J. Cell Set. 41, 65-74 (1980) 65Printed in Great Britain © Company of Biologists Limited igSo

LIGHT-INDUCED PIGMENT AGGREGATION IN

CULTURED FISH MELANOPHORES: SPECTRAL

SENSITIVITY AND INHIBITORY EFFECTS OF

THEOPHYLLINE AND CYCLIC

ADENOSINE-3',5'-MONOPHOSPHATE

YUKO WAKAMATSU,* SATORU KAWAMURAf ANDTORU YOSHIZAWAf* Biological Laboratory, Yoshida College, Kyoto University, Kyoto 606, Japan, andf Department of Biophysics, Faculty of Science, Kyoto University, Kyoto 606,Japan

SUMMARYAbout 20 % of xiphophorin fish (Xiphophorus maculatus) dermal melanophores which were

cultured in monolayer for 5 to 10 days responded to light directly, exhibiting reversible melano-8ome aggregation within a few minutes. In order to determine the spectral sensitivity for thelight-response, absorbance changes due to the light-induced melanosome aggregation weremeasured on single melanophores in a range of wavelengths from 370 to 550 nm using arecording microspectrophotometer. The melanophores showed maximum sensitivity to a wave-length of about 410 nm and very little to those longer than 500 nm. io~* to io~' M theophyllineand io~* M cAMP had inhibitory effects on the light-response.

INTRODUCTION

Light-responses are a ubiquitous phenomenon in animal life and many types ofsuch response are known, for example, phototaxis, photoperiodism, vision, and photo-control of biological rhythms. The nature of photoreceptors and the mechanisms ofthe responses, however, are little understood yet except for vision (Erlanger, 1976;Menaker, 1976). Difficulty in elucidating extra-retinal photoreception stems from alack of suitable experimental systems to study the response at the cellular level.

Dermal melanophores of lower vertebrates and invertebrates are a type of effectorcell which are responsible for colour changes of the body by aggregation and dispersionof melanosomes (melanin-containing organelles) in the cytoplasm. Although the light-responses of the melanophores are usually mediated by hormones and/or the nervoussystem through photoreceptor organs (Bagnara & Hadley, 1973), some melano-phores are known to respond to light directly (light-sensitive melanophores) in diverseanimals (van der Lek, 1967; Coohill & Fingerman, 1976; Gras & Weber, 1977).The light-sensitive melanophores may be an appropriate material for investigation ofextra-retinal photoreception at the cellular level, because the response can be easilydetected by visible movements of melanosomes in the cytoplasm.

66 Y. Wakamatsu, S. Kawamura and T. Yoshizavoa

In a previous paper, one of the authors (Wakamatsu, 1978) reported that some ofthe melanophores of platyfish (Xiphophorus maculatus) cultured in vitro responded tolight directly by reversible melanosome aggregation. The purpose of the presentstudy is to determine the spectral sensitivity curve of the light-sensitive melanophores,as a guide to finding the photoreceptive pigment and elucidating the mechanism of thelight-response. Since melanosome movements in melanophores of lower vertebratesare regulated by intracellular levels of cyclic adenosine-3',5'-monophosphate (cAMP)(Bitensky & Burnstein, 1965; Novales & Fujii, 1970; Bagnara & Hadley, 1973), theeffects of theophylline and cAMP on the light-response were examined.

MATERIAL AND METHODS

Cultures of melanophores

A domesticated black variety of platyfish {Xiphophorus maculatus) was purchased from acommercial source and bred in the laboratory. From 14 to 16 specimens about 2 months oldwere sterilized by 1:10 000 sodium merthiolate in calcium and magnesium-free Dulbecco'sphosphate-buffered saline (CMF-PBS) for 2 to 5 min and then rinsed twice in CMF-PBS.The integuments from the dorsal to lateral part were taken off, together with the scales, andrinsed in CMF-PBS in a 20-ml Erlenmyer flask. Using a modification of Ozato's method (1977),the tissues were dissociated by incubating in 2 ml dissociating solution containing 0-125%trypsin, 0-06 % collagenase, and 2 % bovine serum albumin in CMF-PBS for 15 min at 28 °Cand then shaking the flask on a gyratory shaker (35 rev/min) for 15 min at the same temperature.The cell suspension thus obtained was filtered through a stainless-steel mesh. The filtrate wascentrifuged at 100 g for 5 min after addition of 15 ml of culture medium, composed of Leibo-vitz's medium (L-15, GIBCO, New York) supplemented with 10 % foetal calf serum (Micro-biological, Bethesda), ioo units penicillin/ml, ioo/tg streptomycin/ml, and 2 fig fungizon/ml.The cells were resuapended in 2 ml of the medium, inoculated on a plastic sheet, 22 x 20 mm(LUX, Newbury Park) in a culture dish (Falcon, Oxnard, 35 x 10 mm), and incubated at 28 °Cin air. The procedure was repeated 3 times. The medium was renewed 2 days after theinoculation in order to remove unattached cells and debris. No melanophore was light-sensitiveinitially, but s to 10 days after starting the culture, about 20 % of the melanophores showed lightsensitivity (Fig. iA, B). The light-sensitive melanophores were in the dispersed state in thedark. On illumination, the melanosomes aggregated within a few minutes. When the melano-phores were brought into the dark, the melanosomes dispersed again within 3-5 min. Thus,the light-response was reversible. At this stage, the plastic sheet was taken out of the dish,placed upside down on a few drops of the medium on a quartz slide and sealed with whiteVaseline. This was used for measuring the spectral sensitivity.

Measurement of the spectral sensitivity

A dark-adapted light-sensitive melanophore was exposed to monochromatic stimulatinglight in a range of wavelengths from 370 to 550 nm for about 3 min. The dispersed melano-somes aggregated if the light was effective. Then the melanophore was kept in the dark for 3-7min, during which it recovered the original dispersed state. During and after exposure to light,absorbance changes of the melanophore, due to melanosome movements, were continuouslyrecorded by a microspectrophotometer (Shimazu MPS 5000 and its attached microscope).The experimental apparatus is shown in Fig. 2. Red light of 640 nm wavelength was used as themeasuring light, because it was confirmed to be ineffective in causing the light-response inpreliminary experiments. The light from a halogen lamp (12 V, 50 W) (LSM) passed throughthe sample (SA) on the stage (ST) of the microscopic apparatus placed in the sample compart-ment of the spectrophotometer and was focussed onto the image of a light-sensitive melanophorewith a diameter of about 100 /tm on a screen (SC) which was placed in front of a photomulti-plyer (PM). By replacing the screen with another one having a pin hole (PH), which was nearlyequal in size (5 mm in diameter) to the image of the melanophore magnified by the objective

Light-sensitive fish melanophores 67

Fig. 1. Light-response of a cultured melanophore from the integuments of youngplatyfish (Xiphophorus maculatus). The melanophore was in the dispersed state inthe dark (A) and in the aggregated state in the light (B). The bar represents 100 /im.

: < * "

OC

PM

^ - 1 c=b=3 F

-PH

SC

Grating monochromator

Recording spectrophotometer

Fig. 2. Schematic diagram of the apparatus for measurement of the spectral sensi-tivity of the cultured melanophores. E, eye; F, cut-off filter (> 610 nm); G, grating;HM, half mirror; LSM, light source for measurement; LSS, light source for stimu-lation; MlP M,, M3, mirrors; OB, objective; OC, ocular; PC, photocell; PH, pin hole;PM, photomultiplier; SA, sample; SC, screen; SH, shutter; ST, stage; , measur-ing light; , monitoring light; , stimulating light.

(OB) ( x 50), the absorbance changes, which can be taken as a measure of melanosome move-ments in the melanophore, could be measured. For light stimulation of the melanophore, agrating monochromator (JASCO, MFP-i) with a xenon lamp (Ushio, 50 V, 2 kW) (LSS) wasused. The stimulating light beam was reflected upwards with a half-mirror (HM) and broughtonto the sample (SA). A cut-off filter (F) (> 610 nm) was set to prevent the stimulating lightfrom reaching the photomultiplyer (PM). The monochromatic light used for light stimulationhad a half width of 15 nm and its energy was measured by a silicon photocell (Hamamatsu

68 3'". Wakamatsu, S. Kazoamura and T. Yoshizawa

TV, S780-5BQ) (PC) which was calibrated by a standard lamp. The melanophore couldbe observed through an ocular (OC) by removing the cut-off filter (F), using white Light (moni-toring light), which was obtained from the light source for stimulation (LSS) by replacing thegrating (G) with a mirror (Mt). Heat was removed by a water layer (10 cm) placed in the lightpath.

Effects of theophylline and cAMP

Effects of theophylline and cAMP on melanosome movements in the light-sensitive melano-phores were examined with or without the light stimulus at 420 nm. A plastic sheet to which thelight-sensitive melanophores adhered was placed upside down on the cavity (5 mm in diameter)of a perfusion dish which was made of transparent plastic and sealed with grease. Media con-taining io~7 to io~* M theophylline and io"4 M CAMP were applied to the melanophores throughthe capillaries of the perfusion dish using a Perista minipump (Atto, SJ-1211H). The effectswere expressed as absorbance changes of measuring light at 640 nm.

RESULTS

Spectral sensitivity of the light-sensitive melanophores

It was confirmed in preliminary experiments that stimulation at near 420 nm wasmost effective on melanosome aggregation in the light-sensitive melanophores. Beforemeasuring the spectral sensitivity of the light-response, the dose-response curve of

8

1 0

0-8

0-6

0-4

0 2

nn

A

/

- y/ \ i i i i

B •— s* •

r

-/

20 40 60 80 100 0 20 40Light intensity at 420 nm, %

60 80 100

Fig. 3. Typical dose-response curves of light intensity for stimulation at 420 nm andabsorbance changes at 640 nm due to melanosome aggregation in 2 light-sensitivemelanophores. The absorbance change in some cases increased linearly with thelight intensity (A); in other cases, it reached a plateau (B).

light intensity at 420 nm to the absorbance change at 640 nm was measured. In mostcases, the absorbance change increased linearly with the light intensity, which wasadjusted by inserting some neutral filters (Fig. 3 A). But in some cases, the absorbancechange reached a plateau due to the saturation of melanosome aggregation at a certainlight intensity (Fig. 3B). For measuring the spectral sensitivity, we used intensitieswhich lay in the linear portion of the dose-response curve. Thus, the absorbance

Light-sensitive fish melanophores 69

change can represent the sensitivity of the melanophore to stimulating light at variouswavelengths. In view of possible fluctuation of the light-sensitivity because of un-known factors during the course of the experiment, alternate stimulations at 420 nmand an experimental wavelength were carried out. The absorbance change caused bystimulation at 420 nm was used as a standard for the sensitivity at another wavelength.

An example of the measurement of spectral sensitivity is shown in Fig. 4. In thecase of 400 nm, about 4 s after the onset of stimulation, absorbance began to decrease,and continued to decrease during stimulation for 3-5 min. About 4 s after the end of

Wavelength, nm

420

400

420

0-1

— — • 1 1 —

~ ' — - ^ .

1

0

— • - ^

• — .

1

— —

2

-

^ — — .

3Time,

'

— - — "

4min

^-

5

-—

6

460

- 420

Fig. 4. Typical records of absorbance changes at 640 nm due to melanosome move-ments in a light-sensitive melanophore which was stimulated with light at 420 nm andan experimental wavelength alternately. Downward-pointing arrow, light on; upward-pointing arrow, light off.

stimulation, a gradual increase of absorbance was observed and within 3 min theabsorbance recovered to the original level. Similar patterns of the absorbance changewere observed on stimulation at other wavelengths. The time-courses of the absor-bance change were somewhat varied in each melanophore.

Cases in which the sensitivity to 420-nm stimulation showed a large fluctuationduring the experiments were omitted. Results from 5 series of experiments were used.The relative sensitivity (SA) of the light-sensitive melanophore to light at a certainwavelength (A) can be expressed as the ratio of the absorbance change (at 640 nm)(A O.D.A) per unit number of photons of the light stimulation at a certain wavelength(A") to that at 420 nm (AO.D.«°/JV4ai).

AO.D.420/^420

AO.D.A/(energy of stimulating light at wavelength A xA)AO.D.420/(energy of stimulating light at 420 nm x 420)

70 Y. Wakamatsu, S. Kawamura and T. Yoshizawa

In this equation, the absorbance change (AO.D.) was estimated from the differencebetween the arithmetic mean values of absorbances before a stimulation and after itsrecovery, and the absorbance just before the end of the stimulation.

The relative sensitivities to light at various wavelengths are plotted in Fig. 5. Wave-lengths from 400 to 420 nm were highly effective but those longer than 500 nm wereonly very slightly effective. The spectral sensitivity curve showed only one peak, atabout 410 nm.

1-2-1

1

3801

4001

4201 1

440 460Wavelength,

1480

nm

1500

1520

1540

00

Fig. 5. A spectral sensitivity curve of the light-sensitive melanophores. The relativesensitivities at various wavelengths were determined on the basis of the sensitivityat 420 nm (open circle).

[Theoph.]M

10"*

10

10"

After 1 h washing

in

r?o30s

Time

Fig. 6. Typical records showing effects of theophylline on a light-sensitive melano-phore. The melanophore was exposed to theophylline from io~7 to io"1 M and 10 minlater stimulated with light at 420 nm for 45 s. Downward-pointing arrows, light on;upward-pointing arrows, light off.

Light-sensitive fish melanophores 71

Effects of theophylline and cAMP

The effects of theophylline, an inhibitor of phosphodiesterase, on the light-sensitivemelanophores were examined. A typical experiment is shown in Fig. 6. A light-sensitive melanophore was exposed to io~7 to io~2 M theophylline in the dark and10 min later stimulated with light at 420 nm for 45 s. The light-induced melanosomeaggregation was completely inhibited by io~2 and io~3 M theophylline, was parti-ally inhibited by io"4 M, and was apparently not inhibited by io~5 to io~7 M. Thelight-sensitivity of the melanophore recovered completely after washing out the

J30s

1M CAMP

Time

Fig. 7. A typical record showing effects of cAMP on a light-sensitive melanophore.When io"4

M CAMP was applied to the melanophore in the aggregated state bystimulation with light at 420 nm, the absorbance increased owing to melanosomedispersion. By turning off the light (upward-pointing arrows), increase of the absor-bance was accelerated and by turning on again (downward-pointing arrow), it wasinhibited again.

theophylline with standard medium for 1 h. This shows that the effects of theophyllinewere not due to irreversible change to the melanophore. These results suggest thatthe accumulation of cAMP in the light-sensitive melanophore may inhibit the light-induced melanosome aggregation. To ascertain this, the effect of cAMP was examineddirectly. A light-sensitive melanophore was stimulated with light at 420 nm, resultingin complete melanosome aggregation. Then io~4 M cAMP was applied to the melano-phore under continuous light stimulation at 420 nm. Remarkable melanosome dis-persion was observed within 1 min. Moreover, when light stimulation was discon-tinued during dispersion, some acceleration was observed. When the light was appliedagain, dispersion was partially inhibited (Fig. 7). The same acceleration and partialinhibition were also observed when io~* M theophylline was applied (data not shown).

72 Y. Wakamatsu, S. Kawamura and T. Yoshizawa

DISCUSSION

The spectral sensitivity of melanosome aggregation of the light-sensitive melano-phores from X. maculatus was measured. The melanophores showed high sensitivityto light between 400 and 420 nm, with a peak at about 410 nm, and very low sensitiv-ity to light of wavelengths longer than 500 nm. These results suggest that a yellowpigment exists in the melanophores as a photoreceptive substance which playsa role as a trigger to lead to melanosome aggregation, since the melanophores weredissociated and in culture, and therefore free from the systemic control of the nervoussystem and hormones. The spectral sensitivity curve of the light-sensitive melano-phores is similar to the absorption spectra of some pteridines in the visible region ofthe spectrum. Several kinds of pteridines exist in fish melanophores fHama, 1963;Matsumoto, 1965; Henze, Rempeters, & Anders, 1977), one of which may be a pos-sible photoreceptive pigment in the melanophores, but further biochemical studies arenecessary to ascertain this.

Spectral sensitivities of light-sensitive melanophores have been measured in someother species. The melanophores of the fiddler crab, Uca pugilator, are sensitive tolight in the near-ultraviolet region (300 to 400 nm), exhibiting melanosome dispersionon stimulation (Coohill, Bartell & Fingerman, 1970). In a sea urchin, Diadema setosum,blue light (450 to 500 nm) is effective in causing dispersion of melanosomes withmaximum effectiveness at 470 nm (Yoshida, 1957). Melanophores of the tail fin ofXenopus laevis are most sensitive to light of 425 nm in the light-induced melanosomeaggregation (van der Lek, 1967). Thus, effective wavelengths to induce melanosomemovements and the direction of induced movements are different among species.It is interesting, however, that the melanophores of the tail fin of Xenopus laevis aresimilar in their light-response to those of X. maculatus described here.

Melanosome movements in melanophores of lower vertebrates are believed to beregulated by intracellular levels of cAMP (Bitensky & Burnstein, 1965; Novales &Fujii, 1970; Bagnara & Hadley, 1973); that is, melanosomes disperse at a high leveland aggregate in a low one. The present study revealed that theophylline, an inhibitorof phosphodiesterase, inhibited in dose-dependent fashion the light-induced melano-some aggregation. The inhibitory effect was also exhibited by cAMP. These resultssuggest that light may act by decreasing intracellular levels of cAMP by activatingphosphodiesterase. Recently, the light-activation of various enzymes has been reported(see for review, Erlanger, 1976, and Hug, 1978). In visual process, the light-activationof phosphodiesterase was revealed in rod outer segments of amphibia and mammals(Bitensky et al. 1975). Cohen's report (1974) also seems to suggest the light-activationof phosphodiesterase resulting in decrease of cAMP levels in Phycomyces blakes-leeanus cells. Thus, the light-activation of this enzyme may be a common phenome-non in photosensitive cells.

In general, melanosome movements in fish melanophores are controlled by adrener-gic nerves. That is, the melanosomes aggregate in the presence of epinephrine and theaggregation is inhibited by a-adrenergic blocking drugs such as dibenamine (Fujii &Novales, 1972; Bagnara & Hadley, 1973). The light-sensitive cultured melanophores

Light-sensitive fish melanophores 73

also aggregate on application of epinephrine (Wakamatsu, 1978). Dibenamine in-hibited the response to epinephrine, but did not inhibit the light-induced melanosomeaggregation (Wakamatsu, unpublished observation). These facts suggest that lightcontrols the melanosome movements without mediation of the adrenergic receptorsand acts directly on phosphodiesterase enzyme systems in the light-sensitive melano-phores.

The light-sensitive melanophores in culture will be useful as one of the simplestsystems for study of molecular mechanisms of signal transduction from receptor toeffector.

We wish to express our thanks to Dr Kenjiro Ozato of Kyoto University for invaluable sug-gestions about fish cell culture and for reading the manuscript.

REFERENCES

BAGNARA, J. T. & HADLEY, M. E. (1973). Chromatophores and Color Change. Englewood Cliffs:Prentice-Hall.

BITENSKY, M. W. & BUBNSTEIN, S. R. (1965). Effects of cyclic adenosine monophosphate andmelanocyte stimulating hormone on frog skin in vitro. Nature, Land. 208, 1282-1284.

BITENSKY, M. W., MIKI, N., KEIRNS, J. J., KEIRNS, M., BARABAN, J. M., FREEMAN, J., WHEELER,M. A., LACY, J. & MARCUS, F. R. (1975). Activation of photoreceptor disk membrane phospho-diesterase by light and ATP. In Advances in Cyclic Nucleotide Research, vol. 5 (ed. G. I.Drummond, P. Greengrad & G. A. Robison), pp. 213-240. New York: Raven Press.

COHEN, R. J. (1974). Cyclic AMP levels in Phycomyces during a response to light. Nature,Lond. 251, 144-146.

COOHILL, T. P., BARTELL, C. K. & FINGERMAN, M. (1970). Relative effectiveness of ultravioletand visible light in eliciting pigment dispersion directly in melanophores of the fiddler crab,Uca pugilator. Physiol. Zool. 43, 232-239.

COOHILL, T. P. & FINCERMAN, M. (1976). Comparison of the effects of illumination on themelanophores of intact and eyestalkless fiddler crabs, Uca pugilator, and inhibition of theprimary response by cytochalasin B. Experientia 32, 569—570

ERLANCER, B. F. (1976). Photoregulation of biologically active macromolecules. A. Rev.Biochem. 45, 267-283.

FUJII, R. & NOVALES, R. R. (1972). Nervous control of melanosome movements in vertebratemelanophores. In Pigmentation: Its Genesis and Biologic Control (ed. V. Riley), pp. 315-326. New York: Appleton-Century-Crofts.

GRAS, H. & WEBER, W. (1977). Ligh-induced alterations in cell shape and pigment displace-ment in chromatophores of the sea urchin Centrostephanus longispinus. Cell Tiss. Res. 182,165-176.

HAMA, T. (1963). The relation between the chromatophores and pterin compounds. Ann.N. Y. Acad. Sci. 100, 977-986.

HENZE, M., REMPETERS, G. & ANDERS, F. (1977). Pteridines in the skin of xiphophorine fish(Poeciliidae). Comp. Biochem. Physiol. 56B, 35-46.

HUG, D. H. (1978). The activation of enzymes with light. In Photochemical and PhotobiologicalReviews, vol. 3 (ed. K. C. Smith), pp. 1-33. New York: Plenum Press.

MATSUMOTO, J. (1965). Role of pteridines in the pigmentation of chromatophores in cyprinidfish. Jap. J. Zool. 14, 45-94.

MENAKER, M. (ed.) (1976). Extraretinal photoreception. Symposium on extraretinal photo-reception in circadian rhythms and related phenomena. Photochem. Photobiol. 23, 213-3°6.

NOVALES, R. R. & FUJII, R. (1970). A melanin-dispersing effect of cyclic adenosine monophos-phate on Fundulus melanophores. J. cell. Physiol. 75, 133-136.

OZATO, K. (1977). Mitotic activity of differentiated goldfish erythrophores in culture. J. CellSci. 26, 93-99.

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74 Y. Wakamatsu, S. Kawamura and T. Yoshizawa

VAN DER LEK, B. (1967). Photosensitive Melanophores. Dissertation for University of Utrecht,Netherlands.

WAKAMATSU, Y. (1978). Light-sensitive fish melanophores in culture. J. exp. Zool 204, 299—304-

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[Received 30 March igyg-Revised 20 July 1979)