2000-Fujii The regulation of motile activity in fish chromatophores.pdf

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Copyright Pigment Cell Res 2000PIGMENT CELL RES 13: 300319. 2000Printed in Irelandall rights resered ISSN 0893-5785

Review

The Regulation of Motile Activity in Fish Chromatophores

RYOZO FUJII1

Department of Biomolecular Science, Faculty of Science, Toho Uniersity, Miyama, Funabashi, Chiba 274-8510, Japan

*Address reprint requests to Dr. Ryozo Fujii, 3-22-15, Nakaizumi, Komae, Tokyo 201-0012, Japan. E-mail: [email protected]

Received 3 March 2000; in final form 31 March 2000

times, they are also useful in courtship and mutual communi-Chromatophores, including melanophores, xanthophores, ery-

throphores, leucophores and iridophores, are responsible for cations among individuals of the same species, leading to an

the revelation of integumentary coloration in fish. Recently, increased rate of species survival. Such strategies are realized

by complex mechanisms existing in the endocrine and/orblue chromatophores, also called cyanophores, were added to

nervous systems. Current studies further indicate that somethe list of chromatophores. Many of them are also known to

paracrine factors such as endothelins (ETs) are involved inpossess cellular motility, by which fish are able to change their

these processes. In this review, the elaborate mechanismsintegumentary hues and patterns, thus enabling them to exe-

cute remarkable or subtle chromatic adaptation to environ- regulating chromatophores in these lovely aquatic animals are

described.mental hues and patterns, and to cope with various ethological

encounters. Such physiological color changes are indeed cru-

cial for them to survive, either by protecting themselves from Key words: Melanophore, Erythrophore, Xanthophore, Leu-

cophore, Iridophorepredators or by increasing their chances of feeding. Some-

blue chromatophores in callionymid fish, naming them

cyanophores (5). Thus, six kinds of chromatophores are

now known in poikilothermic vertebrates. Various combina-

tions of these chromatophore species in various proportions

realize various hues in certain regions of the integument,

thus enabling animals to adapt to environmental conditions

for their survival (2).

In order to effect such chromatic strategies, poikilother-

mic animals also make good use of the cellular motile

activities of pigment cells. Namely, the rapid physiological

color changes have elaborately evolved during the long

history of evolution. The colorations and color changes,

thus obtained, constitute critically important strategies to

avoid attack by predators and to obtain prey more easily for

survival. On many occasions, furthermore, delicate and

subtle changes in hues and patterns, thus realized, are usedfor communication with conspecifics. These phenomena are

especially remarkable in bony fish. The extraordinarily so-

INTRODUCTION

We joyfully appreciate beautiful colors and patterns dis-

played by many species of animals. Such integumentary

colors are dependent on the presence of pigment cells in

the skin (13). We know that in homeothermal verte-

brates (mammals and birds), melanocytes producing

melanin are the sole pigment cells responsible for their

coloration. By contrast, various types of pigment cells, as

well as pigmentary substances, are involved in the col-

oration of lower animals that include poikilothermal verte-

brates and invertebrates. These pigment cells have

inclusively been called chromatophores. If we deal solely

with vertebrates, at least five kinds of chromatophores are

present, namely, melanophores (black or brown), xan-

thophores (ocher or yellow), erythrophores (red), leu-

cophores (whitish), and the iridophores (metallic or

iridescent). This nomenclature is now widely accepted,which the present author has also endeavored to establish

for a long time (3, 4). In addition, we recently discovered

Abbreiations ACTH, adrenocorticotropic hormone; ET, endothelin; MC, melanocortin; MCH, melanin-concentrating hormone; MC-R,MC receptor; MSH, melanophore-stimulating hormone; MT, melatonin; MT-R, MT receptor; NAT, N-acetyl transferase; NE, nor-epinephrine; PG, prostaglandin; POMC, proopiomelanocortin; PRL, prolactin; SL, somatolactin1 R. Fujii is now at 3-22-15, Nakaizumi, Komae, Tokyo 201-0012, Japan.

Pigment Cell Res. 13, 2000300


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phisticated properties of their chromatic systems that we

now observe have certainly developed during evolution of

more than 400 million years (2).

The motile activities of chromatophores are dependent on

the intracellular presence of motor-proteins, namely tubulin,

dynein and kinesin. Current understanding about the cellu-

lar motility per se of vertebrate chromatophores has been

reviewed elsewhere (3, 6, 7).

It has been a fairly long time since we have published a

review relevant to the present title (8). On this occasion

therefore, the author tried to outline his views on the current

status of studies concerning the regulation of the motileresponses of fish chromatophores.

REVELATION OF COLORS

In homeothermal vertebrates, melanin-containing organelles

(melanosomes) are synthesized in melanocytes that reside in

the basal layer of the epidermis, and are transferred into

epidermal cells. The darkness of the skin is responsible for

the absorption of light by these recipient cells for the most

part. As a group of poikilotherms, teleosts possess

melanophores as homologues of melanocytes. Like

melanocytes, they are dendritic cells, but extend a number of

cellular projections almost parallel to the plane of the skin.

In teleosts, melanophores are mostly found in the dermis

and are often called dermal melanophores. Sometimes, how-

ever, melanophores are also found in the epidermis, but the

melanosomes are mostly kept confined within the cells, and

aggregate into the perikaryon or disperse throughout the

cytoplasm in response to various signals, as do dermal

melanophores (3).

In many species of fish, melanophores take principal part

in physiological color changes, but there also exist other

kinds of dendritic chromatophores in the skin, i.e. xan-

thophores, erythrophores, cyanophores and leucophores.

Pigmentary organelles contained within them are now called

xanthosomes, erythrosomes, cyanosomes and leucosomes,

respectively (3, 5), and are inclusively called chromatosomes.Excepting for the light-scattering leucosomes, they are light-

absorbing. The melanosomes effectively absorb light rays

within the entire range of visual spectrum, but other chro-

matosomes absorb rays of complementary color to that the

cells exhibit. Leucosomes, by contrast, scatter light rays of

wider wavelengths. Thus, leucophores look whitish when

illuminated by incident light (3).

Although very commonly existing in whitish or silvery

parts of the skin, iridophores are rather peculiar chroma-

tophores, because they are usually non-dendritic and do not

contain colored organelles (3). Instead, stack(s) of transpar-

ent thin crystals of guanine are present in the cytoplasm.

The thin crystals are called reflecting platelets, since they

are strongly light-reflecting owing to their very high refrac-

tive index (of no less than 1.83). Within a stack of them,

higher reflectivity can be achieved as a result of the multiple

thin-film interference phenomenon. As for detailed descrip-

tions about the optics of iridophores, our previous articles

can be referred to (2, 3). In iridophores that are responsible

for silvery glitters and whiteness of side and belly skin, the

platelets are arranged in a stack, to exhibit the multi-layer

thin-film interference phenomenon of the ideal type. Such

iridophores are immotile cells, and are not directly involved

in the physiological color changes.

By contrast, iridophores in some teleostean species have

cellular motility, which plays a predominant role in their

fascinating color changes (3). These iridophores contain

stacks of very thin platelets, and in a given stack, the

distance between platelets is very uniform. Simultaneous

changes in the distance between platelets in a stack result in

changes in the light-reflecting characteristics. Naturally, the

optical treatment of the multi-layer interference system

should be far from that of the ideal system. When thedistance increases, the motile iridophores reflect light of

longer wavelengths. When the spacing between the platelets

decreases, conversely, the spectral peak shifts towards

shorter wavelengths. The former response was designated

the LR response, being an abbreviation of the Longer-

wavelength light-Reflecting response, while the latter one is

called the SR response, an abbreviation for Shorter-wave-

length light-Reflecting response (3, 9). In later sections,

these terms will frequently be employed to describe the

reaction of motile iridophores.

Motile iridophores with dendritic processes have recently

been described in some gobiid fish, including the dark

sleeper goby Odontobutis obscura obscura (10). As with

iridophores of many amphibians, reflecting platelets aggre-

gate into the perikaryon or disperse to dendritic processes in

response to neural or hormonal stimuli (1). When the

platelets aggregate in the perikaryon, the cells appear bluish

in color. However, the same cells look yellowish when the

platelets are dispersed. The bluish tone is considered to be

due to the gradual formation of organized piles of platelets

during their aggregation (11).

Each chromatophore is a small entity, usually containing

a single kind of pigmentary material or stack(s) of light-

reflecting platelets. When differently colored chroma-

tophores are distributed in the skin, the resulting color

appears to be a mixture of different colors. By making good

use of the divisionistic effects, the fish can exhibit a numberof intermediate hues almost at will (2, 3). Although simpler

than those in anuran skin (1), dermal chromatophore units

are found in the skin of colorful specimens such as bluish

damselfish (2, 3).

Under the epidermis of fish, there are wide extracellular

spaces of rather uniform thickness, composed mainly of

collagen fibrils. The dermal chromatophores are usually

present below this compact collagenous layer, and are not in

direct contact with the bottom of the epidermis. Parallel

collagen fibrils form a thin sheet, and several sheets are

arranged as lamellae, but the fibrils within alternating sheets

run approximately perpendicular to those in adjacent ones.

Resembling plywood, the lamellar structure apparently rein-

forces the thin integument, and protects underlying fragile

chromatophores (3, 12). The laminated collagenous struc-

ture can also be assigned another important role since its

architectural features closely resemble those of the stroma

(substantia propria) of the vertebrate cornea. The latter, of

course, is extremely transparent to light, in addition to its

mechanical rigidity. The attained transparency of the struc-

ture overlying the chromatophores must be of great impor-

Pigment Cell Res. 13, 2000 301


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tance for animals in executing effective chromatic responses

(2, 3, 12).

When animals are on land or in the air, the light reflectiv-

ity at the very surface of the body covering may not be less

than 2.4%, while that for animals in the water can be

calculated to be 0.022% (13). Those values were based on

the assumption that the refractive index of the body surface

material is 1.37, the value being adopted from that represen-

tative of the cytoplasm of living cells. Being normally kera-

tinized, squamous or cuticulized, the coverings of terrestrial

animals may have refractive indices higher than 1.37, and

thus, the light reflectivity should be somewhat higher thanthe value given above. The strikingly smaller value for

aquatic animals reflects the fact that the uppermost epider-

mal cells are normally unkeratinized and alive. In this way,

the reflectivity at the body surface of an aquatic animal is

practically negligible. Under such morphological situations,

the colors due to the states of chromatophores are clearly

visible from the outside.

Working on the ice goby, Leucopsarion petersii, Goda and

Fujii (13) reported a special case of the role of

melanophores in the color revelation. As the common name

signifies, even adult specimens of this fish are transparent,

but a small number of melanophores and xanthophores

were found in the skin. In addition, very large melanophores

exist deep inside the body, namely in the peritoneum and

near the vertebrae. They are clearly visible from outside, and

are responsive to various agents. Apparently these

melanophores do not belong to dermal cells, but have

definite roles in the chromatic responses.

ENVIRONMENTAL FACTORS THATDIRECTLY INFLUENCE CHROMATOPHORES

Several physical factors, and sometimes chemical ones, from

the environment affect chromatophores. Most such stimuli

are perceived by sense organs and are brought to the central

nervous system, where the information is processed to yieldappropriate chromatic reactions from the animals. Some

factors, however, directly influence chromatophores. We

have reviewed many of these in a recent article (14), and in

the present article therefore, recent results of interest are

mainly dealt with.

Direct Effects of Light on Chromatophores

Physiological color changes in animals are frequently cate-

gorized into two types (15). One type is the so-called pri-

mary color response, in which chromatophores respond

directly to incident light. The other type is the secondary

color response, in which the chromatophores are controlled

by the nervous and/or endocrine systems. The primary color

responses are mainly observable during the embryonic and

larval stages until the time when chromatophores are not yet

under the control of endocrine and/or nervous systems. It

has often been observed that when chromatophores are

denervated, or when a blinded or a blindfolded fish is

examined, even normal chromatophores respond to light

directly.

Using melanophores from embryos, larvae or young black

platyfish, Xiphophorus maculatus, Wakamatsu (16) reported

that some melanophores in culture responded to light by

aggregating melanosomes, although all the melanophores

were initially light-insensitive. The spectral sensitivity peak

stood at about 410 nm (17). By contrast, melanophores

from larvae of the rose bitterling, Rhodeus ocellatus, re-

sponded to light by dispersing melanosomes, whereas the

melanosomes aggregated in the dark (18). The effective

wavelength of the light was around 420 nm (19). Observing

the responses of melanophores on scales plucked from adult

dark chubs (Zacco temmincki), Iga and Takabatake (20)found that the light dispersed pigment by acting directly on

the cells, although the sensitivity differed among individuals.

Using the melanophores of adult medaka, Oryzias latipes,

that had been cultured for more than 1 day, Negishi (21)

confirmed the direct responsiveness of the melanophores to

light. The most effective wavelength for the induction of

melanosome dispersion in medaka was close to 415 nm,

while melanophores of dark chubs showed a maximum

spectral sensitivity at about 525 nm (22).

Chromatophores other than melanophores have also been

studied for their responsiveness to light: for example, the

leucophores of Oryzias responded to light by dispersing

their light-scattering inclusions (23). Motile iridophores in

the lateral stripes of the neon tetra, Paracheirodon innesi,

show the LR response to light (24, 25). Xanthophores in

adult specimens of medaka were also found to respond to

light by xanthosome aggregation, and the effective wave-

length was around 400 nm (26). While examining the effect

of light on adult Oryziaschromatophores, Oshima et al. (27)

recently found that both innervated and denervated xan-

thophores responded to light (9000 lx) within 30 s by

pigment aggregation, and that the response was not medi-

ated through -adrenoceptors. The maximum spectral sensi-

tivity was about 410420 nm, and the effect was reversible.

Responsiveness was higher in summer than in winter and

Ca2+ ions and calmodulin were not involved in the re-

sponse. Their conclusion was that photoreception by visualpigment that absorbs light at 410420 nm increases phos-

phodiesterase activity, resulting in a decrease in cytosolic

cyclic AMP levels, finally leading to the xanthosome

aggregation.

Using the Nile tilapia, Oreochromis niloticus, Oshima and

Yokozeki (28) recently reported that either innervated or

denervated erythrophores responded directly to light of

defined wavelengths by pigment aggregation or dispersion.

In spectral regions between 400 and 440 nm and also

between 550 and 600 nm, erythrosomes aggregated, whereas

their dispersal was accelerated around 470530 nm. These

results suggest the coexistence of three kinds of visual

pigments in tilapia erythrophores.

Other Physical Factors

Some environmental factors other than light influence chro-

matophores either indirectly or directly, but because of their

relatively low importance, such factors have only rarely been

investigated, and accordingly, data are rather scanty. We

consider, however, some of them to be menaces to fish. For



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example, UV rays may injure pigment cells and impair the

activity of nerve fibers that control cellular responses, espe-

cially for fish living in shallow waters. Hydrostatic pressure

should have influences on deep-sea fish, especially when they

show diurnal vertical migration for feeding. Low tempera-

tures normally reduce cellular motility. Osmolarity and pH

of the water in which they dwell should be other relevant

physical factors; for example, fish that migrate between

inland and sea waters must face drastic changes in osmolar-

ity. If we consider that homeostatic mechanisms are func-

tioning in vivo, the internal milieu around chromatophores

may not be directly influenced by them. When needed, wecan experimentally examine the effects of these factors, and

the results of such studies have actually provided important

knowledge about the physiology of pigment cells (14). Since

many of these factors have recently been reviewed (14), they

will not be further discussed here.

Chemical Factors

That some environmental chemical substances directly affect

chromatophores seems to be unlikely, because, unlike other

cells constituting the body, chromatophores are rigidly pro-

tected from the invasion of chemicals. Being different from

terrestrial animals, where layers of keratinized cells cover

the body, part of the living cell membrane, that directly

faces the environmental watery phase of the outermost

epidermal cells and the occluding junctions between those

cells, functions as a diffusion barrier in fish.

Fish possess various chemosensory organs for feeding and

reproduction (30). The perceived chemical information is

integrated in the central nervous system to arouse certain

ethological responses, as in the cases of other sensations. It

is known, however, that some chemicals, as solutes in the

water surrounding the animals, can be taken up, affecting

the chromatophores directly. The most interesting instance

may be melatonin (MT). Immersing pencilfish (Nannostomus

beckfordi) in aquarium water containing MT, Reed (31) first

observed the phenomenon, and further developed a biologi-cal assay for MT. We have also been able to observe the

effects of MT by immersing fish in MT-containing water for

analysis of circadian chromatic responses, as well as for

characterization of MT-receptors (R) (3234). Owing to its

high lipid solubility, MT can affect the state of chroma-

tophores by invading the body rather easily, probably

through the gill epithelium. By selecting less polarized

molecular species, we may be able to study the effects of

various substances on chromatophores in vivo.

Considering that signaling mechanisms, both in odor

perception by the olfactory epithelium and in chroma-

tophores, are commonly G protein-coupled, Karlsson et al.

(35) recently examined the in vitro effects of odorants on

melanophores of the cuckoo wrasse, Labrus ossifagus.

Among some odorants tested, cinnaldehyde and -ionone

were found to have melanosome dispersing actions. Later,

Lundstrom and Svensson (36) actually tried to use

melanophores on a Labrus scale for odor sensing. Although

odorant molecules are relatively nonpolar, whether they can

penetrate the skin to influence chromatophores in vivo still

remains to be tested.

HORMONAL REGULATION OFCHROMATOPHORE MOVEMENT

Information perceived by lateral eyes and other sense organs

is transferred via the optic nerve to the central nervous

system, where it is integrated to yield adequate adaptive

chromatic reactions via endocrine, paracrine and neural

routes.

A number of principles are involved in the regulation of

chromatophore motility in fish. In order to facilitate the

understanding of the system for regulating chromatophores

therefore, consider the scheme shown in Fig. 1. This dia-

gram was drawn primarily to demonstrate the systems con-

trolling dendritic chromatophores of the light-absorbing

type that include melanophores, xanthophores and ery-

throphores. The diagram may also be practically applicable

to novel blue chromatophores (cyanophores) (5), although

certain modifications may be needed. On the other hand,

because of the different optical properties the regulatory

systems for light-scattering or reflecting chromatophores are

naturally somewhat different from those for light-absorbing

chromatophores. Therefore, although some parts are quite

analogous, the above diagram cannot be applied as it stands

to control systems for leucophores or motile iridophores.

Nuclear receptors have sometimes been shown to be

involved in the control of pigmentation in fish, but theireffects are always on morphological color changes (3, 4). It

may be pointed out here that cell-surface receptors are

exclusively concerned with systems controlling physiological

color changes, except in the case of nitric oxide (NO), which

will be briefly touched upon later.

Requiring complicated analyses, studies on mechanisms

regulating the production and release of pigment-motor

hormonal substances still remain to be investigated for the

most part, and therefore, the author did not try to review

those herein. With reference to outcomes from other fields

of studies, such as on mammals, amphibians, etc., the

mechanisms may hopefully be elucidated in the near future.

In this section therefore, the roles played by several hor-

monal principles that affect chromatophores areenumerated.

Melanophore-Stimulating Hormone

Among several hormonal principles known to control fish

chromatophores, melanophore-stimulating hormone (MSH)

produced by the intermediate lobe of the pituitary must be

the most widely known. Some readers, especially those who

are working in medically oriented fields, may wonder why

the term melanocyte-stimulating hormone is not employed

here. As noted previously, the term melanocyte is not

popularly employed by zoologists who are working with

poikilothermal animals, and instead, melanophore has long

been the common expression among them. Consequently,

the term has been cut in the hormones designation. In fact,

MSH induces very rapid dispersion of melanosomes within

melanophores (physiological color changes), in addition to

its other role in morphological color changes, i.e. stimulat-

ing the proliferation of melanophores and melanization

within them (1, 4). In any case, the effects of MSH are more

remarkable on melanophores than on melanocytes. Fortu-



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nately, the abbreviated form of the hormone, MSH, is

common, and thus we have practically no trouble in using

two different expressions in zoological and medical fields.

Even at the present time incidentally, MSH has still been

called melanotropin or intermedin rather frequently. In

general, the former has been used to indicate more inclu-

sively the peptide hormones that affect pigmentation, even

of invertebrates. It is thus desirable to rearrange the relevant

terms in order to avoid confusion.

Among molecular species of MSHs, -MSH (an acetyl-

tridecapeptide amide) is believed to have a major role both

in the regulation of chromatophores in lower vertebratesincluding fish (Fig. 2), and in melanocytes in homeotherms.

Namely, the structure of MSH may have been conserved for

a long time since the emergence of vertebrates. Among

rather primitive fish, somewhat modified peptides have been

reported, although we are still unaware that such structures

are the ancient forms of -MSH or not. To date, some

molecular species of-MSHs have also been reported (Fig.

2). As to whether the -forms are functional in color

changes in vivo, further study is needed. All MSHs are now

understood to be derived from a multi-functional precursor

called proopiomelanocortin (POMC).

A vast number of earlier studies on the action of MSH on

fish chromatophores was initially reviewed by Pickford and

Atz (37), and later, Fujii (4) and Fujii and Oshima (8)

summarized more recent work. Visconti et al. (38) recently

reported that -MSH effectively disperses pigment in

melanophores of an elasmobranch fish, using the skin of the

freshwater ray, Potamotrygon reticulatus. The actions of

MSH are not restricted to melanophores: The peptide has

frequently been reported to disperse xanthosomes and ery-

throsomes in bright-colored chromatophores in teleosts (8,

37, 3941).

Recently, studies on motile iridophores have made much

progress (3): It was shown that those of the blue damselfish

type and of the neon tetra type responded to -MSH by theSR response, but only when very strong solutions were

applied (9, 25). In the blue damselfish (Chrysiptra cyanea),

they were completely irresponsive (42). Motile iridophores

of the dendritic type, existing in some gobiid fish, responded

to MSH by aggregation of light-reflecting platelets (43, 44).

Apparently, such responses contribute to the darkening of

skin. Concurrent responses to MSH of light-absorbing chro-

matophores and iridophores function cooperatively to real-

ize effective dark-to-pale (and reverse) changes in the skin.

Usually, the direction of responses of light-absorbing

chromatophores, comprising of melanophores, xan-

thophores and erythrophores, and that of light-scattering

chromatophores, i.e. leucophores, are reciprocal (3, 8). For

Fig. 1. Diagram showing the regulatory system for motile activities of melanophores and other light-absorbing chromatophores in teleosts.Explanations for abbreviations in the figure are arranged in order from left to right. -A-R, -adrenoceptor; NE, norepinephrine; mACh-R,muscarinic acetylcholine receptor; ACh, acetylcholine; MCH-R, MCH receptor; Epi, epinephrine; ATP, adenosine 5-triphosphate;-MT-R,-MT receptor; PRL cell, prolactin-producing cell; MCH, melanin-concentrating hormone; -ET-R, -ET receptor; AL, anterior lobe ofhypophysis; cAMP, cyclic adenosine 3,5-monophosphate; cGMP, cyclic guanosine 3,5-monophosphate; IP3, inositol-1,4,5-trisphosphate;PRL, prolactin; AS-R, adenosine receptor; MSH cell, MSH-producing cell; PIH cell, PRL-release inhibiting hormone-secreting cell; IL,intermediate lobe of hypophysis; PRL-R, PRL receptor; MIH cell, MSH release-inhibiting hormone; MC-R, melanocortin receptor; -MSH,-melanophore-stimulating hormone;-A-R, -adrenoceptor; PL, posterior lobe of hypophysis; ET, endothelin; MT, melatonin; MCH cell,MCH-producing neuron in hypothalamus; -ET-R, -ET receptor; -MT-R, -melatonin receptor; NO, nitric oxide.



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Fig. 2. Amino acid sequences of MSHs and MCH determined hitherto from fish [modified from Fujii and Oshima (8)].

example, sympathetic cues signal the aggregation of pig-

mented chromatosomes and the dispersion of light-reflectingleucosomes. Rather unexpectedly however, leucophores of

the medaka O. latipes responded to -MSH by leucosome

dispersion (40). Namely, in Oryzias the direction of leu-

cophore response is similar to that of the light-absorbing

chromatophores, the phenomenon being rather paradoxical.

Such seemingly odd processes may have evolved in order to

realize the delicate skin hues and patterns required for

adaptation to environmental conditions.

The action of MSH on fish melanophores has been shown

to be mediated by receptors that are specific to the peptide

(3, 8, 45). It was shown that MSH receptors require extra-

cellular Ca2+ ions for their action on melanophores (46).

Working on Oryzias xanthophores and leucophores and on

Xiphophorus erythrophores, Oshima and Fujii (41) further

showed that the peptide does not act to disperse chromato-

somes unless the bathing medium contains Ca2+ ions. It is

interesting that, among a number of hormonal and neural

substances signaling motile responses of fish chroma-

tophores, MSH is the only one that requires the presence of

extracellular Ca2+ ions. Those ions are probably required

for formation of the complex between the MSH molecule

and the regulatory subunit of the receptor.

Responses of motile iridophores of the dendritic type of

the dark sleeper goby to other signaling molecules, such as

NE, are analogous to Oryzias leucophores (43, 44), and the

principal second messenger is thought to be cyclic AMP. In

these iridophores therefore, MSH may signal platelet aggre-gation by decreasing adenylyl cyclase activity resulting in

the decreased levels of cAMP. This may be an unusual mode

of action for MSH.

On the basis of their functions, we are now urged to

classify MSH receptors into two large groups. To date, the

categorization of adrenoceptors into - and -forms has

already been established. Namely, the nucleotide-cyclase

inhibiting receptors are prefixed by , while those activating

the enzyme are designated . According to that principle,trials have already started to subclass receptors mediating

motile responses of chromatophores, such as those for MT

(32), and others including melanin-concentrating hormone

(MCH) and endothelins (ETs; cf. relevant sections in this

article). In the case of MSH receptors, the same yardstick

can not be applied unfortunately, because agonistic

molecules have already been endowed with the names of-

or -MSH. However, it might be possible to use -MC-R

and -MC-R for this purpose (see Fig. 1). In any case, the

above-mentioned novel MSH receptors of Odontobutis

melanophores should be treated using a different term when

we need to distinguish them from the conventional MSH

receptors.

ACTH

Although its role in physiological color change has not yet

been established, adrenocorticotropic hormone (ACTH) has

also been shown to be melanosome-dispersing (3, 4). This

fact is understood when we recall that the ACTH molecule

includes the amino acid sequence of MSH (Fig. 2). Other

brightly-colored chromatophores respond to ACTH as well.

For example, chromatosomes in xanthophores of the mud-

sucker gobyGillichthys mirabilis (47) and of the goldfish (48)

disperse in response to the peptide. Erythrophores in cul-

tures of the swordtail Xiphophorus helleri respond similarly

(39).

The receptor mediating the action of MSH has long been

called the MSH receptor. In the endocrinology of

homeotherms, the term MCn-R, where MC is the abbrevia-

tion of melanocortin accompanied by an Arabic number,

has become widely employed to express both the receptors

for MSH and ACTH. Since the cloning of the correspond-

ing receptors in poikilothermal vertebrates has not yet been

fruitful, such expressions have not yet become popular. We



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presume that in the near future nomenclature for related

receptors may be revised, in view of a more firm standpoint.

Prolactin

Prolactin (PRL), another peptide hormone produced by the

anterior lobe of the pituitary, was first shown to affect

chromatophores by Sage (47), who detected its ability to

disperse pigment in xanthophores of the mudsucker

Gillichthys, with resultant yellowing of the fish. Using two

highly purified molecular species of PRL from the Mozam-

bique tilapia Oreochromis mozambicus (tPRL177 andtPRL188), Oshima and her associates examined their effects

on chromatophores of the Nile tilapia, O. niloticus, and

recognized that the peptides had little, if any, melanosome-

aggregating effects on melanophores, but that tPRL177 had

the distinct action to disperse pigment in xanthophores (49).

They further investigated the chromatosome-dispersing ef-

fects of tPRL177on xanthophores of the Nile tilapia and the

rose bitterling (Rhodeus ocellatus ocellatus), and on ery-

throphores of tilapias, swordtails (X. helleri) and paradise

gobies (Rhinogobius giurinus), and were able to further

detect seasonal changes in the responsiveness of ery-

throphores to the hormones. Based on these observations,

they concluded that the enhanced PRL action on ery-

throphores in the breeding season must be deeply involvedin expressing nuptial coloration (50). Dispersion of chro-

matosomes may be linked to the synthesis of brightly-col-

ored pigments, namely, their sparse distribution within the

perikaryon may release the Golgi-endoplasmic reticulum

system to synthesize more chromatosomes, by unfastening

the product inhibition, which would result in the generation

of the conspicuous hues for courtship.

As mentioned above, PRL seems to have rather limited

effects on teleostean melanophores (50), but Visconti et al.

(38) recently reported that PRL darkens the skin of a

freshwater ray (P. reticulatus) effectively, suggesting its ac-

tive role in elasmobranch coloration, although further com-

parative examinations are needed.

Somatolactin

Somatolactin (SL) is a novel teleostean pituitary hormone

belonging to the growth hormone-prolactin (PL) family

(51). Various molecular forms have already been cloned,

which have more than 200 amino acids (52). Using the red

drum, Sciaenops ocellatus (Sciaenidae), Zhu and Thomas

(53) found that the increase of SL in the plasma is associ-

ated with the aggregation of melanophore inclusions. How-

ever, their results to date are rather confusing, necessitating

further analyses for establishing SLs participation in

pigmentation.

Melanin-Concentrating Hormone

The presence of a hormone antagonizing the action of MSH

had long been a matter of controversial opinion. Strong

suggestion of the hypothalamic origin of such a principle

was first presented by Enami (54), who named it

melanophore-concentrating hormone (MCH). As a neu-

rosecretory hormone, it is transferred from the hypothala-

mus to the posterior lobe of the pituitary from which it is

secreted (55). Baker and her colleagues tried to characterize

it (56), and finally Kawauchi et al. (57) succeeded in isolat-

ing it from the pituitary glands of the chum salmon

Oncorhynchus keta. It is a cyclic heptadecapeptide with a

disulfide bond (Fig. 2), and it is now called melanin-con-

centrating hormone, because what concentrates are not

melanophores, but melanin-carrying organelles.

Nagai et al. (58) reported that motile melanophores of all

teleostean species they tested responded to MCH by aggre-

gation of melanosomes, as the name implies. The action of

MCH is mediated by a specific receptor (5961). It shouldbe emphasized, however, that the definite action of MCH

has been shown only in teleosts: In amphibians and reptiles,

melanophores responded to that hormone by dispersing

melanosomes, and the sensitivity was much lower than that

in fish (62). The biological significance of MCH in eliciting

color changes in lower vertebrates has recently been well

documented by Baker (63) who naturally devoted much

space about its action on fish chromatophores.

Chromatophores other than melanophores responded

similarly to MCH (14, 60, 61). For example, Oshima et al.

(60) showed that swordtail erythrophores and medaka xan-

thophores responded well to MCH by chromatosome aggre-

gation. Motile iridophores of the blue damselfish,

Chrysiptera cyanea, were among the few instances of chro-

matophores that are refractory to MCH (64), those iri-

dophores being regulated solely by nerves.

In contrast, light-scattering organelles in leucophores of

medaka dispersed in response to MCH, but much higher

concentrations of the hormone were needed (60). Further, in

contrast to its pigment aggregating action, extracellular

Ca2+ ions were needed, as for the melanosome-dispersing

action on amphibian melanophores. Thus, it was once

thought that the pigment-dispersing action of MCH might

be mediated by MSH receptors.

Castrucci et al. (61) examined the action of MCH on

melanophores of the Brazilian eel (Synbranchus marmora-

tus), and reported that at lower concentrations it aggregatedmelanosomes, whereas at higher concentrations it dispersed

them. Applying higher concentrations of MCH to

melanophores of the mailed catfish Corydoras paleatus and

the Nile tilapia, O. niloticus, Oshima and her associates also

observed that the melanosome aggregation was followed by

re-dispersion, and that Ca2+ ions were necessary for the

latter process (65, 66). As mentioned above, MSH receptors

require external Ca2+ for their action, and therefore, a

dense population of MSH receptors on the cell membrane

might have been concerned with this process. An alternative

explanation was recently put forward by Oshima who as-

sumes that there are two types of receptors for MCH (65)

that exist commonly on melanophores, medaka xan-

thophores and swordtail erythrophores. The first type of

MCH receptor would mediate pigment aggregation at phys-

iological concentrations, while those of the other type of

MCH receptor on the membranes of medaka leucophores

and of amphibian melanophores would mediate dispersion

of pigment, but only when the agonist concentration is very

high, and would require extracellular Ca2+ ions. In

melanophores of the Brazilian eel, both types of receptors



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9/20

PARACRINE FACTORS

In higher vertebrates, several paracrine factors have been

shown to regulate the physiological responses of effector

cells. Such paracrine systems may also be operating in lower

animals, because they seem be the most primitive means of

communication among cells. In fish, such factors might

include prostaglandins (PG), angiotensin II, ETs,

bradykinin, somatostatin, and other neuropeptides, includ-

ing intestinal hormones, etc. To date, however, few reports

have appeared that demonstrate such processes in the chro-

matic systems of fish.

Opioid peptides

Opioid receptors are present in the brain, as well as in the

peripheral tissues, of vertebrates. Since they have been

shown to inhibit liberation of transmitters from nerve termi-

nals, similar roles of these neuropeptides in modulating the

primary effects of endocrine or nervous cues of chroma-

tophores could be expected.

Suggesting a possible role of opioid peptides in the secre-

tion of MSH, Satake (79) demonstrated that an intracranial

injection of naloxone, a specific inhibitor of opiate recep-

tors, induced aggregation of pigment in goldfish xan-

thophores. The effect was antagonized by

methionine-enkephalin (met-E). Next, Levina and Gordon

(80) showed that melanophores and xanthophores of ze-

brafish (Brachydanio rerio) responded to MSH and to met-E

by chromatosome dispersion, and that the effect of met-E

developed later and faded more slowly. Naloxone inhibited

the action of met-E, and the involvement of a central

mechanism was suggested in the met-E-induced darkening

of the skin. Recently, Carter and Baker (81) reported that

either the pars distalis or the neurointermediate lobe of the

pituitary actually contains substantial opiate activity. To

date, however, little information is available about the role

of opioid peptides in regulating chromatophores in fish.

Eicosanoids

Among physiologically active eicosanoids, PGs are of much

interest, because they are regarded to be important factors

in modifying the regulation of hormonal, as well as neural

signaling to effector cells. In fact, they have frequently been

shown to influence activities of various autonomically regu-

lated effectors via paracrine signaling. As early as 1974,

Abramowitz and Chavin (82) noted that PGs elicited disper-

sion of pigment in melanophores of black goldfish in vitro.

Further investigations along this line, however, have been

unexpectedly meager. It is therefore, desirable to know

whether these and related fatty acid derivatives take part in

modulating chromatophore responses.

Endothelins

It has recently been shown that human keratinocytes pro-

duce ETs, which can act as strong mitogens, as well as

melanogens, for human melanocytes (83). Keratinocytes and

adjacent melanocytes may form the paracrine linkage for

ET. Working on teleostean fish, Fujii and his associates

found that ET induced motile responses of most chroma-

tophores in the teleosts examined (84), and that their actions

were dose-dependent. The pharmacological properties of ET

receptors possessed by melanophores (85), erythrophores,

xanthophores (86), and motile iridophores (unpublished ob-

servations) resemble those of ETB described in mammalian

tissues. The direction of responses to ET of these chroma-

tophores coincides with that of the responses to sympathetic

stimuli via -adrenoceptors. In addition to cyclic AMP,

inositol 1,4,5-triphosphate (IP3) has already been found to

work as another second messenger mediating the aggrega-

tion of pigment, at least in some chromatophores (87, 88).

Therefore, the process of signaling in the response to ET ofthese chromatophores might be analogous to those disclosed

in mammalian tissues, including human melanocytes (89).

ETs, by contrast, disperse leucosomes in leucophores of

the medaka, O. latipes (90). The pharmacological properties

of ET receptors of leucophores resemble mammalian ETB,

as in other chromatophore species of fish. On the other

hand, Lerner and his associates (91), while working on

melanophores of the African clawed toad Xenopus laeis,

reported that ET dispersed melanosomes mediated by ETCreceptors. The direction of the pigmentary response to ET

was identical to that in Oryziasleucophores, but opposite to

that observed in most teleostean chromatophores (3, 84

86). Lerners group (92) also reported that an increase in the

cytosolic levels of IP3 correlated with melanosome disper-

sion in Xenopus melanophores, which in terms of the direc-

tion of melanosome displacement, was quite opposite to that

reported by us in fish (87). The involvement of IP3in motile

responses of leucophores has not yet been studied. In con-

sideration of past results on the common roles of second

messengers in teleost chromatophores (3, 14), however, it is

likely that IP3 also mediates the aggregation of leucosomes.

Namely, ET receptors of leucophores might mediate the

dispersion of leucosomes via decreases in the intracellular

levels of IP3. Thus, ET receptors of leucophores are quite

different from Xenopus ETC, and also from those of other

kinds of chromatophores of teleosts examined to date. Ten-

tatively, we named the ET receptors of leucophores -ETreceptors, and those of light-absorbing cells -ET recep-

tors. The adoption of the prefixes and is based on the

terminology of some pigment-motor substances that have

reciprocal actions on chromatophores, as touched upon

previously. In Fig. 1, which exhibits the general regulatory

system for motile activities of light-absorbing chroma-

tophores in teleosts, both -ET and -ET receptors are

incorporated. ET may be secreted as a paracrine factor to

modify the actions of the known nervous or hormonal

principles.

Working on an elasmobranch species (P. reticulatus),

Visconti et al. (38) recently reported that ETs were not able

to induce either skin lightening or darkening. Thus,

melanophores of this species may be unresponsive to ET.

Since ET has definite actions on teleostean chromatophores,

further comparative studies are needed in lower fish.

Imokawa et al. (83, 89) showed that in humans, kerati-

nocytes are the source of ET. Very recently, the secretion of

ET from goldfish epidermal cells in culture has been re-

ported (93), and thus, the possible source of ET for chroma-

tophore responses might be sought there. Epidermal



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11/20

Fig. 4. Diagram showing the chromatic nervous pathways frommelanosome-aggregating center to melanophores in fish. Originallydescribed in the minnow, P. laeis, by von Frisch (99).

variety of-blockers developed thereafter, many later work-

ers have come to the same conclusion (3, 8, 102). Employing

radiolabeled NE, Kumazawa and Fujii (103) actually

showed that NE is released from nervous elements in re-

sponse to nervous stimuli. Current investigations, further-

more, suggest that the firing rate of postganglionic

sympathetic fibers needed to maintain melanophores in an

intermediate state of pigment aggregation in vivo is rela-

tively low, being about 1 Hz. A higher firing rate results in

a more aggregated state, while a lower frequency, or a

cessation, of impulses causes dispersion of pigmentary or-

ganelles (101, 102).Several researchers have attempted to determine the sub-

type of -adrenoceptors on chromatophores. Some have

reported that 2-agonists are more effective than 1-ago-

nists, and that transmission is more easily blocked by 2-

blockers than by 1-blockers (104, 105). Those workers

naturally came to the conclusion that the pigment-aggregat-

ing adrenoceptors are of the a2 type, and that cyclic AMP is

functioning as a second messenger. Recently, Mayo and

Burton (106) stated that adrenoceptors possessed by

melanophores of the winter flounder, Pleuronectes (syn-

onym:Pseudopleuronectes) americanus, are mostly of the a2

subtype.

Working on melanophores of the cuckoo wrasse, L. os-

sifagus, Svensson et al. (107) recently succeeded in cloning

most 2-adrenoceptors for the first time among varieties of

receptors mediating chromatophore movements. The de-

duced amino acid sequence of the peptide sequence showed

4757% homology with human 2-adrenoceptors. Together

with data from forthcoming cloned receptors, the results

may afford important data for receptor mechanisms, as well

as for understanding the phylogenetic relationships among

species in the large class, Osteichthyes.

At least in some species the aggregation of pigment may

be triggered by an increase in levels of Ca2+ ions in the

cytosol (108 110). In addition, Fujii et al. (87) recently

demonstrated the involvement of inositol 1,4,5-trisphos-

phate (IP3) in the aggregation of pigment in tilapiamelanophores. In many different cell types, IP3 has been

shown to induce the release from intracellular storage com-

partments of Ca2+ ions into the cytosol. Moreover, we are

now aware that 1-adrenergic stimuli activate phospholipase

C, which catalyzes the production of IP3. These observa-

tions indicate that in addition to 2-adrenoceptors, 1-

adrenoceptors are functional at least in some cases. In fact,

a remarkable aggregation of pigment takes place in response

to l-agonistic stimuli, and 1-type adrenolytics always have

inhibitory effects on that process.

Chromatophores other than melanophores have also been

shown to be under the control of the sympathetic system.

For instance, erythrophores of the swordtail, X. helleri(40)

and those of the squirrelfish Holocentrus ascensionis (108)

have been shown to be under the influence of the nervous

system. Comparing the physiological characteristics of xan-

thophores with those of melanophores and leucophores on

scales of the medaka O. latipes, Iwata et al. (111) showed

that xanthophores responded in quite the same manner as

melanophores. Therefore, the nervous mechanisms con-

trolling xanthophores seem to be analogous with those of

their study (96) is plausible, further detailed examinations

are needed to present more precise neuronal connections.

Recently, Grove (97) wrote an interesting review relevant

to this subject, including several historical and rather little

known outcomes, to which readers can refer with interest.

Sympathetic Innervation

The peripheral nervous mechanism controlling fish chroma-

tophores has a long history of investigation. Earlier works

indicated that chromatophores of lower fish, including elas-

mobranches, are also under the control of the nervous

system, in addition to the hormonal regulation (15). Nowa-

days, however, they are regarded as predominantly under

the control of endocrine systems (4, 8, 38, 69, 98). In bony

fish, by contrast, a strong participation of the nervous

control of chromatophores has been shown repeatedly (3, 8,

98).

Several researchers have tried to follow the tracts of

chromatic fibers from the center. As an example, a diagram

based on earlier descriptions by von Frisch (99) on the

melanin-aggregating nervous pathways in the minnow,

Phoxinus laeis, is exhibited here (Fig. 4). This scheme is still

applicable to any teleostean species without major modifica-

tions. The diagram shown as Fig. 3 (96) is the modern

version of the von Frisch original.

Apparently, von Frisch himself anticipated the presenceof the antagonistic melanin-dispersing fibers that run

alongside the aggregating fibers, but could not observe them

physically. Later workers occasionally tried to depict the

pathways, such as that presented by von Gelei (100), who

also worked on the same species ofPhoxinus minnow, but

as already touched upon above, the presence of such fibers

has been disproven.

If electrical stimulation of nerve fibers to the skin gives

rise to motile responses of chromatophores existing down-

stream, we can safely believe that those cells are under the

control of the nervous system. As far as we are aware, most

melanophores of teleosts are innervated by such nerves, and

their mode of innervation has been analyzed (29, 101).

Since innervation to chromatophores has been thought to

be sympathetic postganglionic, the peripheral neurotrans-

mitter that signals chromatophores was justifiably supposed

to be adrenergic. Observing the effects of an adrenergic

antagonist, dibenamine, Fujii (67) first demonstrated the

adrenergic nature of transmission to melanophores.

Dibenamine is known to block -adrenoceptors, and thus,

the transmission could be regarded as -adrenergic. Using a



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melanophores. In general, however, the nervous influences

on erythrophores and xanthophores seem to be weaker

compared with melanophores.

In leucophores, nervous stimulation induces the reverse

movement, namely, the dispersion of light-scattering or-

ganelles (112, 113). In their study, Iwata et al. (111) further

showed that melanophores and the leucophores existing

nearby are under the control of the same fibers. The recep-

tors concerned are of the -adrenergic type (114, 115). Later

pharmacological analyses by Yamada (116) indicated that

those subtype of the receptors is 1. Iga (117) noticed that

under the blockade of -adrenoceptors, leucosomes aggre-gated in response to catecholamines, and concluded that the

response was mediated by adrenoceptors of the type.

Later, Morishita and Yamada (118) characterized these

receptors to be of the 2 type. It remains to be determined

whether receptors of this type actually function in vivo.

Recently, Iga and Mio (119) discovered leucophores in the

skin of the dark-banded rockfish Sebastes inermis, and

reported that adrenergic mechanisms controlling leucosome

movements are fundamentally the same as those ofOryzias.

Motile iridophores of the non-dendritic type responded to

nervous stimulation by the LR response (9, 42). In dendritic

iridophores of the goby type, platelets disperse into pro-

cesses upon nervous stimulation (10, 11).

By means of autoradiography using radiolabeled NE,

Yamada et al. (120) succeeded in visualizing the pattern of

adrenergic innervation on melanophores of the medaka O.

latipes clearly. They also demonstrated the pattern of inner-

vation to erythrophores of the swordtail, X. helleri (121).

Using medaka, Sugimoto and Oshima (122) showed that

dark background adaptation resulted in increased numbers

of melanophores and xanthophores along with denser net-

works of varicose fibers around those chromatophores, and

that reverse changes occurred in white background adapted

fish. It was further shown that, after long-term adaptation

to a white background, the responsiveness of melanophores

to NE was reduced (123). For a better understanding about

the coupling of the morphological to the physiological colorchanges, further examinations are naturally needed.

Cholinergic Transmission to Melanophores

Working on two catfish species belonging to the family

Siluridae (order: Siluriformes), Fujii and his associates

found that peripheral transmission to melanophores is

cholinergic, notwithstanding the fact that postganglionic

fibers to the effector cells are sympathetic in the usual

manner. The common Japanese catfish, S. asotus (124), and

the translucent glass catfish, K.bicirrhis(78) were the species

examined. Replacing -adrenoceptors entirely, cholinocep-

tors of the muscarinic type play an exclusive role in trans-

ducing nervous signals to the melanophores. Since they

belong to two remote genera, we presume other species in

this family may also be controlled in the same way. Surveys

have been made to examine the presence of cholinoceptors

in other catfish families within the order Siluriformes. It was

found that, in families close to Siluridae, melanophores are

often endowed with adrenergic and cholinergic receptors,

both of which mediate the aggregation of melanosomes

(125). In these fish, the neurally evoked aggregation of

pigment is mediated by -adrenoceptors, as it is in many

common teleosts. Thus, the physiological roles of these extra

cholinoceptors in those fish still remain to be solved.

Recently, Hayashi and Fujii (126) discovered that some,

but not all, melanophores of two species belonging to the

genusZacco(family: Cyprinidae, order: Cypriniformes) pos-

sess muscarinic cholinoceptors that also mediate

melanosome aggregation. That was the first report to de-

scribe the presence of cholinoceptors on chromatophores in

fish species other than those which belong to the order

Siluriformes.Making use of selective antagonists for muscarinic recep-

tors, Hayashi and Fujii (127) characterized the muscarinic

cholinoceptors possessed by melanophores of the glass

catfish,K.bicirrhis, and the mailed catfish,C.paleatus, to be

of the M3 subtype.

Until the present time, no reports have appeared about

the existence of such cholinoceptors of chromatophores

other than melanophores.

True and Co-Transmitter Interactions

It was first suggested by Fujii and Miyashita (128) that

adenosine or adenine nucleotides might take part in con-

trolling pigment dispersal in fish chromatophores. They

found that non-cyclic adenylyl compounds, which were used

as control compounds, were even more effective than cyclic

adenosine 3,5-monophosphate (cAMP) in dispersing pig-

ment in melanophores of guppies. Using guppies and silurid

catfish, Miyashita et al. (129) extended this pharmacological

analysis and came to the conclusion that the pigment-dis-

persing action of these nucleotides was mediated by

adenosine receptors since those effects could easily be antag-

onized by methylxanthines, specific blockers of adenosine

receptors.

Working on melanophores of tilapias, Kumazawa et al.

(130) detected the apparent liberation of ATP from chro-

matic nerves in response to electrical stimulation. Theyconcluded that ATP is released as a co-transmitter from

postganglionic sympathetic fibers together with the true

transmitter, NE. The concurrent release of the true transmit-

ter and co-transmitter from the fibers to chromatophores

has been confirmed in experiments with radiolabeled com-

pounds (103, 131).

The peripheral nervous mechanism, as characterized to

date, is shown schematically in Fig. 5. The true transmitter,

NE, acts to induce a rapid aggregation of melanosomes via

mediation by -adrenoceptors on the membrane. Most NE

molecules are quickly removed by being taken back up into

the nervous elements. The remainder is either removed via

the general circulation or is inactivated by catecholamine

O-methyltransferase (COMT) and monoamine oxidase

(MAO). ATP released concurrently with NE is dephospho-

rylated by ATPase and then by 5 -nucleotidase in the synap-

tic cleft. The resultant nucleoside, adenosine, survives for

some time there and functions to reverse the influence of the

true transmitter, namely, to cause the re-dispersion of pig-

ment via specific receptors for adenosine on the effector

membrane. Most of the nucleoside is finally removed by



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being taken back up into presynaptic nervous elements, and

the remainder is carried away by the circulation.

One important aspect of the proposed dual-transmitter

theory is that there is a substantial difference, in terms of

action, between the true transmitter and the co-transmitter.

The effect of NE disappears very quickly, while that of the

co-transmitter lasts longer. After the cessation of nervous

excitation, the latter can effectively reverse the action of the

former. Rapid dispersion of pigment after nervous excita-

tion is realized in this way. The rapid changes observable in

living animals may also be controlled by the same mecha-

nism. An identical explanation has been presented for theregulation of melanophores of the blue damselfish C.cyanea

(64) and the blue-green damselfish Chromis iridis (9). The

motile responses of amelanotic melanophores of medaka are

also regulated in the same way (132).

Recent studies on medaka indicate that leucophores re-

spond to adenosine by dispersion of leucosomes (133). Spe-

cific adenosine receptors of the A2 type mediate this

response. However, the direction of the movement of leuco-

somes in response to the co-transmitter is the same as that

elicited by the true transmitter. In fact, the recovery from

the effect of NE occurs very slowly.

The involvement of the dual-transmitter system in the

control of motile iridophores may be analogous to that of

melanophores. The motile iridophores of blue damselfish

(64), blue-green damselfish (9) and neon tetras (25) respond

to adenine derivatives of adenine with the SR response,

which is the opposite of the LR response elicited by -

adrenergic stimuli.

Feedback Inhibition of Transmitter Release

Using the tilapia O. niloticus, Oshima (134) succeeded in

showing that adenylyl compounds, including adenosine and

ATP, inhibit the release of adrenergic transmitter, possibly

by decreasing the rate of entry of Ca2+ ions into presynap-

tic portions of the fibers. Since these nucleotides are thought

to be released as the co-transmitter from the sympathetic

fibers (cf. above subsection), such a feedback inhibitory

mechanism is a kind of autocrine mechanism. Strangely,

neither inhibition via 2-adrenoceptors nor acceleration via

-adrenoceptors of the outflow of the transmitter has been

proven to date.

Relationship to Chromatic Patterns

We know well that chromatic patterns of the integument arevery important for the survival of animals in their habitat

(3). Some chromatic patterns are practically stationary. Very

frequently, such patterns change under various ethological

conditions. Among such changes, slower ones, such as those

that take place during ontogeny, are brought about by

morphological color changes, but faster changes in patterns

are due to physiological color changes. For example, in-

volvement of the pineal gland secretion, MT, in circadian

pattern changes in pencilfish has already been mentioned.

More rapid changes needed for adapting to background

patterns or for intraspecific communication can only be

realized through the activities of the nervous systems. Past

studies have indicated that changes are due to differential

neural commands to chromatophores or to groups of chro-matophores. In practice, there is a limited number of preset

patterns. On the basis of the coarseness of the background

texture, the central nervous system selects an appropriate

pattern (2, 8). Naitoh et al. (135) studied the chromatic

adaptation of the common freshwater goby, Rhinogobius

brunneus, to black and white checkerboard backgrounds,

and found that numerous nerve fibers control integumentary

chromatophores differentially and in a coordinated manner.

Several species of tilapias have recently been widely em-

ployed for analyzing communicatory functions of various

Fig. 5. Diagram showingtransmission from sympatheticpostganglionic fibers tochromatophores in which both trueand co-transmitters are involved.COMT, catecholamineO-methyltransferase; MAO,monoamine oxidase; NE,norepinephrine;-A-R,-adrenoceptor; Gi, inhibitoryG-protein; ATP, adenosine5-triphosphate; AC, adenylyl cyclase;cAMP, cyclic adenosine3,5-monophosphate; AMP, adenosine5-monophosphate; AS, adenosine;AS-R, adenosine receptor; Gs,stimulatory G-protein; AC, adenylylcyclase; IS, inosine [Modified fromFujii and Oshima (8)].



14/20

pigmentary patterns, and many interesting results have been

obtained (136). Working with a mouth-brooder tilapia,

Haplochromis burtoni, Muske and Fernald (137) recently

showed that nervous cues control the very rapid appearance

or disappearance of the facial stripe, the eyebar, which

signals territorial ownership and aggressive intent in males.

Nervously controlled pattern changes in flatfish that show

rapid adaptability to background patterns are famous, and

are another good example to mention, and indeed, a num-

ber of investigations have been done on these interesting fish

(8, 15). In their recent series of studies, Burton and his

associates put forward integrative analyses on the patterningof the winter flounder, P. americanus. (106, 138, 139). This

species of flatfish has a dark band, general background and

white spot components with different responsiveness in vivo

to stress. For example, measurement in vitro showed that

melanophores in the white spots showed a much higher

concentration threshold to NE than that for the other two

pattern components. They also found that the spot

melanophores responded to increased K+ concentration

and to electrical nervous stimulation faster than other com-

ponents, and further, that the inhibitory influences of -

adrenergic blockers differed among pattern components.

Based on those data, they concluded that the differential

activity associated with patterning includes a peripheral

neuroeffector component, part of which is directly associ-

ated with melanophores.

That the endocrine system takes part in pattern formation

seems difficult to understand because hormonal substances

go everywhere in the body rather homogeneously via the

general circulation. However, the involvement of MT in

such processes has recently been disclosed, as mentioned

before. Thus, the situation has become a challenge to analy-

sis, but presumably, correlated management of chroma-

tophores by both the endocrine and the neural systems can

elicit elaborate changes in patterns.

Effects of Nerve CuttingSeverance of chromatic nerve-fibers en route to chroma-

tophores naturally results in the interruption of central tonic

influences on effector cells, namely the darkening of the

downstream zone. Based on their observations of such

denervated dark bands in some teleostean and elasmobranch

fish, Parker and his colleagues came to the conclusion that

the response was caused by the repetitive firing of putative

parasympathetic melanin-dispersing fibers at the cut ends of

axons (15). For a number of reports relevant to this prob-

lem, readers can refer to a monograph by Parker (15). As

already mentioned, however, this double innervation the-

ory has not been supported, but the phenomenon itself

provides various important clues for understanding various

mechanisms of the effector systems (3, 14, 140).

Among the phenomena taking place after the denervation

of chromatophores, hypersensitization to some pigment-mo-

tor substances is worth mentioning again, because some new

observations have appeared. Using the goby C. gulosus,

Fujii (67) had already described his quantitative results on

the hypersensitivity of melanophores to epinephrine, NE

and also to MT. Karlsson et al. (141), while working on the

cuckoo wrasse L. ossiphagus, noted that after putting

melanophores in culture, they became hypersensitized to

-adrenergic stimuli, and they concluded that such effects

were due to denervation. Employing scales plucked from

individual medaka that had been adapted to a dark back-

ground for 10 days, Sugimoto (123) reported that the re-

sponsiveness of melanophores to NE significantly increased.

His conclusion was that the depressed sympathetic nervous

activities during the dark-background adaptation might

have affected the cells like denervation. Fujii and Oshima

(14), however, think that the hypersensitivity may be related

to the loss of spontaneously released adenylyl co-transmitterfrom sympathetic fibers, since the co-transmitter is now

known to antagonize the action of the true transmitter

either in vivo or in vitro, as described above. The physiolog-

ical significance of denervation hypersensitization is still

unclear.

SIGNAL TRANSDUCTION ACROSSCHROMATOPHORE MEMBRANE

Signal transduction studies up to the 1990s have been

previously reviewed by Fujii (3, 14), and Nery and Castrucci

(88) have recently reviewed work dealing with signaling

mechanisms in chromatophores in poikilothermal animals.A relevant article by Oshima (65) will also appear soon, and

therefore, the author will summarize his views on this topic,

only considering some recent works.

Membrane Potential Changes

Like smooth muscle cells, chromatophores of fish are under

the control of the sympathetic nervous system. Rather

strangely, however, their motile activities seem to be inde-

pendent of the electrical activities of the surface membrane

since Tetrodotoxin did not affect the motility of

melanophores per se (29). Action potentials therefore, are

not required for triggering cellular motility. Working on

denervated skin pieces, or on those in which the liberationof neurotransmitters was blocked, Fujii and Taguchi (70)

showed that melanosome-aggregating and dispersing agents

induced motile reactions of melanophores quite normally

when the cells were in saline in which Na+ ions were totally

replaced with an equimolar amount of K+ ions. Under such

conditions, the cell membrane should have been completely

depolarized. These results indicate that ionic fluxes across

the membrane and resultant changes in the membrane po-

tential are irrelevant to the cellular responses. Meanwhile, it

has become clear that in chromatophores of many species,

even the presence in the extracellular space of Ca2+ ions is

not required for their motility (3, 14, 70, 87). Namely, Ca2+

inflow or Ca2+ potential may not be involved in the motile

responses. The sum of these observations shows that

voltage-dependent ionic channels are not involved with the

responses.

All chromatophores in vertebrates have been shown to be

of neural crest origin, and thus, they are categorized as

so-called paraneurons (3). It is rather strange therefore,

that their cell membrane is not electrically excitable. As

briefly touched upon below, results on signal transduction



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across the cell membrane show that receptors that mediate

pigment-motor responses are G protein-coupled. Since that

process may not require membrane potential changes, the

irrelevance of electrical activities across chromatophore

membranes can be understood smoothly.

As a rather exceptional case for the role of the cell

membrane, we should mention again the erythrophores of

holocentrid squirrelfish. Luby-Phelps and Porter (108) re-

ported that the aggregation of pigment in erythrophores of

H. ascensionis definitely depend on extracellular Ca2+ ions.

Depolarization of the cell membrane due to K+-rich

medium may open voltage-dependent Ca2+ channels, allow-ing the inflow of ions to initiate pigment aggregation. The

sequence is quite different from that disclosed in

melanophores and other chromatophores (3, 14, 142). We

have recently observed the responses of erythrophores of

squirrelfish belonging to the family Holocentridae (order

Beryciformes), including the crowned squirrelfish,Sargocen-

tron diadema, and the soldierfish species, Myripristis ran-

dalli, and we have obtained results fundamentally identical

to those described above (108) (unpublished observations).

It should also be mentioned here that before the finding

of the peculiar responses of squirrelfish erythrophores by

Luby-Phelps and Porter (108), Iga (143) had already re-

ported that xanthophores of the medaka, O. latipes, are

directly responsive in pigment aggregation to increased con-

centrations of K+ ions. Confirming Igas observations,

Oshima et al. (144) recently concluded that Ca2+ ions

penetrate the cytosol through the voltage-dependent chan-

nels, which lower the levels of cAMP by inhibiting adenylyl

cyclase. Incidentally, in melanophores and other chroma-

tophores, K+ ions have been shown not to act directly on

the cells, but rather on nervous elements to release neuro-

transmitters which in turn aggregate pigment (3, 142). These

results suggest that there are some exceptional cases among

bright-colored chromatophores, across the cell membrane of

which depolarization takes place to allow Ca2+ influx,

resulting finally in pigment aggregation. As for other kinds

of chromatophores, no reliable data have been publishedthat indicate their existence.

At the beginning of their assignment to take part in

physiological color changes, the membranes of chroma-

tophores in ancient fish might have been excitable because

they had a common origin with neurons. Being different

from many electrically excitable cells, the chromatophores

have not been required to exert such quick reactions. We

also know that a large amount of ionic flux requires much

energy to recover the ionic distribution across the cell

membrane. Our conclusion therefore, is that erythrophores

or xanthophores of the Holocentrus type may belong to a

more primitive type of chromatophore, retaining ancient

physiological properties.

Cyclic AMP

Undoubtedly, cAMP is the major second messenger in

chromatophores. As early as 1970, Novales and Fujii (145),

while working on split tail-fin pieces of Fundulus killifish,

succeeded in detecting a melanosome-dispersing effect of

extracellularly applied cAMP. In order to obtain more

direct evidence for the role of cAMP, Fujii and Miyashita

(128) injected the nucleotide iontophoretically into guppy

melanophores and could observe the dispersal of

melanosomes. Using a photolabile caged cAMP, Furuta et

al. (146) recently succeeded in detecting its melanosome-dis-

persing effects on Oryzias melanophores. Detection by

means of radioimmunoassay of an increase in the level of

cAMP in Xiphophorus melanoma cells (147) and in guppy

melanophores (148) provided results in accordance with this

concept. Thereafter, many reports on the role of AMP in

dispersing pigment, not only in melanophores, but also in

xanthophores, erythrophores and leucophores, have ap-peared, and have been reviewed several times (3, 14, 73, 88).

Those reviews also deal with current understanding of the

mechanisms involved in the process of signal transduction.

The first step in the motile response of the chromatophore

is the binding of the first messenger, i.e. a hormonal or

neuronal substance, to corresponding receptors which con-

stitute regulatory subunits of adenylyl cyclase. The informa-

tion is then signaled via a GTP-binding protein, either Gs or

Gi, to the catalytic subunit of adenylyl cyclase. The increase

in cytosolic levels of cAMP is due to the heightened activity

of this subunit, leading finally to the pigment dispersion.

The reverse process, i.e. the aggregation of pigment, is

triggered by a decrease in the level of the nucleotide, which

results from decreased activity of the catalytic subunit via

Gi.

It has generally been accepted that the mechanisms of

action of catecholamines and peptide hormones are cAMP-

dependent. For example, we have already treated adreno-

ceptor-mediated dispersion of pigment in a relevant section,

and the adrenoceptors involved are mixtures of 1 and 2(76). The latest results along this line on fish chroma-

tophores include those on MCH. Using melanophores of the

Nile tilapia,O. niloticus, Oshima and Wannitikul (66) exam-

ined the signaling mechanism for MCH. Based on their

results obtained by employing various inhibitors, they con-

cluded that cAMP is the second messenger involved.

It is interesting to know that even in nervously evokedpigment aggregation, cAMP is the major second messenger.

Recent results are in agreement with the view that the

subtype of adrenoceptors concerned is of the a2 subtype,

which work to diminish adenylyl cyclase (104, 105, 107).

Ca2+ and IP3

As mentioned before, the cell membrane of common chro-

matophores is quite resistant to changes in the external ionic

composition. Meanwhile, Luby-Phelps and Porter (108) pre-

sented their results on erythrophores of the squirrelfish H.

ascensionis in which an influx of external Ca2+ ions is

required for the aggregation of pigment. Using a Ca2+

ionophore, they further manipulated the intracellular con-

centration of Ca2+ ions, and showed that the response was

dependent on the concentration of ions. Working on ery-

throphores of the same material, and after permeabilizing

the surface membrane of the cells by treatment with a Brij

surfactant, McNiven and Ward (149) found that free Ca2+

ions at 100 mM induced the aggregation of pigment,

whereas lowering of the concentration to 10 nM caused



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dispersion. Both processes were ATP-dependent but cAMP-

independent. Furthermore, Oshima et al. (144) recently

showed that K+-induced aggregation of pigment in xan-

thophores of medaka is accompanied by Ca2+ entry in the

cytosol. One should remember, however, that chroma-

tophores represented by squirrelfish erythrophores and

medaka xanthophores are rather peculiar.

Fujii and Taguchi (70) showed that external Ca2+ ions

were not required for movement of pigment per se. Subse-

quently, the absence of a requirement for extracellular Ca2+

ions has been demonstrated in many types of chroma-

tophores (3, 14, 42, 87, 150, 151). By electron-microscopiccytochemical localization of Ca2+ ions, Negishi and Obika

(109) showed that in melanophores of medaka, an increase

in the cytosolic level of Ca2+ ions was associated with the

aggregation of melanosomes in the perikaryon, whereas the

Ca2+ level was much lower in cells with dispersed pigment.

Employing fluorescent Ca2+ indicators, Oshima et al. (110)

found that an increase in the intracellular level of free Ca 2+

ions occurred after NE stimulation of platyfish (X. macula-

tus) melanophores, which had been dissociated and sus-

pended in saline. Since the possible dynamics of Ca2+ ions,

calmodulin, cyclic nucleotide phosphodiesterase, etc., have

been recently reviewed by us (14) and also by Nery and

Castrucci (88), further explanation is not given here.

Confirming the irrelevance of extracellular Ca2+ ions in a

study of tilapia (O. niloticus) melanophores in culture, Fujii

et al. (87) recently concluded that D-myo-inositol 1,4,5-

trisphosphate (IP3) functions as another second messenger

for the aggregation of pigment (Fig. 1). It may take part in

transducing adrenergic signals via 1-adrenoceptors at least.

IP3 is generally known to cause the liberation of Ca2+ ions

from their intracellular storage compartments. In

melanophores too, IP3may act via the release of Ca2+ ions

from such compartments within the cell. Elements of

smooth endoplasmic reticulum are potent candidates, since

such compartments exist abundantly in the cytoplasm (3,

152). Readers interested in the signaling mode of IP3should

refer to the original descriptions (87) or to later explanations(3, 14, 88).

Although cAMP may be the major second messenger in

chromatophores, both the cAMP and IP3-Ca2+ systems

probably interact cooperatively to move chromatosomes. It

is suggested that slower responses are mediated by decreases

in levels of cyclic AMP, while faster ones are realized by the

IP3-Ca2+ system.

CONCLUSION AND PERSPECTIVES

It must be astonishing for many readers to know that in

fish, so many hormonal, neural and even paracrine factors

are involved in the regulation of motile activities of chroma-

tophores in the skin. Naturally, those chromatophores are

endowed with various receptors and other devices for receiv-

ing numerous cues, either from intrinsic or from external

sources. The author believes that several additional novel

principles may be found in the near future that regulate

chromatophore motility, necessitating repeated additions of

sections in forthcoming review articles in relevant fields.

Among chromatophores, melanophores usually play the

most important part in generating the remarkable and yet

subtle changes in hues or shades, as well as in color patterns.

Thus melanophores, among several types of chroma-

tophores, usually possess more species of receptors than

other cells do. It is interesting to point out that

melanophores are the closest homologues to melanocytes of

homeotherms, to which humans belong. Without a system

for cellular motility, the activities of melanocytes are con-

trolled in a simpler manner. It may safely be said therefore,

that fish possess a much more sophisticated chromatic sys-

tem than we do. We now know that fish and mammalsstarted separate ways of evolution more than 400 million

years ago. In mammals, the pigment cell system may have

become simplified, in other words, they have devoluted. For

example, no definite role of the nervous system in regulating

melanocyte function has been proven.

Why have fish evolved to possess chromatophores with so

many kinds of receptors to sense signals? The answer can be

sought in the crucial roles of chromatophores in survival

strategies of the animals. In their habitat, they actually

employ variously defined types of colorations and patterns

such as for cryptic and aposematic purposes (2). It should

be emphasized here that such colors and patterns are very

often changeable, and function to cope with various etho-

logical stressors. In order to actualize such abilities,

exquisitely fine-tuned mechanisms for controlling chroma-

tophores have thus evolved.

The molecular structures of simple hormonal substances,

such as catecholamines, MT and even smaller peptides such

as -MSH, have been well conserved both in Pisces and in

Mammalia, although their physiological assignments are

more or less modified. As for the larger peptides, sequences

and even the number of amino acids are fairly different

between the two classes of vertebrates. It is interesting to

point out here that even MSHs show considerable molecular

diversity, especially among more anciently emerged fish, a

fact that should be due to their longer phylogenetic history

of evolution. Different from the situation in mammals whichis much more advanced, the cloning of receptor peptides on

fish pigment cells still remains mostly unexplored, the sole

result obtained hitherto being an 2-adrenoceptor on

melanophores of a teleostean species L. ossifagus (107). It is

evident, however, that, having a larger number of amino

acids, receptor molecules should have diverged beyond our

expectations.

In past studies classifying receptors for fish chroma-

tophores, pharmacological analyses have been the main

approach. In those investigations, agonists and/or antago-

nists for receptors developed for therapeutic uses in humans

have mostly been employed. For rough categorization, such

approaches are appropriate, and have certainly been fruitful.

However, we should now consider that, since they separated

long ago, receptors in fish and mammals constitute two

distinct groups, each of which have diverged independently.

Therefore, the structures, as well as the pharmac

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2000-Fujii The regulation of motile activity in fish chromatophores.pdf