Camp. Biorhem. Physrol. Vol. 88A, No. I, pp. 15-20, 1987 0300.9629187 $3.00 + 0.00 Printed in Great Britain 0 1987 Pergamon Journals Ltd

a-MSH (MELANOCYTE STIMULATING HORMONE) AND

MCH (MELANIN CONCENTRATING HORMONE) ACTIONS

IN BUFO lCTERICUS ICTERICUS MELANOPHORES

ELISABETH N. FERRONI and ANA MARIA DE LAURO CASTRUCCI

Departamento de Fisiologia Geral, Instituto de Biociencias, Universidade de SLo Paulo, C.P. I1 176, SLo Paulo, Brasil. Telephone: (011) 210-2122

(Received 24 September 1986)

Abstract-l. The darkening actions of MCH (melanin concentrating hormone), c(-MSH and the synthetic analog [Nle4, o-Phe’]-a-MSH on the toad, Bufo ictericus ictericus, melanophores were studied regarding the role of calcium in the hormone receptor coupling, signal transduction and intracellular pigment translocation.

2. In the absence of external calcium, MCH and both melanotropins still elicit maximal skin darkening. 3. Verapamil, a calcium-channel blocker, completely abolishes the a-MSH-induced response and

partially inhibits MCH-induced darkening, although the calcium carrier, ionophore AZ3,s7, was unable to promote any pigment translocation.

4. Since darkening responses promoted by cyclic nucleotides proceeded normally in the presence of verapamil and extracellular calcium was not necessary for melanotropin dispersing action, it is suggested that the blocking activity obtained with verapamil is probably due to an impairment of the CaZ+- dependent adenylate cyclase activity.

5. Reversal of melanotropin-induced darkening could be obtained with melatonin, in both normal and Ca’+-free Ringer, whereas MCH darkening is reversed by melatonin only in the absence of calcium.

6. The results seem to indicate that calcium is not required for hormone receptor binding and pigment migration, whereas it is specifically needed for signal transduction.

INTRODUCTION

It has been reported that there is a specific calcium

requirement for LX-MSH (melanocyte stimulating hor- mone) darkening action in the lizard Anolis car- olinensis (Vesely and Hadley, 1971) in the toad Scuphiopus couchi (Vesely and Hadley, 1976) and in the frogs Xenopus laevis (van de Veerdonk, 1976) Rana berlandieri and Rana catesbeiana (Vesely and Hadley, 1979). It has been suggested that this divalent cation is specifically required for hormone-receptor binding and signal transduction, since darkening induced by intracellular messengers, such as cyclic nucleotides, proceeds in the absence of external cal- cium (Vesely and Hadley, 1976; Hadley et al., 198 1 a).

We have previously reported (Ferroni and Cas- trucci, 1987) that the toad (Bufo ictericus ictericus) skin bioassay constitutes an assay as sensitive to melanotropins as the classical frog (Rana pipiens) skin bioassay. As MCH, a lightening hormone in teleosts surprisingly mimics LX-MSH in darkening frog and lizard skins (Wilkes et al., 1984; Ide et al., 1985) as well as toad skins (Ferroni and Castrucci, lot. cit.), we decided to study the mechanism of MCH action regarding the role of calcium ions, as com- pared to a-MSH and its prolonged acting analog [N1e4, D-Phe’]-a-MSH.

MATERIALS AND METHODS

Changes in skin color in response to hormonal stimu- lation were measured as changes in light reflectance from the outer (epidermal) surface of the skin using a Photovolt Photoelectric Reflection Meter (Photovolt Corporation, New York), as originally described for the frog skin bio-

assay (Schizume et al., 1954). An increase in reflectance represents skin lightening whereas a decrease in reflectance indicates skin darkening. Male toads (Bufo ictericus icter- icus) were collected in the vicinity of Slo Paulo, Brasil. Back skin as well as thigh and leg skin were excised from pithed animals. Square pieces were individually mounted on metal rings and held in place by outer plastic rings. The skins were then rinsed in Ringer’s solution (NaCl 6.5 g/l; KC1 0.14 g/l; CaCl, 0.12 g/l; NaHCO, 0.2 g/l) for 2 hr. During this time, a slow aggregation of melanosomes within melanophores occurs, resulting in light green skins. The excess solution was poured off, leaving 10 ml of solution in each beaker. An initial reflectance value was obtained for groups of skins. Succeeding average values for each group of skins were recorded as percentage changes above or below the initial base reading.

Verapamil, melatonin and calcium ionophore A2r,s7 were obtained from the Sigma Chemical Co., St. Louis. Dibenamine was obtained from Smith, Kline and French Laboratories, Philadelphia. G(-MSH, [Nle4, o-Phe’]-a:-MSH and MCH were synthesized in Dr V. J. Hruby’s laboratory, University of Arizona, Tucson, Arizona.

All the agents were added in 0.1 ml amounts to 10 ml of bathing solution. Concentrations of hormones are expressed as the final molar concentrations in the bathing medium.

Calcium-free Ringer was made by substituting CaCI, for 1 mM EDTA (ethylenediaminetetraacetic acid).

RESULTS

In the toad skin bioassay, [N1e4, D-Phe’]-a-MSH is

about 10 times more potent than a-MSH, whereas

MCH is about 600 times less active (Fig. I), similarly

to the potencies determined in the frog (Ranapipiens) bioassay (Sawyer et al., 1982; Wilkes et al., 1984).

Although various aspects of melanotropin mech-

15

16 ELISABETH N. FERRONI and ANA MARIA DE LAURO CASTRUKI

0 I2 II IO 9 e 7 6

- lag ii oncentrotion] (Ml

Fig. 1. Dose-response curves to c(-MSH (0) mle4, Et-Phe’]-ct-MSH (0) and MCH (v), as determined by the in &TO toad (8. ictericus) skin bioassay. Each value represents the mean f SE, darkening response

of the skins (N = 12) at the concentrations noted.

anism of action have been clarified, the MSH-like neither a-MSH nor MCH actions were affected by a activity that MCH has shown in amphibian skins I-hr incubation in IO-’ M Dibenamine. needs further study. In the lizard (Vesely and Hadley, 1971, 1976) and

It has been suggested that a-adrenergic receptors in frog (Vesely and Hadley, 1979; Sawyer et al., 1983) frog melanophores might be structurally related to skin bioassays, Ca *+ is required for a-MSH action, c(-MSH receptors, since the cc-blocker Dibenamine since the darkening response is blocked in the absence inhibits the melanotropin-induced response (Goid- of extracellular calcium. However, in the toad skin man and Hadiey, 1970). In the toad skin bioassay, bioassay the darkening responses to a-MSH, wle’,

U Nofmol Ringer

D Co*+-tfas Rintpf

Fig. 2. Bar graph showing that the maximal darkening responses to a-MSH, [Nle4, D-Phe7]-a-MSH and MCH are not impaired by the absence of external calcium. Furthermore, the maximal response to MC.33

is significantly enhanced in calcium-free Ringer (N = 20).

a-MSH and MCH actions on Bufo melanophores 17

50.

40.

v normal Ringer

v Go’+ -free Ringer

, 1

9 6 7 6 5

- logCMCH1 M

Fig. 3. Dose-response curves to MCH in normal Ringer(V) and in calcium-free Ringer (V) as determined by the same toad (B. icfericus) skin bioassay. Each value represents the mean f SE, darkening response

of the skins (N = 7) at the concentrations noted.

o-Phe’]-cr-MSH and MCH still proceed normally after a I-hr exposure to calcium-free Ringer (Fig. 2). Furthermore, the maximal response of the system to MCH is significantly enhanced in the absence of the cation, although the dose-response curve is not affected (Fig. 3). However, using verapamil, a calcium-channel blocker, the cc-MSH-induced re- sponse was completely blocked and MCH-induced darkening was only half-maximal (Fig. 4). These results seem, at first, controversial, since the ability of verapamil to block the melanotropin responses would suggest a calcium influx promoted by the hormone-receptor binding. If this was the case, vera- pamil should also impair the cyclic nucleotide dark-

-I- ,

ening actions and should be able to reverse the established darkening induced by the melanotropins. On the contrary, CAMP and dibutyril CAMP dark- ening activities were not significantly diminished by verapamil (Fig. 5) and also, the maximal response to MCH could not be reversed by further application of verapamil or removal of extracellular calcium (data not shown). Furthermore, ionophore A,,,,,, known to increase intracellular levels of calcium was not able to induce either darkening or lightening of the skins.

The only agonist that was found to be able to lighten cr-MSH and [Nle4, o-Phe’]-cl-MSH-darkened skins was melatonin (Fig. 6) and the reversal was only partial, although it normally proceeded in both nor-

v.15

T N-IO

_L . IO

0 Control

q Veropomil5slO”M

ID Veropomll IO-‘M

N=5

N.IO

d-M SH IO-‘t.4 *

MCH d6u

Fig. 4. Inhibitory effect of verapamil on maximal darkening responses to a-MSH and MCH determined by the toad (B. icfericus) skin bioassay.

ELISABETH N. FERRONI and ANA MARIA DE LAURO CASTRUCCI

i

-- cRMP IO-~ M Db CAMP 10-3H

c] Ringer Control

B Veropomil t0-4M

Fig. 5. Darkening responses elicited by cyclic AMP and dibutyril cyclic AMP in normal Ringer and in presence of

verapamil (N = IO).

ma1 and calcium-free Ringer. On the other hand, the darkening elicited by MCH was reversed by the indoleamine only in the absence of calcium (Fig. 7).

DISCUSSION

Earlier reports have suggested that the melano- tropin receptor and a-adrenoceptor could be com-

ponents of the same structural membrane unit or, at least, be closely related structures, since Dibenamine. an a-adrenergic blocker, inhibited a-MSH darkening action in Runa skin (Goldman and Hadley, 1970). Bufi melanophores do not posssess adrenergic recep- tor, either CI or p type (Ferroni and Castrucci, 1987) and since Dibenamine did not affect the darkening response to X-MSH, the result lends support to the hypothesis proposed by Goldman and Hadley.

As previously mentioned, calcium has been re- ported to be necessary for MSH activity in various melanocyte systems (Sawyer et al., 1983) whereas melanosome translocation per SC would not have such a requirement. since prostaglandins, cyclic nucleo- tides and methylxanthines still elicited dispersion in Ca’+-free Ringer. Whether calcium is essential for MSH-receptor coupling and for adenylate cyclase activation, or just for one of the two, could not be clarified in previous works, although de Graan ri ui. (1982a) have proposed two Ca’ ’ sites, one associated with MSH-receptor binding and the other with the subsequent intracellular event in Xenops lurcis mela- nophores. The toad skin bioassay has proved to be a good model for separately analysing each step, as melanotropin--receptor binding in this system does not require extracellular calcium. Although vera- pamil blocked both MCH and LX-MSH darkening actions, suggesting a calcium influx for pigment migration, darkening responses were not affected by the lack of external calcium. Therefore the im- pairment of MCH- and ~-MSH-induced responses caused by verapamil cannot be due to calcium channel blockade but is most probably due to the inhibitory activity verapamil might exert upon Cal+-dependent adenylate cyclases (Cheung, 1982). Supporting this suggestion, ionophore Az2,X7 did not

melatotun

1 1

30‘ w 90 120’ I50

Time (min)

Fig. 6. Reversal of darkening responses to GI-MSH and mle4, D-Phe’]-a-MSW by melatonin, as determined in the in vim toad (B. ictericus) skin bioassay. Each point represents the mean + SE response of the skins

at the times noted.

G(-MSH and MCH actions on Bufo melanophores 19

MCH

I

I’

I

I # melotonin

I

ON ormol Ringer

v co2’ -free Ringer

30’ 9b* 120’ 150’

Time (mini

Fig. 7. Reversal of MCH-induced darkening response by melatonin in calcium-free Ringer (v), whereas in normal Ringer (V), the indoleamine was ineffective (N = 13).

elicit darkening responses in the toad melanophores unlike the response de Graan et al. (1982b) have shown in Xenopus melanophores. Cyclic AMP and dibutyril cyclic AMP activities were not significantly diminished in the presence of verapamil. These results strongly suggest that calcium is specifically required for the transduction signal of melanotropins in Bufo melanophores.

Hadley et al. (198 1 b) have proposed a hypothetical model in which calcium ions are extruded from the pigment cell or pumped into the endoplasmic reticu- lum during melanosome dispersion. Our results are in agreement with this proposal for the responses elic- ited by both MCH and cr-MSH are enhanced in the absence of external calcium.

The fact that melatonin was able to reverse the darkening actions of both a-MSH and [Nle4, D-Phe’]-a-MSH but not of MCH is rather inter- esting. As increasing concentrations of melatonin promoted the same degree of lightening, a dose- response curve could not be obtained, suggesting that the indoleamine is not acting through specific recep- tors. Nevertheless the fact that MCH-induced dark- ening was reversed by melatonin, only in conditions where calcium was absent, might be a first clue to a better understanding of the MSH-like activity of MCH in tetrapods.

Acknowledgements-This work was partially supported by FAPESP, grants 84/1967-8 and 86/0251-4 (Brasil). The authors wish to thank Dr Victor J. Hruby for the gift of the peptides (design and synthesis supported by N.I.H., grant MA 17420).

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