Pigment cell signalling for physiological color change

Camp. Biuchelu. Physinl. Vul. 118A, No. 4, pp. 1135-l 144, 1997 Gq+ght 6 1997 Elsrwrr Science Inc. All rights reserved.

ISSN 0300~9629/97/$17.00 PII [email protected]~29(97)ooo45.5

ELSEVIER

REVIEW

Pigment Cell Signalling for Physiological Color Change

Luiz Eduurdo Maia Nery” and Ana Maria de Laura Castrucci DEPARTAMENTO DE FISI~LOGIA, INSTITUTO DE BIOCI~NCIAS, UNIVERSIDAIIE IIE Sio PALILO, SAo PA~~LO, BRASIL.

Communicated by Dr. Vera Va1, Editorial Board

ABSTRACT. The cellular signalling pathways participating in physiological color change are reviewed, particu-

larly in crustaceans, teleosts, amphibians, and reptiles. This review is an attempt to summarize what is known

and to raise some hypotheses about basic questions still to he elucidated. The first picture that emerges from

the literature is that the transduction pathways are identical in the Lrarious types of chromatophores of a single

species, except for the iridophore. The CAMP-dependent pathway has been well conserved throughout evolution;

CAMP increase is the pigment dispersion signal whereas the nucleotide decrease leads to granule aggregation.

On the other hand, the Ca+‘-dependent pathways evoke pigment aggregation in teleosts and crustaceans, and

dispersion in amphibians and probably reptiles as well. Another interesting point is the ultimate convergence

of the signalling pathways of different agonists inducing the same response in one chromatophore type. A hyporh- esis is raised about why different chromatophores behave differently in the absence of agonists, that is, why some are punctate, whereas others are stellate. COMP BIOCHEM PHYSIOI. 118A;4: 113% 1144, 1997. 0 1997 Elscvier

Science Inc.

KEY WORDS. Calcium, CAMP, cellular signalling, chromatophore. physiological color change, protein-kinase

A, protein-kinase C. protein phosphorylation

INTRODUCTION

Animal body coloration is generally determined by integu- mental pigment cells. Most animals exhibit a changeless or slowly changeable color pattern. The latter may vary according to ontogenetic, dietary, or seasonal determinants. These sl<)w and long lasting changes, so-called morphologi-

Address rrpint requests tu: Ana Maria de L. Castrucci, Dept. Fisiol- ogia, Inst. BloclOncias, Universidade de %o Paula, SHo Pa& CEP 05508-900, Braail. Tel. (5511) 818-7610; Fax (5511) 818-7422; E-mail: [email protected].

*Permanent addrex Laborat5tio de Zootisiologia, Departamrnto de CGnclas FlsiolGgicas, Fund@, Universidade do Rio Grande, Ric> Grande, RS, CEP 96201-900, Bras&

Abbreuuztions-ACTH, adrenocurticotropic hormone; CAMP, cyclic adenosme 1’, i’-monuphosphate; ATP, adenosine triphosphate; BAPTA, 1,2-hls(2-aminophcn~lxy) ethane-N,N,N,hJ-tetraacetic acid; 8-Br-CAMP. S-hnlmo-alien~)sine 3’. 5’qclic monophosphate; CZM, calmidizolium; D600, methoxyverapamil hydrochloride; DEAE-cellulose, diethylamino- ethyl-cellulose; EC,,. effective concentration for half-maximal response; EHNA. erythro-9-[3-(2-hydtroxynonyl)] adenine; ETc, endothelin-C receptor; H-7, [I -( 5-isoquinolinesulfonyl)-2-methylpiperazine]; H-89, {N-[2- ((paTa-hr(~mtlcinnamyl) amino) ethyl]-5-isoquinolinesulfonamide); IP,, inosm>l ( 1,4,5) trisphcxphate; MCH, melanin concentrating hormone; alpha-M%, alpha-melanocyte-stimulating hormone; PCH, pigment concentrating hormone; PDH, pigment dispersing hormone; PKA, CAMP-dependent protein kinase; PKC, protein kinase C; PPZB, protein phosphatase 2B; R(I 31-8220, {l-[~-(arnidinothio) propyl-1 H-indoyl-3-y&3-1( l- methyl-lH-indoyl-3-yl)-maleimide-methane sulfate]; TPA, phorhol 12s my&ate 1 ?-acetate; 4-beta-TPA, 4-beta-phorbol 12-myristate 13-acetate; W-7, N-(6-aminohexyl)-5-chlon,-l-naphthalenesulfonamide.

Received 27 June 1996; revised 10 March 1997; accepted 8 April 1997.

cal color change, mainly result from altered pigment cell number and/or amount of pigment within the cells, and it will not be discussed in this review. Many animals also possess the ability to rapidly change color, usually as an imme- diate response to environmental stimuli, such as hack- ground color, light intensity, or changing social context. This color alteration, named physiological color change, is promoted by the bidirectional translocation of pigment granules within stellate pigment cells, except for the cepha- lopod molluscans.

Pigment cells, or chromatophores, may be grouped according to their pigment color and internal structures. Mel- anophores possess black or brown granules of melanin or ommochromes, erythrophores have red color, provided by carotenoid vesicles and pteridin granules, xanthophores possess yellow color, due to a diverse proportion of carot- enoids and pteridins, and leucophores and iridophores both have colorless purines. Pigment is usually stored in granules or, in iridophores, into thin, flat microplatelets. which con- fer upon these cells an iridescent color due to light retlec- tion.

The granule centrifugal translocation is the pigment dispersion out into the cellular projections, whereas pigment aggregation refers to the centripetal movement which con- centrates granules in the perinuclear region, emptying the cell projections. The knowledge of the mechanism(s) un- derlying pigment migration is still incomplete and contro-

1136 L. E. M. Nery and A. M. de Lauro Castrucci

versial, but it is well established that granule motion depends upon microtubule and microfilament stability, and requires the participation of the endoplasmic reticulum [for review, see (23,65,81,87)].

The control of pigment migration may be exerted by the nervous, as well as the endocrine system. The relative participation of each regulatory system varies among the animals’ groups and within each group, with no clear phylogenetic relationship. The vertebrate alpha-MSH (melanocyte-stimulating hormone), melanin concentrating hormone (MCH), melatonin and catecholamines, and the crustacean neuropeptides pigment concentrating hormone (PCH) and pigment dispersing hormone (PDH) are the most intensively studied pigment cell agonists.

Concerning the cellular signalling pathways participating in the chromatophore responses to these agonists, several studies have been performed since the classical work of Bi- tensky and Burstein (8), who first demonstrated that CAMP promoted pigment dispersion in Rana pipiens melanophores. However, after more than 30 years, some basic relevant questions remain to be elucidated, such as: 1) Are the signal transduction pathways identical in the various chromatophore types (i.e., melanophores, erythrophores, leucophores, etc.) of a single species? 2) How well were these signalling pathways preserved throughout evolution? 3) Are there any late convergent steps in the signalling pathways of the various agonists that induce the same response in one chromatophore? and 4) Why are some unstimulated chromatophores, such as amphibian melanophores and teleostean leucophores, punctate (aggregated pigment), whereas others, such as amphibian leucophores and teleostean melanophores, are stellate (dispersed pigment)?

The present review on cell signalling for physiological color change aims to discuss answers to the above questions or, at least, to raise some hypotheses. One should bear in mind that among the many poikilothermic vertebrates and invertebrates able to display color change, just a few species of decapod crustaceans, teleosts and anuran amphibians, and the reptile Anolis carolinensis have been studied, thus limiting phylogenetic generalization.

CELLULAR SIGNALLING FOR PIGMENT DISPERSION

The first studies on the cellular signalling of dispersing agonists aimed to investigate the role of CAMP. The utilization, either in viva or in isolated skins, of methylxanthines, CAMP, and its permeable derivatives, demonstrated that pigment dispersion in melanophores of both amphibians and reptiles (8,13,32,36,49), as well as in melanophores, erythrophores, and leucophores of crustaceans and teleosteans (22,37,58,64,71,76), could be mediated by CAMP. These results were confirmed in amphibian and teleostean cultured melanophores (53,82,93) and in experimental pro- cedures involving CAMP intracellular injection in melano-

phores of the teleost, Lebistes reticuIatus (24), and of the anuran, R. pipiens (3 1). Nevertheless, only in 1980, Miya- shita and Fujii (54) were able to demonstrate that alpha- MSH, the most conspicuous vertebrate melanophore dispersing agonist, promoted an increase in CAMP content of cultured melanophores of the teleost L. reticulatus. More recently, Potenza and Lerner(75) also monitored the CAMP increase in amphibian melanophores, not only in response to alpha-MSH, but to catecholamines acting on beta-adrenoceptors as well.

de Graan et al. (17,18) described alterations in the phosphorylated state of a 53kDa protein (~53) concomitantly with the alpha-MSH-induced pigment dispersion in the anuran Xenopus laeuis. Furthermore, the phosphorylation could be also promoted by forskolin, a potent adenylate cyclase activator. And, ~53 exhibited a positive reaction with the antiserum anti-beta-tubulin. Lynch et al. (46,47) demonstrated, in the goldfish Carussius auratus xanthophores, that a 57kDa protein (~57) was phosphorylated in response to 8-Br-CAMP or ACTH, a 39-mer peptide, identical in the 1-13 sequence with alpha-MSH. ~57 was associated with the carotenoid granule, and its kinase was located in the cytosol. Accordingly, Rozdzial and Haimo (79) showed that p57 was phosphorylated during CAMP-induced pigment dispersion in partially-lysed melanophores of the teleost Tilapia mossumbica (now Oreochromis mossumbicus), and that the process might be inhibited by PKA-specific blockers. A few years later, the binding of the dephosphorylated 57kDa protein to the cytoskeleton, and its dissociation upon phosphorylation was proved in goldfish xanthophores (72,73).

Since the p57 phosphorylation played such an important role in pigment dispersion of teleost chromatophores, Yang et al. (94) investigated whether this protein might serve as a substrate to kinases other than PKA. They demonstrated that all the phosphorylation sites of ~57 were exclusively phosphorylated by PKA. In sequence, Yu et al. (95) demon strated, in digitonin permeabilized xanthophores, that ATP and PKA, or its catalytic subunit, were sufficient to promote p57 phosphorylation and to loosen the granules, which started moving. However, the cytosol was required for full dispersion. Based on these data, the authors suggested that pigment dispersion was a biphasic process, and that the ~57 phosphorylation accounted just for the first phase. A little later, Rodionov and co-workers (78), using nocodazol, a potent microtubule-disrupting drug, and kinesin, a mechano- chemical ATPase responsible for the sliding of particles along microtubules, suggested that CAMP increase in cultured melanophores of the teleost, Gymnocorymbus ternetzi,

would lead to the loosening of the central pigment aggregate, allowing kinesin to translocate the granules into the cell projections.

A general pattern (Fig. 1A and B) emerges from these data: Regardless of the species, chromatophore type, or dispersing agonist, pigment dispersion is induced by the initial rise in CAMP, which activates PKA which in turn phospho-

Pigment Cell Signalling 1137

PIGMENT DISPERSION

A CRUSTACEAN AND TELEOSTEAN CHROhtATOPHORES

0 a

-

B .&lFWBIAN AND REPTILIAN CHROMATOPHORES

+h -D ‘7-l PLC

0' IC -m--q

Ca+*

PROTEIN PHOSPHORYLATION PROTEIN PHOSPHORYLATION

FIG. I. Cellular signalling pathways for pigment dispersion in chromatophores, except for the iridophores. (A) Protein phosphorylation required for pigment dispersion in crustacean and teleostean chromatophores is only dependent on PKA. (8) Protein phosphorylation required for pigment dispersion in amphibian and reptilian chromatophores is dependent on PKA or PKC. a: Agonist; AC: adenylate cyclase; CAMP; cyclic adenosine 3’,5’-monophosphate; S-AMP: adenosine 5’-monophosphate; ATP:adenosine triphosphate; Ca+‘: calcium; Gs: stimulatory G protein; Gq protein; DAG: diacylglycerol; IC: intracellular compartment; IP,: inositol (1,4,5) t&phosphate; PDE: phosphodiesterase; PIP2: phosphatidylinositol (4,s) biphosphate; aPKA: active protein kmase A; iPKA: inactive protein kinase A; PKC: protein kinase C; PLC: phospholipase C; RI and RI: receptors.

rylates a granule-bound 53-57kDa protein. In the phosphorylated state, this protein detaches from the cytoskeleton, and the granules are then free to be carried by kinesin to the cell periphery.

But is CAMP the unique second messenger for pigment dispersion within chromatophores? Unlike most chromatophores, the iridophores bear guanine crystals arranged in radially organized reflecting platelets (23). According to Oshima et al. (68), increasing the distance between platelets leads to a spectrum shift of the reflected light, from yellow and green to blue and violet, whereas the decrease causes the color reversal. Surprisingly, the addition of CAMP, 8- Br-CAMP, or forskolin to skin iridophores of the teleost Chrysiptera cyunea (66), and to cultured iridophores of the teleost Odontobutis obscuru (48), promoted a lightening response, i.e., platelet aggregation, rather than dispersion. Could it be that a CAMP-independent kinase would be responsible for phosphorylation in iridophores or, could the phosphorylation be associated to platelet aggregation in these cells? Could such a difference be an adaptation to the fact that the iridophore pigment is not contained in granules? Up to date, these questions remain unanswered.

Could the iridophores be unique pigment cells in respect to the general pattern? Some authors (63,90) verified that the Ca’+ ionophore A23187 elicits pigment dispersion in

amphibian melanophores, similar to what was observed by Hadley (35) in Anolis curolinensis melanophores. More recently, Sugden and Rowe (85) were able to inhibit melatonin-induced pigment aggregation in X. Levis melanophores by 4-beta-TPA, a potent PKC activator which mimics the diacylglycerol action. Additionally, the 4-beta-TPA effect was blocked by a specific PKC inhibitor, Ro 31-8220, whereas the reversal of melatonin-induced aggregation by alpha-MSH was not affected. It was demonstrated that another PKC stimulating agent, TPA, induced a dose-dependent pigment dispersion in X. laeuis melanophores, exhib- iting a ECjc = 4.4 X lo-’ M. In order to demonstrate that an increase in CAMP was not required for the TPA-induced dispersion, the authors expressed the murine bombesin receptor in X. k&s melanophores, by electroporation with pJG3.6BR. Following bombesin treatment, the melanophores accumulate IPI, but not CAMP, nevertheless, pigment granule dispersion was achieved. In a similar way, these cells, once transfected with the cDNA of substance P receptor, known to be coupled to phospholipase C, responded with pigment dispersion when challenged by substance P (50). Xenopus Levis melanophores also possess an ETc receptor for endothelin-3, which promoted a transient pigment dispersion (ECw = 2.4 X lo--’ M), followed by desensitization (41). In Rana catesbeiana melanophores, en-


dothelin-l acted as a partial dispersing agonist, whereas in

Bufo icrericus melanophores, it had no effect (20). In mam-

malian non-pigment cells, endothelin is known to activate

phospholipid-dependent pathways (5; Fig. 1B). McClintock

et al. (5 1) demonstrated that the dispersing responses to

MSH, serotonin, vasoactive intestinal peptide, oxytocin,

and calcitonin gene-related peptide beta were inhibited by

H-89, a PKA inhibitor, but not by Ro 31-8220. But, inter-

esting enough, the activation of the endogenous ETc recep-

tor, and of the transfected bombesin and substance P recep-

tors, all leading to dispersion as well, were inhibited by Ro

3 l-8220. Therefore, the substrate phosphorylation required

for pigment dispersion in X. Levis melanophores may be

catalyzed either by PKA or PKC (Fig. 1B).

Unlike amphibians, teleostean melanophores respond to

endothelin-1 with pigment aggregation (20,ZS). Thus, it is

possible that in teleosteans, the phosphorylation required

for the initial phase of dispersion is solely dependent upon

CAMP (Fig. lA), whereas in amphibians and reptiles both

PKA and PKC may lead to pigment dispersion (Fig. 1B).

Up to now, there are no reports of the participation of ki-

nases other than PKA in the process of pigment dispersion

in crustacean chromatophores.

CELLULAR SIGNALLING FOR PIGMENT AGGREGATION

Once pigment dispersion has been triggered by the increase

in CAMP, it might be expected that pigment aggregating

agonists would induce a decrease of CAMP content (Fig.

2A and B). Supporting this view, Yamada and Iwakiri (93)

demonstrated that imidazole, a phosphodiesterase activat-

ing agent, promoted granule aggregation in teleost (Oryrias

Iatipes) leucophores. And Negishi et al. (60) reported a 30%

decrease in CAMP content upon epinephrine-induced pig-

ment aggregation in cultured melanoma cells of the teleosts

Xiphophorus maculatus and Xiphophorus heleri. A similar per-

centage of CAMP reduction was also determined in nor-

epinephrine-treated melanophores of the teleost L&us

ossifagus (4), and in epinephrine-treated melanophoroma

P4-E.12 cells (15).

Pertussis toxin, a potent Gi-protein blocker, inhibited

norepinephrine-promoted granule aggregation in melano-

phores of the teleosts L. ossifagus (40), 0. latipes (59), and

Cmssius auratus (57). These data demonstrated the partici-

pation of a Gi-protein-coupled receptors in the aggregating

responses elicited by catecholamines (Fig. 2A).

Melatonin, the pineal gland indoleamine, is a pigment

aggregating agonist of amphibians, and its effect on X. Levis

melanophores may also be blocked by pertussis toxin

(84,92). In fact, a high affinity melatonin receptor has been

recently cloned from X. lamis melanophores, and demon-

strated to belong to the family of G-protein coupled recep-

tors (19). In the toad, B. ictericus, melanophores melatonin

exhibited the typical aggregating effect, and inhibited

alpha-MSH- or forskolin-induced dispersion, but not dibu-

tyryl-CAMP dispersing action (21). These results suggest

that the activation of melatonin receptors leads to adenyi-

ate cyclase inhibition and a decrease in CAMP. However,

pertussis toxin was ineffective on those responses to melato-

nin. Grundstrom et al. (34) had previously demonstrated

that pertussis toxin inhibited the aggregating action of ex-

ogenous norepinephrine applied to L. ossifagus melano-

phores. But, strangely enough, the toxin did not affect the

aggregation elicited by the electrical stimulation of the sym-

pathetic nerve, despite total blockade of both responses by

yohimbine, a potent alpha?-adrenoceptor inhibitor. A possi-

ble explanation for these contradictory results was provided

by Yung et al. (96) in human embryonic kidney cells,

transfected with the cDNA of X. Levis melatonin receptor.

In the transfected cells, melatonin inhibition of chnrionic

gonadotrophin-induced CAMP increase was, as expected,

blocked by pertussis toxin. However, if these cells were co-

transfected with the cDNA of protein Gz alpha-subunit,

melatonin inhibition of the response to the gonadotrophin

was no longer sensitive to the toxin. It becomes clear, there-

fore, that both catecholamines and melatonin may act

through a pertussis toxin-insensitive Gi-protein in verte-

brate melanophores, leading to adenylate cyclase inhibi-

tion.

Furthermore, melatonin activity on fish melanophores

does not always result in pigment aggregation. Denervated

melanophores of Chasmichthys grdosus, Oreochromis niloticus,

and Fundulus heteroclitus display differential sensitivity to

the indoleamine; some cells respond with full pigment ag-

gregation, others are totally unresponsive (26,27). In addi-

tion, melatonin may elicit granule dispersion in melano-

phores of the dark stripes, and aggregation in cells located in

the light stripes of Nunnostomus heckfordi (62). The authors

postulated the presence of two distinct melatonin receptors,

alpha mediating aggregation and beta mediating dispersion.

Going back to Yung and colleagues’ results (96), cells co-

transfected with the cDNAs of melatonin receptor and ad-

enylate cyclase II responded to melatonin with CAMP in-

crease, rather than decrease. If some teleost melanophores

possessed endogenous adenylate cyclase II, they might re-

spond to melatonin with an increase in CAMP, and there-

fore, with pigment dispersion.

Pigment aggregation resulting from CAMP decrease

should occur due to a protein dephosphorylation caused by

PKA inactivation and phosphatase activity (Fig. 2A and

B). In fact, Rozdzial and Haimo (79) demonstrated a 30-

50% decrease of p57 phosphorylation after epinephrine-

induced pigment aggregation in the teleost, 0. mossambictls,

melanophores. Both p57 dephosphorylation and granule ag-

gregation were inhibited in the presence of the phosphatase

blocker, beta-glycerophosphate. They also demonstrated

the specificity of the phosphatase, since the pigment aggre-

gation was prevented when p57 was thiophosphorylated.

Beckerle and Porter (7) suggested that dynein, a mechano-

Pigment Cell Signalling

A CRUSTACEAN AND TELEOSTEAN CHROMATOPHORES

1139

PIGMENT AGGREGATION

B AMPHIEmNANDREPTILIAN CHROMATOPHORES

Q a

PROTEIN DEPHOSPHORYLATION PROTEIN DEPHOSPHORYLATION

FIG. 2. Cellular signalling pathways for pigment aggregation in chromatophores, except for the iridophores. (A) Protein dephosphorylation required for pigment aggregation in crustacean and teleostean chromatophores is dependent on a serinelthreonine protein phosphatase which becomes active when PKA is inactive, or by PKC or Ca+‘/cahnod& complex positive regulation. In addition, a possible cross-talk between these pathways is suggested (see text for more details). (B) Protein dephosphorylation required for pigment aggregation in amphibian and reptilian chromatophores is dependent on a serinekhreonine protein phosphatase, which is active only under PKA inactivation. a: Agonist; AC: adenylate cyclase; CAMP: cyclic adenosine 3’,5’-monophosphate; 5’.AMP: adenosine 5’.monophosphate; ATP: adenosine triphosphate; Ca+“: calcium; Ca+‘/CaM: calciumlcahnodu- lin complex; Gi: inhibitory G protein; Gq: Gq protein; DAG: diacylglycerol; IC: intracellular compartment; II?,: inositol(1,4,5) t&phosphate; PDE: phosphodiesterase; PIP2: phosphatidylinositol (4,s) biphosphate; aPKA: active protein kinase A; iPKA: inactive protein kinase A; PKC: protein kinase C; PLC: phospholipase C; PP: serinelthreonine protein phosphatase; RI and RI: receptors. ---+ active path; - - + inactive path; -?+ putative cross-talk.

chemical ATPase, played a role in the granule aggregation of the teleost, Holocentrus ascensionis, melanophores. Exper- imental data pointing to dynein participation were obtained with the blockade of pigment aggregation in 0. niloticus permeabilized melanophores by the dynein inhibitors, EHNA, and vanadate (67).

In view of the above results, it is presently proposed that the activation of a Gi-protein-coupled receptor inhibits adenylate cyclase, leading to PKA inactivation (Fig. 2A and B). Under this condition, a specific phosphatase may de- phosphorylate ~57, allowing the dynein transporting action on the pigment granules, which then aggregate around the nucleus.

tained in the erythrophores and melanophores of the teleosts H. ascensionis (45), 0. latipes (61) and Xiphophorus maculatus (70), and in the erythrophores of the macruran crustacean Mucrobrmhium ~otiuna (9). However, the ionophore was ineffective in the teleost melanophoroma P4-E. 12 cells (15) and in L. reticulatus (55) melanophores. Fur- thermore, in disagreement with previously reported results (61), Namoto (59) found no aggregating activity of CaLi. ionophore in 0. latipes melanophores. The fact that Il’, was able to aggregate tilapia. (0. niloticus) permeabilized melanophores (29), brought further light to the matter, as IF’, is well known to open Ca ‘+-channels of intracellular compart- ments (Fig. 2A).

Again, one should inquire if CAMP reduction is the only Among teleost pigment aggregating agonists, catechol- way to induce chromatophore pigment aggregation. Inter- amines were the first candidates as causal agents in a sudden estingly from this point of view, several authors have dem- rise in cytosolic Ca’+. The teleost chromatophore may pos- onstrated that pigment aggregation within crustacean and sess one or both alpha- and beta-adrenoceptor types. Cate- teleostean chromatophores is calcium-dependent. One of cholamine binding to the chromatophore alpha-adrenocep- the first results to indicate calcium participation was the tor triggers pigment aggregation, except for the iridophores ionophore A23 187 aggregating effect in the crayfish, P&Z- (48). The well studied mammalian alpha-adrenoceptors are monetes pugio, erythrophores (43). The same result was ob- subtyped into alpha, and alpha, subgroups, based on three

1140 L. E. M. Nety and A. M. de Lauro Castrucci

main lines of evidence ( 11): (a) selective affinity of antagonists; (b) receptor amino acid sequences; and (c) cell signalling pathway coupled to the receptor, of particular interest in this review. Once activated, all vertebrate alphar-adrenoceptors elicit an increase in the intracellular Ca*+ concentration (12), whereas alpha]-adrenoceptors’ signalling pathways may vary, although most comprise adenylate cyclase inhibition (44). To date, only alpha*-adrenoceptors have been pharmacologically characterized in teleostean chromatophores (for instance, 4,56). However, as already mentioned by Fujii and Oshima (25), the participation of alpha,-adrenoceptors in pigment aggregation of some teleost species needs to be revisited in light of the data discussed below.

Luby-Phelps and Porter’s study (45) was one of the first to investigate the role of a cytosolic Ca*+ rise in catecholamine-induced pigment aggregation. They reported that D600 and papaverin, blockers of type L Ca*+-channels, depressed the aggregating response to epinephrine in H. uscen-

sionis. However, controversial results have been reported for two other teleostean species in melanophores previously loaded with a lipophilic derivative of the Ca*+-chelator, BAPTA. In 0. mossambicus (89), BAPTA derivative de- creased the response to epinephrine, and in Pteroghyllum scalare (BO), the blocker inhibited the transient Ca*+ rise promoted by epinephrine, but did not impair granule aggregation. The latter authors also reported the lack of Ca*+- ionophore effects.

Other controversial results concern CaL+/calmodulin participation (Fig. 2A) in the aggregating response of teleost chromatophores to catecholamines. In cultured Xiphophorus naculatus melanophores and erythrophores (70), the norepinephrine-induced rise in cytosolic Ca*+, and the simulta- neous pigment aggregation are prevented by the Ca’+/calmodulin inhibitor, W-7. This result was further confirmed in C. cyanea melanophores (67), and in addition to other Ca*+/calmodulin antagonists, trifluorperazine and CZM, in cultured 0. mossambicus melanophores (89). Nevertheless, Sammark et al. (80) reported that W-7 and CZM had no effect on epinephrine-induced aggregation in Pterophyllum scalare melanophores.

Thaler and Haimo (89) investigated what role Ca’+/ calmodulin complex might play in the catecholamine- activated pathway. These authors described the inhibitory effect of 10e6 M okadaic acid, a selective phosphatase antag- onist, on epinephrine-induced pigment aggregation in whole and in partially lysed 0. mossambicus melanophores. Furthermore, the pigment aggregating ability, lost with the total melanophore lysis, was recovered with the addition of PPZB, a Cal+-dependent phosphatase, known as calcineurin, and blocked with the antibody anti-PPZB. In con- firmation, the authors were able to demonstrate dermal and melanophore localization of PP2B by immunoblotting and immunofluorescence, respectively.

Although PKC is the main effector of Ca’+-dependent signalling pathways, the few investigations using the kinase antagonists, such as H-7 (3,67), were not able to demon strate PKC involvement in the catecholamine-induced granule aggregation of teleosts.

In general, one might then say that the Cal+ rise and Ca*+/calmodulin formation in erythrophores and melanophores of some teleostean species are relevant signals for the activation of a Ca’+-dependent phosphatase, which is probably the ultimate event for catecholamine-promoted aggregation. In other species, the aggregating response to catecholamines apparently solely depends on adenylate cyclase inhibition.

MCH, another teleost pigment aggregating agonist, is a cyclic heptadecapeptide, produced in the hypothalamus and released by the neurohypophysis. MCH has been proved to aggregate melanophore, erythrophore, and xanthophore granules (42,68,69). To date, only two independent investigations on MCH signalling transduction were carried on in teleosts. In isolated skin of Synbrunchus marmoratus, it has been reported that the phospholipase inhibitors, parabro- mophenacyl bromide, and neomycin sulphate, as well as the PKC antagonists, dibucaine and H-7, depressed the melanophore aggregating response to MCH (2). The authors have also demonstrated that MCH should promote a protein dephosphorylation, as beta-glycerophosphate, a phosphatase blocker, inhibited MCH-induced pigment aggregation as well. In addition, the same group (14) had previously reported that extracellular Ca*+ was not required for MCH action. In isolated L. ossifugus melanophores, Svensson et al. (86) observed a 35% reduction in CAMP in response to MCH treatment. These authors did not investigate whether a phospholipid-dependent pathway might also be being activated in this species. At the moment, therefore, two possi- bilities may be raised: (a) there could be two diverse MCH receptors in teleost pigment cells; and (b) the activation of MCH receptor could activate PKC and, as a late event, activate a phosphatase, as well as decrease CAMP, through both pathways’ cross-talks (Fig. 2A), as already known to occur in many mammalian cells in response to agonists other than MCH. In this way, the Ca*+/calmodulin-dependent isoforms of phosphodiesterase and the Cal+-inhibited isoforms of adenylate cyclase might well be the cross-talk effecters (for a review, see 6, 16). This hypothesis can not be discarded as a possible explanation for the controversial literature on the Ca*+ role in the aggregating responses to catecholamines.

PCH is the only known pigment aggregating agonist of crustacean chromatophores. This octapeptide is produced and secreted by the neurosecretory complex, X-organ-sinus gland. The absence of extracellular Ca*+ or the presence of verapamil, a blocker of the L type Cal+-channel, inhibits PCH aggregating activity (10,52). Further studies are needed to bring clarity to the signalling mechanism of PCH.

Pigment Cell Signalling 1141

In summary, in teleosts and crustaceans (Fig. 2A), unlike

amphibians and reptiles (Fig. 2B), pigment aggregation may

be elicited by cytosolic Ca ‘+ increasing agonists, leading to

a protein dephosphorylation due to the final activation of

specific phosphatases, simultaneously or not to a CAMP de-

crease promoted by a cross-talk. In addition, the exclusive

decrease of CAMP induced by the activation of a Gi-protein

coupled receptor is the alternative signalling for pigment

aggregation in all mentioned groups (Fig. 2A and B).

CONCLUSIONS AND PERSPECTIVES

In this section, we will try to build a general picture of pig-

ment cell signalling, and perspectives for future investiga-

tion, focusing on the questions initially raised. One of the

most basic questions-are the transduction pathways iden-

tical in the various chromatophore types of a single spe-

cies-can be answered yes, except for iridophore. This per-

spective opens a line of research for investigating why

iridophores behave differently.

Another central question is related to the evolution of

cell signalling. The CAMP-dependent signal pathway has

been well conserved throughout evolution. In all groups in

which pigment cell signalling has been investigated, includ-

ing crustaceans, the increase of CAMP and the resulting

PKA activation is the common event leading to granule

dispersion. Accordingly, the nucleotide decrease and PKA

inactivation allow pigment aggregation. However, if one

looks at the Ca’+-dependent pathways, Ca2+ signal evokes

pigment aggregation in teleosts and crustaceans, unlike am-

phibians and probably reptiles, in which intracellular Ca”

rise promotes granule dispersion. What might have changed

in the retrapod chromatophore to favour this opposite func-

tion for Ca” ? In amphibians, as already mentioned, the

ultimate target for phosphorylation/dephosphorylation is a

53kDa protein, whereas in teleosts, its size is 57kDa. Would

these proteins be isoforms of a single family, but with dis-

tinct sites for phosphorylation? Are the phosphorylation

sites exclusive substrates for serine/threonine kinases? Fu-

ture investigations are urged to focus on the protein kinases’

and protein kinase substrates’ characterization for a better

understanding of the various signalling systems.

Among vertebrates, the teleosts exhibit a diverging pic-

ture of cell signalling, but what about the evolutionary an-

cestral groups, cyclostomes and elasmobranchs? To our

knowledge, there are no data about pigment cell signalling

in these groups. But it has been reported that the non-

stimulated melanophores assume the punctate (aggregated

pigment) state as in the tetrapods, and they are very sensi-

tive to alpha-MSH, but unresponsive to MCH [for review,

see (83,91)]. Further studies need to be done in order to

shape the phylogenetic history of pigment cell signalling in

vertebrates. In this regard, one might ask how mammals fit

into this framework? Although mammals lost the ability to

display physiological color change, do they use the same

pathways for morphological color change? Apparently,

PKA-, as well as PKC-dependent phosphorylations, are re-

sponsible for melanin synthesis in both normal and trans-

formed melanocytes (1,30,39). In this sense, one might as-

sume that the amphibian/reptile signalling for color change

has been preserved in mammals. Looking at the overall pic-

ture, it seems that the neopterygeans (holosteans and tele-

osts) constitute a divergent group from the vertebrate line

in regard to the unstimulated state of chromatophore, and

to the signalling pathways leading to pigment transloca-

tions.

Another question we have tried to answer was “are there

any late convergent steps in the signalling pathways of dif-

ferent agonists that induce the same response in one chro-

matophore?” Again, except for the iridophore, all pigment

cells in every studied group respond to any dispersing ago-

nist with the phosphorylation of a granule-bound protein,

resulting in the kinesin-induced motion of pigment gran-

ules. The responses to aggregating agonists depend upon the

dephosphorylation of the same protein, allowing then dyn-

ein action as the motile carrier agent.

And finally, another interesting question is why do differ-

ent chromatophores behave differently in the absence of

agonists, that is, why are some punctate (aggregated pig-

ment), whereas others are stellate (dispersed pigment)? For

instance, melanophore pigment of btachyuran crustaceans,

amphibians and reptiles, and leucophore granules of teleosts

are aggregated in the unstimulated state, whereas the other

chromatophores of the same animal are punctate. Since te-

leostean and amphibian melanophores equally respond to

CAMP increase with a granule-bound protein phosphoryla-

tion, one could hypothesize that unstimulated chromato-

phores would assume the dispersed or the aggregated state

depending upon, respectively, a higher <or lower basal CAMP

content. The higher CAMP basal content would assure the

phosphorylated state of the substrate, in the absence of ago-

nists. For the teleosts L. ossifagus (86) and C. auratus (57),

there have been reported 50.4 -C 2.8 pmol/mg protein and

57.4 2 3.8 pmol/mg protein, respectively, whereas values

between 60 and 70 pmol/mg protein were determined in X.

laeeris (33,74,75). It should also he pointed that most assays

for CAMP determine total CAMP and do not differentiate

between free and bound forms. Therefore, comparing the

basal cAMP levels of telenstean and amphibian melano-

phores, no striking difference is observed.

In the 197Os, two PKA isoforms were identified through

DEAE-cellulose chromatography (77). They have identical

subunits C, but differ in the structures of subunit R (38,88).

These PKAs exhibit very similar properties, hut different

CAMP affinities. In order to obtain 50% of the maximal

activity of PKA I, a 2-fold higher concentration of CAMP

was required, as compared to PKA II (77). These data might

suggest that the state assumed by non-stimulated chromato-


phores would depend on the PKA isoform expressed by the cell; in the stellate cells, the substrate would then be phosphorylated by a PKA active in the presence of basal levels of CAMP, whereas the other chromatophores’ PKA would require an increase in CAMP content, in order to become active. Further investigation is necessary to test the above hypothesis.

In conclusion, although the knowledge of pigment cell signalling for physiological color change has had a remark- able advance in the last decade, many important aspects still remain to be elucidated, such as the protein kinases’ and the ultimate effecters’ characterization, that will neces- sarily take advantage of the molecular biology tools.

This work has been partially supported by the Conselho National de

Ciincia e Tecnologia do Bras& Financiadora de Estudos e Projetos,

and Funda@o de Amparo & F’esquisa do Es&o a!e Silo Puulo. LEMN

is a fellow of the Courd.ena@o de Aperfeisoamento de Pessoal de Nivel

SuperiorlPrograma Institutional de Capacitqiio de Docentes. We are

deeply thankful to Dr. Wade C. Sherbrooke for the critical review of

the manuscripr.

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Pigment cell signalling for physiological color change

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