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The Calcium Dependence of Pigment Translocation in Freshwater Shrimp Red Ovarian Chromatophores.



Abstract. The roles of calcium in cell signaling consequent to chromatophorotropin action and as an activator of mechanochemical transport proteins responsible for pigment granule translocation were investigated in the red ovarian chromatosomes of the freshwater shrimp Macrobrachium olfersii. Chromatosomes were perfused with known concentrations of free Ca [++] ([10.sup.-3] to [10.sup.-9] M) prepared in Mg [++]-EGTA--buffered physiological saline after selectively permeabilizing with 25 [mu]M calcium ionophore A23187 or with [10.sup.-8] M red pigment concentrating hormone (RPCH). The degree of pigment aggregation and the translocation velocity of the leading edges of the pigment mass were recorded in individual chromatosomes during aggregation induced by RPCH or A23187 and dispersion induced by low Ca [++]. Aggregation is Ca [++] dependent, showing a dual extracellular and intracellular requirement. After perfusion with reduced Ca [++] ([10.sup.-4] to [10.sup.-9] M) RPCH triggers partial aggregation ([approx] 65%), although the maximum translocation velocities ([approx]16.5 [mu]m/min} and velocity profiles are unaffected. After aggregation induced at or below [10.sup.-5] M Ca [++], spontaneous pigment dispersion ensues, suggesting a Ca [++] requirement for RPCH coupling to its receptor, or a concentration-dependent, Ca [++] -induced Ca [++] -release mechanism. The Ca [++] -channel blockers Mn [++] (5 mM) and verapamil (50 [mu]M) have no effect on RPCH-triggered aggregation. An intracellular Ca [++] requirement for aggregation was demonstrated in chromatosomes in which the Ca [++] gradient across the cell membrane was dissipated with A23187. At free Ca [++] above [10.sup.-3] M, aggregation is complete; at [10.sup.-4] M, aggregation is partial, followed by spontaneous dispersion; below [10.sup.-5] M Ca [++], pigments do not aggregate but disperse slightly. Aggregation velocities diminish from 11.6 [pm] 1.2 [mu]m/min at 5.5 mM Ca [++] to 7.4 [pm] 1.3 [mu]m/min at [10.sup.-4] M Half-maximum aggregation occurs at 3.2 X [10.sup.-5] M Ca [++] and half-maximum translocation velocity at 4.8 X [10.sup.-5] M Ca [++] Pigment redispersion after 5.5 mM Ca [++] -A23187-induced aggregation is initiated by reducing extracellular Ca [++] slight dispersion begins at [10.sup.-7] M, complete dispersion being attained at [10.sup.-9] M Ca [++]. Dispersion velocities increase from 0.6 [pm] 0.2 to 3.1 [pm] 0.5 [mu]m/min. Half-maximum dispersion occurs at 7.6 X [10.sup.-9] M Ca [++] and half-maximum translocation velocity at 2.9 X [10.sup.-9] M Ca [++]. These data reveal an extracellular and an intracellular Ca [++] requirement for RPCH action, and demonstrate that the centripetal or centrifugal direction of pigment movement, the translocation velocity, and the degree of pigment aggregation or dispersion attained are calcium-dependent properties of the granule translocation apparatus.


Chromatic adaptation in the decapod Crustacea is brought about by the differential translocation of colored pigment granules contained within specialized, multicellular effectors known as chromatosomes (McNamara, 1981). The mechanisms responsible for the centripetal and centrifugal movement of the granules within the constituent chromatophores are regulated by small peptide hormones of neurosecretory origin, often specific and antagonistic for each pigment cell type, but generally termed pigment concentrating (PCH) and pigment dispersing (PDH) chromatophorotropins (Josefsson, 1983; Rao and Fingerman, 1983; Fingerman, 1985).

The signal transduction pathways and intracellular enzymatic cascades activated by these peptides are poorly known in the Crustacea, although they are fairly well established in vertebrate groups like the teleosts, amphibians, and reptiles, which also exhibit color changes regulated by (neuro)endocrine and neural mechanisms (reviews in Novales, 1983; Nery and Castrucci, 1997; Tuma and Gelfand, 1999). Both intracellular free [Ca.sup.++] concentrations and cyclic nucleotides have been implicated in the second messenger systems that respectively activate the pigment aggregating and dispersing mechanisms in the Crustacea (Fingerman, 1969; Rao and Fingerman, 1983; Nery et at., 1997, 1998). However, it is difficult to establish generic regulatory mechanisms because the two principal groups studied, the brachyuran crabs and the caridean shrimps, differ considerably in their pigmentary responses to the same effector agents. To illustrate, chromatosome pigments spontaneously disperse in shrimp epidermal preparations in vitro (Fingerman et al., 1975; Lambert and Fingerman, 1979; McNamara and Taylor, 1987; Tuma at al., 1993), but they aggregate in crab preparations, in the absence of agonists (Lambert and Fingerman, 1976; Kulkarni and Fingerman, 1986). Intracellular [Ca.sup.++], increased by the calcium ionophore A23187 and calcium-containing salines, causes pigment aggregation in shrimps (Lambert and Fingerman, 1979; Britto et al., 1990; McNamara and Ribeiro, 1999) but dispersion in crabs (Quackenbush, 1981). In shrimps, cyclic AMP disperses red chromatophore pigments (Fingerman, 1969; Nery at al., 1998), but in crabs it is either without effect or also disperses chromatophore pigments (Quackenbush, 1981; Rao and Fingerman, 1983). However, cyclic GMP also disperses black and white but not red chromatophore pigments in crabs (Quackenbush, 1981) and has no effect on shrimp red chromatophores (Nery at al., 1998). Clearly, unequivocal signal transduction pathways that modulate the molecular motors responsible for pigment aggr egation and dispersion are yet to be established in the Crustacea as a whole.

Alterations in intracellular calcium concentrations appear to play a major role in the signaling transduction pathway or effector mechanism regulating the mechanochemical protein motors responsible for pigment translocation in caridean chromatophores. Red pigment concentrating hormone (RPCH) induces the aggregation of dispersed pigments, but its action is inhibited or impaired in [Ca.sup.++]-free media (Lambert and Fingerman, 1979; McNamara and Taylor, 1987; Britto et al., 1996) and in the presence of verapamil, an L-type [Ca.sup.++] channel blocker (Britto et al., 1996). In contrast, dispersion of A23187- or RPCH-aggregated pigments can be induced in [Ca.sup.++]-free saline (Lambert and Fingerman, 1978, 1979; McNamara and Ribeiro, 1999). However, unlike certain teleost chromatophores in which pigment aggregation is also [Ca.sup.++]-dependent (Luby-Phelps and Porter, 1982; McNiven and Ward, 1988; Kotz and McNiven, 1994), in crustacean chromatophores the origin and role of [Ca.sup.++] in pigment translocation is unclear, and no estimates are available of changes in intracellular [Ca.sup.++] concentration during pigment movements.

Using red ovarian chromatosomes from Macrobrachium olfarsii, a freshwater caridean shrimp, the present study investigates the origin and calcium-dependent nature of pigment aggregation and dispersion induced by RPCH and A23187. The intracellular free [Ca.sup.++] associated with activation of the respective granule translocation mechanisms is also estimated with the aid of a [Mg.sup.++]-EGTA buffer system.

Materials and Methods

Immature, female freshwater shrimp, Macrobrachium olfarsii, presenting small, translucent ovaries, were collected from the Pa[acute{u}]ba River in S[tilde{a}]o Paulo State, Brazil, and maintained under a natural photoperiod in about 100 1 of river water (salinity [less than] 0.5%, temperature [approx] 23[degrees]C) in 250-1 tanks: they were fed celery, beetroot, carrot, and minced beef or chicken.

The ovarian chromatosome preparation employed has been described in detail previously (McNamara and Ribeiro, 1999). Briefly, after dissection, the red chromatosomes were gravity perfused (0.7 ml/min) in an acrylic microperfusion chamber (150 [mu]1 volume) and observed at 320X by reflected and transmitted light using a Wild M10 stereoscopic microscope coupled to a Sony DXC-151A CCD video camera and Trinitron monitor.

An ocular graticle was used to quantify the diameter of the pigment mass in chromatosomes of 220-240 [mu]m diameter at 2-mm intervals. The data were converted to percent maximum dispersion, and the translocation velocity (in micrometers per minute) of the leading edge of the pigment mass was calculated, for each interval, according to McNamara and Ribeiro (1999).

Perfusion salines were prepared based on ionic data from McNamara at al. (1990) for M. olfarsii. The control saline contained (in millimoles): [Na.sup.+] 177, [K.sup.+] 5, [Ca.sup.++] 5.5, and [Mg.sup.++] 1 as chlorides ([approx]195).

Salines of specific low [Ca.sup.++] concentration were prepared using a [Mg.sup.++]-10 mM EGTA buffer system. For RPCH- and A23187-induced pigment aggregation, the respective [Ca.sup.++] and [Mg.sup.++] necessary to furnish final free [Ca.sup.++] concentrations of [10.sup.-3] to [10.sup.-9] M, while holding free [Mg.sup.++] at 1.03 mM, were calculated using the MCalc computer program based on Fabiato (1988). For pigment dispersion in previously A23187-perfused preparations, the [Ca.sup.++] and [Mg.sup.++] given by Luby-Phelps and Porter (1982) were used to provide final free [Ca.sup.++] concentrations of [10.sup.-7], [10.sup.-8], and [10.sup.-9] M at a final free [Mg.sup.++] of 1.3 mM. In the [Ca.sup.++]-free saline (residual [Ca.sup.++] [approx]7 X [10.sup.-11] M), [Ca.sup.++] was substituted by 27.5 mM choline chloride, and 2 mM EDTA was added.

All salines contained 0.5% DMSO (dimethyl sulfoxide; Sigma, MO), 2.5 mM [Na.sub.2][HCO.sub.3], and 2 mM glucose (osmolality prior to DMSO, 354 [pm] 0.7 mOsm/kg [H.sub.2]O) and were adjusted to pH 7.4. Calcium ionophore A23187 (free acid, Sigma, MO), dissolved in DMSO, was used at a final concentration of 25 [mu]M. Red pigment concentrating hormone (Peninsula Laboratories Inc., CA), dissolved in distilled water or 10% DMSO, was used at a final concentration of either 10 or 30 nM. Verapamil hydrochloride and Mn[Cl.sub.2] (Sigma, MO) were dissolved in DMSO or distilled water and used at final concentrations of 50 [mu]M (Hille, 1992) and 5 mM (Zhang et al., 1997), respectively. All experiments were performed at room temperature ([approx]23[degrees]C). Under these conditions, the chromatosome preparation is functional for at least 3-4 h, and up to five successive cycles of aggregation and dispersion can be induced using A23187 and [Ca.sup.++]-free saline, respectively.

Each experiment was repeated seven times, using measurements from a single chromatosome in each preparation. Since most data were not normally distributed, the treatment effect was evaluated using the Kruskal-Wallis one-way, nonparametric ANOVA, followed by Dunn's test to locate significantly different groups. Correlations between pigment translocation velocities and degree of aggregation or dispersion were evaluated employing Pearson's product moment correlation test (SigmaStat 2.03, SPSS Inc., CA). Concentration-effect curves and the 50% response values were obtained using the dose-response logistic curve-fitting function of SlideWrite 5.0 Plus (Advanced Graphics Software, Inc., CA). All tests were performed with a significance level of P = 0.05.


Calcium dependence of pigment aggregation

Red pigment concentrating hormone. On perfusion with 10 nM RPCH in control (5.5 mM [Ca.sup.++]) saline, the dispersed pigments aggregate rapidly and completely within 24 m (Fig. 1A1). Translocation velocity attains a customary peak of 17.0 [pm] 2.9 [mu]m/min (n = 11) with a small right-hand shoulder, followed by a short plateau of 1.8 [pm] 0.3 [mu]m/min, gradually declining thereafter (Fig. 1B1). RPCH washout induces immediate dispersion, reaching about 90% within about 40 min (Fig. 1A1). Dispersion velocity attains a maximum of 5.6 [pm] 2.5 [mu]m/min (Fig. 1B1).

Perfusion with 10 nM RPCH and the various [Mg.sup.++]-EGTA--buffered [Ca.sup.++]-salines reveals a hormone effect notably dependent on extracellular [Ca.sup.++]. In [10.sup.-3] M [Ca.sup.++], the degree of aggregation and the velocity profile (Fig. 1A2, 1B2) are similar to those in control saline (5.5 mM [Ca.sup.++], Fig. 1A1, 1B1). However, in salines containing reduced [Ca.sup.++], maximum aggregation varies from 90% ([10.sup.-4] M, Fig. 1A3) to 63% ([10.sup.-5] M, Fig. 1A4), 67% ([10.sup.-6] M, Fig. 1A5) and 60% ([10.sup.-9] M, Fig. 1A6), requiring about 13 min. However, the peak translocation velocities ([approx]16.5 [mu]m/min) are unaffected by [[Ca.sup.++]] (P = 0.97), and the velocity profiles (Fig. 1B3-1B6) are virtually identical to that in 5.5 mM [Ca.sup.++] (Fig. 1B1). Strikingly, at [[Ca.sup.++]] [leq] [10.sup.-5] M, the aggregation effect is rapidly reversed and spontaneous pigment dispersion ensues, attaining 80%-100% of initial dispersion (Fig. 1A4-1A6) in the presence of RPCH. The mean disper sion velocity of about 5 [mu]m/min is unaffected by [[Ca.sup.++]] (P = 0.67). Subsequent perfusion with 5.5 mM [Ca.sup.++] + induces full pigment aggregation (Fig. 1A4-1A6), and the translocation velocity profiles (Fig. 1B4-1B6) and peak velocities (P = 0.99) are the same as in preparations perfused directly with 5.5 mM [Ca.sup.++] (Fig. 1B1).

Although the response to RPCH washout is immediate, dispersion is incomplete, reaching a maximum of about 50% (Fig. 1A2-1A6).

Calcium channel blockers. The time course and degree of pigment aggregation in response to 30 nM RPCH are unaffected by a 30-min preincubation with 5 mM [Mn.sup.++] + (Fig. 2A) or 50 [mu]M verapamil (Fig. 3A). The velocity profiles are also typical (Figs. 2B and 3B, respectively, cf. Fig. 1B1) and the maximum velocities ([approx]20 [mu]m/min) are similar (P = 0.13).

Calcium ionophore A23187. Perfusion with control (5.5 mM [Ca.sup.++]) saline causes no net movement of the dispersed pigment mass (initial 10 min of Fig. 4A1-4), although 10-min perfusion with the EGTA-buffered salines containing either [10.sup.-3] (Fig. 4A2), [10.sup.-4] (Fig. 4A3), or [10.sup.-5] M [Ca.sup.++] (Fig. 4A4) induces progressive pigment aggregation (P [less than] 0.001) that attains a maximum of about 25% in [10.sup.-5] M [Ca.sup.++] (Fig. 4A4). Translocation velocities are slow and range around a maximum of [approx]2-4 [mu]m/min (Fig. 4B2-4).

Perfusion with 25 [mu]M A23187 while holding external [Ca.sup.++] at the designated reduced levels leads to complete aggregation within 26 min with 5.5 mM [Ca.sup.++] + (Fig. 4A1) and virtually complete aggregation (90%) at [10.sup.-3] M (Fig. 4A2). Peak aggregation velocities (11.6 [pm] 1.2 [mu]/min, n = 28 [Fig. 4B1] and 9.0 [pm] 3.7 [mu]m/min, n 7 [Fig. 4B2] respectively) are similar (P = 0.39). However, with [10.sup.-4] M [Ca.sup.++], the initial aggregation response (7.4 [pm] 1.3 [mu]m/min, n = 7 [Figs. 4A3, 4B3]) reverses after about 8 min, and slow, steady ([approx]1 [mu]m/min) pigment dispersion ensues (Fig. 4A3, 4B3). With [10.sup.-5] M A23187 induces slight, slow ([approx] 1.3 [mu]m/min) pigment dispersion (Fig. 4A4, 4B4).

Further perfusion with 5.5 mM [Ca.sup.++] produces complete, rapid pigment aggregation (Fig. 4A3, 4A4), regardless of the previous [[Ca.sup.++]] used. Velocity profiles and peak velocities ([approx]11 [mu]m/min, Fig. 4B3, 4B4) do not differ (P = 0.83) from those in preparations perfused directly with 5.5 mM [Ca.sup.++] (Fig. 4B1).

Removal of [Ca.sup.++] ([approx]7 X [10.sup.-11] M [Ca.sup.++], 2 mM EDTA) and A23187 from the perfusate after previous buffering with [Mg.sup.++]-EGTA (Fig. 4A2-4) does not produce the customary full pigment dispersion seen with EDTA alone (cf. Fig. 4A1), possibly due to chelating interference by EGTA.

Dependence of pigment dispersion on reduced calcium

After complete pigment aggregation using 25 [mu]M A23187 and saline containing 5.5 mM [Ca.sup.++], moderately rapid (maximum of 6.8 [pm] 1.7 [mu]m/min [Fig. 5B1]), complete, pigment dispersion can be induced using a [Ca.sup.++]-free saline (residual [Ca.sup.++] [approx] 7 X [10.sup.-11] M, 2 mM EDTA [minutes 90-130 of Fig. 5A1]); removal of the ionophore itself does not induce dispersion.

As the [Ca.sup.++] in the [Mg.sup.++]-EGTA-buffered perfusate is decreased from [10.sup.-7] (Fig. 5A2) to [10.sup.-8] (Fig. 5A3) and [10.sup.-9] M (Fig. 5A4), differential degrees of maximum pigment dispersion are induced, attaining about 80% in the latter. Maximum dispersion velocities increase (0.6 [pm] 0.3, 2.8 [pm] 2.1, and 4.7 [pm] 1.7 [mu]m/min, respectively, [P = 0.03]) with decreasing [[Ca.sup.++]] (Fig. 5B2-4). Further slight dispersion to about 90% can be induced using EDTA-buffered [Ca.sup.++]-free saline (minutes 90-130 of Fig. 5A4).

Relationship between degree of aggregation or dispersion and translocation velocity

Both translocation velocity and the degree to which pigment aggregates respond in a positively correlated, concentration-dependent manner to increasing external free [Ca.sup.++] between [10.sup.-5] M and 5.5 mM (Fig. 6); half-maximum translocation velocity ([V.sub.50]) is reached at 4.8 X [10.sup.-5] M and half-maximum aggregation at 3.2 X [10.sup.-5] M external free [Ca.sup.++]. The velocity and degree of pigment dispersion are also positively correlated and increase in a concentration-dependent manner to decreasing external free [Ca.sup.++] between [10.sup.-7] and [10.sup.-11] M (Fig. 7), with [V.sub.50] occurring at 2.9 X [10.sup.-9] M and half-maximum dispersion at 7.6 x [10.sup.-9] M external free [Ca.sup.++].


The present data obtained with red pigment concentrating hormone (RPCH) and A23187 respectively reveal an extracellular and an intracellular calcium requirement for pigment aggregation in the red ovarian chromatophores of Macrobrachium olfersii.

RPCH requires extracellular [Ca.sup.++] at [geq] [10.sup.-4] M to induce complete pigment aggregation. At lower concentrations ([10.sup.-5] to [10.sup.-9] M [Ca.sup.++]), aggregation is incomplete ([approx] 65%), although the maximum translocation velocities ([approx] 16.5 [mu]m/min and triphasic velocity profiles (McNamara and Ribeiro, 1999) are unaffected, except for minor suppression of the final low-velocity phase. This suggests that [Ca.sup.++] is required for coupling between RPCH and its receptor, much as in neural nicotinic receptors (Ospina et al., 1998; Booker et al., 1998; Liu and Berg, 1999). Incomplete aggregation may result from diminished signal transduction consequent to a reduction in the affinity between RPCH and its receptor at low [Ca.sup.++]. However, the intracellular signal generated is sufficient to transiently trigger the molecular motors responsible for aggregation at full capacity until the [Ca.sup.++]-regulatory mechanisms restore intracellular free [Ca.sup.++] to the resting conc entration, leading to spontaneous dispersion.

Alternatively, an RPCH-activated, [Ca.sup.++] induced/[Ca.sup.++]-release mechanism dependent on the [Ca.sup.++] gradient across the cell membrane (Putney and Bird, 1993; Verkhratsky and Shmigol, 1996) may operate. The reduced [Ca.sup.++] entering the chromatophore through RPCH-activated [Ca.sup.++] channels would be insufficient to promote sustained opening of target smooth endoplasmic reticulum [Ca.sup.++] channels, or to activate protein kinase C-dependent (Abr[tilde{a}]o et al., 1991; Sugden and Rowe, 1992) or [Ca.sup.++]-calmodulin-dependent effector pathways (Lee et al., 1994; Verkhratsky and Shmigol, 1996; Mukhopadhyay et al., 1997), also resulting in transient pigment aggregation. Since the [Ca.sup.++]-channel blockers [Mn.sup.++] and verapamil have no effect on the degree and velocity of pigment aggregation, these putative receptor-activated [Ca.sup.++] channels seem not to be L-type channels (Britto et al., 1996; Katz, 1996; Zhang et al., 1997; Striessnig et al., 1998). Although only a single maxim um blocker dose was employed, and other [Ca.sup.++] channel types may be present, this suggests that the [Ca.sup.++] required to activate the granule transport motors may be intracellular rather than extracellular in origin.

When the limiting effect of low extracellular [Ca.sup.++] bypassed by dissipating the [Ca.sup.++] gradient across the cell membrane with A23187, a second regulatory effect of [Ca.sup.++] appears. Assuming rapid equilibration of cytosolic [Ca.sup.++] with that in the extracellular medium (Lambert and Fingerman, 1978, 1979), both the degree of aggregation and the translocation velocity show [Ca.sup.++] -dependence below about [10.sup.-3] M (Fig. 6). The transient aggregation followed by dispersion at [10.sup.-4] M [Ca.sup.++] may result from decreased intracellular free [Ca.sup.++] consequent to the action of smooth endoplasmic reticulum (Treiman et al., 1998) and plasma membrane [Ca.sup.++]-dependent ATPases (Carafoli, 1991). The mitochondrial electrogenic [Ca.sup.++] uniporter and [H.sup.+] ([Na.sup.+])/ [Ca.sup.++] antiporter (Pozzan et al., 1994), and plasma membrane [Na.sup.+]/[Ca.sup.++] antiporter (Missiaen et al., 1991; Thastrup, 1990) may also act to reduce intracellular free [Ca.sup.++] At still lowe r concentrations ([leq][10.sup.-5] to [approx][10.sup.-11] M [Ca.sup.++]), pigment dispersion ensues (Fig. 7). Thus, the direction of pigment movement, the translocation velocity of the edges of the pigment mass, and the degree of dispersion or aggregation appear to be calcium-dependent properties of the intracellular effector, the pigment translocation apparatus, or both.

Although the translocation mechanisms may depend on very different pathways of signal transduction and activation and on diverse mechanochemical motor proteins, the present data corroborate estimates of the threshold [Ca.sup.++] necessary for pigment movements in teleost chromatophores; no such data are available for crustacean chromatophores. Pigment granules remain dispersed in cultured Xi-phophorus maculatus erythrophores perfused with 10 [mu]M A23187 and EGTA-buffered, [10.sup.-8] M free [Ca.sup.++] at [10.sup.-4] M [Ca.sup.++] erythrosome aggregation is almost complete (Oshima et al., 1988). In isolated Holocentrus ascensionis erythrophores incubated in 10 [mu]M A23187 and EGTA-buffered, external free [Ca.sup.++] below 5 X [10.sup.-6] M, pigment granules are also dispersed; above 5 X [10.sup.-6] M [Ca.sup.++] reversible aggregation is induced (Luby-Phelps and Porter, 1982). Microinjection of 10 [mu]M [Ca.sup.++] also induces erythro-some aggregation (Kotz and McNiven, 1994). In cultured H. ascencionis e rythrophores, stripped of their plasma membranes with Brij 58 detergent to reveal the cytoskeleton, pigment granules aggregate rapidly at [10.sup.-7] M free [Ca.sup.++] in an EDTA-EGTA buffer system; with [10.sup.-8] M [Ca.sup.++] the granules disperse (McNiven and Ward, 1988). Direct measurements using Fura-2 show that intracellular [Ca.sup.++] increases up to 100-fold or more, from about 30 nM in resting erythrophores to about 900 nM in epinephrine-stimulated H. ascencionis erythrophores (Kotz and McNiven, 1994). The present data, showing half-maximum aggregation at 3.2 X [10.sup.-5] M free [Ca.sup.++] and half-maximum dispersion at 7.6 X [10.sup.-9] M free [Ca.sup.++] are consistent with these findings.

Although threshold intracellular [Ca.sup.++] + concentrations for chromatophore pigment aggregation and dispersion remain to be estimated in nearly all crustacean species, pigment translocation in caridean and brachyuran chromatophores is clearly affected by if not dependent on alterations in intracellular [Ca.sup.++]. Lambert and Fingerman (1978) demonstrated A23187-induced (25 [mu]M) aggregation in Palaemonetes pugio red ovarian chromatosomes perfused with Van Harreveld's saline (14 mM [Ca.sup.++]); subsequent perfusion with [Ca.sup.++] -free-5 mM EDTA saline results in intermediate ([approx]45%) pigment dispersion. Native pigment dispersing hormone (PDH, 0.75 [mu]/ml) induces granule dispersion in the red, yellow, and black integumental chromatosomes of Uca pugilator perfused with Pantin's saline (12.7 mM [Ca.sup.++]), as do 10 and 25 [mu]M A23187 (Quackenbush, 1981). Curiously, 10 and 25 [mu]M A23187 not only inhibit the late phase (90-200 min) of PDH-induced pigment dispersion in Uca pugilator red chrom atosomes, but also induce pigment aggregation (Quackenbush, 1981), much as in caridean chromatosomes. Complete aggregation in the polychromatic epidermal chromatosomes of Palaemon affinis is induced by an eyestalk extract delivered in physiological saline containing 10 mM [Ca.sup.++] in [Ca.sup.++]-free saline, pigment aggregation is only partial ([approx]30%; McNamara and Taylor, 1987). In the red integumental chromatosomes of Macro brachium potiuna perfused with 6.5 mM [Ca.sup.++], A23187 ([10.sup.-7] to [10.sup.-4] M) has a concentration-dependent aggregating effect, reversible by [10.sup.-8] M [alpha]-PDH (Britto et al., 1990).

These various data demonstrate that pigment aggregation in the caridean shrimps and pigment dispersion in the brachyuran crabs can be brought about by changes in intracellular [Ca.sup.++] concentrations. The effector pathways of such concentration changes, whether increased by signal transduction or after A23187, are poorly known. Based on rightward shifting of the PCH dose-response curve by inhibitors of intracellular effectors, Nery et al. (1997) have suggested that pigment aggregation in M. potiuna red integumental chromatosomes may involve an inositoltrisphosphate-like cascade coupled to protein phosphatase 1 activation. This latter effect may down-regulate effector protein phosphorylation via the cyclic AMP-protein kinase A signaling pathway (Nery and Castrucci, 1997), given that increased intracellular cyclic AMP also induces pigment dispersion in caridean shrimps (Fingerman, 1969; Lambert and Fingerman, 1978; Nery et al., 1998). Clearly, possible convergence or cross-talk between the [Ca.sup.++] -signa ling and cyclic nucleotide-signaling pathways in crustacean pigment translocation mechanisms requires further investigation.


This study was financed by research grants to JCM (FAPESP 94/5981-7; CNPq 400517/95/7, 303282/84-3) and a postgraduate scholarship to MRR (FAPESP 94/ 4151-0).

(*.) To whom correspondence should be addressed. E-mail:

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Publication:The Biological Bulletin
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Date:Jun 1, 2000
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