

Melanin-dispersing effect of adenosine 3',5'-monophosphate on amphibian melanophores.

Because of conflicting prior reports of the effect of 3″,5″-AMP (adenosine 3″,5″- monophosphate) on frog melanophores, the effect of this substance has been studied on amphibian melanophores. Although lower concentrations produce some darkening, 10 HIm is required to markedly darken isolated frog skin or expand tissue cultured salamander embryonic melanophores. The darkening of frog skin by 3″,5″-AMP was not sodium dependent. The darkening produced by combinations of 3″,5″-AMP with melanocyte-stimulating hormone (MSH) or caffeine was equal to the sum of the darkening produced by these agents alone. The results were discussed in terms of the possibility that 3″,5″- AMP may be the “second messenger” for MSH and caffeine action on melanophores. (Endocrinology81: 283, 1967)

Chromatophores and permeability characteristics of frog skin

1. 1. To test eventual relations between chromatophore regulation and permeability characteristics, eleven pharmacological substances capable of acting on frog skin permeability have been studied. 2. 2. It is demonstrated that chromatotrophic substance action on chromatophores is not a mere action on cellular permeability characteristics. 3. 4. Melanocyte-stimulating hormone (MSH) increases greatly the electrical potential difference (p.d.) which exists spontaneously between the two faces of the skin. On the contrary, the net water flux when the skin is submitted to osmotic pressure difference is not modified. 4. 4. Na ions are necessary to MSH and atropine action on chromatophores. No other ion can substitute for Na; this fact suggests that Na ions could participate directly in biochemical reactions responsible for the observed phenomena. 5. 5. Implications of these results on Novale's theory (1959) are discussed.

The effects of sodium, chloride, and calcium concentration on the response of melanophores to melanocyte-stimulating hormone (MSH)

Further studies have been conducted upon ionic factors influencing intermedin (MSH) action on the frog skin melanophore. The sodium requirement for MSH action is “absolute”, since none of a large series of monovalent cations are able to replace sodium. Replacement of chloride by other anions results in an inhibition of the response to MSH. In calcium-free media, there is an enhanced response to MSH. Furthermore, EDTA was found to darken frog skin. These results are discussed in terms of the hypothesis that MSH action involves an increase in the sodium content of the melanophore, followed by melanin dispersion.

On the action of melanocyte stimulating hormone

On the action of melanocyte stimulating hormone

Occurrence of melanocyte stimulating hormone (MSH) in a transplantable pituitary tumor.

SummaryStrain 2 adrenotropic tumors of LAF1 mice were assayed for MSH, ACTH and vasopressin. The concentration of MSH was approximately that of the normal mouse pituitary. ACTH and vasopressin concentrations were very low. The possible relationship of MSH and ACTH was discussed. The adrenotropic tumor-bearing animal should be advantageous in the study of the action of MSH in the mammal.The authors are indebted to Dr. Jacob Furth for supplying the tumor-bearing mice for transplantation and to Dr. Aaron B. Lerner for a standard preparation of MSH.