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A sensitive bioassay for melanotropic hormones using isolated medaka melanophores
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
70, 127-132 (1988)
A Sensitive Bioassay for Melanotropic Hormones Medaka Melanophores
Using Isolated
SUMIKO NEGISHI ,* ICHIRO KAWAZOE,~ AND HIROSHI KAWAUCHI? * Department Endocrinology,
of Biology, Keio University, Yokohama School of Fisheries Sciences, Kitasato
223, Japan, University,
and fLaboratory Sanriku, Iwate
of Molecular 022-01, Japan
Accepted November 14, 1987 Melanophore-stimulating hormones (MSHs) from chum salmon cause pigment dispersion in isolated melanophores of medaka, a teleost. The in vitro medaka melanophore bioassay that responded to light with pigment dispersion and to the dark with pigment aggregation was utilized for measuring the activity of melanotropic hormones. cx-MSH I was the most potent melanophore-dispersing agent tested. The minimal dose for the induction of pigment dispersion was lo-l5 M a-MSH I, IO-l3 M N-des-acetyl(Ac)-a-MSH, and 10-l’ M O-MSH I, respectively. The melanosome-dispersing activity of B-MSH I was enhanced about 40% by salmon N-acetyl-endorphin I (N-AC-EP). The results suggest that N-AC-EP may act as an enhancer for the activity of certain MSHs. The present bioassay provides a unique method for determining the biological activity of melanotropic peptides. o 1988 Academic Press, Inc.
Melanophore aggregation of teleost is generally caused by adrenergic stimulation, melatonin (Fujii and Oshima, 1986), or melanin-concentrating hormone (MCH) (Kawauchi et al., 1983; Wilkes et al., 1984; Oshima et al., 1985, 1986; Kawazoe et al., 1987; Castrucci et al., 1987). Pigment dispersion is regulated by melanophorestimulating hormone (MSH) (Fujii and Miyashita, 1982; Iga and Takabatake, 1982; Fujii and Oshima, 1986). Previous attempts to develop teleost melanophore bioassay have met with mixed success. Melanophores of the medaka (Oryzias latipes) seem to be insensitive to MSH in an in vitro skin bioassay (Negishi and Obika, 1980). As melanophores of separated medaka scales usually exhibit pigment dispersion in physiological saline, they must first be aggregated by adrenalin or MCH, in order to determine the dispersing activity of MSH. Isolated melanophores were then studied in an attempt to overcome these problems. Isolated melanophores of the medaka are capable of responding to light with pigment dispersion
and to the dark with aggregation, without hormonal pretreatment (Negishi, 1985). Therefore, isolated melanophores may be suitable for studying the dispersing effects of MSH. This paper describes the development of a sensitive bioassay for MSH using isolated medaka melanophores. Three kinds of MSH, a-MSH I, N-des-Ac-cx-MSH I, and @MSH I, isolated from the chum salmon pituitary, were used to test melanophorestimulating activity. Synthetic mammalian (x-MSH was also utilized in order to examine the activity. Chum salmon N-AC-Ep I was tested, since the role of this hormone has not been demonstrated for fish chromatop hores . MATERIALS
AND METHODS
Melanophores
of the wild type (BBRR) medaka, were isolated from scales by treatment with 0.25% collagenase type II (Cooper Biomedical) and 0.15% trypsin (Merck) dissolved in a physiological saline at 20”-25” for 45 to 60 min (Obika, 1976). The isolated melanophores were cultured in Leibovitz’s medium (L-15, GIBCO) containing 5% fetal calf serum Oryzias
latipes
127 0016~6480/88 $1.50 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
128
NEGISHI,
KAWAZOE,
(FCS, GIBCO) in Falcon dishes at 25” for 2-6 days. Melanophores were examined by inverted microscope, and the diameter of each cell was measured by an ocular micrometer. Illumination was kept as dark as possible in order to avoid melanosome dispersion induced by light. The magnitude of pigment dispersion in each cell was expressed as a percentage of the diameter of fully dispersed pigment granules measured under light. Chemicals were dissolved in a physiological saline solution with the following composition: 130 mA4 NaCl; 2.7 miU KCl; 1.8 n&f CaCl,; 5.5 mM Dglucose; 5.0 mM Tris-HC1 (pH 7.4). Propranolol (Sigma) was added to some assay medium at a concentration of 0.1 mM. Melanophore-stimulating hormones; a-MSH I, N-des-Ac-a-MSH I, and B-MSH I, and N-AC-EP I were prepared from chum salmon pituitary glands as described (Kawauchi et al., 1984; Takahashi et al., 1984). They were purified to homogeneity by reversephase HPLC on a TSK-gel ODS-120T column with a linear gradient of acetonitrile and 0.1% trifluoroacetic acid. Synthetic a-MSH was purchased from Sigma Chemical.
AND
KAWAUCHI
activities of salmon MSHs and synthetic (x-MSH at a dose of lo-l3 M. The minimal effective dose of these hormones were estimated to be about lo-l5 M CX-MSH I, lo-l3 M N-des-Ac-cx-MSH I, lo-” M P-MSH I, and lo-l3 M synthetic (x-MSH. Figure 6 shows the effect of N-AC-EP I on melanophore-stimulating activity. NAC-EP I itself caused no pigment dispersion at a concentration of lo- l1 M, but enhanced the activity of lo- l1 M P-MSH I by 40%. The synergistic activity was not observed for wMSH at a concentration of lo- 13-10- l5 M . DISCUSSION
The potency of MSH in Oryzias melanophores has remained unclear by using in vitro skin bioassay (Negishi and Obika, 1980). However, present results show that RESULTS MSHs are highly potent in dispersing pigMelanophores were fully dispersed in the ments of isolated melanophores. They reculture medium under 600 lux light (Fig. la) spond to MSHs in a dose-dependent and and completely aggregated in the dark reversible manner at concentrations of within 40 min (Fig. lb). P-MSH I (10m9 A4) lo- “-lo- l5 M . brought about melanosome dispersion The melanophore-stimulating activity of within 5 min despite a complete absence of MSHs have usually been analyzed in frog light (Fig. lc). Subsequent removal of the or lizard skin (Vesely and Hadley , 1979; hormone by rinsing with saline resulted in Kawauchi et al., 1984; Castrucci et al., centripetal migration of melanosomes 1984), fishes (Fujii and Miyashita, 1982; Iga within 20 min. Figure 2 shows that un- and Takabatake, 1982; Castrucci et al., treated melanophores remained aggregated 1987), and cultured melanophores (Ide et as long as they were kept in the dark. Fol- al., 1985), in which the effective doses of lowing irradiation with light, the cultured MSHs are distributed between 10m6 and melanophores dispersed gradually and at- lo- lo M. It is, therefore, surprising that tained full dispersion after 10 min. synthetic (x-MSH brought about pigment Figure 3 shows the effect of a P-adrenerdispersion at lo- l3 M and cx-MSH I isolated gic blocker, propranolol, on pigment dis- from chum salmon did at lo-l5 M in our persion by @MSH I. Melanophore dispersystem. The reason that chum salmon sion induced by P-MSH is not affected by (x-MSH I was lo2 times more potent than propranolol. synthetic a-MSH may result from the difFigure 4 demonstrates the melanosomeference in the purity of both hormones. dispersing activity of wMSH from chum Previous work showed that melanosome salmon at several doses. The results show a aggregation in the dark was reversed by exan inhibdose-dependent response between lo- I1 tracellular CAMP or theophylline, itor of phosphodiesterase (Negishi, 1985). and lo-l5 M (x-MSH I . Figure 5 shows the melanin-dispersing Thus, melanosome aggregation in medaka
A SENSITIVE
BIOASSAY
FOR
MSH
FIG. 1. Re sponses of isolated medaka melanophores to light, darkness, and /3-MSH. (a) Melanophores dispel -sed in culture medium under room light, 600 lux. (b) Melanophores aggreg ,ated in complete d arknes ;s for 40 min. (c) Melanophores dispersed in lop9 M P-MSH I for 5 min in the dark. Bar indicat es 200 cLm.
129
130
NEGISHI,
KAWAZOE,
AND
KAWAUCHI
I 30
I 60 Time
1 90
(mid
2. Responses of isolated medaka melanophores to P-MSH I or illumination. Top horizontal bar indicates darkness (black) or light at 600 lux (white). P-MSH I (low9 M) was added after 40 min, then washed away by exchanging media. Each point represents the mean & SE of results from 16 cells. FIG.
may be caused by a decrease in intracellular levels of CAMP. It is generally accepted that MSH brings about pigment dispersion in melanophores through the increase in the intracellular CAMP (Abe et al., 1969; Goldman and Hadley, 1969; Bagnara and Hadley, 1973; Fujii and Oshima, 1986). Therefore, it seems reasonable to propose that medaka melanosome dispersion by MSHs is caused by the enhancement of the intracellular levels of CAMP.
It is probable that MSHs at low concentrations can bring about melanosome dispersion in the dark by regulating intracellular CAMP. Pigment aggregation could then be regulated by relatively small changes in intracellular CAMP. This is supported by our observation that CX-MSH, even at 10V6 M, is only slightly active when lo- lo M adrenalin was present in the media (Negishi, unpublished data). Adrenalin is thought to act by decreasing the intracellular levels of CAMP (Abe et al., 1969; Colley
r2
Prop
PMSH
01
I 10
I 20 Time
I
I
I
30 (mid
40
50
FIG. 3. Effect of propranolol (prop) on melanophore-stimulating activity of P-MSH I. Melanophores were aggregated in the dark for 30 min, then further aggregated by pulse-treatment of 10e4 A4 propranolol for 10 min. @MSH I (lo-* M) was then added. Each point represent the mean +- SE of results from 14 cells.
0
I
L
I
10
5
Time
(mid
4. Effects of wMSH I from chum salmon on isolated medaka melanophores. a-MSH I: (0) lo- l3 M; (0) lo-l4 A4; (A) lo-l5 A4; (Cl) lo-l6 M. Each point represents the mean + SE of results from at least 15 cells. FIG.
A SENSITIVE
I 5
0’
BIOASSAY
I 10
Time
(mid
FIG. 5. Comparison of various MSHs on darkaggregated melanophores. MSHs (lo-l3 M) were added to aggregated medaka melanophores. (0) a-MSHI; (0) ZV-des-Ac-a-MSH I; (0) B-MSH I; (A) synthetic a-MSH. Each point represents the mean + SE of results from 17-19 cells.
and Hunt, 1974; Negishi et al., 1982). The rapid paling response of medaka may be controlled by the release of catecholamines such as noradrenalin (Bagnara and Hadley, 1973; Fujii and Oshima, 1986). If MSH functions as a hormonal regulator of melanosome dispersion in medaka, it would be expected to act at very low concentrations as shown in the present result, accompanied by a decrease of catecholamines, as the darkening response occurred. It is 100
2 C
.-0 ul G .4 u
50
:
OL
, 5 Time
I 10 (mid
6. EP I-enhanced melanophore-stimulating activity of B-MSH I. (0) EP I 10-i’ M + B-MSH I 10-l’ M; (0) B-MSH I 10-l’ M; (A) EP I 10-l’ M. Each point represents the mean + SE of results from at least 21 cells. FIG.
FOR
131
MSH
thought that MSHs may be particularly important in color changes of juvenile fish when the nervous system is underdeveloped (Bagnara and Hadley , 1973). Our results show that chum salmon NAC-EP I enhanced melanosome-dispersing activity of P-MSH I in the isolated melanophores bioassay. This suggests that NAC-EP I might play a supplementary role for P-MSH I in medaka. This result is consistent with that from Rana pipiens (Carter and Shuster, 1978), but inconsistent with that from Rana berlandieri (Novales and Novales, 1979). Considering that B-EP and B-MSH are both derived by the enzymatic cleavage of the P-lipotropin prohormone (Hunter and Baker, 1979; Kawauchi, 1983; Kawauchi et al., 1984), this enhancing activity of N-AC-EP I for B-MSH I could be coordinately regulated. The physiological bases for this synergistic activity remain to be elucidated. The light-sensitive melanophores of medaka provide a valuable bioassay which may be useful to analyze these and other modifiers of melanophore activity, since present in vitro melanophore bioassay is remarkably sensitive for determining the activity of melanotropic peptides. REFERENCES Abe, K., Butcher, B. W., Nicholson, W. E., Baird, C. E., Liddle, R. A., and Liddle, G. W. (1969). Adenosine 3’,5’-monophosphate (cyclic AMP) as the mediator of the actions of melanocyte stimulating hormone (MSH) and norepinephrine on the frog skin. Endocrinol. 84, 362-368. Bagnara, J. T., and Hadley, M. E. (1973). “Chromatophores and Color Change.” Prentice-Hall, Englewood Cliffs, NJ. Carter, R. J., and Shuster, S. (1978). B-Endorphin potenciates pigmentary activity. J. Invest. Dermatol. 70, 228-229. Castrucci, A. M. L., Hadley, M. E., and Hruby, V. J. (1984). Melanotropin bioassays: In vitro and in vivo comparisons. Gen. Comp. Endocrinol. 55, 104-111. Castrucci, A. M. L., Hadley, M. E., and Hruby, V. J. (1987). A teleost skin bioassay for melanotropic peptides. Gen. Comp. Endocrinol. 66, 374-380. Colley, A. M., and Hunt, E. L. (1974). Cyclic AMP and adrenergic receptors in Gambusia afinis me-
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lanophores response in vitro. Gen. Comp. Endocrinol. 23, 154-163. Fujii, R., and Miyashita, Y. (1982). Receptor mechanisms in fish chromatophores. V. MSH disperses melanosomes in both dermal and epidermal melanophores of a catfish (Parasilurus asotus). Comp. Biochem.
Physiol.
7lC,
l-6.
Fujii, R., and Oshima, N. (1986). Control of chromatophore movements in teleost fishes. 2001. Sci. 3, 13-47. Goldman, J. M., and Hadley, M. E. (1969). The beta adrenergic receptor and cyclic 3’,5’-adenosine monophosphate: Possible role in regulation of melanophore responses of the spadefoot toad, Scaphiopus
couchi.
Gen.
Comp.
Endocrinol.
13,
151-163. Hunter, C., and Baker, B. I. (1979). The distribution of opiate activity in the trout pituitary gland. Gen. Comp. Endocrinol. 37, 11 l-l 14. Ide, H., Kawazoe, I., and Kawauchi, H. (1985). Fish melanin-concentrating hormone disperses melanin in amphibian melanophores. Gen. Comp. Endocrinol.
58, 48-90.
Iga, T., and Takabatake, I. (1982). Action of melanophore-stimulating hormone on melanophores of the cyprinid fish Zacco temminski. Comp. Biothem.
Physiol.
C73,
51-55.
Kawauchi, H. (1983). Chemistry of proopiocortinrelated peptides in the salmon pituitary. Arch. Biochem.
Biophys.
227, 343-350.
Kawauchi, H., Kawazoe, I., Adachi, Y., Buckley, D. I., and Ramachandran, J. (1984). Chemical and biological characterization of salmon melanocytestimulating hormones. Gen. Comp. Endocrinol. 53, 3748. Kawauchi, H., Kawazoe, I., Tsubokawa, M., Kishida, M., and Baker, B. I. (1983). Characterization of melanin-concentrating hormone in chum salmon pituitaries. Nature (London) 305, 321-333. Kawazoe, I., Kawauchi, H., Hirano, T., and Naito, N. (1987). Characterization of melanin concen-
AND
KAWAUCHI
trating hormones in teleost hypothalamus. Comp.
Endocrinol.
65, 423-43
Gen.
1.
Negishi, S. (1985). Light response of cultured melanophores of a teleost adult fish, Oryzias latipes. J. Exp.
Zool.
236, 327-333.
Negishi, S., Masada, M., Wakamatsu, Y., Ohoka, T., and Obika, M. (1982). Epinephrine-induced changes in the cyclic nucleotide content of fish melanoma cells. Gen. Comp. Endocrinol. 47, 8% 93. Negishi, S., and Obika, M. (1980). The effects of melanophore-stimulating hormone and cyclic nucleotides on teleost fish chromatophores. Gen. Comp. Endocrinol. 42, 471-476. Novales, R. R., and Novales, B. J. (1979). Endorphins supersensitize frog skin melanophores to isoproterenol, but subsensitize them to a-melanocyte-stimulating hormone. Gen. Comp. Endocrinol. 39, 481-489. Obika, M. (1976). Pigment migration in isolated fish melanophores. Annot. Zool. Japan. 49, 157-163. Oshima, N., Kasukawa, H., Fujii, R., Wilkes, B. C., Hruby, V. J., Castrucci, A. M. L., and Hadley, M. E. (1985). Melanin concentrating hormone (MCH) effects on teleost (Chrysiptera cyanea) melanophores. J. Exp. Zool. 235, 175-180. Oshima, N., Kasukawa, H., Fujii, R., Wilkes, B. C., Hruby, V. J., and Hadley, M. E. (1986). Action of melanin-concentrating hormone (MCH) on teleost chromatophores. Gen. Comp. Endocrinol. 64, 381-388. Takahashi, A., Kawauchi, H., Mouri, T., and Sasaki, A. ( 1984). Chemical and immunological characterization of salmon endorphins. Gen. Comp. Endocrinol. 53, 381-388. Vesely, D. L., and Hadley, M. E. (1979). Ionic requirement for melanophore stimulating hormone (MSH) action on melanophores. Comp. Biochem. Physiol.
A62, 501-508.
Wilkes, B. C., Hruby, V. J., Sherbrooke, W. C., Castrucci, A. M. L., and Hadley, M. E. (1984). Synthesis and biological actions of melanin concentrating hormone. Biochem. Biophys. Res. Commun.
122, 613-619.
AND
COMPARATIVE
ENDOCRINOLOGY
70, 127-132 (1988)
A Sensitive Bioassay for Melanotropic Hormones Medaka Melanophores
Using Isolated
SUMIKO NEGISHI ,* ICHIRO KAWAZOE,~ AND HIROSHI KAWAUCHI? * Department Endocrinology,
of Biology, Keio University, Yokohama School of Fisheries Sciences, Kitasato
223, Japan, University,
and fLaboratory Sanriku, Iwate
of Molecular 022-01, Japan
Accepted November 14, 1987 Melanophore-stimulating hormones (MSHs) from chum salmon cause pigment dispersion in isolated melanophores of medaka, a teleost. The in vitro medaka melanophore bioassay that responded to light with pigment dispersion and to the dark with pigment aggregation was utilized for measuring the activity of melanotropic hormones. cx-MSH I was the most potent melanophore-dispersing agent tested. The minimal dose for the induction of pigment dispersion was lo-l5 M a-MSH I, IO-l3 M N-des-acetyl(Ac)-a-MSH, and 10-l’ M O-MSH I, respectively. The melanosome-dispersing activity of B-MSH I was enhanced about 40% by salmon N-acetyl-endorphin I (N-AC-EP). The results suggest that N-AC-EP may act as an enhancer for the activity of certain MSHs. The present bioassay provides a unique method for determining the biological activity of melanotropic peptides. o 1988 Academic Press, Inc.
Melanophore aggregation of teleost is generally caused by adrenergic stimulation, melatonin (Fujii and Oshima, 1986), or melanin-concentrating hormone (MCH) (Kawauchi et al., 1983; Wilkes et al., 1984; Oshima et al., 1985, 1986; Kawazoe et al., 1987; Castrucci et al., 1987). Pigment dispersion is regulated by melanophorestimulating hormone (MSH) (Fujii and Miyashita, 1982; Iga and Takabatake, 1982; Fujii and Oshima, 1986). Previous attempts to develop teleost melanophore bioassay have met with mixed success. Melanophores of the medaka (Oryzias latipes) seem to be insensitive to MSH in an in vitro skin bioassay (Negishi and Obika, 1980). As melanophores of separated medaka scales usually exhibit pigment dispersion in physiological saline, they must first be aggregated by adrenalin or MCH, in order to determine the dispersing activity of MSH. Isolated melanophores were then studied in an attempt to overcome these problems. Isolated melanophores of the medaka are capable of responding to light with pigment dispersion
and to the dark with aggregation, without hormonal pretreatment (Negishi, 1985). Therefore, isolated melanophores may be suitable for studying the dispersing effects of MSH. This paper describes the development of a sensitive bioassay for MSH using isolated medaka melanophores. Three kinds of MSH, a-MSH I, N-des-Ac-cx-MSH I, and @MSH I, isolated from the chum salmon pituitary, were used to test melanophorestimulating activity. Synthetic mammalian (x-MSH was also utilized in order to examine the activity. Chum salmon N-AC-Ep I was tested, since the role of this hormone has not been demonstrated for fish chromatop hores . MATERIALS
AND METHODS
Melanophores
of the wild type (BBRR) medaka, were isolated from scales by treatment with 0.25% collagenase type II (Cooper Biomedical) and 0.15% trypsin (Merck) dissolved in a physiological saline at 20”-25” for 45 to 60 min (Obika, 1976). The isolated melanophores were cultured in Leibovitz’s medium (L-15, GIBCO) containing 5% fetal calf serum Oryzias
latipes
127 0016~6480/88 $1.50 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
128
NEGISHI,
KAWAZOE,
(FCS, GIBCO) in Falcon dishes at 25” for 2-6 days. Melanophores were examined by inverted microscope, and the diameter of each cell was measured by an ocular micrometer. Illumination was kept as dark as possible in order to avoid melanosome dispersion induced by light. The magnitude of pigment dispersion in each cell was expressed as a percentage of the diameter of fully dispersed pigment granules measured under light. Chemicals were dissolved in a physiological saline solution with the following composition: 130 mA4 NaCl; 2.7 miU KCl; 1.8 n&f CaCl,; 5.5 mM Dglucose; 5.0 mM Tris-HC1 (pH 7.4). Propranolol (Sigma) was added to some assay medium at a concentration of 0.1 mM. Melanophore-stimulating hormones; a-MSH I, N-des-Ac-a-MSH I, and B-MSH I, and N-AC-EP I were prepared from chum salmon pituitary glands as described (Kawauchi et al., 1984; Takahashi et al., 1984). They were purified to homogeneity by reversephase HPLC on a TSK-gel ODS-120T column with a linear gradient of acetonitrile and 0.1% trifluoroacetic acid. Synthetic a-MSH was purchased from Sigma Chemical.
AND
KAWAUCHI
activities of salmon MSHs and synthetic (x-MSH at a dose of lo-l3 M. The minimal effective dose of these hormones were estimated to be about lo-l5 M CX-MSH I, lo-l3 M N-des-Ac-cx-MSH I, lo-” M P-MSH I, and lo-l3 M synthetic (x-MSH. Figure 6 shows the effect of N-AC-EP I on melanophore-stimulating activity. NAC-EP I itself caused no pigment dispersion at a concentration of lo- l1 M, but enhanced the activity of lo- l1 M P-MSH I by 40%. The synergistic activity was not observed for wMSH at a concentration of lo- 13-10- l5 M . DISCUSSION
The potency of MSH in Oryzias melanophores has remained unclear by using in vitro skin bioassay (Negishi and Obika, 1980). However, present results show that RESULTS MSHs are highly potent in dispersing pigMelanophores were fully dispersed in the ments of isolated melanophores. They reculture medium under 600 lux light (Fig. la) spond to MSHs in a dose-dependent and and completely aggregated in the dark reversible manner at concentrations of within 40 min (Fig. lb). P-MSH I (10m9 A4) lo- “-lo- l5 M . brought about melanosome dispersion The melanophore-stimulating activity of within 5 min despite a complete absence of MSHs have usually been analyzed in frog light (Fig. lc). Subsequent removal of the or lizard skin (Vesely and Hadley , 1979; hormone by rinsing with saline resulted in Kawauchi et al., 1984; Castrucci et al., centripetal migration of melanosomes 1984), fishes (Fujii and Miyashita, 1982; Iga within 20 min. Figure 2 shows that un- and Takabatake, 1982; Castrucci et al., treated melanophores remained aggregated 1987), and cultured melanophores (Ide et as long as they were kept in the dark. Fol- al., 1985), in which the effective doses of lowing irradiation with light, the cultured MSHs are distributed between 10m6 and melanophores dispersed gradually and at- lo- lo M. It is, therefore, surprising that tained full dispersion after 10 min. synthetic (x-MSH brought about pigment Figure 3 shows the effect of a P-adrenerdispersion at lo- l3 M and cx-MSH I isolated gic blocker, propranolol, on pigment dis- from chum salmon did at lo-l5 M in our persion by @MSH I. Melanophore dispersystem. The reason that chum salmon sion induced by P-MSH is not affected by (x-MSH I was lo2 times more potent than propranolol. synthetic a-MSH may result from the difFigure 4 demonstrates the melanosomeference in the purity of both hormones. dispersing activity of wMSH from chum Previous work showed that melanosome salmon at several doses. The results show a aggregation in the dark was reversed by exan inhibdose-dependent response between lo- I1 tracellular CAMP or theophylline, itor of phosphodiesterase (Negishi, 1985). and lo-l5 M (x-MSH I . Figure 5 shows the melanin-dispersing Thus, melanosome aggregation in medaka
A SENSITIVE
BIOASSAY
FOR
MSH
FIG. 1. Re sponses of isolated medaka melanophores to light, darkness, and /3-MSH. (a) Melanophores dispel -sed in culture medium under room light, 600 lux. (b) Melanophores aggreg ,ated in complete d arknes ;s for 40 min. (c) Melanophores dispersed in lop9 M P-MSH I for 5 min in the dark. Bar indicat es 200 cLm.
129
130
NEGISHI,
KAWAZOE,
AND
KAWAUCHI
I 30
I 60 Time
1 90
(mid
2. Responses of isolated medaka melanophores to P-MSH I or illumination. Top horizontal bar indicates darkness (black) or light at 600 lux (white). P-MSH I (low9 M) was added after 40 min, then washed away by exchanging media. Each point represents the mean & SE of results from 16 cells. FIG.
may be caused by a decrease in intracellular levels of CAMP. It is generally accepted that MSH brings about pigment dispersion in melanophores through the increase in the intracellular CAMP (Abe et al., 1969; Goldman and Hadley, 1969; Bagnara and Hadley, 1973; Fujii and Oshima, 1986). Therefore, it seems reasonable to propose that medaka melanosome dispersion by MSHs is caused by the enhancement of the intracellular levels of CAMP.
It is probable that MSHs at low concentrations can bring about melanosome dispersion in the dark by regulating intracellular CAMP. Pigment aggregation could then be regulated by relatively small changes in intracellular CAMP. This is supported by our observation that CX-MSH, even at 10V6 M, is only slightly active when lo- lo M adrenalin was present in the media (Negishi, unpublished data). Adrenalin is thought to act by decreasing the intracellular levels of CAMP (Abe et al., 1969; Colley
r2
Prop
PMSH
01
I 10
I 20 Time
I
I
I
30 (mid
40
50
FIG. 3. Effect of propranolol (prop) on melanophore-stimulating activity of P-MSH I. Melanophores were aggregated in the dark for 30 min, then further aggregated by pulse-treatment of 10e4 A4 propranolol for 10 min. @MSH I (lo-* M) was then added. Each point represent the mean +- SE of results from 14 cells.
0
I
L
I
10
5
Time
(mid
4. Effects of wMSH I from chum salmon on isolated medaka melanophores. a-MSH I: (0) lo- l3 M; (0) lo-l4 A4; (A) lo-l5 A4; (Cl) lo-l6 M. Each point represents the mean + SE of results from at least 15 cells. FIG.
A SENSITIVE
I 5
0’
BIOASSAY
I 10
Time
(mid
FIG. 5. Comparison of various MSHs on darkaggregated melanophores. MSHs (lo-l3 M) were added to aggregated medaka melanophores. (0) a-MSHI; (0) ZV-des-Ac-a-MSH I; (0) B-MSH I; (A) synthetic a-MSH. Each point represents the mean + SE of results from 17-19 cells.
and Hunt, 1974; Negishi et al., 1982). The rapid paling response of medaka may be controlled by the release of catecholamines such as noradrenalin (Bagnara and Hadley, 1973; Fujii and Oshima, 1986). If MSH functions as a hormonal regulator of melanosome dispersion in medaka, it would be expected to act at very low concentrations as shown in the present result, accompanied by a decrease of catecholamines, as the darkening response occurred. It is 100
2 C
.-0 ul G .4 u
50
:
OL
, 5 Time
I 10 (mid
6. EP I-enhanced melanophore-stimulating activity of B-MSH I. (0) EP I 10-i’ M + B-MSH I 10-l’ M; (0) B-MSH I 10-l’ M; (A) EP I 10-l’ M. Each point represents the mean + SE of results from at least 21 cells. FIG.
FOR
131
MSH
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