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Cyclic guanosine monophosphate signaling cascade mediates pigment aggregation in freshwater shrimp chromatophores.

Introduction

Crustaceans exhibit adaptive chromatic responses to alterations in background color as a consequence of the bidirectional movement of the brightly colored pigment granules present in their pigmentary effectors, or chromatosomes. Located within the epidermis and in the fibrous capsules surrounding the internal organs, chromatosomes consist of 2 to 12 uninuclear chromatophores, which are irregular cells constituted by a perikaryon and one or two peripherally ramified cell extensions through which the pigment granules translocate (McNamara, 1981). Antagonistic, neurosecretory peptide hormones, the chromatophorotropins, produced and released by the eyestalk X-organ/ sinus gland complex and other neurohemal organs, regulate such pigment movements in decapod crustaceans (Rao, 1985). Red pigment concentrating hormone (RPCH) induces the aggregation of red and other dark pigments in caridean chromatophores (Fernlund and Josefsson, 1968, 1972; Josefsson, 1983), and red pigment dispersing hormone (RPDH) produces pigment dispersion (Rao and Fingerman, 1983; Fingerman, 1987).

Chromatophorotropins produce pigment aggregation by triggering a series of intracellular events that as yet are not well established, although pigment aggregation kinetics (McNamara and Ribeiro, 1999) and a role for increased cytoplasmic free [Ca.sup.2+] in pigment aggregation (Lambert and Fingerman, 1977, 1978; Nery et al., 1997; McNamara and Ribeiro, 1999, 2000; Ribeiro and McNamara, 2007) are well known. Just how the signal transduction cascade initiated by RPCH binding to its receptor triggers pigment translocation is far from clear. Cyclic nucleotide second messengers clearly play a role in mediating pigment translocation in crustacean chromatophores (Fingerman, 1969; Rao and Fingerman, 1983; Nery et al., 1997, 1998; Nery and Castrucci, 1997); however, few studies have been performed, and the findings so far conflict. To illustrate, pigment aggregation in caridean chromatophores appears to be associated with a decrease in cyclic adenosine monophosphate (cAMP) (Fingerman, 1969; Nery et al., 1997) and an increase in cytosolic free [Ca.sup.2+] (McNamara and Ribeiro, 2000). Conversely, increased cAMP causes pigment dispersion in Palaemonetes vulgaris chromatophores (Fingerman, 1969); Nery et al., (1998) have demonstrated that db-cAMP--the lipid-soluble cAMP analog--and forskolin--an adenylate cyclase activator--induce pigment dispersion in Macrobrachium potiuna epidermal chromatophores. However, in Macrobrachium olfersi ovarian chromatophores, db-cAMP does not induce significant pigment translocation (Ribeiro and Mc-Namara, 1997). Cyclic guanosine monophosphate (cGMP) does not stimulate pigment aggregation in Palaemonetes pugio ovarian chromatophores (Lambert and Fingerman, 1979), and the lipid-soluble analog, db-cGMP, does not cause pigment aggregation in M. potiuna epidermal chromatophores (Nery et al., 1998). However, in M. olfersi ovarian chromatophores, db-cGMP does induce complete and reversible pigment aggregation with kinetics identical to those of RPCH (Ribeiro and McNamara, 1997). Clearly, a role for cGMP in the pigment aggregation cascade must be evaluated.

Cyclic GMP production can be catalyzed by plasma-membrane-receptor guanylyl cyclases or by soluble, cytosolic guanylyl cyclases (Wong and Garbers, 1992; Schmidt et al., 1993; Giannella, 1995). The plasma-membrane guanylyl cyclases (GC-A, -B and -C), which are single-span receptors, possess a highly variable, extracellular ligand-binding domain, a transmembrane domain, and an intracellular protein kinase-like domain, together with a highly conserved, catalytic cyclase domain (Wong and Garbers, 1992). Such receptors are activated by atrial natriuretic peptides and heat-stable bacterial enterotoxins, and their extracellular domains contain cysteine residues that are highly conserved across many species, suggestive of conserved structure and function (Foster et al., 1999; see also Pavloff and Goy, 1990, and Schulz, 1992, for reviews of vertebrate and invertebrate GC's and phylogenies). Cytosolic guanylyl cyclases (GC-S) are heterodimeric, heme-containing proteins found across the animal kingdom, including mammals, fish, arthropods, molluscs, echinoderms, and nematodes (Koesling and Friebe, 1999; Fitzpatrick et al., 2006), and are activated by vasodilatory agents like sodium nitroprusside and nitroglycerine (Murad, 1994; Wong and Garbers, 1992). Nitric oxide (NO) is a powerful GC-S activator that binds to the heme group (Ignarro, 1990; Schmidt et al., 1993; Murad, 1994; Muller, 1997), leading to cGMP production (Wong and Garbers, 1992; Mittal, 1995) and stimulating protein kinase G activity (Lowenstein and Snyder, 1992). NO is generated from L-arginine (Wong and Garbers, 1992; Murad, 1994; Mittal, 1995; Michel and Feron, 1997) by nitric oxide synthase (NOS), an enzyme found in both soluble and particulate forms, which can be either induced or constitutively regulated by [Ca.sup.2+]/calmodulin (Ignarro, 1990; Lowenstein and Snyder, 1992). Originally identified in mammalian tissues, NO has been identified in the signaling cascades of many invertebrates such as coelenterates, platyhelminths, nematodes, annelids, molluscs, arthropods, echinoderms, and urochordates (Martinez, 1995; Midler, 1997). In pigment cells, NO causes aggregation in amphibian melanophores (Nilsson et al., 2000, 2001) and dispersion in fish melanophores (Hayashi and Fujii, 2001). Interestingly, other crustacean neuropeptides like crustacean hyperglycemic hormone (CHH) and Molt-inhibiting Hormone (MIH) released from the X-organ/ sinus gland complex produce their effects through increased cGMP generated via a membrane receptor GC (Lee et al., 2007a, b; Zheng et al., 2008) or also by cytosolic GC in the case of MIH (Lee et al., 2007a).

The signaling cascade activated by RPCH/receptor coupling in caridean chromatophores is obscure. To evaluate the participation of cGMP in pigment aggregation in ovarian chromatophores, we employed cell-permeant dibutyryl cGMP and agents that increase cGMP synthesis via GC-S stimulation through NO release, and inhibitors that reduce cytosolic cGMP titers by inhibiting GC-S. Our findings reveal that cGMP likely mediates RPCH-triggered pigment aggregation in caridean shrimp chromatophores, another example of the action of an X-organ neurosecretory peptide via the cGMP cascade.

Materials and Methods

Young adult, female freshwater shrimp, Macrobrachium olfersi (Wiegmann, 1836), possessing small, immature ovaries, were collected from the marginal vegetation of the Pauba River on the Atlantic coast of Sao Paulo State, Brazil (IBAMA/DIREN permit #18/2002) and held in tanks containing 80 1 of aerated river water. The shrimps were fed daily with chopped spinach, carrot or beetroot, and raw beef or chicken. Physiological experiments were conducted as described in McNamara and Ribeiro (1999). Briefly, each ovary containing dorsal chromatosomes was sectioned transversally and the anterior half transferred to a small, acrylic chamber (154-[micro]l volume) and gravity perfused (0.7 ml/min) with shrimp saline.

Two physiological salines based on M. olfersi hemolymph (McNamara et al., 1990; McNamara and Ribeiro, 1999) were used: (i) normal calcium saline (NCS) (364 [+ or -] 1.9 mOsm/kg [H.sub.2]O, n = 25), containing (in mmol [l.sup.-1] ) [Na.sup.+] 180, [K.sup.+] 5, [Ca.sup.2+] 5.5. and [Mg.sup.2+] 1 as chlorides ([approximately equal to]198); and (ii) km calcium, EDTA-buffered saline (LCS-EDTA) (383 [+ or -] 0.3 mOsm/kg [H.sub.2]O, n = 5), [Ca.sup.2+] substituted by 5.6 mmol [l.sup.-1] choline chloride in 2 mmol [l.sup.-1] EDTA (ethylenediamine tetra-acetic acid, Sigma, Missouri) (residual [Ca.sup.2+] 9.21 X [10.sup.-11] mol [l.sup.-1]). LCS was used initially to assess possible [Ca.sup.2+]-dependence of the cGMP cascade, and at the end of each experiment, to evaluate both the degree of aggregation reversibility and integrity of the dispersion mechanism since low [Ca.sup.2+] induces complete pigment dispersion under control conditions (McNamara and Ribeiro, 2000). Both salines also contained 2.5 mmol [l.sup.-1] [Na.sub.2][HCO.sub.3], 5 mmol [l.sup.-1] HEPES, and 2 mmol [l.sup.-1] glucose, and were adjusted to pH 7.4.

Red pigment concentrating hormone (RPCH, [10.sup.-8] mol [l.sup.-1], Peninsula Laboratories Inc., San Carlos, CA) was used to induce standard pigment aggregation. To evaluate the effect of increased concentrations of cytosolic cyclic guanosine monophosphate (cGMP) on pigment aggregation, we employed dibutyryl 3' :5'-cyclic guanosine monophosphate (db-cGMP, 10 [micro]mol [l.sup.-1], Sigma, Missouri), a lipid-per-meant cGMP analog; sodium nitroprusside (SNP, 0.5 [micro]mol [l.sup.-1], Sigma, Missouri; Almond and Paterson, 2000; Rupin et al., 2000; Chen et al., 2001) and 3-morpholinosydnonimine (S1N-1, 100 [micro]mol [l.sup.-1], Sigma, Misssouri; Elphick et al., 1993; Mathy-Hartert et al., 2000; Mayhan, 2000). Both cytosolic guanylyl cyclase activators have been used successfully in crustacean systems (see Scholz, 2001, for review). Escherichia coli heat-stable enterotoxin (STa, 1 [micro]mol [l.sup.-1], Sigma, Missouri; Giannella, 1995; Krause et al., 1997) was used as a stimulator of the type-C plasma membrane guanylyl cyclase receptor (Wong and Garbers, 1992; Mayhan, 2000; Chen et al., 2001; see also Schulz, 1992, for review). Dose-response curves were generated for SNP and SIN-1, the concentrations used being based on the above-cited studies. These two experiments were performed in the dark, owing to the light sensitivity of these agents.

To evaluate the effect of decreased intracellular cGMP concentrations on pigment aggregation, we used zinc protophorphyrin IX (ZnPP-IX, 30 [micro]mol [l.sup.-1] Sigma, Missouri; Drummond and Kappas, 1981; Serfass and Burstyn, 1998) and 6-anilino-5,8-quinolinedione (LY83583, 10 [micro]mol [l.sup.-1], Sigma, Missouri; Fleming et al., 1991; Volk et al., 1997). These are both cytosolic guanylyl cyclase inhibitors, the latter used successfully in scallop ciliary photoreceptors (Gomez and Nasi, 2000).

The effect of all reagents on pigment translocation was quantified by measuring pigment distribution in the cell extensions of chromatosomes 180 to 220 [micro]m in diameter, at 2-min intervals, against an ocular graticle. The perfused preparation was observed at 200 X, employing both transmitted and incident light in a Wild M10 stereoscopic microscope coupled to a Sony DXC-151A CCD video camera and Trinitron monitor (Sony Corporation, Tokyo). Pigment dispersion of 100% is defined as the maximum degree of pigment dispersion throughout the chromatophore cytoplasm 10 min after perfusion with NCS. Pigment aggregation of 100% is defined as the minimum degree of pigment dispersion (which equals the maximum degree of pigment aggregation) encountered after a 30-min RPCH perfusion under similar control conditions; it constitutes that state in which no pigments remain in the chromatophore cell extensions. The cell body alone contains the aggregated pigment mass, the diameter of which is used to calculate the minimum degree of pigment dispersion (see McNamara and Ribeiro, 1999). Each experiment was repeated 7-9 times, using a single chromatosome in each preparation. All experiments were performed at room temperature ([approximately equal to] 23 [degree]C). Such perfused preparations allow up to five cycles of aggregation and dispersion verified using A23l87-induced aggregation and LCS washout (see McNamara and Ribeiro, 2000). Although aggregation and dispersion become attenuated after the third cycle, translocation velocity is mainly unaffected. Only the first pigment aggregation/dispersion cycle was used here.

Statistical analyses were performed using SigmaStat 2.03 (SPSS Inc., Chicago, IL) and Statgraphics 6.0 Plus (Statistical Graphics Corp., Herndon, VA). Since the data generated were usually not normally distributed, treatment effects on the degree of pigment aggregation and translocation velocity were evaluated using the Mann-Whitney U test or Kruskal-Wallis one-way ANOVA, followed by Dunn's test to locate significantly different groups. Effects and differences were considered significant at P = 0.05. Data are presented in the text as the mean [+ or -] SEM (n), and where necessary, dose response curves were fitted with sigmoid curves using the sigmoidal function of the graphical application SlideWrite 5.0 (Advanced Graphics Software, Inc., Encinitas, CA).

Results

The effects of the various reagents on pigment aggregation/dispersion in the red ovarian chromatophores were compared with standard pigment aggregation induced by red pigment concentrating hormone (RPCH) and standard pigment dispersion induced by RPCH washout (McNamara and Ribeiro, 1999). Normal, calcium-containing (5.5 mmol [l.sup.-1] [Ca.sup.2+]) saline was used unless otherwise stated.

RPCH- and db-cGMP-induced pigment aggregation

RPCH at [10.sup.-8] mol [l.sup.-1] produced complete pigment aggregation within 22 min (Fig. 1A), with a typical velocity profile consisting of a brief peak of rapid translocation (17.0 [+ or -] 2.9 [micro]m/min, n = 11) followed by a low velocity plateau (1.8 [+ or -] 0.3 [micro]m/min, n = 66) (Fig. 1B). RPCH washout led to 88% pigment dispersion (Fig. 1A) with a maximum velocity of 5.6 [+ or -] 2.5 [micro]m/min (n = 11) (Fig. 1B) (McNamara and Ribeiro, 1999).

[FIGURE 1 OMITTED]

Dibutyryl guanosine 3';5'-cyclic monophosphate (db-cGMP, 10 [micro]mol [l.sup.-1]) likewise triggered complete pigment aggregation with a time course (22 min) (Fig. 1A) and velocity profile (peak velocity 16.6 [+ or -] 1.5 [micro]m/min, n = 10; plateau velocity 2.1 [+ or -] 0.4 [micro]m/min, n = 50) identical to RPCH (Fig. IB) (Mann-Whitney test, P > 0.05). Remarkably, db-cGMP washout produced complete (97%) pigment dispersion within just 10 min (Fig. 1A), maximum translocation velocity (13.2 [+ or -] 2.3 [micro]m/min, n = 10) reaching more than twice that of RPCH washout (Mann-Whitney test, P < 0.05, Fig. 1B). However, db-cGMP induced pigment aggregation only in NCS ([Ca.sup.2+] 5.5 mmol [l.sup.-1]); in LCS/EDTA ([Ca.sup.2+] 9 X [10.sup.-11] mol [l.sup.-1]) there was no response to db-cGMP perfusion (Fig. 1A). These findings show that db-cGMP mimics RPCH well in a [Ca.sup.2+]-dependent manner.

[FIGURE 2 OMITTED]

Stimulation of the type-C plasma membrane receptor guanylyl cyclase

Escherichia coli heat-stable enterotoxin (STa, 1 [micro] mol [l.sup.-1]) induced minor pigment aggregation on its own ([approximately equal to] 17%, Fig. 2A) but had no effect on pigment aggregation induced by RPCH (peak velocity 14.3 [+ or -] 2.7 .[micro]m/min, n = 8; plateau velocity 4.3 [+ or -] 0.8 [micro]m/min, n = 32; Mann-Whitney test, P > 0.05) (Fig. 2B). STa washout (30 min) produced minimal dispersion ([approximately equal to] 10%), although subsequent perfusion with LCS-EDTA led to 80% pigment dispersion (Fig. 2A). Apparently cGMP production in these chromatophores does not involve a membrane-located GC receptor.

Activation of cytosolic guanylyl cyclase

To establish the pharmacological concentrations necessary to stimulate cytosolic cGMP production in freshwater shrimp chromatophores, we generated dose-response curves for the soluble guanylyl cyclase activators sodium nitroprusside (SNP) and morpholinosydnonimine (SIN-1), which are nitric oxide donors (Fig. 3). The pigment aggregation responses to SNP ([10.sup.-9] to [10.sup.-3] mol [l.sup.-1] ) were fit with a sigmoid curve ([R.sup.2] = 0.995; [EC.sub.50] = 2.9 X [10.sup.-7] mol [l.sup.-1] Fig. 3). Although 10 [micro] mol [l.sup.-1] SNP was the lowest concentration to induce complete pigment aggregation, this response was not consistent (data not shown). Aggregation during time-course experiments using 10, 50, or 100 [micro] mol [l.sup.-1] SNP was also limited but did increase with decreasing SNP concentrations. Thus, we employed 0.5 [micro] mol [l.sup.-1] SNP in the time-course experiments. A sigmoid curve was also fit to the aggregation response to SIN-1 ([10.sup.-9] to [10.sup.-3] mol [l.sup.-1] ) ([R.sup.2] = 0.923; [EC.sub.50] = 5.2 X [10.sup.-7] mol [l.sup.-1] , Fig. 3). Although the maximum aggregation response possible was [approximately equal to]50%, we employed 100 [micro] mol [l.sup.-1] SIN-1.

[FIGURE 3 OMITTED]

In time-course experiments, both 0.5 [micro] mol [l.sup.-1] SNP and 100 [micro] mol [l.sup.-1] SIN-1 induced moderate pigment aggregation by themselves ([approximately equal to] 30% and [approximately equal to]40%, respectively, Fig. 4A) with peak velocities of 7.6 [+ or -] 2.4 [micro]m/min (n = 8) and 6.1 [+ or -] 1.4 [micro]m/min (n = 8), respectively (Fig- 4B), both less than control aggregation velocities (Mann-Whitney test, P < 0.05). Neither GC-S activator affected RPCH-induced pigment aggregation (peak velocities of 17.2 [+ or -] 1.8 [micro]m/min, n = 8, and 12.2 [+ or -] 1.9 [micro]m/min, n = 8, respectively; plateau velocities of 2.8 [+ or -] 0.4 [micro]m/min, n = 32, and 3.0 [+ or -] 0.4 [micro]m/min n = 25, respectively; Mann-Whitney test, P > 0.05, Fig. 4B). SNP and SIN-1 washout induces pigment dispersion of 25% and 20%, respectively, and subsequent perfusion with LCS/EDTA led to pigment dispersion of 70% and 80%, respectively (Fig. 4A). These data show that activation of GC-S contributes to the aggregation response. Differential SNP- and SIN-1-dependent increases in cGMP may account for the different washout times and dispersion responses. Pigment dispersion in carideans is regulated by cAMP antagonistically to cGMP-regulated aggregation (Nery et al., 1997, 1998; Nery and Castrucci, 1997): the cAMP signaling cascade may be simultaneously inhibited by increased cGMP, leading to variable dispersion kinetics as downstream events reverse.

[FIGURE 4 OMITTED]

Inhibition of cytosolic guanylyl cyclase

Zinc protophorphyrin IX (ZnPP-IX, 30 [micro] mol [l.sup.-1] ) and 6-anilino-5,8-quinolinedione (LY83583, 10 [micro] mol [1.sup.1]) did not induce pigment aggregation on their own (Fig. 5A). However, they did partially inhibit RPCH-triggered pigment aggregation ([approximately equal to] 35%) and [approximately equal to] 22%, respectively). Peak velocities of the inhibitor plus RPCH were 8.8 [+ or -] 1.9 [micro]m/ min (n = 8) and 14.5 [+ or -] 2.0 [micro]m/min (n = 7), respectively, followed by plateau velocities of 2.4 [+ or -] 0.9 [micro]m/min (n = 16) and 2.1 [+ or -] 0.7 [micro]m/min (n = 42), respectively. All these translocation velocities were slower than control (RPCH alone) velocities (Mann-Whitney test, P < 0.05, Fig. 5B). These findings also show that GC-S contributes cGMP to the aggregation response. Washout of ZnPP-IX and LY83583 did not induce pigment dispersion, activation of which may entail a cGMP-dependent step. Since sGC may still be inhibited 20 min after washout, only incomplete dispersion ensues.

[FIGURE 5 OMITTED]

Discussion

The present findings unequivocally reveal that the intracellular signaling cascade by cyclic guanosine monophosphate (cGMP) participates in pigment aggregation in the red ovarian chromatophores of Macrobrachium olfersi. The cell-permeant analog dibutyryl 3':5'-cyclic guanosine monophosphate (db-cGMP) perfectly mimics the action of the native neuropeptide red pigment concentrating hormone (RPCH) in terms of both pigment translocation kinetics and reversibility. Stimulation by sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1) of the cytosolic guanylyl cyclase system to produce endogenous cGMP likewise induces pigment aggregation ([approximately equal to]40%) that also follows the typical kinetics of pigment translocation. Inhibition of this same cyclase by zinc protophorphyrin IX (ZnPPIX) and 6-anilino-5,8-quinolinedione (LY83583) reduces RPCH-triggered pigment aggregation by [approximately equal to]30% and also decreases the velocity of rapid phase translocation. Close inspection of Figure 1 shows that the translocation response to db-cGMP is qualitatively better resolved compared to that of RPCH. Aggregation follows a clearly steeper curve (Fig. 1A), and the washout response is extremely rapid over a very brief period (Fig. 1B). This suggests that the binding of RPCH to its receptor and particularly its release are slower events than is motor activation by the cGMP cascade, revealing that receptor/RPCH events may govern the time course of response rather than the intracellular signaling cascade itself.

That cGMP plays a role in the movement of crustacean chromatophore pigments is not a novel finding. Both cGMP and db-cGMP directly induce dose-dependent pigment dispersion in crab (Uca pugilator) black, white, and red chromatophores in vitro, and they inhibit dispersion in red chromatophores but enhance dispersion in black and white chromatophores induced in vitro by a partially purified eyestalk hormone (Rao and Fingerman, 1983). However, unstimulated pigment granule distribution in brachyuran black chromatophores in vitro is exactly the opposite of that in the corresponding chromatophores of palaemonid shrimps: in crabs, such pigments naturally aggregate, but in palaemonids they disperse. Thus, in these two crustacean taxa, the opposing molecular motors that putatively effect pigment granule translocation (Boyle and McNamara, 2008) are apparently coupled to the same signaling pathway; that is, in crab black chromatophores, the cGMP cascade leads to centrifugal pigment dispersion, possibly via the microtubule-based motor kinesin; but in shrimp chromatophores, the same cascade is coupled to centripetal pigment aggregation via the actin-based motor myosin (McNamara and Ribeiro, 1999; Boyle and McNamara, 2006). That [db-]cGMP is without effect in the two other palaemonid species investigated to date may reflect the fact that Lambert and Fingerman (1979) employed non-permeant cGMP in Palemonetes pugio, whereas Nery et al. (1998) used an epidermal preparation in Macrobrachium potiuna. The heavy overlying cuticle and tightly adjoined epidermal cells may restrict reagent diffusion to the chromatophores, a possible explanation for this lack of effect. Further, epidermal chromatophores are ultrastructurally quite distinct from internally located pigmentary effectors: their cell extensions contain abundant microtubules (McNamra, 1981), whereas microtubules in internal chromatophores are few and microfilaments in internal chromatophores are few and microfilaments predominate (McNamara, 1981; McNamara and Sesso, 1982; McNamara and Ribeiro, 1999).

Calcium also mediates pigment movements in crustacean and vertebrate chromatophores (Rao and Fingerman, 1983; McNamara and Taylor, 1987; Britto et al., 1996; Nery and Castrucci, 1997; Nery et al., 1997). Both the degree and velocity of pigment aggregation are [Ca.sup.2+] requirement for RPCH binding (McNamara and Ribeiro, 1999). Further, complete pigment aggregation requires [Ca.sup.2+] originating both extracellularly and from the smooth endoplasmic reticulum (Ribeiro and McNamra, 2007). It is thus particularly relevant that db-cGMP triggers pigment aggregation in ovarian chromatophores only when [Ca.sup.2+] (NCS, 5.5 mmol [l.sup.-1] [Ca.sup.2+]) is present in the perfusate. In low-calcium saline (residual [Ca.sup.2+] 9.21 x[10.sup.-11] mol [l.sup.-1]), db-cGMP is without effect, revealing not only that RPCH-induced aggregation results necessarily from both an increase in cytosolic [[Ca.sup.2+]] and cGMP production but that the cGMP cascade is [Ca.sup.2+] -dependent, possibly at the guanylyl cyclase step. Thus, there seems to be at least one point of regulatory cross-talk between the [Ca.sup.2+] and the cGMP cascades in these cells, and cytosolic [[Ca.sup.2+]] may well modulate the latter at diverse downstream nexuses. Since the distinct pigment types in the monochromatic chromatophores that constitute polychromatic chromatosomes that constitute polychromatic chromatosomes (McNarma, 1981) can migrate simultaneously in opposing direction, granule translocation may be coupled to distinct signaling cascades or to different surface-located motors (Boyle and McNamara, 2006). That both cGMP and [Ca.sup.2+] can effect pigment movement independently in monochromatic red ovarian chromatophores, although apparently converging on a single motor type and/or translocation direction, corroborates this idea.

Our findings strongly suggest that the cGMP cascade has its origin in the activation of cytosolic guanylyl cyclase (GC-S) since GC-S activation by nitric oxide (NO), a well-known effect (Katuski et al., 1977), induces pigment aggregation. SNP and SIN-1, both NO donors, induced [approximately equal to] 40% pigment aggregation, while the GC-s inhibitors, ZnPP-IX and LY83583, both inhibited RPCH-triggered aggregation by [approximately equal to] 30%. Escherichia coli enterotoxin (Sta) that stimulates membrane-located guanylyl cyclase does not induce per se or enhance RPCH-triggered pigment aggregation, which excludes the likelihood of membrane-located cGMP production. Such data find support in other pigment cell systems. NO induces pigment aggregation in melanophores of the clawed toad Xenopus laevis (Nilsson et al., 2000), while L-Name (N-nitro-L-arginine methyl ester), an NO synthase inhibitor, and ODQ (1H-(1,2,4) oxadiazolo (4,3-a) quinoxalin-1-one), a GC-S inhibitor, reduce pigment aggregation. In teleost melanophores, NO donors like NOR1, MSD, SNP, and GTN, and 8-Br-cGMP induce pigment dispersion rather than pigment aggregation (Hayashi and Fujii, 2001). These findings provide support for the role of GC-S-generated cGMP in pigment translocation and constitute further evidence that the same signaling pathways are coupled to different molecular motors in different taxa, both vertebrate and invertebrate. Again, [Ca.sup.2+]-dependent regulation of NO synthase may constitute another point at which cross-talk information flows between the [Ca.sup.2+] and the cGMP cascades, ultimately regulating GC-S activity.

It is unclear exactly how cGMP and [Ca.sup.2+] might activate the pigment-aggregating mechanism. Smooth muscle contraction and relaxation are regulated by myosin light chain kinase (MLCK) and phosphatase (MLCP) (Surks et al., 1999), and MLCP itself is regulated by protein kinase G. During contraction, increased intracellular [Ca.sup.2+] activates the [Ca.sup.2+]/calmodulin-dependent MLCK, which in turn activates myosin ATPase (Surks etal., 1999). During relaxaction, decreased intracellular [Ca.sup.2+] inactivates MLCK, followed by myosin light chain dephosphorylation by MLCP (Surks et al., 1999). In shrimp chromatophores, as in smooth muscle, putative myosin phosphatase activity may be regulated by cGMP-dependent kinases activated in response to increased cytosolic cGMP as a consequence of stimulated GC-S.

There is substantial evidence that pigment dispersion in shrimp red chromatorphores is mediated by activation of the cyclic adenosine monophosphate (cAMP) cascade (see cyclic adenosine monophosphate (cAMP) cascade (see Nery et al., 1998). Thus, two opposing cyclic nucleotide pathways may simultaneously regulate the activity of the respective molecular motors responsible for aggregation and dispersion in a tug-of-war manner--increased cGMP and [Ca.sup.2+] and decreased cAMP promoting pigment aggregation, and increased cAMP and decreased cGMP and [Ca.sup.2+] producing dispersion. The resulting state of intracellular pigment distribution at a given instant may thus reflect the degree of activation of downstream, cascade-specific regulators like protein kinases G, C, and A and the calcium/calmodulin-dependent kinase, and the extent of phosphorylation of molecular motors like myosin and kinesin (see Boyle and McNamara, 2006, 2008) and their specific kinase-dependent regulators.

Acknowledgments

This study constitutes part of a doctoral thesis submitted by MRR to be postgraduate program in Comparative Biology (DB/;FFCLRP, USP), and was financed by research grants to JCM (FAPESP 2000/04588-2, CNPq 40017/95-7, CNPq 303282/8-3; 304174/2006-8), and a doctoral scholarship to MRR (CAPES). We thank the Centro de Biologia Marinha, USP, for providing logistical support during collecting expeditions.

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MARCIA REGINA RIBEIRO AND JOHN CAMPBELL McNAMARA *

Departamento de Biologia, FFCLRP, Universidade de Sao Paulo, Ribeirao Preto, 14040-901, Sao Paulo, Brazil

* To whom correspondence should be addressed, at Departamento de Biologia, FFCLRP, Universidade de Sao Paulo, Av Bandeirantes, 3900 Ribeirao Preto 14040-901, SP, Brazil. E-mail: mcnamara@ffclrp.usp.br

Abbreviations: cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; db-cGMP, dibutyryl 3':5' -cyclic guanosine monophosphate; GC-S, soluble guanylyl cyclase; LCS, low-calcium saline; LY83583, 6-alilino-5, 8-quinolinedione; NCS, normal calcium saline; NO, nitric oxide; RPCH, red pigment concentrating hormone; SIN-1, 3-morpholinosydnonimine; SNP, sodium nitroprusside; STa, Escherichia coli heat-stable enterotoxin; ZnPP-XI. zinc protoporphyrin IX.

Received 22 April 2008; accepted 22 October 2008.
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