Synthesis and biological actions of melanin concentrating hormone

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

Vol. 122, No. 2, 1984

Pages 613-619

July 31, 1984

SYNTHESIS AND BIOLOGICAL ACTIONS OF MELANIN CONCENTRATING HORMONE Brian C. Wilkes , Victor J. Hruby*, Wade C. Sherbrooke °, Ana M. de L. Castrucci +, and M.E. Hadley + Departments of

Chemistry, °Ecology and Evolutionary Biology and +Anatomy

U n i v e r s i t y of A r i z o n a , Tucson, A r i zo n a 85721 Received June 20, 1984 SUMMARY: A melanin (melanosome) concentrating hormone, MCH, was synthesized and the methodology for its synthesis is detailed. This heptadecapeptide, J H-Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val-OH, stimulated melanosome concentration (centripetal aggregation) within melanophores of all species of teleost fishes studied. Melanosome aggregation in response to MCH was not blocked by Dibenamine as was the response to norepinephrine (NE), demonstrating that melanosome aggregating responses to MCH and NE are mediated through separate receptors. Melanosome aggregation in response to MCH was reversed by an equimolar concentration of ~-melanocyte stimulating hormone (~-MSH). In contrast, MCH stimulated melanosome dispersion (centrifugal movement) within melanophores of a frog (Rana pipiens) and a lizard (Anolis carolinensis). Therefore, MCH exhibits both melanosome concentrating and dispersing actions depending upon the species studied.

A putative recently

melanin

isolated

decapeptide, GIu-VaI-OH,

from

granule the

(melanosome)

salmon

pituitary

concentrating hormone, gland

(i).

This

MCH, was

cyclic

hepta-

H-Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trpwas

reported

to

stimulate

the

in

vitro

centripetal

movement

(aggregation) of melanosomes within melanophores of several species of teleost fishes.

The isolation and characterization

of this

factor substantiated

the

early observation of Enami that such a hormone existed and might play a role in the chromatic responses

(color changes)

(2).

that

MCH

is a peptide

of certain species of teleost fishes

is synthesized

stored and released by the neurohypophysis MCH and documented

of

the

teleost hypothalamus

synthesis

(e.g., a frog and a lizard) of

the

peptide

and

and

We synthesized the putative

its contrasting actions on melanophores

e-~ tetrapod vertebrates aetails

in the

(3,4).

(5).

provide

of teleost fishes We now report the

further

data

on

the

biological actions of the hormone. MATERIALS AND METHODS Melting points were determined on a Thomas-Hoover melting point apparatus and are uncorrected. Thin-layer chromatography (TLC) was performed on Silufol plates (Kavalier, Czechoslovakia) using the following solvent systems: A) l-butanol/HOAc/H20 (4:1:5, upper phase only); B) l-butanol/HOAc/pyridine/H20

0006-291X/84 $1.50 613

Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(15:3:10:12); C) l-butanol/pyridine/HOAc/HoO (5:5:1:4); D) 2-propanol/25% aqueous NHa/HoO (3:1:1); E) amyl alcohol/py~idine/HgO (7:7:6). Detection was by iodine ~ap~r and ninhydrin. Optical rotations w~re performed on a Rudolph Research Polarimeter (Autopol) at the mercury green line (546 nm). Electrophoresis was performed on Whatman 1 chromatography paper using an instrument by Gilman at a potential drop of 20 v/cm at pH 5.9 and pH 2.5. Detection was by Sakaguchi spray (6). Amino acid analyses were obtained on a Beckman 120C amino acid analyzer following hydrolysis for 22 h at II0°C with 4 M methanesulphonic acid containing 0.2% 3-(2-aminoethyl)indole (7). No corrections were made for destruction of amino acids during hydrolysis. High pressure liquid chromatography (HPLC) was performed on a Spectra-Physics SP-8700 instrument with a SP-8400 detector at 214 nm using a Vydac C18 column (25 X 0.46 cm). Aqueous trifluoroacetic (0.1%) acid was the buffer with acetonitrile as the organic modifier. N~t-Boc protected amino acids and amino acid derivatives were purchased from Vega Biochemicals (Tucson, AZ), Penninsula Laboratories (San Carlos, CA), Bachem (Torrence, CA), or were prepared using published procedures. Before use, all amino acid derivatives were tested for homogeneity by TLC in solvent systems A, B, and C, by melting points, and by the ninhydrin test (8). Solvents used for chromatography were redistilled prior to use. MCH was synthesized using Boc-Val-Merrifield resin prepared as reported (9) with an amino acid substitution of 0.47 mmol/g resin as determined by standard methods (i0). N ~ -Boc amino acid derivatives were coupled to the resin using a three-fold excess of each amino acid, a 2.4 fold excess of dicyclohexylcarbodiimide (DCC) and a 2.4 fold excess of l-hydroxybenzotriazole (HOBT). Removal of the N~-Boc protecting group was accomplished with 45% trifluoroacetic acid (TFA) in dichloromethane (CH2C12) containing 2% anisole. A cycle for the incorporation of each amino acid derivative consisted of the following: i) washing the resin with three 20 ml portions of CH2CI 2, 1 min per wash; 2) washing with two 30 ml portions of EtOH, 2 min per wash; 3) washing with two 20 ml portions of CH2CI~, 1 min per wash; 4) cleavage of the N <-Boc protecting group with 45% TFA in C~9C12, one wash for 2 min, one wash for 20 min; 5) washing with three 20-ml portions of CH2CI 2, one min per wash; 6) neutralize the resin with two 30-ml portions of i0~ Giisopropylethylamine in CH2CI 2, 2 min per wash; 7) washing with two 20-ml portions of CHIC12, one min per wash; 8) addition of the next N ~ - B o c amino acid derivative along with DCC and HOBT in 30 ml DMF. Coupling of each amino acid derivative was complete in thirty to sixty minutes as monitored through the ninhydrin test except where noted. After coupling of the final amino acid derivative, the resulting peptide resin was dried in vacuo. Starting with 2.10 grams Boc-Val-resin (i.0 mmol total) the following N ~ -Boc amino acids were coupled (in order of their coupling): Glu(Bzl); Trp(For); Cys(DMB); Pro; Arg(Tos); Tyr(2,6-Cl2-Bzl) ; Val; Arg(Tos); Gly; Val. At this point the resulting protected MCH_ ._-resin was dried and weighed (4.30 I-I g). Val-10 required a 4 hour coupling, an~ Arg-9 required a 4 hour coupling and a 2 hour repeat coupling to proceed to completion. To 1.08 g (0.25 mmol) of the peptide resin were coupled: Met; Cys(DMB); Arg(Tos); Met; Thr(Bzl); Asp(Bzl). The resulting peptide-resin was dried and weighed (1.32 g). An HF cleavage of the peptide from the resin was accomplished using the procedure of Matsueda (ii). The reaction mixture was washed with 3 X 30 ml EtOAc and the product e x t r ~ t e d with 3 X 30 ml 30% HOAc and lyophilyzed giving the crude [Cys(H) , Cys(H) ]-MCH. The crude product was diluted in one liter deaerated H_O Z and the free sulfhydryl groups were oxidized as described (12). The solution was then lyophilized giving 300 mg crude MCH. Purification was best accomplished using carboxymethylcellulose ion exchange chromatography as previously described (1,13). This product was further purified using preparative HPLC giving an overall yield of 14%; amino acid analysis; Trp(1), 1.00; Arg(3), 3.00; Asp(l), 1.06; Thr(1), 1.03; Glu(1), 1.14; Pro(l), 1.05; Gly(1), i.i0; Cys(2) 1.93; Val(3), 2.70; Met(2), 2.04; Tyr(1), 1.02; Tlc (A) 0.00; (B) 0.87; (C) 0.55; (D) 0.73; (E) 0.54; HPLC K z= 3.0 (72% TFA/28% CH3CN,

614

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2.0 ml/min), K = 8.33 (76% TFA/24% CH3CN, 2.0 ml/min).

[~i]

-8.0 ° (C = 0.20

in 10% HOAc); electrophoresis 9.4 cm (pH 2.5, 2H); 5.4 cm (pH 5.9, 2H). The bioassays of MCH utilized whole skin preparations from two species of mailed (armored) catfish, family Loricariidae. Two to six skins could be obtained from each individual fish depending upon size. Skins were mounted on metal rings and held in place by an outer plastic ring as in the frog (14) and lizard (15) skin bioassays that have been described elsewhere. Tyrode's solution (i) was utilized with fish skins whereas an amphibian Ringer was used for the frog skin bioassay. The ~-MSH utilized was synthesized as already described (16).

RESULTS AND DISCUSSION Addition of the synthetic MCH to skins of the teleost fishes, Hypostomus sp.

and Pterygoplichthys

melanosome

aggregation

relatively

slow and was

medium

lacking

the

sp.,

resulted

within

reversible when

peptlde

in a lightening of the skins due to

melanophores.

(Fig.

i).

The

lightening

response

the bathing medium was changed Norepinephrine

(NE),

a

was to a

catecholamine

known to cause melanosome aggregation in most fish melanophores, also lightened the skins, an effect which was again reversible by rinsing the skins in medium lacking

the agent

effective When

NE

response,

than and

(Fig.

the

MCH

I).

peptide

were

At in

equimolar

stimulating

employed

at

concentrations, melanosome

concentrations

giving

the rate of the aggregating response to NE

NE was much

aggregation maximal

(Fig.

less i).

lightening

(10-5M) was much faster

than that to MCH (IO-7M)( unpublished data). When skins were incubated in Dibenamine, an ~-adrenoceptor antagonist, the response results

to NE, were

but not

obtained

to synthetic MCH, by

Rance

and

was

Baker

inhibited using

(Fig.

another

2).

50 rinse

# i

40

~

MCH (IO'TM)

NE (IO'TM)

/'qI

30

o ¢x o o

. ~

•

o

20

/I _....:_.. I

i ~ • ~

T

I0

;o I

0

20

40

60

80

i00

120

Time (minutes) Fig. i. In vitro lightening response of Hypostomus sp. skins to equlmolar concentrations of MCH ( • ) and NE ( <> ) and subsequent redarkening following the removal (rinse) of the hormones. Each value represents the mean, ± S.E., response of the skins (N=6) in the presence or absence of the hormones.

615

Similar

~-adrenoceptor

Vol. 122, No. 2, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

)'-!

60

• ~t 40

//

30 co m Do = o

50

~

_

c~

i

o~

=° £.~, 30

_

o

~

~

o

20

L.

u ~

• MCH I0"7M

~ ~. 20

(after 60'Dibenamme) ~'= elO

•

0

NE

T /

107M

(after 60'Dibenomlne)

I0

:0 .ol0

J

MCH 0

60

70 80 Time (minutes)

90

0

~-MSH

I E

I

I

I

I

20

40

60

80

I

i00

Time(minutes)

In vitro lightening response of Hypostomus sp. skins to MCH ( 0 ) after 60 min treatment with Dibenamine; the response to NE ( <> ) was completely blocked by the alpha-adrenoceptor antagonist. Each value represents the mean, ± S.E., response of the skins (N=6) to the hormones.

Fig. 2.

Fig. 3.

In vltro_Izghtening response of Pterygoplichthys sp. skins to (I0 M), followed by a darkenlng response to~-MSH (10-7M). Each value represents the mean, i S.E., response of the skins (N=6) to the hormones.

MCH

antagonist, response

phentolamine;

to pituitary

aggregation

in

which

extracts

response

to

did (17).

the

not

affect

These

two

melanosome

results

agonists

is

aggregation

indicate

mediated

in

that melanosome through

separate

receptors. In addition, MCH

is

the

these data suggest

result

of

direct

that the response of the melanophores

interaction

with

the

cell,

and

not

to

mediated

indirectly through the release of a catecholamine. Fish skins maximally lightened by MCH darkened almost to the original base (time zero) values when ~-MSH was added at an equimolar concentration Lightening induced by NE was also readily reversed by ~ M S H We

have

melanophores helleri,

determined from

Carassius

Hypostomus

all

that

species

auratus,

MCH of

stimulates fish

Lebistes

sp. and Pterygoplichthys

that

have

reticulatus,

sp.).

(data not shown).

melanosome

we

(Fig. 3).

aggregation

studied

Pimephales

in

(Xiphophorus promelas

(5),

In all cases, the minimal effective

dose of the heptadecapeptide was at 10 -9 - Io-IOM concentrations. Addition of MCH to frog skins did not induce a skin lightening response as was

observed

in

fish,

but

rather,

it

caused

a

dose-dependent

resulting from melanosome dispersion within melanophores. obtained using the skins of the lizard Anolis carolinensis. 1/600th

as potent

as ~ - M S H

in

the

frog 616

and

lizard

darkening

Similar results were MCH was only about

skin bioassays

(5).

The

Vol. 122, No. 2, t 9 8 4

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

50

• O

~-MSH (4xI0"~°M) MCH (10"6M)

.o

E

g~ 3o ~-~ 20

E

_

~°c. e

10

o

g

~

to ' =

Fig. 4.

darkening replaced

/ r ~)

l

30

l

60

91o

,

response

of

frog

by fresh Ringer

The

l

150

l

IB0

,

210

240

In vitro darkening response of Rana pipiens skins to MCH ( < > ) and =<-MSH ( O ) at the concentrations noted. Each value represents the mean, -+ S.E., response of the skins (N=6) in the presence or absence of the hormones.

skins

to

MCH

in the absence

was

present

results

concentrating

utilizing

hormone,

a

MCH,

reversed

of the peptide

darkening of lizard skins was also reversible

melanin

l

120 Time (minutes)

when

the

(Fig.

4).

medium

was

MCH-induced

(data not shown).

synthetic

preparation

demonstrate

the

of

potent

a

putative

actions

of

the

peptide on stimulating melanosome

aggregation within teleost melanophores.

The

present

previous

the

results~ along

salmon pituitary

with

the

isolation

(i~ provide strong support

of

the

for a possible

peptide

from

role of the peptide

in the control of color changes in fishes. In many species of teleost within

integumental

fishes

melanophores

it is clear that melanosome

is controlled by sympathetic

It is likely that the rapid color changes of some species regulated by the release of NE.

On the other hand,

aggregation

neurons

of teleost

(18,19). fishes

is

slower paling responses

in

some species may be controlled by the release of a pituitary MCH and subsequent humoral

delivery

mechanisms

to

integumental

of melanophore

control

melanophores. are involved

It

is

possible

that

in the chromatophore

both

responses

of some species of fishes. Most

interesting

dispersion

within

is

the

melanophores

observation of

both

a

that frog

MCH and

stimulates a

lizard.

(Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2) action of MCH on teleost melanophores the effects of the two structurally same

receptor

or

whether

~-MSH

teleost

fishes.

necessarily

or

infer

through

separate

another The

fact

that

it will be important

~ -MSH

reverses

the

to determine whether

different peptides are mediated through the receptors.

melanotropin that

melanosome Since ~ - M S H

~MSH

There

regulates reversed

normally

617

plays

is

no

evidence

melanosome

the any

action

of

as

to

dispersion

in

MCH

physiological

need role

not in

Vol. 122, No. 2, 1984

melanophore

control

melanotropin

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in

regulates

teleosts.

There

melanosome

is

good

dispersion

elasmobranch fishes (e.g., sharks, 2 0 ) .

evidence, within

however,

that

melanophores

a of

Whether a melanotropin receptor also

exists in some or all species of teleost fishes and whether this receptor and a melanotropin, such as MSH, play a role in color change remains undetermined. Our initial interest in the reported primary structure of MCH related to the possibility that the peptide might antagonize the actions of ~-MSH.

The

existence of such an antagonist would be of great importance in studies on mechanisms

of ~-MSH

action.

It may

be

possible

to

synthesize

structural

analogues of MCH that possess more or less MCH- or MSH-like activity.

It will

be important to determine the essential structural and conformational features of the peptide that are required for receptor binding and signal transduction that lead to melanosome translocation within melanophores.

This will require

classical structure-activity studies involving deletions and/or substitutions of amino acids comprising the primary structure of the peptide. major

goal

of

our

current

research.

Such

structure-activity

This is a studies

may

provide important insights into the evolution of the hormones and receptors regulating pigment granule movements within melanophores.

ACKNOWLEDGEMENT Supported in part by U.S. Public Health Service grant AM17420 and by National Science Foundation grants PCM-8112200 and PCM-8110708. Dr. A.M. / . Castrucci is a fellow of the Conselho Nacional de Desenvolvimento Tecnologlco e 1 Cientifico of Brasil, grant 200 430/82.

REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. i0. Ii. 12.

Kawauchi, H., Kawazoe, I., Tsubokawa, M., Kishida, M. and Baker, B.I. (1983) Nature 305, 321-323. Enami, M. (1955) Science 121, 36-37. Rance, T. and Baker, B.I. (1979) Gen. Comp. Endocrinol. 37, 64-73. Baker, B.I. and Rance, T.A. (1983) Gen. Comp. Endocrinol. 50, 423-431. Wilkes, B.C., Hruby, V.J., Castrucci, A.M.L., Sherbrooke, W.C. and Hadley, M.E. (1984) Science, 224, 1111-1113. Weber, C.J. (1930) J. Biol. Chem. 86, 217. Spackman, D.H., Stein, W.H. and Moore, S. (1958) Anal. Chem. 30, 1190-1206. Kaiser, E., Colescott, R.L., Boissinger, C.D. and Cook, P.I. (1970) Anal. Biochem. 34, 595-598. Gisin, B.F. (1973) Helv. Chim. Acta 56, 1476-1482. Gisin, B.F. (1972) Anal. Chim. Acta 58, 248-249. Matsueda, G.R. (1982) Int. J. Pept. Protein Res. 20, 26-34. Hope, D.B., Murti, V.V.S. and du Vigneaud, V. (1962) J. Biol. Chem. 237, 1563-1566.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Wilkes, B.C., Sawyer, T.K., Hruby, V.J. and Hadley, M.E. (1983) Int. J. Pept. Protein Res. 22, 313-324. Shizume, K., Lerner, A.B. and Fitzpatrick, T.B. (1954) Endocrinology 54, 553-560. Castrucci, A.M.L., Hadley, M.E. and Hruby, V.J. (1984) Gen. Comp. Endocrinol., in press. Yang, Y.C.S., Hruby, V.J., Heward, C.B. and Hadley, M.E. (1980) Int. J. Pept. Protein Res. 15, 130-138. Rance, T.A. and Baker, B.I. (1978) J. Endocrinol. 77, 47P. Iwata, K.S. and Fukuda, H. (1973) Response of Fish to Environmental Changes; (Chavin, W., ed.) Ch. C° Thomas, Springfield, II., pp. 316-341. Fujii, R. and Novales, R.R. (1969) Am. Zool. ~, 453-463. Parker, G.H. (1948) Animal Colour Change and their Neurohumours,. Cambridge University Press and MacMillan Co., New York..

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