The effect of white light on normal and malignant murine melanocytes: A link between opsins, clock genes, and melanogenesis.

The skin possesses a photosensitive system comprised of opsins whose function is not fully understood, and clock genes which exert an important regulatory role in skin biology. Here, we evaluated the presence of opsins in normal (Melan-a cells) and malignant (B16-F10 cells) murine melanocytes. Both cell lines express Opn2, Opn4--for the first time reported in these cell types--as well as S-opsin. OPN4 protein was found in a small area capping the cell nuclei of B16-F10 cells kept in constant dark (DD); twenty-four hours after the white light pulse (WLP), OPN4 was found in the cell membrane. Despite the fact that B16-F10 cells expressed less Opn2 and Opn4 than Melan-a cells, our data indicate that the malignant melanocytes exhibited increased photoresponsiveness. The clock gene machinery is also severely downregulated in B16-F10 cells as compared to Melan-a cells. Per1, Per2, and Bmal1 expression increased in B16-F10 cells in response to WLP. Although no response in clock gene expression to WLP was observed in Melan-a cells, gene correlational data suggest a minor effect of WLP. In contrast to opsins and clock genes, melanogenesis is significantly upregulated in malignant melanocytes in comparison to Melan-a cells. Tyrosinase expression increased after WLP only in B16-F10 cells; however no increase in melanin content after WLP was seen in either cell line. Our findings may prove useful in the treatment and the development of new pharmacological approaches of depigmentation diseases and skin cancer.

Environmental control of biological rhythms: effects on development, fertility and metabolism.

Internal temporal organisation properly synchronised to the environment is crucial for health maintenance. This organisation is provided at the cellular level by the molecular clock, a macromolecular transcription-based oscillator formed by the clock and the clock-controlled genes that is present in both central and peripheral tissues. In mammals, melanopsin in light-sensitive retinal ganglion cells plays a considerable role in the synchronisation of the circadian timing system to the daily light/dark cycle. Melatonin, a hormone synthesised in the pineal gland exclusively at night and an output of the central clock, has a fundamental role in regulating/timing several physiological functions, including glucose homeostasis, insulin secretion and energy metabolism. As such, metabolism is severely impaired after a reduction in melatonin production. Furthermore, light pollution during the night and shift work schedules can abrogate melatonin synthesis and impair homeostasis. Chronodisruption during pregnancy has deleterious effects on the health of progeny, including metabolic, cardiovascular and cognitive dysfunction. Developmental programming by steroids or steroid-mimetic compounds also produces internal circadian disorganisation that may be a significant factor in the aetiology of fertility disorders such as polycystic ovary syndrome. Thus, both early and late in life, pernicious alterations of the endogenous temporal order by environmental factors can disrupt the homeostatic function of the circadian timing system, leading to pathophysiology and/or disease.

The expression of melanopsin and clock genes in Xenopus laevis melanophores and their modulation by melatonin

Vertebrates have a central clock and also several peripheral clocks. Light responses might result from the integration of light signals by these clocks. The dermal melanophores of Xenopus laevis have a photoreceptor molecule denominated melanopsin (OPN4x). The mechanisms of the circadian clock involve positive and negative feedback. We hypothesize that these dermal melanophores also present peripheral clock characteristics. Using quantitative PCR, we analyzed the pattern of temporal expression of Opn4x and the clock genes Per1, Per2, Bmal1, and Clock in these cells, subjected to a 14-h light:10-h dark (14L:10D) regime or constant darkness (DD). Also, in view of the physiological role of melatonin in the dermal melanophores of X. laevis, we determined whether melatonin modulates the expression of these clock genes. These genes show a time-dependent expression pattern when these cells are exposed to 14L:10D, which differs from the pattern observed under DD. Cells kept in DD for 5 days exhibited overall increased mRNA expression for Opn4x and Clock, and a lower expression for Per1, Per2, and Bmal1. When the cells were kept in DD for 5 days and treated with melatonin for 1 h, 24 h before extraction, the mRNA levels tended to decrease for Opn4x and Clock, did not change for Bmal1, and increased for Per1 and Per2 at different Zeitgeber times (ZT). Although these data are limited to one-day data collection, and therefore preliminary, we suggest that the dermal melanophores of X. laevis might have some characteristics of a peripheral clock, and that melatonin modulates, to a certain extent, melanopsin and clock gene expression.

The expression of melanopsin and clock genes in Xenopus laevis melanophores and their modulation by melatonin.

Vertebrates have a central clock and also several peripheral clocks. Light responses might result from the integration of light signals by these clocks. The dermal melanophores of Xenopus laevis have a photoreceptor molecule denominated melanopsin (OPN4x). The mechanisms of the circadian clock involve positive and negative feedback. We hypothesize that these dermal melanophores also present peripheral clock characteristics. Using quantitative PCR, we analyzed the pattern of temporal expression of Opn4x and the clock genes Per1, Per2, Bmal1, and Clock in these cells, subjected to a 14-h light:10-h dark (14L:10D) regime or constant darkness (DD). Also, in view of the physiological role of melatonin in the dermal melanophores of X. laevis, we determined whether melatonin modulates the expression of these clock genes. These genes show a time-dependent expression pattern when these cells are exposed to 14L:10D, which differs from the pattern observed under DD. Cells kept in DD for 5 days exhibited overall increased mRNA expression for Opn4x and Clock, and a lower expression for Per1, Per2, and Bmal1. When the cells were kept in DD for 5 days and treated with melatonin for 1 h, 24 h before extraction, the mRNA levels tended to decrease for Opn4x and Clock, did not change for Bmal1, and increased for Per1 and Per2 at different Zeitgeber times (ZT). Although these data are limited to one-day data collection, and therefore preliminary, we suggest that the dermal melanophores of X. laevis might have some characteristics of a peripheral clock, and that melatonin modulates, to a certain extent, melanopsin and clock gene expression.

Modulation of rhodopsin gene expression and signaling mechanisms evoked by endothelins in goldfish and murine pigment cell lines

Endothelins (ETs) and sarafotoxins (SRTXs) belong to a family of vasoconstrictor peptides, which regulate pigment migration and/or production in vertebrate pigment cells. The teleost Carassius auratus erythrophoroma cell line, GEM-81, and Mus musculus B16 melanocytes express rhodopsin, as well as the ET receptors, ETB and ETA, respectively. Both cell lines are photoresponsive, and respond to light with a decreased proliferation rate. For B16, the doubling time of cells kept in 14-h light (14L):10-h darkness (10D) was higher compared to 10L:14D, or to DD. The doubling time of cells kept in 10L:14D was also higher compared to DD. Using real-time PCR, we demonstrated that SRTX S6c (12-h treatment, 100 pM and 1 nM; 24-h treatment, 1 nM) and ET-1 (12-h treatment, 10 and 100 pM; 24- and 48-h treatments, 100 pM) increased rhodopsin mRNA levels in GEM-81 and B16 cells, respectively. This modulation involves protein kinase C (PKC) and the mitogen-activated protein kinase cascade in GEM-81 cells, and phospholipase C, Ca2+, calmodulin, a Ca2+/calmodulin-dependent kinase, and PKC in B16 cells. Cells were kept under constant darkness throughout the gene expression experiments. These results show that rhodopsin mRNA levels can be modulated by SRTXs/ETs in vertebrate pigment cells. It is possible that SRTX S6c binding to the ETB receptors in GEM-81 cells, and ET-1 binding to ETA receptors in B16 melanocytes, although activating diverse intracellular signaling mechanisms, mobilize transcription factors such as c-Fos, c-Jun, c-Myc, and neural retina leucine zipper protein. These activated transcription factors may be involved in the positive regulation of rhodopsin mRNA levels in these cell lines.

Modulation of rhodopsin gene expression and signaling mechanisms evoked by endothelins in goldfish and murine pigment cell lines.

Endothelins (ETs) and sarafotoxins (SRTXs) belong to a family of vasoconstrictor peptides, which regulate pigment migration and/or production in vertebrate pigment cells. The teleost Carassius auratus erythrophoroma cell line, GEM-81, and Mus musculus B16 melanocytes express rhodopsin, as well as the ET receptors, ETB and ETA, respectively. Both cell lines are photoresponsive, and respond to light with a decreased proliferation rate. For B16, the doubling time of cells kept in 14-h light (14L):10-h darkness (10D) was higher compared to 10L:14D, or to DD. The doubling time of cells kept in 10L:14D was also higher compared to DD. Using real-time PCR, we demonstrated that SRTX S6c (12-h treatment, 100 pM and 1 nM; 24-h treatment, 1 nM) and ET-1 (12-h treatment, 10 and 100 pM; 24- and 48-h treatments, 100 pM) increased rhodopsin mRNA levels in GEM-81 and B16 cells, respectively. This modulation involves protein kinase C (PKC) and the mitogen-activated protein kinase cascade in GEM-81 cells, and phospholipase C, Ca(2+), calmodulin, a Ca(2+)/calmodulin-dependent kinase, and PKC in B16 cells. Cells were kept under constant darkness throughout the gene expression experiments. These results show that rhodopsin mRNA levels can be modulated by SRTXs/ETs in vertebrate pigment cells. It is possible that SRTX S6c binding to the ETB receptors in GEM-81 cells, and ET-1 binding to ETA receptors in B16 melanocytes, although activating diverse intracellular signaling mechanisms, mobilize transcription factors such as c-Fos, c-Jun, c-Myc, and neural retina leucine zipper protein. These activated transcription factors may be involved in the positive regulation of rhodopsin mRNA levels in these cell lines.

Melanopsin triggers the release of internal calcium stores in response to light.

Melanopsin is the photopigment that confers photosensitivity upon intrinsically photosensitive retinal ganglion cells (ipRGCs). This subset of retinal ganglion cells comprises less than 2% of all RGCs in the mammalian retina. The paucity of melanopsin-positive cells has made studies on melanopsin signaling difficult to pursue in ipRGCs. To address this issue, we have established several cell lines consisting of a transformed human embryonic kidney cell line (HEK293) stably expressing human melanopsin. With these cell lines, we have investigated the intracellular rise in calcium triggered upon light activation of melanopsin. Our human melanopsin-expressing cells exhibit an irradiance-dependent increase in intracellular calcium. Control cells expressing human melanopsin, where the Schiff-base lysine has been mutated to alanine, show no responses to light. Chelating extracellular calcium has no effect on the light-induced increase in intracellular calcium suggesting that calcium is mobilized from intracellular stores. This involvement of intracellular stores has been confirmed through their depletion by thapsigargin, which inhibits a subsequent light-induced increase in intracellular calcium. Addition of the nonselective cation channel blocker lanthanum does not alter light-induced rises in intracellular calcium, further supporting that melanopsin triggers a release of internal calcium from internal stores. HEK293 cells stably expressing melanopsin have proven to be a useful tool to study melanopsin-initiated signaling.

Biological activity and binding of estradiol to SK-Mel 23 human melanoma cells.

Patients expressing estradiol receptors in melanoma cells have been reported to have a better prognosis. We therefore decided to investigate the in vitro effects of beta-estradiol and tamoxifen on the growth and tyrosinase activity of SK-Mel 23 human melanoma cells. Twenty-four-hour treatment with 0.4 nM beta-estradiol inhibited cell proliferation in 30% (0.70 +/- 0.03 x 10(5) cells) and increased tyrosinase activity in 50% (7130.5 +/- 376.5 cpm/10(5) cells), as compared to untreated cells (1.0 +/- 0.05 x 10(5) cells and 4769 +/- 25.5 cpm/10(5) cells, respectively). Both responses were completely (100%) blocked by 1 microM tamoxifen. Higher concentrations (up to 1.6 nM) or longer treatments (up to 72 h) did not result in a larger effect of the hormone on proliferation or tyrosinase activity. Competition binding assays demonstrated the presence of binding sites to [2,4,6,7-3H]-beta-estradiol, and that the tritiated analogue was displaced by the unlabeled hormone (1 nM to 100 microM, Kd = 0.14 microM, maximal displacement of 93%) or by 10 microM tamoxifen (displacement of 60%). Beta-estradiol also increased the phosphorylated state of two proteins of 16 and 46 kDa, after 4-h treatment, as determined by Western blot. The absorbance of each band was 1.9- and 4-fold the controls, respectively, as determined with Image-Pro Plus software. Shorter incubation periods with beta-estradiol did not enhance phosphorylation; after 6-h treatment with the hormone, the two proteins returned to the control phosphorylation levels. The growth inhibition promoted by estradiol may explain the better prognosis of melanoma-bearing women as compared to men, and open new perspectives for drug therapy.

Biological activity and binding of estradiol to SK-Mel 23 human melanoma cells

Patients expressing estradiol receptors in melanoma cells have been reported to have a better prognosis. We therefore decided to investigate the in vitro effects of á-estradiol and tamoxifen on the growth and tyrosinase activity of SK-Mel 23 human melanoma cells. Twenty-four-hour treatment with 0.4 nM á-estradiol inhibited cell proliferation in 30 percent (0.70 ñ 0.03 x 10(5) cells) and increased tyrosinase activity in 50 percent (7130.5 ñ 376.5 cpm/10(5) cells), as compared to untreated cells (1.0 ñ 0.05 x 10(5) cells and 4769 ñ 25.5 cpm/10(5) cells, respectively). Both responses were completely (100 percent) blocked by 1 æM tamoxifen. Higher concentrations (up to 1.6 nM) or longer treatments (up to 72 h) did not result in a larger effect of the hormone on proliferation or tyrosinase activity. Competition binding assays demonstrated the presence of binding sites to [2,4,6,7- H]-á-estradiol, and that the tritiated analogue was displaced by the unlabeled hormone (1 nM to 100 æM, Kd = 0.14 æM, maximal displacement of 93 percent) or by 10 æM tamoxifen (displacement of 60 percent). á-estradiol also increased the phosphorylated state of two proteins of 16 and 46 kDa, after 4-h treatment, as determined by Western blot. The absorbance of each band was 1.9- and 4-fold the controls, respectively, as determined with Image-Pro Plus software. Shorter incubation periods with á-estradiol did not enhance phosporylation; after 6-h treatment with the hormone, the two proteins returned to the control phosphorylation levels. The growth inhibition promoted by estradiol may explain the better prognosis of melanoma-bearing women as compared to men, and open new perspectives for drug therapy.

The role of calcium, calcium-activated K+ channels, and tyrosine/kinase in psoralen-evoked responses in human melanoma cells.

8-Methoxy psoralen (8-MOP) exerts a short-term (24 h) mitogenic action, and a long-term (48-72 h) anti-proliferative and melanogenic action on two human melanoma cell lines, SK-Mel 28 and C32TG. An increase of intracellular calcium concentration was observed by spectrofluorometry immediately after the addition of 0.1 mM 8-MOP to both cell lines, previously incubated with calcium probe fluo-3 AM (5 micro M). The intracellular Ca2+ chelator BAPTA/AM (1 micro M) blocked both early (mitogenic) and late (anti-proliferative and melanogenic) 8-MOP effects on both cell lines, thus revealing the importance of the calcium signal in both short- and long-term 8-MOP-evoked responses. Long-term biological assays with 5 and 10 mM tetraethylammonium chloride (TEA, an inhibitor of Ca2+-dependent K+ channels) did not affect the responses to psoralen; however, in 24-h assays 10 mM TEA blocked the proliferative peak, indicating a modulation of Ca2+-dependent K+ channels by 8-MOP. No alteration of cAMP basal levels or forskolin-stimulated cAMP levels was promoted by 8-MOP in SK-Mel 28 cells, as determined by radioimmunoassay. However, in C32TG cells forskolin-stimulated cAMP levels were further increased in the presence of 8-MOP. In addition, assays with 1 micro M protein kinase C and calcium/calmodulin-dependent kinase inhibitors, Ro 31-8220 and KN-93, respectively, excluded the participation of these kinases in the responses evoked by 8-MOP. Western blot with antibodies anti-phosphotyrosine indicated a 92% increase of the phosphorylated state of a 43-kDa band, suggesting that the phosphorylation of this protein is a component of the cascade that leads to the increase of tyrosinase activity.

The role of calcium, calcium-activated K+ channels, and tyrosine/kinase in psoralen-evoked responses in human melanoma cells

8-Methoxy psoralen (8-MOP) exerts a short-term (24 h) mitogenic action, and a long-term (48-72 h) anti-proliferative and melanogenic action on two human melanoma cell lines, SK-Mel 28 and C32TG. An increase of intracellular calcium concentration was observed by spectrofluorometry immediately after the addition of 0.1 mM 8-MOP to both cell lines, previously incubated with calcium probe fluo-3 AM (5 µM). The intracellular Ca2+ chelator BAPTA/AM (1 µM) blocked both early (mitogenic) and late (anti-proliferative and melanogenic) 8-MOP effects on both cell lines, thus revealing the importance of the calcium signal in both short- and long-term 8-MOP-evoked responses. Long-term biological assays with 5 and 10 mM tetraethylammonium chloride (TEA, an inhibitor of Ca2+-dependent K+ channels) did not affect the responses to psoralen; however, in 24-h assays 10 mM TEA blocked the proliferative peak, indicating a modulation of Ca2+-dependent K+ channels by 8-MOP. No alteration of cAMP basal levels or forskolin-stimulated cAMP levels was promoted by 8-MOP in SK-Mel 28 cells, as determined by radioimmunoassay. However, in C32TG cells forskolin-stimulated cAMP levels were further increased in the presence of 8-MOP. In addition, assays with 1 µM protein kinase C and calcium/calmodulin-dependent kinase inhibitors, Ro 31-8220 and KN-93, respectively, excluded the participation of these kinases in the responses evoked by 8-MOP. Western blot with antibodies anti-phosphotyrosine indicated a 92 percent increase of the phosphorylated state of a 43-kDa band, suggesting that the phosphorylation of this protein is a component of the cascade that leads to the increase of tyrosinase activity.

Comparative EPR and fluorescence conformational studies of fully active spin-labeled melanotropic peptides.

Similar to melanocyte stimulating hormone (alpha-MSH), its potent and long-acting analogue, [Nle(4), D-Phe(7)]alpha-MSH, when labeled with the paramagnetic amino acid probe 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac), maintains its full biological potency, thus validating any comparative structural investigations between the two labeled peptides. Correlation times, calculated from the electron paramagnetic resonance signal of Toac bound to the peptides, and Toac-Trp distances, estimated from the Toac fluorescence quenching of the Trp residue present in the peptides, indicate a more rigid and folded structure for the potent analogue as compared to the hormone, in aqueous medium.

Psoralen activity and binding sites in melanotic and amelanotic human melanoma cells.

The biological activity and specific binding sites of 8-methoxypsoralen (8-MOP) are assayed using two human melanoma cell lines, melanotic SK-Mel 28 and amelanotic C32TG. Long-term (72 hr) treatment with 8-MOP at a concentration of 10(-4)M results in an increase in melanogenesis and a decrease in proliferation, similar in both cell lines. Daily exposure of these cells to ultraviolet A (UVA) irradiation (1.28 mJ/cm(2)) does not enhance the response to the compound. Daily pulse application (30 min daily) of 8-MOP does not promote any response. However, in combination with UVA, 8-MOP pulse treatment becomes as effective as the long-term treatment. A decrease in cell proliferation in the constant presence of 8-MOP is not coupled with apoptosis, since no increase in the number of apoptotic nuclei was observed after the treatment. The flow cytometry indicates that 8-MOP arrests the cells at the G0/G1 phase, irrespective of the presence or absence of UVA light. In view of the lack of epidermal growth factor (EGF) receptors in both cell lines, it is not likely that such an arrest is associated with the down-regulation of EGF receptors by 8-MOP. It is noted that this compound elicits a biphasic cell response, since cell proliferation increases after the first 24-hr treatment, whereas it decreases in the subsequent 48 hr and thereafter. Competition binding assays using 3H-8-MOP disclosed: 1) the specific binding of the compound in both cell lines occurs in the presence or absence of UVA light, and 2) a higher binding rate at low concentrations of the compound is in SK-Mel 28 (72%) rather than C32TG (58%) cells. The competition assays in the presence of UVA suggest a possible occurrence of covalent bindings between psoralen and receptor, as DNA covalent binding accounted to only 3-5% of the total binding in both cell lines.

Possible role of non-classical chromatophorotropins on the regulation of the crustacean erythrophore.

Two neuropeptides, the pigment dispersing hormone (PDH) and the pigment concentrating hormone (PCH), are well known to respectively promote centrifugal and centripetal granule translocation in the freshwater shrimp Macrobrachium potiuna erythrophores. Herein, we demonstrate for the first time the effects of crustacean non-classical chromatophorotropins on the pigment migration in M. potiuna erythrophores. Although proctolin, 20-hydroxyecdisone (20HE), and melatonin were ineffective, the crustacean cardioactive peptide (CCAP) was a full agonist, inducing pigment dispersion in a dose-dependent manner with EC(50) of 9.5. 10(-7) M. In addition, concentrations of CCAP lower than the minimal effective dose (10(-8) and 10(-7) M) decreased the PCH-induced aggregation, shifting rightward the dose-response curve (DRC) to PCH 2.2- and 29-fold, respectively. Surprisingly, melatonin (10(-7) and 10(-6) M) also shifted to the right 8.7- and 46.5-fold, respectively, the DRC to PCH. In conclusion, our data demonstrate that besides PCH and PDH, CCAP and melatonin also regulate the pigment migration within the crustacean erythrophore. J. Exp. Zool. 284:711-716, 1999.

Elasmobranch color change: A short review and novel data on hormone regulation.

Skins of Potamotrygon reticulatus are light in color in vitro, exhibiting punctate melanophores. Alpha-Melanocyte stimulating hormone (EC(50) = 4.58 x 10(-9) M) and prolactin (EC(50) = 1.44 x 10(-9) M) darken the skins in a dose-dependent manner. The endothelins ET-1, ET-2 and ET-3, and the purines, ATP, and uracil triphosphate (UTP) were not able to induce either skin lightening or darkening. Forskolin and the calcium ionophore A23187 promoted a dose-dependent darkening response, whereas N(2), 2'-O-dibutyryl guanosine 3'-5'-cyclic monophosphate (db cyclic GMP), phorbol-12-myristate-13-acetate (TPA), and 1-oleoyl-2-acetyl-sn-glycerol (OAG) were ineffective. The maximal response obtained with the calcium ionophore A23187 was only 76% of maximal darkening. These results indicate that the cyclic adenosine 3'-5'-monophosphate (cAMP) pathway is probably involved in the pigment dispersion of P. reticulatus melanophores. Other experiments should be done to further investigate how cytosolic calcium may be physiologically increased, and the existence of a putative cross-talk between calcium and cAMP signals. In conclusion, the only hormones effective on P. reticulatus melanophores were prolactin and alpha-MSH. No aggregating agent has been shown to antagonize these actions. Prolactin effect on elasmobranch melanophores adds a novel physiological role to this ancient hormone. J. Exp. Zool. 284:485-491, 1999.

Effects of continuous exposure to light on behavioral dopaminergic supersensitivity.

BACKGROUND: This study examines the effects of long-term continuous exposure to light on dopaminergic supersensitivity induced by repeated treatment with haloperidol in rats. METHODS: Spontaneous general activity in an open-field (SGA) and stereotyped behavior induced by apomorphine (SB-APO) or amphetamine (SB-AMP) were used as experimental parameters. Rats were allocated to four groups in each experiment: saline-treated animals kept under a 12-hour light/dark cycle (LD) or 24-hour light/light cycle (LL), and 2 mg/kg haloperidol-treated animals kept under the above cycles. Plasma corticosterone concentration was also measured by radioimmunoassay in saline-treated rats kept under a LD or LL cycle. RESULTS: All the behavioral parameters used showed the development of central dopaminergic supersensitivity in rats kept under both cycles. Continuous exposure to light enhanced SGA and SB-AMP in both saline- and haloperidol-treated rats, but did not modify SB-APO. Animals kept under the LL cycle presented an increased plasma corticosterone concentration. CONCLUSIONS: Our results suggest that continuous exposure to light leads to an increase in dopaminergic function in both normal and "supersensitive" rats. This effect seems to be mediated by a presynaptic mechanism possibly involving corticosterone actions.

First synthesis of a fully active spin-labeled peptide hormone.

For the first time in the electron spin resonance (ESR) and peptide synthesis fields, a fully active spin-labeled peptide hormone was reported. The ESR spectra of this alpha-melanocyte stimulating hormone (alpha-MSH) analogue (acetyl-Toac0-alpha-MSH) where Toac is the paramagnetic amino acid probe 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid, suggested a pH-independent conformation and a more restricted movement comparatively to the free Toac. Owing to its equivalent biological potency in a skin pigmentation assay as compared to the native alpha-MSH and its unique characteristic (paramagnetic, naturally fluorescent and fully active), this analogue is of great potential for investigation of relevant physiological roles reported for alpha-MSH.

Physiological color change in the bullfrog, Rana catesbeiana.

Adults of Rana catesbeiana maintained for 4 days in 12:12 light/dark regimen exhibited a rhythmic color change of 24 hr. Under constant light, however, the rhythm disappeared, and the reflectance values gradually became greater, that is the animals became lighter. Under constant darkness, the rhythm was also abolished, but the animals tended to a darker color. On black background the skin darkening proceeded at a faster rate as compared to the skin lightening of animals adapting to a white background. The difference in color change rate suggests that the darkening responses are probably mediated by an increase in a circulating hormone, whereas skin lightening probably results from the serum level decrease of the same hormone. Most certainly, this hormone is alpha-MSH, as the in vitro assays demonstrated its high potency as a full darkening agonist (EC50 = 9 x 10(-10) M). Prolactin (EC50 = 7.7 x 10(-8) M) and endothelins 2 (EC50 = 1.3 x 10(-6) M) and 3 (EC50 = 4.8 x 10(-7) M) were also full agonists, but 100- to 1000-fold less potent than alpha-MSH. Isoproterenol, in the absence or presence of dibenamine, and endothelin-1 also elicited darkening responses in a dose-related manner, but reaching only 23% and 35% of the maximal darkening, respectively. Isoproterenol darkening effect was completely blocked by propranolol, confirming its action through beta-adrenoceptors. These results, taken together with the lack of lightening activity of norepinephrine on alpha-MSH-darkened skins, suggest that R. catesbeiana melanophores do not possess very active beta-adrenoceptors and lack alpha-adrenoceptors. On the other hand, the lightening agonist melatonin elicited only half-maximal dose-dependent reversal of MSH-induced darkening. Our results suggest that the chromatic rhythm is not endogenous, and most likely is determined by the light/dark cycle effect on alpha-MSH secretion.