Adrenergic Receptors Mediating Pigment Movements In
Chromatophores of the Medaka, Oryzias latipes

Fumihiro MORISHITA

Zoological Institute , Faculty of Science,
Hiroshima University , Hiroshima 730 ,JapanPublications of the Thesis(1)

Subtypesofbeta

adrenergic

receptors

mediatingpigment dispersion in chromatophores of the medaka,
Oryzias latipes.
Mo rishita , F., H. Katayama and K. Yamada (1985)
Compo Biochem. Physiol.,

81C: 279-285.

(This covers the Chapter III of the Contents)(2)

Responses

oftheOryzias latipes,

melanophoresofto adrenergic drugs:

involvement of alpha2

themedaka,evidence foradrenergic receptors

mediat-ing melanin aggregation.
Morishita, F.

(1987)

Compo Biochem. Physiol.,

88C: 69-74.

(This covers the Chapter IV of the Contents)(3)

Subtype of alpha adrenoceptors mediating leucosome
aggregation in medaka leucophores.
Morishita, F. and K. Yamada (1989)
J. Sci. Hiroshima Univ., Ser. B, Div. 1,

33:99-112.

(This covers the Chapter V of the Contents)

Contents

I.

General introduction - - - - - - - - - - - - - - 1-4

II .

Materials and methods - - - - - - - - - - - - -III .

SubtypesIV .

receptors

mediatingpigment dispersion in chromatophores - - -

9-29

The

of

subtypebetaofadrenergic

5-8

alpha

adrenergic

receptors

mediat-l n g melanosome aggregation in melanophores - - 30-44

V.

The

subtypeofalphaadrenergic

receptors

mediat-ing leucosome aggregation in leucophores - - - 45-62

VI .

General discussion - - - - - - - - - - - - - - 63-69

VII .

Summary - - - - - - - - - - - - - - - - - - -70-75

VIII . Acknowledgements - - - - - - - - - - - - - - - 76-77

IX .

References - - - - - - - - - - - - - - - - - - 78-92

I .

General introductionManytele os t

fis h e s

rapidl yc h a n ge

t h eirbody

tints o r col or pa tter n s i n re spons e to bac kg r ound hue sor va r ious st r esses.

Such changes of t in t spatte r ns

physiologicalarecalledcolo rand colo rchangesandoccur due to translocation of pigment granules wi thin
speciali zed

colored cells,

and fins of the animals

chromatophores,(cf.

Parker,Inscales1948;

Bagnara andl . e.

melanophores

Hadley , 1973; Fujii and Oshima, 1986).
Three

types of chromatophores,

xanthophores and leucophores,

areusually found to bepresent in scales of the medaka, Oryzias latipes. Amongthese , the cells mainly responsible for the body tintsofanimalsaremelanophores

and

Both

leucophores.

centrifugaltranslocationofpigment

granulesmelanophores(dispersionofmelanosomes)

andwithincentri-petal migration of pigment within leucopho r es (aggr e gationofleucosomes)

bring

about

a

da r kening

of

body

ti n t , while the reverse , i . e. melanosome aggregation ofmelanophores

and leucosome

dispersionofleucophores ,cause body paling .
Previous physiological and histological investigations have suggested that both melanopho r es and leucophores of the medaka receive the same adrenergic lnnervation and that theepinephrineal. ,1984) .

neurotransmitter released is nor-

(Iwata et al.,
In

1981;

physiological

Iga,

1983;

saline ,Yamada etmelanophores

usually maintain a fully melanosome-dispersed state and2leucophores are in a fully leucosome-aggregated state.
Nerve-mediated stimuli,
potassium

lons,

or

such

as

of

same

cause

the

stimulimelanophores,

while

leucosomeandinducenorepinephrine

exogenous

melanosome-aggregation
time ,

electric currents

at

the

dispersionofleucophores (Miyoshi, 1957; Iga, 1969, 1978).
On

themedakaother hand,

havechromatophores

receptors

pharmacologicalrevealedmelanophores are alpha in nature
dispersion

beta

natureIn

1977;

Yamada,

of

(Iga,

1968;

In

Obika,

However,inthose which

leucophores

1976;

on

adrenergic

and thatleucosomes1980).

that

aggregation of melanosomes

which mediate

mediateresearches

Iga

subsequent

et

are
al.,

investiga-

tions have demonstrated that alpha adrenergic receptors
are also present in leucophore,

which mediate aggrega-tion ofandleucosomes( Iga,

1979)

that

also possess beta adrenergic receptors ,tion causes melanosome dispersion

melanophores

whose stimula-(Komatsu and Yamada,1982) .
Thus,

notwithstanding

thefindings

lanophores

and leucophores

of

the

same

adrenergic

innervation

phorespossess both alpha

tors,

they

respond

to

and
nerve

norepinephrineIncompletely

each

This

impliesother.

adrenergic

receptors

and

for

3

medaka

that

beta

the

or

the

chromato-

adrenergic

oppositetheboth me-recel vethesestimuli

that

that

recep-

exogenous

directions

toaffinities

ofneurotransmitter,norepinephrine,differ between melanophores and leuco-

phores.
The

alm of thepresent

study wasto analyse

how

the receptor mechanism is involved in the regulation ofsuch

different

responses

of

medakachromatophores

toidentical stimuli .
In mammalian adrenergic tissues,

depending on theaffinities for specific adrenergic agonists and antagonists ,

both

alpha

and

beta

adrenergic

classified into two subtypes ,( Starke ,1981 ; Johannson ,( Lands et al . ,--receptorsarei . e . alpha-l and alpha-2

1984)

and beta-l and beta-2

1967 ; Petrack and Andrew ,1976;

Barnett

et al ., 19 7 8 ; Minneman et al . , 1979), respectively.
Therefore , in the present experiment , according to
the criteria for mammalian tissues ,subtypes of adren-

ergic receptors in both melanophores and leucophores ofthe medaka were investigated .
that the subtypes
mediateofalpha adrenergic

dispersion ofphores and leucophores
tively (Chapter III ),The results demonstrated
receptors

whichpigment granules within melanoarebeta-2and beta-l ,respec-and the subtypes of alpha recep-

tors

mediating aggregation ofpigment granules

wi thin

both

of

are

naturethesechromatophores(Chapters IV and V)
ferencebetweenalpha-2and showed thatmelanophores

and

In

the observed dif-leucophores

in

re-

sponse to identical stimuli is due to such differencesin the subtypes of receptors in the cells.

4

II .

Materials and methods

5Pre pa r a ti on an d p r o ce du r e
Exp erime n t s were perfor med wit h chromato pho res
scales o f t he medaka, Or yz i as latip e s
scales were isolated

f r oman t e r o-do r sal

body trunks of the animals.
directly to drugs
narilyused,

incubated inacteic acid)(w i ld type).

1nThe

region oftheTo

e xpos e

ch r oma t opho r es

isolatedscaleswe r e10 mM EDTA

prelimi-(ethylenediaminetetra-in physiological saline(128 mM NaCl,

2.5mM KCl, 1.8 mM CaC1 , pH 7.3 with 5.0 mM Tris-HCl)
2

for

60 min at 4.0°C and agitated toremovethecells which overlie the chromatophores.
phores used here were

exclusively theepithelial

The chromatodenervated ones

to exclude any indirect influence of the drugs throughpre-junctional elements.

Denervation was secured after

maintaining the isolated scales in physiological salin e
for mo r e than 15 hr at room temperatureThe

response

electrically by a
the

eye

p1ece

of melanophores

wasmeasu r ed photo-

CdS photoconductive

cell mounted onofmicroscopeaccordingdescribed by Nagahama and Katayama
onaneletronic

magnitudeofthe(Yamada, 1980).

polyrecorder(1982)(TOA,

me t hodand r eco r ded

EPR-221A).

melanosome-aggregation

expressed as a percentage of the full

the

The

responsechange

1n

was
light

transmittance, using a value for maximal aggregation as
100 and that for full dispersion as zero.
Forthroughobservation
aofdark-field

leucophores,

reflected

epi-illumination

6

light

microscope

The response of the leuco-(Olympus, BHA-NE) was used.

phores was represented by percentage changes in apparent length of a given process of the cells,
measured wi th the

aid

of

an

ocularlength of the process at full

which wasmicrometer.

The

leucosome dispersion was

taken as 100 and that at the fully aggregated state as
zero.

Drugs used
Adrenergic

agonists

and antagonists

used wereasfollows:
Catecholamines. l-Norepinephrine hydrochloride (Sigma),
l-epinephrine bitartrate(Tokyo Chemical)

anddl-iso-proterenol hydrochloride (Tokyo Chemical).
Beta agonists.

Salbutamol(Sigma)and terbutaline sul-phate (Fujisawa Pharmaceutical).
Beta

antagonists.

Propranololhydrochloride(Sigma),metoprolol tartrate (Ciba-Geigy) and atenolol (Sigma).
Alpha agonists. Methoxamine hydrochloride (Nippon Shinyaku),l-phenylephrinehydrozoline
chloridehydrochloride(Sigma),oxymetazolinehydrochloride(Sigma),naphazolinehydrochloride(Sigma),tetra-

clonidinehydro-hydrochloride(Sigma)and(Sigma),tramazolinehydrochloride (Boehringer Ingelheim-Tanabe).
Alpha antagonists.
corynanthinechloride

Phentolamine mesylatehydrochloride(Tokyo Chemical)(Sigma),and7(Ciba-Geigy),

prazosinyohimbinehydro-hydrochloride(Sigma) .
All the drugs were prepared forstocks at 1 mM indeionized water except norepinephrine hydrochloride andepinephrine

bitartrate,

which

were

dissolved

ln

0.5%

sodium metabisulphite, and prazosin hydrochloride which
was dissolved in

1% ethanol.

Alpha-MSH(melanophore-

stimulating hormone) was also used, which was dissolved
and stored in 100 mM aqueous solution of ascorbic acidat 50 WM.

Each stock solution was diluted with physio-

logical saline immediately before use.
All the experiments were carried out at 20-25°C.

Statistics
Statistical analyses were performed with Student'sttest and E < O. 01 wasused ascance.

8

the

level

of

signifi-

III .

Subtypes of beta adrenergic receptors mediating
pigment dispersion in chromatophores9Dermaladrenergic
Finnin,melanophores
innervation

1972;

Yamadaofteleost(Falck etetal.,fishes

al.,

1984;

receive

1969;
also

Reed

cf.

and

Parker,1948; Bagnara and Hadley, 1973; Fujii and Oshima, 1986)
and both electric currents

andpotassiumions

inducemelanosome aggregation within the cells through stimulation of

their nervous

and Novales,
Miyashita,(Fuj ii,

Kinosita and Ueda,

1969;

1975;

elements

Iga,

1959;

1970;

Moreover,1975).

Fuj ii

Fujii

and

has

been

it

suggested that the released neurotransmitter is norepinephrine
Novales,the(Fujii,

1961;

Scheline,

1963;

Fujii

and

1972) and that adrenergic receptors mediating

aggregationof(Iga, 1968 ; Grove,melanosomes

are

of

alpha

1969; Fernando and Grove,

Fujii and Miyashita,1975).

cological studies have

Meanwhile,nature1974a, b;

recent pharma-suggested the presence of beta

adrenergic receptors which mediate dispersion of melanosomesin the melanophores

of

the

phyllum eimekei (Reed and Finnin,angelfish,

1972)

Ptero-and the guppy,

Lebistes reticulutus (Miyashita and Fujii, 1975). Lately,using

specific

radioligands,(1982)

have demonstrated the

ceptors

autoradiographically

KomatsuandYamadalocalization of beta reInmelanophores

of

the

medaka .
In contrast,

leucophores,

anotherfish

chromato-

phores, have been known to behave in quite the opposite
way to melanophores; they respond to both nerve-mediat-10

ed stimuli,

i.e.

electric currents and potassium ions,and exogenous norepinephrine with leucosome

dispersion

throughreceptorsthe(Miyoshi,mediation

1952;

spite of thestimuli,

it

of

beta

Iga et al.,difference
has

been

in

adrenergic

1977;
the

Yamada,responsesuggestedthat1980).
toIn

identical

melanophores

andlecuophores receive the same sympathetic postganglionicfibers(Iwata et al.,1984).

Hence,

1981;

Iga,

1983;

Yamada etal.,we may wonder whether identical stimuli

can induce such opposite

responses

by thesechromato-

phores.
In mammalian adrenergic tissues, beta receptors incells are classified into two subtypes, i.e. beta-l andbeta-2,

accroding to theirnists and antagonists(Lands et al.,Andrew,

1967;1979).

In

severalsympathomimetic

leucophore

Barnett et
fish

al.,

species,

pigment,

affinity foramlnesYamada

1967;

1978;

based

specific

Petrack andMinneman

on

the

ago-

et

al.,

potencyof

to cause dispersion of(1980)

hassuggestedthat

adrenergic receptors which mediate the cell response of
the medaka are beta-l In nature.

However,no such at-

tempts have been made on the subdivision of beta receptors in fish melanophores.

Therefore,

In the present

experiment, to define more precisely the nature of subtypes of beta adrenergic receptors in melanophores andleucophores,
specifictheagonists

responses
and

of

these

antagonists

11

chromatophores
of

beta

to

receptors

were investigated.
theadrenergic

sion

response

The results have demonstrated thatreceptorsof

mediatingmelanophoresaremelanosome-disperbeta-2

mediating leucosome-dispersion response ofare beta-I.

12

and

those

leucophores

Results

Effects of catecholamines and propranolol on melannophores
First,

to determine the melanosome-dispersing

ac-tions of catecholamines on melanophores, the effects of
norepinephrine,

epinephrine and isoproterenol wereamined under the influence of

propranolol,

renergic antagonist (Fig. 1).

Norepinephrine, with theconcentration at 1 nM,aex-beta ad-alone induced a moderated mela-

nosome aggregation, i.e. about 40% of the full aggregation which was obtained with the amine at 10
la) .

~M

(Fig.

Propranolol at 1 WM had almost no effect on

responseofmelanophores

tonorepinephrine.

the drug considerably augmented the

responsethe

However,ofcells

to both 5 nM epinephrine and 10 wM isoproterenol, while
each of the amines induced about a half maximal aggregation of melanosomes (Fig.
Figure 2 showsthelb and c).

relationshipof melanosome-aggregation

response

of

of

the

magni tudemelanophorestothe concentration of catecholamines ln the presence orabsenceof propranolol.

Under

the

influence

of

pro-

pranolol, the degree of aggregation response to norepinephrine

slightly

decreased

andresponse curve shifted tohigheramine

theconcentration

(Fig.

2a),

but

the

difference(P > O. 60) .

On

the

other

hand,

13

concentration-

the

was

of

the

insignificant

curvesforbothepinephrine and isoproterenol shifted to lower concentrationsin the presence

concentrations of the(ECofpropranolol

aminesto causeand50%

effective

aggregation) of melanosomes decreased significantly

50

from 2.1 to 1.4 nM and from 8.6 to 3.1~M,(~<0.005)

respectively(Fig. 2b and c).

Effects of beta-2 agonists
The effects of beta-2

agonists,salbutamol whichis also known to be a beta-l antagonist and terbutaline
wereexamined on

responses

of

both

melanophores

leucophores to catecholamines (Fig. 3).
at 10 nM induced a nearly full

andNorepinephrine

aggregation of melano-somes, but the level of aggregation decreased to about
60% under the

influence of1

salbutamol~M(Fig. 3a).

The effect of the drug was completely abolished by thepresence of 1

~M

propranolol.

Salbutamol alone had noeffect on melanophores as can be seen in the figure. On
the other hand,

leucophores

100

nM

isopro-terenol with full dispersion ofleucosomes

as

well

to 10 WM norepinephrine and the

response was markedly

suppressed by the presence

respondedofsalbutamol,

thoughasthedrug had no intrinsic effect on the cells (Fig. 3b).
Terbtaline, another beta-2 agonist used,
had asimilar effect on both rnelanophores

phores to that of salbutamolline reduced both the

4).

and

leuco-

Thus,

terbuta-melanosome-aggregation

response

14

(Fig.

at 10 WM

of melanoph ore s t o no repi n ephrine
some-dispe r s i on re spons e o f(Fig .

4a ) and leuco-l e u cop h ores toi sop r ot ere-nol (Fig. 4b), respect i vely.
Sucheffectsofbeta-2a gonis t s

we rer eve r sibl eand the chromatophores could re spond to cat e cholamines
as usual after rinsing with normal saline.
The relation between the magnitude of the chromatophore response and the concentration of catecholaminesI

As is apparent from the figure,is shown in Figure 5.

concentration-response curves for both melanophores andleucophores

shiftedtohigherconcentrationsof

The EC

amines in the presence of beta-2 agonists.

the
SO

of

norepinephrine for aggregation of melanosomes increased
from 2. 7 to 9. 4 nM (P < O. 01) and to 13. 7 nM

(P < 0 . 001 )in the p r esence of 1 WM salbutamol and 10

WM t erb u t a -

line

(Fig.

Sa),

while

the

EC

SO

of

isoprote r enol

fo rleucosome dispersion increased from 24.8 t o 93.3 nM (P<0.001) and to 158.0 nM (P<O.Ol) under th e influ e n ce of
the

drugs

beta-2

(Fig.

Sb),

r espectively.

Theeffects

agonists

wereconcentration-dependent

and

of
the

responses of both melanophores to 10 nM no r epineph r ine
and leucophores

to 100nM

depressed by the presence

isoprote r enol

we re

of

at

the

drugs

totally

concentra-

tions higher than 100 WM.

Effects of beta-l antagonists
The effects of specific beta-l antagonists,

15

meto-

prolol

and atenolol,

These

were examined.

drugs

had

virtually no effect on the pigmentary state of melanophores

which

had been

dispersed

norepinephrine-treatedphores in the presence of7 a) •

normalThen,aggregated by norepinephrine.
examined oninsalineorthe effects were
aggregatedisoproterenol

melano-(Figs.

6a andIsoproterenol at 100 nM induced a marked decreasein the level of the melanophore response to 10 nM norepinephrine,

but the action waspresence of 0.5
other hand,

never affected by the

metoprolol or 1

~M

although the~Mantagonists

atenolol.
had noOn theintrinsic

effect on leucophores, they significantly (P < 0.01) inhibitied the leucosome-dispersion response of the cells
toisoproterenol

effects

of

both

6b

and

7b).

antagonistsonleucophores(Figs.

The

inhibitory
could

be

removed completely through washing in normal saline.
Figure 8 shows the influence of beta-l antagonists
on the concentration-response curve for
persion response of the
The

EC

SO

for

the

leucophores

control

curve

was

leucosome-dis-

to isoproterenol.
14.0

nM,

but

it

shifted to 192.0 nM (P < 0.001) and to 63.4 nM (P< 0.01)
in the presence of 0.5
respectively.

The

~M

metoprolol and 1

effects

of

these

~M

drugs

atenolol,
were

also

concentration-dependent and the leucophore responseto

100 nM isoproterenol was abolished completely either by
metoprolol

at concentrations

atenolol higher than 100

~M.

16

higher

than

10

~M

or

by

Discussion

It has been known that catecholamines
aggregationofthroughmediationthemelanosomes(Iga, 1968; Grove,

alpha

fish

the

melanophores

adrenergic

receptors

1969; Fernando and Grove,

1974a, b;

Fujii and Miyashita,ofwithininduce1975).

The present results indi-cated that all the catecholamines examined also have amelanosome-aggregating

actionmedaka and that propranolol,ofmelanophores

of

the

a beta adrenergic antago-nist, augument the action of the amines except that of
norepinephrine.

Thissuggeststhatusually both epi-

nephrine and isoproterenol have a melanosome-dispersing
action on melanophores

Inaggregatingthat

actionandaddi tion to the melanosomepropranolol

augments

theaggregation response of melanophores to catecholamines
throughinhibi tion of beta adrenergic

receptors

mediate melanosome dispersion within the cells.

which
More-

over, the present results demonstrated that the beta-2
agonists,salbutamol

andterbutaline,

competitively

suppressed the melanophore response to norepinephrine.
This indicates that the beta adrenergic receptors whichmediate melanosome
beta-2.
cally

Thus,
inhibitnorepinephrine

dispersion withinmelanophores

aresalbutamol and terbutaline antagonistithebymelanosome-aggregating
theiractionmelanosome-dispersingofactions

on the cells through mediation of beta-2 receptors. Al-

17though the effects of beta-2 antagonists were not examined here, the finding that beta-l antagonists did notinterfere with the dispersion response of melanophores
to isoproterenol sUbstantiates indirectly the presenceof beta-2 receptors in the cells.
With regard to the leucophores of the medaka,
renergic

receptors which mediate

leucosomeare suggested to be beta-l (Yamada, 1980).
resul ts

showed

this,

e.

l .

that

both

ad-

dispersion
The present

salbutamol '.

andterbutaline, beta-2 agonists (and which also are beta-l
antagonists),

themselves

effect on the cells,

had

no

leucosome-dispersing

but inhibited thesponse of the cells to isoproterenol.

dispersion

re-The beta-l an-tagonists,

metoprolol and atenolol,

also competitively

inhibited

the

to

which

leucophore

substantiate themediatingresponse

idea that

isoproterenol,

adrenergic

leucosome dispersion wi thin

receptors

leucophoresaresubtype beta-I.
Rapid

paling

in

body

tint

of

teleostfishesismainly due to aggregation of melanosomes within melanophores.

In

some

fish

species,leucophores

concerned with the paling response.
Fundulus

heteroclitus(Foster,on Bachygobius soporaor(Fries,arealso

In vivo studies on

1933;

Fries,

1942)

and

1958)

have

shown thatwhen animals are placed on a

whi te background,phores in the scales and finsrespond with dispersionofleucosomes,

while melanophores

18

leuco-

respond with aggre-

A similar opposite response In

gation of melanosomes.

two chromatophores has been

demonstratedinisolatedtoepinephrine

scalesofFundulus

exogenous

vi troon(Odiorne, 1933) and on those of the medaka to potassiumions (Miyoshi, 1952).
Recent investigations suggest physiologically andhistologically that both melanophores

andleucophores

of

same

adrenergic

themedakaareinnervated by

thefibers and that a putative neurotransmitter released isnorepinephrine (Iwata,
et al.,

1984).

phores

and

--

Then,

et al.,

Iga,

1983; Yamada

the question arises

how melano-

leucophores

usually

ways to identicalstimuli,potassium

exogenous

ions

or

1981;

respondwell

asCarlsson1980).

ananddifferentsuch as electr ic currents,
norepinephrine.

nephrine is generally accepted to beasin

Norepi-a beta-l agonist

alphaagonist(LandsHedberg,

1967;

Minnemanet

As shown in the present study,

al.,

and

1967;

Molinoff,the beta recep-

tors in the medaka melanophores are not beta-l in subtype but are beta-2,

so the neurotransmitter or exoge-

nous

does

norepinephrine

not

act

on

them

but

acts

preferably on alpha receptors to induce aggregation ofmelanosomes.
leucophores
spond

to

the

other

hand,

are exclusively beta-l

norepinephrine

leucosomes.
differenceOnproperly

beta

19

of

and

the

cells

re-

with

dispersionofHere, it can be said that,in the receptor

receptors

subtypes,

through such amelanophores

andleucophores

behave

In

vivo

and

in

in

vitro,

quite

opposite directions to each other.
Lately,theMiyashi ta et al.

presenceof

adenosine(1984)

have demonstrated

receptors

which

Moreover,melanosome dispersion in guppy melanophores.
Kumazawa et al.

(1984)and Kumazawa andmediateFuj ii(1984)

have indicated that adenosine or ATP isreleased as

cotransmitter

the

from

chromatic

nervesofatilapia,

Sarotgerodon niloticus. If so, how are beta-2 receptors
in melanophores concerned with the dispersion of melanosomes?

Miyashita

and

Fujii

(1975)

havesuggestedthat the endogenous beta stimulating amine responsibleformelanosome

dispersion of melanophores

is epineph-rine and that its source is chromaffin cells in adrenal
tissues or in the dermis.
the present case.

The same may be applied toEndogenous

dispersion of melanosomes

epinephrinemay

inducethrough mediation of beta-2

receptors, but there is no real evidence at present tosupport this.
In mammalian heartand 1 ung tissues,

and beta-2

receptors

same cells(Lands et al.,Minneman et al.,

have

1979;

The neurotransmi tter ofboth beta-lbeen known to exist
1967;

Minnemanin theBarnett et al.,

1978;

and

1980).

Molinoff,

sympathetic nervesisnorepi-nephrine and it acts mainly via beta-l receptors, whilecirculating epinephrine may activate

beta-2

causing an additional potentiation

the

20

of

receptors
tissuere-sponses(Carlson and Hedberg,

1976;

Minnemanetal.,

1979; Minneman and Molinoff, 1980).
On the contrary,Ranaesculenta,

LarsenandLarsen,

1981)

1978;

and

(Falck et al.,pipiens

R.

Helle,

in cardiac tissues of the frogs,

the

1966)

and

Hancock

teleosts~.temporaria

et

al.,

1979;

Pleuronectes

and Salmo gairdneri(SteneSten-platessa(Ask etal.,1980), it has been suggested that the main sympathetic
neurotransmitterisepinephrine

which

acts

on

beta-2

receptors and that beta-l receptors are less importantin the tissues and are activated by circulating norepinephrine.
However,

unlikecardiac tissues
of

the

of

chromatic

the

the

resul ts

teleosts,

adrenergic

demonstrated
thenervesnorepinephrine and not epinephrine.

in

the

neurotransmi tterofteleostsis

At present, wether

such a difference in neurotransmitter mechanism between
the chromatophore system and cardiac tissues is due totissue specificity or species specificity is not known.

21

100 (a)

5~ (
0

~

~
1nM NE

5

10

20

NE

25

1pM Proc:-"'0100 (b)

Ol

5~r

d)

....

Ol
Ol

0

<t

30 (min)10}JM NE

~
5nM E

5

10

20

E

25

1pM Pro30 (min)
10pM NE

100 (c)

t

Iso

5

10

20

Iso

1J.JM ProF i g.

1.

Ty pical recordings

25

30 (min)
10!.!M NE

showing the melanosome-aggrega-tion response of d enerv ated melanophores of the medaka to (a) 1 nM
nor e p i n e p hri n e (NE),(b)

5 nM epinephrine(E ) and (c)10 u M iso-protere n ol (Iso) , b efore and after treatment with i uM propranolol(Pr o ).

Ab scissae , ti me i n min .

as a p e rcen tage o f

Ordinates , magnitude of response

the max imal le v el of aggregation that was ob-

t ain ed in r esponse to 10 UM NE .

Note that both records and time

sca les ar e br o k e n a t mar ks for conv enience .

22

100

(a)

~

c

...co
0

50

C)
Q)

...

C)
C)

<

0

I

I

I

Fig. 2 .
(a),

I

I

5 10
1
Concentrat ionof E (nM)51
Con centrationof NE (nM)

I

I

I

1

5 10

50

Con cen t rat ionof I so (!-1M )Relationship of the concentration of norepinephrine

epinephrine

(b)

and

isoproterenol

(c)

to

the

magni tudeofmelanosome-aggregation response of the rnedaka melanophores in th eabsence(open symbols) and the presence

propranolol .

Ordinates,

scale .
point

Abscissae,

is

the

concentration of amines

magni tudemean of 10(solid symbols)
inof aggregation respons emeasurements

vertical bars indicate SDs .

23

indifferentof 111M

logari thmic
(%).

Each

animalsand1.!JM Sal

1pM Pro+Sal~ 100 (b)
c
o
"iii
~

0-

I/)

o

10

15

i

25

Iso
1jJM Sa I

Fig. 3.

Typical recordings showing the effects of salbutamolon chromatophores of the medaka.

(a)Melanosome-aggregationre-sponse of melanophores to 10 nM norepinephrine (NE) under the influence of 1
~M~M

salbutamol (Sal) with or without the presence of 1

propranolol (Pro).

Abscissa, time in min.

of aggregation response (%).

Ordinate, magnitude(b) Leucosome-dispersion response of

leucophores to 100 nM isoproterenol (Iso) in the presence of 1
salbutamol.

Ordinate, magni tudeof response

as apercentage ofthe maximal level of dispersion which was obtained with 10

24

~M

~M

NE.

aR
(a)
~100

02

~ 50
01

"~ 0

~J
5

<t

0

~ 100

(b)

10

10nMNE

\
25

20

30

50 (min)
10pMNE40

NE
1pM Pro+ Ter

NE
10pM Ter

c
0
°Vi

V
Q.
f/)

0

10

25

15

Iso
10.l::!M Ter

Fig. 4.
ine

Typical recordings showing the effects of terbutal-

on chromatophores of the medaka.

(a)Melanosome-aggregation

response of melanophores to 10 nM norepinephrine

(NE)

under

the

influence of 10 WM terbuthaline (Ter) with or without the presenceof 1 WM propranolol (Pro).

(b)

Leucosome-dispersion response of

leucophores to 100 nM isoproterenol (Iso) in the presence of 10 11M
terbutaline.

For explanation of abscissae and ordinates for thisand Figs. 6 and 7, see legend of Fig. 3.

25

100 (b)100 (a)......
~

~

c:

c:

-

.2

~

~
50
C)

Q.

en

~

C)

~

50

en

0)
0)
~

0

5

10

Fig . 5.

(a)

50 100

10

50

500

Concentration of I so ( nM )Concentration of NE (nM)

Relation between

theconcentration of norepi-

nephrine and the magnitude of aggregation response of melanophores
in the absence (open circles) or presence of either 1 UM salbutamo l(solid squares ) or 10 11 M terbutaline(solid triangles).

Ab-

scissa, concentration of norepinephrine (NE) in logarithmic scale.
Ordinate, magnitude of aggregation response (%).

(b) Relationshipo f the concentration of isoproterenol to the magnitude of dispersion responseofleucophorespresence of 1 UM salbutamol(solid triangles).

theabsence(open

circles)or(solid squares) or 10 11M terbutaline

Abscissa , concentration of isoproterenol (Iso)in logarithmic scale .

(%).

in

Ordinate magni tude of dispersion response

Each point in the figure is the mean of 10 measurements indifferent animals and the vertical bars indicate SDs .

26

r::( ~
~

~ 100

0
5
10nM NE

10

i

20

25

30

NE
100nM I so

0 .5JjM Met + Iso

(b)

c
o
'iii 50

v
0.

I/)

is

10

15

Ii

25

I so
0 .5JjM Met

Fig . 6 .

Typical recordings

10pM N E

showing theeffectsof 0.5\.1 M

metoprolol (Met) on the melanosome-aggregation response of melanophores to 10 nM norepinephrine (NE) under the influence of 100 nM
isoproterenol (Iso) (a) and on the leucosome-dispersion respon s eof leucophores to 100 nM isoproterenol (b).

27

~ 100 (8)i50r-~ J

3 0 <5

5--10-'

10nMNE

\

10

25

NE
100nM Iso3b1}JM Ate + Iso~ 100 (b)
c

.2

III

~

III

1~

is

i

25

(min)

Iso
1AJM Ate

Fig. 7.
ateno10l

Typical recordings

10pM NEshowing theeffects of 1

J.1 M

(Ate) on the melanosome-aggregation response of melano-phores to 10 nM norepinephrine (NE) under the influence of 100 nM
isoproterenol (Iso)(a) and on theleucosome-dispersion responseof leucophores to 100 nM isoproterenol (b).

28

100

c:
o
CI)

Qj 50
a.
CI)

o

o

,

5

10

I

50 100Concentr ation of Iso

Fig . 8 .

500
(nM)

Relation between the magnitude of leucosome-disper-sion response of leucophores and the concentration of isoproterenol without (open circles) or with 0.5 WM metoprorol (solid triangles ) or 1 WM atenolol (solid sqares).
i s oproterenol in logarithmic scale .
persion response (%) .

Abscissa, concentration of
Ordinate, magni tudeof dis-Each point is the mean of 10 measurements

in different animals and the vertical bars indicate SDs.

29

IV. The subtype of alpha adrenergic receptors mediating
melanosome aggregation in melanophores

30

Melanophores In scales and fins of teleost fishes
have

been

shown

to

receiveadrenergic

innervation

1968;

Falck et al.,

1969; Reed

(Jacobowitz and Laties,
and Finnin,
putative

1972;

Fujii

and Miyashita,

neurotransmitte r

of

the

suggested to be norepinephrine
1963;

Fujii

and

Novales,

stimuliorexogenous

stratedtoinducemelanophores(Grove,Fujii

Miyashita,

and

of

stimulation

1969;

1961;

have

the

nerve

is

Scheline,
nerve

been

demon-

melanosomes

withinofalphaadrenergic

Fernando and Grove,
Meanwhi le,1975).

andFurthermore,1972).

norepinephrine

throughreceptors

chromatic(Fujii,aggregation

1975)

1974a,

it

has

b;

been

also shown that dispersion of melanosomes within melanophores

occurthroughmediation

receptors (Reed and Finnin,

of

beta

adrenergic

1972; Miyashita and Fujii,

1975; Yamada et al., 1983).
Recent physiological studies on medaka scales havesuggested that melanophores of the medaka also receive
adrenergic
produces

innervation and thatthrough mediation of alpha adrenergic receptors(Iwata1981;

Yamada et ale

Iga,melanosomes

stimulation
cells

al.,

of

nerve

the

et

aggregationthe·U slng1983).

wi thin

3 H-noreplnep
.
h
'
rlne,

(1984) have demonstrated autoradiographi-

cally

the

patternscales

of

the

of

medakainnervation
and

have

to

melanophores

ascertained

that

in
the

neurotransmitter released is norepinephrine.
In the preceding chapter,

31

according to the c rite-ria fo r subc l assi f i c a ti on o f adrenergic rece p t o r s in
mammalian tissues (Lands et al., 196 7; Min n ema n et al. ,1979), it was dete r mined t ha t b et a adre ne rgic re c e p t o r swhich mediate melanosome d i spe r sion o f

medaka melano-phores are beta-2 in the subtype.

Howeve r , at p r esent,

therecep t ors

subtypeofalphaadrenergic

medakaIn mammalian tis-melanophores has been not examined.
sues,

in

alpha adrenergic receptors which mediate various

cell responses are also classified into two subtypes,alpha-landalpha-2,

cells

for

specific

1 9 81;Johansson,

dependingagonists1984).

on

and

the

affini ties

antagonistsof(Starke,In cold-blooded vertebrates,

studies on subtypes of alpha adrenergic receptors which
participate in the melanosome-aggregation
melanophores

arecomparatively

few

response(Bert h e ls e nofandPettinger, 1977; Carter and Shuste r , 1982; Ander sson etal.,1984).

Therefore,

In the pres e nt expe r iment,asan aid to analyse the receptor mechanisms

involved In

the regulation of chromatophore movements,t he subtyp e

of

alpha

adrenergic

receptors

inthe

medakamelano-phores were investigated using a series of alpha adr energic

agonistsandantagonists,

resemblance and difference in the

in

which

also

aff ini ty of cellthetothe adrenergic drugs were examined between the present
melanophores and the other heretofore studied cells.

32

Results

Effects of alpha agonists
All the alpha agonists examined induced a reversible and concentration-dependent melanosomeofmelanophores,

but

the

effecti ve

the maximal effect obtained for

aggregation

concentration

andeach agonist wereob-

served to differ considerably.
Figure 9 showstherelationship ofof melanosome-aggregation
theconcentration ofgatingpotencyofagonists(3.3(30.0 nM),

nM),

oxymetazoline

the

(0.15

(0.70

(8.5

(44.7 nM),l-lM),

mM)

melanophoresEC

following

epinephrine

tramazoline

tetrahydrozoline

andin theoftoThe melanosome-aggre-

agonists.

from the curves were
nephrine

response

the magni tude

50'sestimatedorder:

nM),

norepi-naphazoline

clonidine(55.0 nM),

phenylephrine(37.5l-lM),

methoxamine

(0.74

mM).

and

Tramazoline, methoxamine and oxymetazoline did not induceamaximalagonists

full

aggregation

concentration
showedfullofmelanosomes

examined

(Fig.

even

9).

at

The

melanosome-aggregating

the

other

action,indicating that they are all full agonists.

Effects of antagonists
Effectsofantagonists,

prazosin,

corynanthine,

yohimbine and phentolamine were examined on norepinephrine- or clonidine-induced melanosome-aggregated melan-

33

In

ophores.
agonists
for

these

experiments,

used werenM

norepinephr ine

for

which

respectively,

clonidine,melanosome

50

aggregationofconcentrations

of
1

and

(cf.

)JM

a

full

Fig.

9).

inducedmelanophores

theMelanophores were pretreated with a given concentration
of

each

wi th

antagonist

ei therforagonist

5

in

min,

and

then

the

presenceof

typicalexamples

for

were
a

treatedrespective

antagonist.
Figure 10 showsofprazosinandmelanophores.

yohimbine

Both

on

antagonists

theeffects

norepinephrine-treatedinhibitedaggregation response of the cells tomelanosome-

50 nM norepineph-rine in a concentration-dependent manner.

A full inhi-

bition)JM

wasobtained withyohimbine at

O. 1

)JM,

prazosin

atrespecti ve ly.

10

These

and

with

antagonists

also inhibited the cell response to 1 )JM clonidine in asimilar fashion
shown

here,

responses

to

(Fig.

11).

prazosin

and

the

alpha

other

Although theyohimbineagonists

data

are

not

inhibited

cell

used,

which

in

the inhibitory effect of yohimbine was always observed
to be about 100-fold greater than that of prazosin.
Figure12presents

therelation

betweenconcent-

rations of antagonists and their inhibitory effects on
melanosome-aggregation

response

nM norepinephrine

(a)

and 1

figure,oxymetazolineasaneffect ofalphaagonist

is

also

34

of

melanophores)JM clonidine
whichshown.

(b).

itself
The

is

drug

to

50

In

the

known
had

no

melanosome-aggregating
than 1 jJM

(cf.

Fig.

action

9),

but

at
it

concentrations
inhibited thelowermelano-phore responses to norepinephrine and clonidine just as
being an antagonist. The order of the inhibitory effectof antagonists on thecellresponse

to norepinephrine

was observed to be the same as that of the drugs on thecell response to clonidine.
tions

of

cell

response

85.8 nM,

antagonists

0.84

to

50

jJM,

1.3

for
nM

The

effective

inducing

50%

norepinephrinejJM,

and 11.7

concentra-

inhibition

of

were

nM,

jJM for

14.5

yohimbine,

phentolamine, oxymetazoline, prazosin and corynanthine,
and those to 1 jJM clonidine were 14.0 nM,
jJM, 1.8 jJM, and 24.5 jJM, respectively.

35

0.54 jJM,

1.6

Discussion

At present,

numerous evidence have revealed thatthe adrenergic receptors mediating melanosome aggregation within melanophore of teleost fishes are alpha innature (cf. Bagnara and Hadley, 1973; Fujii and Oshima,1986) .

In the present experiments on

medakaphores,melanosome-aggregatingof

naphazoline and tramazoline,

potencymelano-clonidine,

all known as alpha-2 ago-

nists, were far greater than that of alpha-l agonists,
i. e.

methoxamine,

Furthermore,thephenylephrine
inhibitory

andactiontetrahydrozoline.
ofyohimbine,aspecific alpha-2 antagonist, on melanophore response to
norepinephrine was aboutphentolamine,lOO-foldagreaterlO-fold greater than that ofnonspecif ic
than

that

antagonist,

and wasofandprazosinaboutcorynan-thine, specific alpha-l antagonists, respectively. Such
differences among the inhibitory actions of antagonists
were observed to become far greater when the antagonist
effects were examined on the cell responsetocloni-

dine.

of

these

Criticalestimation

of

the

effects

agents, based on their proven actions in other animals,
demonstrates that the subtype ofalpha adrenergic

re-ceptors involved in the melanosome-aggregation responseof medaka melanophores is probably of alpha-2.

have

Subclassification

of

alpha

first

on

mammalian

been

made

36

adrenergic
tissues,

receptors
in

which

Langer(1974)

proposed that postsynaptic

receptorsbereferred to as alpha-l and the presynaptic receptors as
However,alpha-2.

strated thatthesubsequentalpha-2

reseraches

receptors

havedemon-

also exist

In

the

postsynaptic tissues, and the terms alpha-l and alpha-2
receptors are now being used solely for receptors withdifferent reltive activities and affinities of agonists
and antagonists,

respectively,

irrespective of the 10-

cation (Berthelsen and Pettinger, 1977; Hoffman et al.,1079;

Timmermans and van Zwieten,

sent

study,

denervatedsincemelanophores

ones,

alpha

melanosome-aggregation

used

receptors

response

In the1982).

ofwere

exclusively

involved
the

pre-

the

in

presentcells

are postsynaptic ones.
Inarecent

study

on

scale

melanophores

teleost, Labrus ossifagus, Andersson et ale

of

the

(1984) havedetermined that adrenergic receptors regulating melanosome

aggregation arealpha-2

in nature,demonstratingthat yohimbine inhibits melanophore response to norepinephrine completely but prazosin practically does not.
Carter and Shuster(1982)

have obtained asuIt on skin melanophores of the lizard,
nensis,indicating

that

the

similarAnois caroli-

receptors

involvedmelanosome-aggregation response of the cells
alpha-2.

However,

concentrations
melanophore

of

In

the

alpha-lresponse

present

in

are

also

experiments,

highantagonists

to norepinephrine

37

re-

inhibited
andtheclonidine,respectively,

and besides,

alpha-l agonists could also

induce melanosome aggregation within the cells,
thepotencies

wereconsiderablymelanosome-aggregating
epinephrine,
about

than

that

Furthermore,

low.

of

nonspecific agonists,

10-fold greateragonists.

effectsthoughnorepinephrine

andwere observed to beof

specificalpha-2

These findings suggest that the alpha recep-

tor population of the present melanophores consists ofboth alpha-l and alpha-2 subtypes.

Coexistence of al-pha-l receptors with alpha-2 receptors has been demonstrated in rabbit uterus cells
and rat hepatocytes(Hoffman et al.,(Hoffman et al.,

1979)

Ruffolo1980).

and Waddell (1982) have observed that alpha receptor ofrat

aorta has properties of both alpha-l

receptors.

andalpha-2

However, because of the observed differencein the affinities of melanophores to specific agonists
and antagonists,

the present results indicate that al-pha-2 receptors play a major role in melanosome-aggregation response of the medaka melanophores.
Among alpha agonists
ments,

used in the present experi-

the action of oxymetazoline was observed to beconsiderably different from that demonstrated in othertissues.
oline

Oxymetazoline and clonidine, both are imidaz-

derivatives,

synaptic

alpha(Starke et al.,

act

as

receptors

alpha-2
ofrabbitagonistsonplumonarypreartery1975) and on presynaptic alpha recep-

tors of guinea pig ileum (Wikber, 1978). In rat brains,38

alpha-2 action of oxymetazoline isobserved to begreater than that of clonidine

Jong

(De

and

far

Soudij n,

1981) .

Meanwhile,

Lasch and Jakobs(1979)

have demon-

stratedinplateletsthoughepinephrine

human

that,

inhibits adenylate cyclase activity through stimulationof alpha-2
act

asreceptor,both oxymetazoline and clonidine

antagonists,

reducing

inhibition of enzyme.

theepinephrine-induced

In the present expeiment, cloni-

dine acted as a potent alpha-2 agonist on melanophore,
but oxymetazoline was far inferior in melanosome-aggregating action and besides it acted rather as an antagonist on the cells,

inhibiting the melanosome-aggregat-

ing action of clonidine.
that the nature
the

~f

All

these

findings

suggestalpha-2 receptor differs considera-

bly

among

cells

examined

acts

as

tors,

while it acts on postsynaptic

an agonist on the

and

that

presynaptic

oxymetazoline
alpha-2alpha-2

recep-

receptors

as an antagonist.
Another

imidazoline derivative,

tetrahydrozoline,

is shown to act as an antagonist on alpha-2

receptorsof human platelets

However,

Ruffolo and Waddell(Lasch and Jakobs,(1982)

rahydrozoline

hasaorta,

while

it shows

rabbi taorta.

In

Soudi j n

(1981)

nohave demonstrated that tet-alpha-2-agonist
an alpha-lcontrast

in humanvan Zwieten (1981)1979).

toplateletsIn

ratagonistic

actioninthese,

Jong

and

Timmermans

and

and

activityDein rat vascular smooth muscles have39

demonstrated
agonist.

that

tetrahydrozoline

acts

as

alpha-2

In the present experiments, malanosome-aggre-

gating action of tetrahydrozoline was approximately the
same

as

other

naphazoline

alpha-2

agonists,

and tramazoline.

Allsuggest that the difference in the

such

as

clonidine,

the

above

findings

action of tetrahy-

drozoline on different animal cells is due to the difference in the molecular structure of receptors in thecorresponding cells.

As there is no available evidence

at present, further invistigations is necessary.

40

100

~
c:

o

:;:; 50
ro

0')

...
~

0')
0')

<l:

o
5

Fig.

9.

Relationship

of

the

3

4

-log concentration of agonist(M)

concentrationofadrenergicalpha agonists to the magnitude of melanosome-aggregation responseof the medaka melanophores.
in logarithmic scale.

(%).

Abscissa,

concentration of agonists

Ordinate, magnitude of aggregation response

Each point is the mean of 8 measurements in different ani-mals and vertical bars indicate SEs.

Clo.

clonidine;E,epineph-rine; Mex, methoxamine; Nap, naphazoline; NE, norepinephrine; Oxy,
oxymetazoline;PE.

phenylephrine;

tramazoline.

41

THZ,

tetrahydrozoline;

Tra,

(a)

1::[
~

c

0

co

I

I

0
N E-Pra

10

I

I

30

40

I

I

I

50(mln) 60
10J.lM

0.1 J.lM

C)

~

I

20

10 J.lM

1 J.lM

(b)

;100[
~

50
0

I

I

I

I

I

0
NE--

10

20

30

40

Yoh

Fig. 10.

II,

50 (min) 60
10J.lM1 nM

10nM

0.1 }.1M

Typical recordings showing the inhibitory effects of(a) prazosin (Pra) and (b) yohimbine (Yoh) on melanosome-aggregation response of the medaka melanophores to 50 nM norepinephrine(NE).

Abscissae,

time in min.

Ordinates, magnitude of aggrega-tion response (%).

42

(a)

lOOf
50

~

c:
0

0

I

I

I

I

I

10

20

30

40

Pra

0)

N E
(b)

<t

50

ro

I

0

Clo--

1pM

0 . 1 pM

I

I

I

50 (min) 60
10 pM

10pM

~ lOOf
0

I

I

I

I

I

0

10

20

30

40

Clo-Yoh
N E

Fig.

11.

1 nM

10 nM

I

0.1 pM

Typical recordings showing theof (a) prazosin and (b) yohimbine

I

10 pM

inhibitory effects(Yoh) on melanosome-aggregation

response of the medaka melanophores to 1 WM clonidine
scissae, time.

I

50 (min) 60(Clo).

Ab-

Ordinates, magnitude of aggregation response (%).

43

(a)

100

~

50

c
0
C'O

en
....
en
en

0

Q)

-

9

8

7

I

I

I

8

7

6

5

6

5

4

(b)

<l:

100

0

c
0

....
.c

50

.I:.
C

0

9

4

-log concentration of antagonist (M)Fig. 12.
and (b)Inhibition of (a) norepinephrine (50 nM NE)-induced(1 11M Clo)-induced melanosome-aggregation response of the medaka melanophores by various adrenergic alpha anclonidine

tagonists.

Abscissae,

concentrations of antagonists in logarith-

mic scale.

Ordinates, inhibition of aggregation (%).

is the mean of 8 measurements
bars indicate SEs.

Cory,

in different animals

corynanthine;

Oxy,phentolamine; Pra, prazosin; Yoh, yohimbine.

44

Each point
and vertical

oxymetazoline;

PA,

v.

The subtype of alpha adrenergic receptors mediating leucosome aggregation in leucophores

45

In scales of the medaka,

areusuallyfound to be located just beneath melanophores.

Nerve-

mediated

stimuli,potassium

ions,

exogenous

norepinephrine

aggregation of melanosomes within
at

the

same

withinleucosomes
1969,

time,

1978).

theNotwithstanding

melanophores

Ianddifference

it

has

sympathetic postganglionic fibers

been

receive

of
Iga,

1952;

such

leucophores

whiledispersion(Miyoshi,stimuli,inducemelanophores,
cause

leucophores

response to identicalthatstimuliandcurrents

electric

as

such

and

leucophores

the

in

suggested
the

same

(Iwata et al.,1981;

Iga 1983; Yamada et al., 1984).
On the other hand,

pharmacologicalinvestigations

on medaka chromatophores have revealed that adrenergic
receptors which mediate aggregation of pigment granules
within melanophores and lecuophores are alpha receptors(Iga, 1968; 1979) and that those which madiate dispersionofpigment

are

beta

in

granulesnaturewi thin(Obika,

these

1976;

Iga

chromatophores
et

al.,

1977;

Yamada, 1980; Komatsu and Yamada, 1982).
Intheforegoing

accordingtoadrenergic

receptorsinMinneman

et

al. ,

1967;

the

st udies

criteria

for(Chapter

and

IV),

subclassification

mammalian
al.,

III

tissues

1979;(LandsStarke,

of
et

1981;

Johansson, 1984), I had examined the subtypes of adrenergic receptors in medaka chromatophores and determinedthat beta adrenergic receptors madiating dispersion of46

pigment

granulesbeta-2andinbeta-I,

melanophores

andleucophores

respectively,

and

that

the

adrenergic receptors which mediate melanosome

are

alpha

aggrega-tion in melanophores are alpha-2 in the subtype.
These

findings

suggestthat

the observed differ-ence in response to nerve stimuli and exogenous norepinephrine between malanophores and leucophores is due todifferences in the subtypes of adrenergic receptors andin the affinity of receptors to neurotransmitter,epinephrine.

nor-

However, at present, the subtype of alphaadrenergic receptors,

which mediate leucosome

aggrega-tion in leucophores has not yet been determined. Therefore,

in the present study, using specific agonists andantagonists,

subclassification

of

alpha

receptorslnmedaka leucophores was undertaken as an aid for further
analysisofthereceptormechanisms

regulation of chromatophore responses.

47

involvedintheResults

Effects of catecholamines
First,

leucosome-aggregating effects of represen-tative catecholamines, i.e. norepinephrine, epinephrine
andisoproterenol,onleucophores

wereSince leucophores usually maintain aaggregated
the

state

in

physiologicalamines were examined on

pretreated with alpha-MSH.

investigated.

fully

leucosome-saline,effectsofleucosome-dispersed cells
As shown in Figure 13,

0.1

nM MSH induced about 90% dispersal of leucosomes.
Among the catecholamines examined,andisoproterenol

causedafurther

norepinephrine

increaseinthedegree of cell responses to MSH (Fig. l3a and b). These
effects ofamines weredependent(data notpranolol(5WM),

observed

shown).
a

beta

to

be

concentration-

In the presence of proantagonist,however,theeffects of amines were completely reversed and leucophores responded wi th aggregation of
l3a and b).

Theaggregating

leucosomes

effectsof(Fig.

aminesalso concentration-dependent and leucophores

were

respondedwith full pigment aggregation to 0.5 nM norepinephrine
and 2 WM isoproterenol,
14) .

When phentolamine

was further added,

respectively
(5

WM),

an

(Figs.

l3a,

band

alpha

antagonist,such aggregation response of leuco-

phores to the amines were markedly inhibi ted

and

the

cells soon reached to a leucosome-dispersed state (Fig.

48

13a and b).

induced aggregationofleucosomes

persed leucophores even
(Fig.

13c).

the

in

catecholamine,another

epinephrine,

Meanwhile,in

MSH-treatedabsenceofpropranolol

concentration of

The effective

was more than 1 nM and thefull

leucosome

was obtained at 20 nM

(Fig.

14).

However,

trations higher than 50 nM,

theaggregating

epinephrine

increasedecreased with

in

dis-

the

amine

aggregation
attheconcen-effectofconcentra-tion and the cells assumed a leucosome dispersed state,attaining a(Figs.

fully dispersed state at more

than

0.5

WM

13c and 14).

Simultaneous application of propranolol completely
abolishedthedispersion

high concentrations ofbutiteffectresponse

epinephrine

had noinfluenceof

low concentrations

the

on

the

of

leucophores

(Figs

13c

and

to

14),

leucosome-aggregating
of

amine

(Fig.

14).

The aggregation response of leucophores to epinephrine
was

also

inhibitedbythepresenceofphentolamine(Fig. 13c).
The

leucosome-aggregating

potencyofcatechol-amines and the EC 0s estimated from Fig.
5

14 were in thefollowingorder:

norepinephr ine(27.0 nM)

and isoproterenol

epinephr ine(2 . 1nM),(180.0 nM).

Effects of specific alpha agonists
Effectsofvariousconcentration49

ofprazosin,analpha-l antagonist,and yohimbine,

nist, were examined.
ment,
the

an alpha - 2 antago-

Similar to the foregoing experi-

leucophores were pretreated wi thpresence

effect

of

of

5

each

llM

propranolol,

antagonist

was

0.1 nMduringexamined

MSH

in

which

the

the

cell

on

response to 0.5 llM norepinephrine.
Figure 15 shows typical recordings of the effectsof yohimbine and prazosin.

Both antagonists inhibi ted

the aggregation response of leucophores to norepinephrine in a concentration-dependent manner.

Asevident

from the figure, the effect of yohimbine was about 100fold greater than that of prazosin.
are not

shown,

similarinhibitory

Ai though the data
effectsofantago-nists were observed on the cell responses

toisopro-terenol and epinephrine, respectively.
The

relation

betweenconcentrations

and

the

inhibitory effects of antagonists on the cell response
to norepinephrine is shown in Figure 16.

Theinhibi-tory potency and the effective concentration of antagonists for inducing 50% inhibition of the cell response
estimated from the figure were in the following order:
yohimbine

(28.2

nM),

phentolamine

(0.49

llM)

and

methoxamineandprazosin (4.2 llM).

Effects of specific alpha agonists
Effectsofalpha-lagonists,

phenylephrine, and alpha-2 agonists, clonidine and oxy-50

metazoline,

wereexaminedon

MSH-induced

leucosome-

Figure 17 shows typical record-

dispersed leucophores.

ings of the effects of methoxamine and clonidine.
isapparent

from

the

figure,potency of the agonists

was

As

leucosome-aggregating

practicallyunchangedinthe presence of propranolol.
Figure 18 shows the relationship of the magnitudeof leucosome-aggregation response of leucophores to theconcentrations of agonists.

The

leucosome-aggregating

potency of agonists and theEC's estimated from50

curves were in the following order: clonidine (0.4oxymetazoline(0.59

phenylephrine (3.3

methoxamine

~M),

~M).

51

(2 •2

~M)

the
~M),

and

Discussion

Leucophores in scales of the medaka usually maintain a fully leucosome-aggregated state in physiological saline.
cal

Heretofore physiological and pharmacologi-

investigations

amlnesinducehave

demonstrated

dispersion

of

that

leucosomesincatecholthe

through mediation of beta adrenergic receptors

cells(Obika,1976; Iga et al., 1977; Yamada, 1980), while MSH causes
leucosomedispersion

via(Negishi

and

1980;

Fu j ii,
nous1985).
cyclicObika,

Besides,
AMP,

acti vationof

Yamada,1982;

methylxanthinesactivator of adenylate cyclase)
leucosomes(Obika,Oshimaandit has been shown that exoge(inhibitors

cyclic nucleotide phosphodiesterase)of

MSH - receptors

1976;

of

the

and forskolin(analso induce dispersion

NegishiandObika,

1980;

Yamada and Iwakiri, 1982; Namoto and Yamada, 1987).
Working on theophylline-treated leucosome-dispersed medaka
under
produceleucophores,

the

Iga

( 1979 )has

influenceof

propranolol

aggregationofleucosomes

observedthatcatecholamines
and

that

this

catecholamine effect is completely abolished by further
addition of dibenamine, an adrenergic alpha blocker. He
concluded from the results that alpha adrenergic receptors which mediate leucosome aggregation are present inmedaka leucophores,in addition to beta adrenergic re-

ceptors mediating leucosome dispersion.

52

In the present

experiment, to maintain leucophores in a leucosome-d i spersed state,

the cells were pretreated withstead of theophylline.

Insuch cells,

MSH

in-

catecholamines

also induced aggregation of leucosomes in the presenceof propranolol, and the effects of amines were markedly
inhibited by the coexistence of phentolamine. The orderof leucosome-aggregating potency of catecholamines obtained in the present study well coincided
has been observed byfindings

also

Iga

strongly(1979).

Thus,

substantiated

with

the

the

that

present

presenceofalpha adrenergic receptors in medaka leucophores, which
are associated with aggregation of leucosomes.
The present resul ts
prazosin,analpha-lalpha-2 antagonist,

revealed further

antagonist,

and

that

both

yohimbine,competitively inhibited theanleuco-

some-aggregating actions of catecholamines on MSH-dispersed leucophores and thatyohimbine

was

about

prazosin.

Besides,and phenylephrine,
oxymetazoline,
dispersed

thelOa-foldinhibitory potency ofgreaterthanboth alpha-l agonists,
and alpha-2 agonists,clonidine andleucosomeaggregation

leucophores

regardlessofabsence of propranolol.
potency of alpha-2

ofmethoxamine

induced

the

that

in

MSH-

presenceor

However, leucosome-aggregating

agonists

was observed tola-fold greater than that of alpha-lbeagonists.

about
These

results clearly indicate that the subtype of alpha adrenergic receptors which mediate53

leucosome-aggregation

response of medaka leucophores is alpha-2.
As described in Chapter IV,
adrenergic

receptors

mediatingthesubtype of alphamelanosome

aggregation

wi thin medaka melanophores are also alpha-2.

However,

the affinity of the receptors

to agonists

somewhatdifferent

and melanophores.

betweenleucophores

is

For

melanophores, the order in potency of catecholamines to
induce melanosome aggregation is:
nephrine and isoproterenol,that obtained forinleucophores,

epinephrine and isoproterenol.

norepinephrine,

contrast
i. e.

to

epi-

the

order

epinephr ine,nor-

In addi tion,oxymeta-zoline acted as a potent alpha agonist on leucophores,
while theagent itself had no effectand rather acted as an antagoists,
responses to catecholamine.

onmelanophores

inhibiting the cell

Such adifferencein theaction of oxymetazoline on alpha-2 adrenergic receptors
has been shown in various other tissues.

Oxymetazoline

acts as an alpha-2 agonist on presynaptic alpha receptors of both rabbi tpulmonary artery1975) and guinea pig ileum(Wikberg,(Starke1978),

etal.,

and on rat

brains (De Jong and Soudijn, 1981), while it acts as analpha-2

antagonist

onJakobs,1979).

these

All

human

platelets

findings(Laschsuggest

that

and
the

nature of alpha-2 receptors differs considerably among
the cells examined.
In the present study,

epinephrine,

trations (less than 50 nM),

54

at low concen-

itself could induce aggre-gation of leucosomes In MSH-dispersed leucophores evenin the absence of propranolol, while it caused a leucosome dispe rsion at higher concentrationsnM).

(more than 50

Since epinephrine is generally known to act

asboth alpha and beta agonists on adrenergic tissues,it

may beacted

said that,
on

alpha

at

lower concentrations,

receptors

andleucosomes within leucophores,

induced

the

amine

aggregationofwhile at higher concen-

trations, it acted on beta receptors and caused a dispersionofleucosomes

overcoming

mediated action.

55

the

alpha

receptor-

100

50

0
MSH

O.lnM
0 .5}JM

N E
Pro
P A

5}JM
5JJM

100

,....

f!.
.........
c

50

0
1Il
L..

Q.I

a.

.-1Il
0

0
MSH

O.lnM
2 pM

Iso
Pro
P A

5pM
5}JM

100

50

0
MSH

O.lnM
20nM

E
Pro
P A

Fig. 13.

O.2}JM
5pM
5}JM

Typical recordings showing the effects of (a) nor-epinephrine (NE), (b) isoproterenol(Iso)and (c) epinephrine(E)on the leucosome-dispersed medaka leucophores treated with 0.1 nM
MSH, before and during application of 5 WM propranolol (Pro) and 5
WM phentolamine (PA).

Abscissae, time in min.

of leucosome dispersion.

56

Ordinates,

degree

?\

\
\

\
\

\
\
\
\

\

o

9---"09876-log cone. of ca techolami nes (M)
Fig.

14.

Relationship of the concentrations of epinephrine(E), norepinephrine (NE) and isoproterenol(Iso) to the magnitudeof leucosome-aggr egation response of MSH-treated dispersed leucohores in the presence (solid line) and absence (broken line for E)
of 5

~M

propranolol.

logarithmic(%) •scale.

Each point

Abscissa, concentration of catecholamines in
Ordinate,

is

the

mean

magni tude of aggregation

responseofdifferent

20

animals and ver tical bars indicat SEs.

57

measurements

in

100

a)

lllliD50..........

0

0

0"-

c

MSH
N E

0

Yoh

'-'

O.lnM
0.5pM
lOnM

50nM

500nM

Vl
~

<l1

a.

100

Vl

.0

50

o
MSH
N E
Pra

Fig.

15.

Typical recordings showing theof (a) yohimbine (Yoh) and (b) prazosin (Pra)inhibi tory

on leucosome-aggre-

gation response of MSH-treated dispersed leucophores

to 0.5

norepinephrine (NE) in the presence of 5 ).1M propranolol
not indicated in figure,wi th MSH).

Abscissae,

propranolol wastime in min.

some dispersion (%).

58

effects

appliedOrdinates,).1M(althoughsimul taneously

degree of leuco-

100........

;f!.
'-'

c

0

.0
.J::.

50

C

a
8

Fig.
of

16.

inhibitory

phentolamine

756-log conc. of antagonists (M)Relation between the concentration and thedegreeeffects(Yoh),

(PA)

and

of

alpha

antagonists,

prazosin

(Pra)

on

yohimbine

leucosome-aggregation

response of MSH-treated dispersed leucophores to 0.5 )1 M norepinephrine in the presence of 5 )1M propranolol.

Abscissa,

tration of antagonists in logarithmic scale.

Ordinate,

inhibi tion

20

(%).

Each point is

the mean ofdifferent animals and vertical bars indicate SEs.

59

concen-degree ofmeasurementsin100 a)50...-..

0-...0

0.1 nM

MSH

0

'-'"

cJ)

5~M

5}JM

'-

P A

a.
cJ)
.-

10 0 b )

(1)

2~M

2l;!M

Clo
Pro

c

0

0

50

0

0 .1 nM

MSH
Mex

10 ~M
5 !;!M

101:!M

Pr o

5 pM

P A
Fig.

17 .

Typicalclonidine

and

(b)

leucophores
(Pro)

and

methoxamineb efore and5

11M

recordings

duringphentolamine

showing(Mex)onapplication(PA) .

the

MSH-treatedof5

Abscissae,

Ordinates , degree of leucosome dispersion (%) •60

effects

11M

of

(a)

dispersed
propranolol

time

in

min.

100

;!-

.........

c
0
",

0'1

50

dI

....

0'1
0'1

4:

a
7 6 5
-log conc. of agonists (M)

Fig.

18.

Relationship of theconcentration of

alpha

ago -

nists, clonidine (Clo), oxymetazoline (Oxy), methoxamine (Mex) andphenylephrine(PE) to the magnitude of leucosome-aggregation re-sponse of MSH-treated dispersed leucophores.
tration of agonists in logarithmic scale.
aggregation response(%).

Abscissa,

concen-Ordinate, magnitude ofEach point is the mean of 10 measure-ments in different animals and vertical bars indicate SEs.

61

VI.

General discussion

62

In the wild type of the medaka,

both melanophores

and leucophores are present in scales and are involvedin the control of physiological color changes.
investigations

have

leucophores of theinnervation

al.,

have

medakaand that

norepinephrineetsuggested1984).

thatphoreshaveboth alpha( Iga,

1968;

Obika,

the

same

al.,

1983;

1981;

Iga,

pharmacological

and

1976;

two

beta
Iga

types

chromato-

adrenergic

receptors

al.,exogenous

aggregation of melanosomes

and

the

same

time

Yamadaofetinduceisresearches

1977;

Yamada,However, notwithstanding these findings,
orandadrenergic
releasedthese

ly nerve-mediated stimuliatmelanophores

neurotransmi tter

Besides,demonstrated1980).

recei vethe(Iwata etthat

Recentnorepinephr ine

wi thindispersion

ofstudy,

analyseusual-

melanophores

leucosomes

wi thinleucophores.
Inthepresent

tothe

receptormechanisms involved in the regulation of chromatophore
responses

more

receptors

inprecisely,

medakathesubtypes

chromatophores

of

were

adrenergic

examinedac-cording to the criteria for subclassification of adrenergic receptors in mammals.
that

the

pigment

beta

receptors

granuleswi thinare beta-l and beta-2,

The

which

results

mediateleucophores

respectively

anddemonstrated

dispersionofmelanophores(Chapter III),

andthat the alpha receptors mediating aggregation of pigment granules within these two chromatophores are both63alpha-2 in nature (Chapters IV and V).

However, affin-

ities of the alpha-2 receptors for catecholamines wereobservedtodifferconsiderably

betweenmelanophores

and leucophores, i.e. the receptors in melanophores are
more sensitive to norepin ephrine than epinephrine ,but

the reverse is the case for those in leucopho res .
In mammalian adrenergic tissues, norepineph ri n e isgenerally
alpha

acceptedagonisttobebeta-l(Landsetal.,agonist1967;

asCarlssonadrenergic

receptors

inmedakanot beta-l but are beta-2

insubtype,mitterorexogenous

norepinephrine

andmelanophores
the

does

as

Since theHedberg, 1976; Minneman and Molinoff, 1980).
beta

well

are

neurotransnot

act

on

them, but acts selectively on alpha-2 receptors to induce

aggregation of melanosomes.

the subtypes of beta receptors
clusively

beta-landtheOn the

in

alpha

other

leucophores

receptorsareasmentioned

above,

theaffinityex-responsible

for leucosome aggregation are alpha-2 in nature.
ever,

hand,

of

How-

alpha-2

receptors of leucophores for norepinephrine is comparatively low so that the cells properly respond to neurotransmitter or exogenous norepinephrine with dispersionofleucosomesThen,throughmediationofbeta-lit can be concluded that throughreceptors.

such adiffer-ence in the receptor subtypes,

melanophores and leuco-

phoresquite

behaveindirections

other.

64

oppositetoeachMiyashita and Fujii(1975)

have demonstrated thatmelanophores of the guppy possess both alpha and beta
adrenergic receptors and suggested that the endogenous
beta amine responsible for dispersion of melanosomeslSepinephrine and that its source is chromaffin cells in
adrenal tissues or in the dermis.

The same may also be

applied to the present melanophores, ' since the subtype
of beta receptors in the cells is beta-2

and epineph-rine but not norepinephrine had a melanosome-dispersing
With respect to leucophores, epinephrine, but

potency.
notnorepinephrine

could

induceanaggregationofleucosomes in the MSH-treated dispersed cells eveninthe absence of beta antagonist (Chapter V).
epinephrine
nervesSince nor-is the neurotransmi tter of the adrenergic

innervating

medakachromatophores,

it

can

be

said that, in vivo, the neurotransmitter solely induces
melanosome

aggregation of melanophores

throughmedia-

tion of the alpha-2 receptors and at the same time dispersion of leucosomes within leucophores via activation
Meanwhile,of the beta-l receptors.
nephrine

may

cause

melanosome

phores through mediation of
aggregation of

theendogenous

dispersion
beta-2ofepi-melano-receptors

andleucosomes wi thin leucophore acting on

the alpha-2 receptors.
In various adrenergic tissues of mammals, stimulation

of

beta

adrenergic

receptors

has

been

shown

to

activate adenylate cyclase and induce proper functions65

of cells through increase in the intracellular level ofc y c 1 i cAMP

( Rob is 0 n e t

a 1 .,

1967 ;

Mi nne rna n e t

a1 . ,

1979; Cerione et al., 1983, 1984), while stimulation ofalpha receptors, especially alpha-2 receptors, inhibits
the enzyme activity vice versa (Lasch and Jakobs, 1979;
Burns et al., 1982).
In

fish

demonstrated
wi thin

species,toexogenous

induceacyclicdispersion

epinephrine-treated(NovalesandCarassius

auratus(Abramowitz

andMiyashita,

1976)(Fujiiandof

Fujii,

1970),

Chavin,Furthermore,

i mm uno ass a y me tho ds,

Neg ish i

eta1 .

demonstrated in melanoma cells

aggregatedcells

cyclic
is

AMP

of

the

the

medakausing radio-( 19 81 ,1 9 8 2)h ave

hybrid

fish

that

a

content

significantlyofgoldfish,1974),

and1980).

intracellularbeenmelanosomesthe(Negishi and Obika,

the

has

aggregated melanophores

Fundulus

guppy

AMP

of

less

non-treated cells and that theophylline,

epinephr inethan

that

of

which induces

dispersion of melanosomes, causes a remarkable increase
Andersson et al.

in cellular cyclic AMP.

(1984)

havealso observed that norepinephrine induces a significant
reduction of cyclic AMP content in Labrus melanophores.
Namoto and Yamada
that

Li +,

(1983)

known asanand Namoto

inhibi tor of(1985)

have

adenylateshown

cyclase(Duosa and Hetcher, 1970; Wang et al., 1974; Ebstein etal.,1976),

inducesmelanosomemelanophores and that Mg

2+

aggregationand Mn66

2+

ofmedaka, known as acceler-ators of the enzyme
et al.,(Drummond and Duncan,

1970;

Halmi

1974), produce inhibition of melanosome-aggre-

gation response to norepinephrine or Li+.

Forskolin, apotentiatorand Linder,ofadenylatecyclase(Metzger

1981; Seamon et al., 1981; Sano et al., 1983), has also
been shown to induce a dispersion of melanosome within
norepinephrine-treated( Andersson

etmelanophoresal.,aggregated1984)(Namoto,melanophores

In.+
Ll -treatedmedakaIn

leucophores

of

and

1987).

Labrusthe

medaka,it has been also observed that both exogenous

cyclicAMPand methylxanthines,

theophylline

such(Yamada and Iwakiri,(Namoto and Yamada,

1987)

as

1982),

caffeineandand forskolincause a dispersion of leuco-

somes, while Li+ inhibits leucosome-dispersion responseofthe1987).

cells
All

to

isoproterenol

thesefindings(Namotoand

Yamada,together withthepresent

results suggest that stimulation of alpha-2 adrenergic
receptors

in

fish

cyclaseacti vi ty

response
content,andmelanophores

inhibits

adenylatecausesamelanosome-aggregation

through

reductionofcellularcyclicAMP

whilestimulationofbeta-2

receptorsinleucophores activates the enzyme and induces dispersionof leucosomes by elevating theintracellular levelofcyclic AMP.
Recentinvestigations

revealed thattive

adenylatein hormonecyclaseon

mammalianandneurotransmitter-sensi-

systems,

67

guaninetissueshave

nucleotide-binding

regulatory

important

role

and

adenylate1984).
the

in

In

of

(G-proteins)the communication

cyclasebeta

stimulatory

binding

proteins(c f .

adrenergic
G-p rotein

agonist

with

play

betweenRodbell,an

receptors

1980 ;

Gilman,receptor-mediatedsystems

(G s)

the

is

involved

receptors

and

the

activat s

Gs,

which stimulates the adenylate cyclase activity,

wh ilein alpha-2 adrenergic receptor-mediated responses ,inhibitory G-protein

(Gi)

is

involved,

thestimulation ofwhich mediates an inhibition of the enzyme.

From thesubtype analogy of adrenergic receptors between medaka
chromatophores and mammalian tissues, it
assumedthattheadrenergic

tentativelylS

regulationofpigment

movements in medaka chromatophores may be mediated byadenylatecyclasebinding proteins,
shown

to

Gs

catalyze

augument

thePfeuffer ,1978 ;

systemadenylatecyclaseand(lAP) ,

is known to blockthethroughtoxin

ADP-ribosylation

activating protein

ductionthese

Choleraand Gi.

theGillincludingofactivity

Meren ,

1978),

two

GTP-

has

b en

Gs

and

( Cassel
while

to
and

islet-

one of the pertussis toxins,

receptor-mediated signal

inhibition

of

the

adenylatetransc yclas e

activity by the ADP-ribosylation of Gi(Katada and Ui,

1981 ,ale1982) .

Recently ,Karlssonet(1985)

have

demonstrated that alpha-2 receptor-mediated melanosomeaggregation response of Labrus melanophores is markedly
inhibited by the presence of lAP and have suggested the68invol vement
However,

of

the

Gi

in

the

involvement

signaloftransduction

GTP-binding

system.

proteins,

especially that of Gs, in the receptor-mediated regulation of adenylate cyclase In fish chromatophores still
remaine to be studied.

69

VII.

Summary70

Melanophores

andleucophoresinscalesofthemedaka, Oryzias latipes (wild type), have been known toreceive the same sympathetic postganglionic fibers andthe released neurotransmitter has been suggested to be
norepinephrine.

Moreover, these two types of chromato-phores have been shown to possess both alpha adrenergic
receptors which mediate aggregation of pigment granules
and

beta

receptors

However,

nervoususually

induce

each

which

mediatestimulianor

opposi tepigment

exogenous
response

dispersion.

norepinephrine

of

the

cells

to

other,

i.e.

aggregationofmelanosomes

withinmelanophores

anddispersionofleucosomes

withinleucophores.
Inthepresent

study,

toanalysethe

receptormechanisms involved in the regulation of such opposite
responses of medaka chromatophores,

subtypes of adren-

ergic receptors in the cells were determined according
to the criteria for subclassification of the receptors
Inmammalianaminesandtissues,
specific

using

representati ve

adrenergic

agonists

catechol-andantago-nists.
In the first series of experiments
subtypes of beta adrenergicbothmelanosome(Chapter III),

receptors responsibleformelanophores

anddispersionofleucosome dispersion of leucophores were examined.
Both norepinephrine, epinephrine and isoproterenol
inducedanaggregationof71

melanosomes

wi thinmelano-phores

andpropranolol,

a

beta

antagonist,

augmented

the action of the amines except that of norepinephrine.
Thissuggest

that

both

epinephrine

andisoproterenol

have a melanosome-dispersing action on melanophores in
addition to the melanosome-aggregating action and thatpropr~nolol

augments

theaggregation

cells to the amines throughresponseoftheinhibition of beta adren-

ergic receptors.

Salbutamol and terbutaline,

specificbeta-2competitively

agonists,some-aggregation

response

nephrine,

while

metoprolol

and

the

of

inhibitedthemelano-melanophores

tonorepi-

specific

atenolol,

did

not

beta-lantagonists,

interfere

wi th

the

melanosome-dispersion response of the cells to isoproterenol.

These findings indicate that the subtype ofbeta adrenergic receptors which mediate the dispersionof melanosomes within melanophores is beta-2.
with regard to leucophores, all the catecholamines
examined produced a dispersion of leucosomes within thecells.

Meanwhile,beta-2

agonists,salbutamol

andterbutaline which are also known to be alpha-l antagonists, had no leucosome-dispersing effect on the cells,
but inhibited the leucosome-dispersion response of the
cells

to

beta-lantagonists,

leucophore

isoproterenol,and metoprolol

also

response tocompetitively

isoproterenol,

andatenolol,inhibitedtheindicatingthatthe beta adrenergic receptors mediating leucosome dispersion of leucophores are beta-l in subtype.

72

In thenextseriesofexperiments(ChapterIV),the subtype of alpha adrenergic receptors which mediate
the melanosome-aggregation response of melanophores was
examined.

Both specific alpha-l agonists, methoxamine,

phenylephrine and tetrahydrozoline, and specific alpha2

agonists,

inducedan

However,clonidine,naphazoline

aggregation

the

of

and

melanosomesmelanosome-aggregatingtramazoline,

in

the

potency

cells.
ofthealpha-2 agonists was about 10-fold greater than that ofthealpha-lagonists.

norepinephrine

Among catecholamines examined,

was

about

melanosome-aggregating
Furthermore,the

5-foldpotency

inhibitory

greaterthanactionintheepinephrine.

ofyohimbine,aspecific alpha-2 antagonist,on melanosome-aggregation

responseof

norepinephrine

10-fold

greatermelanophores
thanspecific antagonist,

to

that

of

and waswasphentolamine,

about

a

about 100- and 1000-foldgreater than that of prazosine and corynanthine,
cific alpha-l antagonists,
ences

among

the

becamefar

greater

respectively.

inhibitory
when

non-

actions

theSuch differ-ofantagonist

spe-

antagonists
effects

examined on the cell response to clonidine.

were

All these

resul ts indicate that thesubtype of alpha adrenergic

receptors

theresponsibleforaggregationofmelano-somes in medaka melanophores is alpha-2.
Inthethird seriesofexperiments(ChapterV),the subtype of alpha adrenergic receptors which mediate

73

the

leucosome-aggregation response ofdetermined.

In

pretreated

wi th

these
MSH

in a

inducedwi thin the cells

an

were

hormone)leucosome-dispersed state.

catecholamines examined,

isoproterenol

wasleucophores(melanophore-stimulating

to maintain the cells
Among theexperiments,

leucophores

norepinephrine

aggregationin the presence of

of

and

leucosomes

propranolol,butepinephrine could induce leucosome aggregation even in
the absence of the antagonist.

Besides, both specificalpha-lagonists,

methoxamine

andalpha-2

agonists,

clonidine

andphenylephrine,

andoxymethazoline,

also

caused leucosome aggregation in leucophores regardlessof thepresence or absence of propranolol.

leucosome-aggregating potency of alpha-2
observed

to

be

about

lO-fold

greater

However,agonists
than

that

was
of

alpha-l agonists. Prazosine, a specific alpha-l antagonist, and yohimbine, a specific beta-2 antagonist, bothcompetitively

inhibitedtheleucosome-aggregation

response of the cells to catecholamines, but the inhibitory potency of yohimbine wasthan that of prazosin.
that

the

mediatesubtype of
the

about

lOO-fold greater

These results clearly indicate

alphaadrenergic

leucosome-aggregation

receptors

response

which

of

leuco-

that

since

phores is alpha-2.
It was concluded based on the results

norepinephrine is known to be a beta-l agonist as well
as

alpha

agonist,theneurotransmitter74

orexogenousnorepinephrine induced aggregation of melanosomes withinthemedaka melanophores

actingselectivelyalpha-2 receptors but not on beta-2 receptors.
other hand,thesubtypesin medaka leucophores
alphaadrenergic

aggregation

were

of

were

beta

adrenergic

responsiblealpha-2.

However,

for

the

the

On thereceptors

exclusively beta-l

receptorsonand theleucosomeaffini tyofalpha-2 receptors in leucophores for norepinephrine wasobserved tophoresbe

could

comparatively

properly

low.

respondto

Therefore,

neurotransmi tterexogenous norepinephrine with dispersion ofthroughmediation of beta-l

receptors.

differencesin the receptor subtypes,

leucophores

of

the

medakacould

quite opposite to each other.

75

leucoorleucosomes

Through · suchmelanophores andbehaveln

directions

VIII.

Acknowledgements

76

The author wishes to express his sincere thanks toProfes s o rK.

Yamadafor

hisinvaluableadviceduringthe cour se of the present study and for his kindness in
prepar i ng the manuscript.
H.

Katayamaa n d Dr .

O.

Thanks are also

Matsushima forcomments.

77

due

their

to

Dr.

valuable

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