Biol. Bull. 210: 121-131. (April 2006)
© 2006 Marine Biological Laboratory
Role of Protein Kinase C, G-Protein Coupled Receptors, and Calcium Flux During Metamorphosis of the Sea Urchin Strongylocentrotus purpuratus
G. Amador-Cano,
E. Carpizo-Ituarte* and
D. Cristino-Jorge
Instituto de Investigaciones Oceanológicas, Universidad Autónoma de Baja California, Apartado Postal 453, Ensenada, B. C. México 22800
* To whom correspondence should be addressed. E-mail: ecarpizo{at}uabc.mx
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Abstract
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Artificial inducers have been used to study signal-transduction pathways involved in metamorphosis of some marine invertebrates. However, the transduction mechanisms for echinoderms have been less explored. In the present study, participation of protein kinase C (PKC), G-protein-coupled receptors (GPCRs), and calcium has been investigated during metamorphosis of the sea urchin Stronglylocentrotus purpuratus. Competent larvae were induced with different drugs that activate (PKC and GP activators, Ca2+ ionophores, and inhibitors of Ca2+ ATPase) or inhibit (PKC, G-protein, and Ca2+ flux inhibitors) metamorphosis. Six of the compounds were effective: the PKC activators TPA and indolactam; the G-protein inhibitors suramin and guanosine; the calcium ionophore A23187, and the calcium ATPase inhibitor thapsigargin. TPA was effective at 0.001 µM; indolactam was effective at 0.001 µM. In the presence of KCl as inducer, the G-protein inhibitor suramin was effective at 10 µM and guanosine at 0.001 µM. In the presence of a bacterial film as inducer, suramin was effective at 50 µM, and guanosine inhibited metamorphosis at 1 µM. A23187 was effective at 5 and 10 µM and thapsigargin at 50 and 100 µM. Our results indicate that GPCRs, protein kinase C, and calcium participate in the metamorphosis of S. purpuratus. These elements of the transduction pathways triggered during metamorphosis may be part of a cascade of signal transduction routes that interact from induction to the end of the morphogenetic events that shape the postlarval form. In addition, according to the results obtained with G-protein inhibitors, the GPCRs may be shared between the artificial (KCl) and natural (biofilm) inducers.
Abbreviations: DAG, diacylglycerol • GPCR, G-protein-coupled receptor • IP3, inositol-1,4,5-tris-phosphate • PKC, protein kinase C • TPA, phorbol-12-myristate-13-acetate
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Introduction
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In marine invertebrates, the stimuli that induce metamorphosis are specific agents, chemical or biological, that are associated with certain natural substrata such as microbial films, potential prey, or algae (Pawlik, 1992; Rodriguez et al., 1993; Hadfield, 1998; Hadfield et al., 2000). In some species, activation of the morphogenetic changes can be triggered, in the absence of natural cues, by several organic and inorganic compounds, which have been used as artificial inducers. These include neuroactive substances such as 
-aminobutyric acid (GABA), 3,4-dihydroxyphenylalanine (DOPA), catecholamines, and choline derivatives, organic and inorganic compounds, and the inorganic ions Mg2+, Li+, NH4+, Cs+, and K+ (Pearce and Scheibling, 1994; Avila et al., 1996; Fleck, 1998; Kawaii et al., 1999; Carpizo-Ituarte et al., 2002).
Artificial inducers have been used to study possible transduction pathways in the metamorphosis of a number of marine invertebrates (Pawlik, 1990; Hadfield, 1998). However, in sea urchins, the mechanisms by which the external signal is detected by the larva, internalized, and transduced into the morphogenetic changes that occur during metamorphosis are poorly understood, although, as in many other invertebrate larvae, the initial response to the metamorphic cue appears to involve a neurosensorial response (Burke, 1983; Bishop and Brandhorst, 2001).
The possible transduction pathways involved in metamorphosis have been investigated in many invertebrate phyla. These include cnidarians (Freeman and Ridgway, 1990; Fleck and Bischoff, 1992; Henning et al., 1996; Fleck, 1997; Leitz, 1997; Siefker et al., 2000; Thieme and Hofmann, 2003), polychaete annelids (Pawlik, 1990, 1992; Ilan et al., 1993; Carpizo-Ituarte and Hadfield, 1998; Holm et al., 1998; Biggers and Laufer, 1999), molluscs (Baxter and Morse, 1987; Morse, 1992; Hadfield, 1998, Hadfield et al., 2000), crustaceans (Clare et al., 1995; Yamamoto et al., 1995; Clare, 1996), echinoderms (Bishop and Brandhorst, 2001, 2003), and ascidians (Bishop et al., 2001). Some of these pathways presumably involve cyclic adenosine-monophosphate (cAMP) and inositol-1,4,5-tris-phosphate/diacylglycerol (IP3/DAG) signaling, in combination with G-protein-coupled receptors (GPCRs), protein kinase C (PKC), and calcium as second messengers. These last three elements are apparently a common theme in signal transduction during metamorphosis of marine invertebrate larvae (but see Holm et al., 1998, for an exception). Recently, the inhibitory system of nitric oxide/cyclic guanosine monophosphate (NO/cGMP) has been documented during metamorphosis of the ascidians Boltenia villosa and Cnemidocarpa finmarkiensis (Bishop et al., 2001), the caenogastropod Ilyanassa obsoleta (Leise et al., 2001), and the sea urchin Lytechinus pictus (Bishop and Brandhorst, 2001, 2003).
In all eukaryotes, a large proportion of the cell-surface receptors are GPCRs. They transduce a wide variety of extracellular signals, including physical, chemical, or biological cues such as light, calcium ions, nucleotides, amino acids, pheromones, lipids, peptides, and glycoproteins (Bai, 2004). Being so abundant as surface receptors and implicated in mammalian and insect olfaction (Reed, 2004), it is not surprising that GPCRs have been presumed to be involved in signal transduction pathways during metamorphosis of marine invertebrate larvae. Hydrozoans and barnacles apparently employ GPCRs of the superfamily of seven-transmembrane-domain to begin metamorphosis (Schneider and Leitz, 1994; Clare, 1996), and some evidence suggests that the same is true for molluscs, in particular abalone (Baxter and Morse, 1987, 1992).
Another common element in the induction of metamorphosis in larvae of marine invertebrates is the activation of PKC. This has been documented as part of the internal signal cascade during metamorphosis in the cnidarians Mitrocomella polydiademata, Cassiopea spp., Aurelia aurita, Chrysaora hysoscella, and Cyanea lamarckii (Freeman and Ridgway, 1990; Fleck and Bischoff, 1992; Siefker et al., 2000), and the crustacean Balanus amphitrite (Yamamoto et al., 1996). Biggers and Laufer (1999) utilized hormones that activate PKC to induce metamorphosis in the polychaete Capitella sp. These authors suggest that the activation of this enzyme modulates calcium and potassium channels during metamorphosis. However, the actual mechanisms by which PKC and calcium regulate metamorphosis in Capitella were not determined.
The role of calcium as a second messenger during metamorphosis has been partially documented in different phyla, including the gastropod Haliotis rufescens (Baxter and Morse, 1987), the polychaete Phragmatopoma californica (Ilan et al., 1993), the brachiopod Terebratalia (Freeman, 1993), the crustacean Balanus amphitrite (Clare et al., 1995; Clare, 1996), the hydrozoan Phialidium gregarium (McCauley, 1997), and the hydroid Tubularia mesembryanthemum (Kawaii et al., 1999). In all of these organisms, the compounds applied affected transduction pathways dependent on cAMP, the participation of the IP3/DAG system, and calcium channels. The specific cascade of events within the studied pathways is not completely understood for any of the transduction pathways involved during metamorphosis of the different organisms mentioned.
The present study sought to determine whether signal-transduction pathways associated with GPCRs and PKC play a role during the metamorphosis of the sea urchin Stronglylocentrotus purpuratus, and whether calcium is participating as a second messenger in these events. Our approach was to test different pharmacological compounds that are known to activate or inhibit these three elements, assuming that (1) activators of PKC, GPCRs, and calcium flux will trigger metamorphosis, and (2) inhibitors of these transduction elements will inhibit the progress of metamorphosis.
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Materials and Methods
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Culture of larvae
Adults of the purple sea urchin Strongylocentrotus purpuratus (Stimpson, 1857) were obtained from the coastal zone of Todos Santos Bay, Baja California, México, immediately transported to the laboratory, and maintained in tanks with flowing seawater until used.
Eggs and sperm were obtained with the method described by Hinegardner (1969), which consisted of an injection of 1 ml of 0.53 M KCl through the peristomial membrane. The organisms were placed individually on glass containers of 100 ml, with the oral hemisphere facing upwards. The containers were previously filled with seawater that had been passed through a 1-µm filter and irradiated with ultraviolet light (FSW). Gametes released by the animals were collected in these containers, and fertilization was completed in vitro using the method described by Strathmann (1987).
Fertilized eggs were placed in 18-1 plastic buckets of with FSW without aeration for 48 to 72 h, the time at which most of the embryos had reached the prism stage. Soon after, they were transferred to 45-1 conical tanks that were filled with FSW at an initial concentration of 2 larvae/ml. The temperature during the culture period was 15–17 °C. The larvae were fed with Rhodomonas spp., and constant aeration was provided. After 26–28 days, competence was determined by the presence of the adult rudiment, which in this stage occupies the left half of the larval body. In competent larvae, the tube feet were clearly visible on the rudiment, and the larvae tended to be near the bottom of the containers (Burke, 1980).
Assay methods
Once the larvae reached competence, they were induced to metamorphosis in the presence of compounds that stimulated or inhibited GPCRs and PKC, or affected calcium flux (see Table 1).
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Table 1 Drugs used to test possible signal transduction pathways during metamorphosis of the sea urchin Strongylocentrotus purpuratus
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We selected 20 to 30 competent larvae that were 26–29 days post-fertilization, on the basis of the size of the adult rudiment and the clearly visible tube feet. The selected larvae were placed in covered multiwell dishes (Falcon 1006) that had previously been prepared with a known volume of FSW and the desired concentration of the drug to be tested. This procedure was followed for each drug, with three replicates for each concentration. Each experiment was repeated twice. In all the experiments, a positive control for metamorphic competence was included; this consisted of FSW with the addition of KCl to a final concentration of 0.1 M. Larvae were allowed to remain in the increased K+ solution for 15 min, after which the solution was replaced with fresh FSW according the method described by Carpizo-Ituarte et al. (2002). The negative control consisted of larvae that remained in FSW during the experimental period. Here we present the results of only one experiment for each drug. The criteria for selecting the results to be included were the best response of the larvae to KCl as a positive control and as a measurement of competence, and the lowest values of spontaneous metamorphosis in FSW as a negative control. However, the results in all the replicate experiments showed the same pattern and statistical differences.
In treatments in which some solvent was used to dissolve the chemical to be tested (e.g., DMSO), an equivalent treatment with the amount of solvent used was included in the experimental design as a solvent control.
In treatments where an inhibiting effect of the drug was expected, the corresponding inhibitor was added 15 min before adding KCl. After this period, KCl was added in the same way as for the positive control. Once the water containing KCl and inhibitor was removed, the inhibitor was added again so that the larvae remained in its presence during the rest of the experimental period.
A series of parallel experiments were performed to test the interaction between G-protein inhibitors (suramin and guanosine) and a bacterial biofilm. In previous experiments carried out in our laboratory, biofilms have been shown to be effective natural inducers of metamorphosis in competent larvae of S. purpuratus. To generate the bacterial films, the same multiwell dishes (Falcon 1006) were placed in a tank containing only adults of purple sea urchins. The dishes remained in the tank for 10 to 12 days. After this period, the dishes were washed with FSW, and 5 ml of fresh FSW was added along with the desired concentration of the inhibitor to be tested (suramin or guanosine). Finally, 20 to 30 competent larvae were added. A positive control to test metamorphic competence was set up in a separate multiwell dish with bacterial film.
To determine whether the bacterial film degraded the G-protein inhibitor, a final experiment was carried out in which 100 µM suramin was incubated for 24 h in the presence and absence of the biofilm before the larvae were introduced. After the solution of suramin was incubated, the medium was used in new multiwell dishes to test its activity with KCl as inducer. A positive control with KCl (0.1 M, 15 min) was included to test for competence, and a negative control for spontaneous metamorphosis consisted of larvae that remained in FSW during the experimental period.
The progress of metamorphosis was examined 24 h after the initial addition of the different compounds tested, with the aid of a stereoscope microscope (Stemi 2000, C. Zeiss). In some experiments (phorbol-12-myristate-13-acetate [TPA], suramin, guanosine), the progress of metamorphosis was recorded at least three times between 6 and 24 h after the initial induction. Larvae were considered metamorphosed if the juvenile, characterized by the presence of the spines and the tube feet, had emerged and the larval epithelium had collapsed and degraded. Postlarvae at this stage were crawling on the bottom of the dish by using their tube feet. Dead larvae or postlarvae were distinguished from live ones by morphological and pigment characteristics. Larvae that were alive at the end of the experiment had actively beating ciliated bands and were swimming. Live postlarvae showed active movements of their primary podia and spines and had visible reddish and greenish pigmentation. Dead juveniles were brown, and their tissues were contracted.
All the chemicals used in the experiments were obtained from Calbiochem. Stock solutions of suramin, guanosine, mastoparan, guanylyl, caffeine, and TMB-8 hydrochloride were prepared in distilled water; K252a, bisindolylmaleimide I, TPA, indolactam V, 1,2 didecanoyl-rac-glycerol, nifedipine, ionophore A23187 free acid, cyclopiazonic acid, and thapsigargin were dissolved in DMSO. Bay K 8644 was dissolved in 100% EtOH.
Statistical analysis
The results are expressed as the number of metamorphosed larvae divided by the total number of inspected individuals. Statistical differences were tested with one-way ANOVA (
= 0.05) or, where homogeneity of variances did not exist, a Kruskal-Wallis ranking test. Multiple pairwise comparisons employed Tukey or Dunn tests when significant differences were initially detected among treatments using ANOVA. All statistical analysis was done using Sigma Stat software, ver. 3.1 (Jandel Scientifics).
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Results
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We tested 16 chemicals that interact with specific signal-transduction pathways. Only six were effective as promoters or inhibitors of metamorphosis (Table 1). The rest of the compounds had no effect or were toxic to the larvae.
Activators of protein kinase C
TPA (phorbol-12-myristate-13-acetate) was the most effective inducer of metamorphosis in the larvae of Strongylocentrotus purpuratus (Fig. 1). At 8 h of exposure to the chemical, the percentage of metamorphosis was highest, 84%, in 0.1 µM, and decreased with lower concentrations to 12% in 0.00001 µM. At 16 and 24 h after the initial induction, the percentage of metamorphosis was close to 100% in the highest concentrations (1 to 0.001 µM) and reached between 20% and 51% in the remaining concentrations (0.0001 and 0.00001 µM). The percentages of metamorphosis obtained in the treatments with TPA at 8 h were significantly different than in the negative control, except at 0.00001 µM (Tukey, P < 0.05); also, concentrations between 0.01 µM and 0.00001 µM were significantly different among themselves (Tukey, P < 0.05) (Fig. 1A). At 16 and 24 h the response leveled out at higher doses (between 1 µM and 0.001 µM), and there were no significant differences between them, including the positive control with KCl (Tukey, P > 0.05) (Fig. 1B, C). At these same time intervals, the results at 0.0001 and 0.00001 µM were not significantly different (Tukey, P > 0.05), but they were significantly different from the rest of the treatments. Mortality in the experiments with TPA was no more than 8% in all treatments, and the DMSO used to dissolve the TPA had no effect on metamorphosis of the larvae. Similar percentages of metamorphosis were obtained in an independent experiment with TPA at 24 h after induction (data not shown).
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Figure 1. Percentage of metamorphosis (± 1 SE) of the sea urchin Strongylocentrotus purpuratus after 8 (A), 16 (B), and 24 h (C) of exposure to different concentrations of TPA as inducer (n = 3). Bars indicate the standard error. Asterisks on top of columns (*) indicate statistical differences (P < 0.05) in treatments compared to negative control (FSW). Treatments include TPA concentrations as indicated on the x axis; KCl, 0.1 M (15 min, then FSW); FSW, filtered and sterilized seawater.
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Indolactam was a less effective inducer of metamorphosis than TPA (Fig. 2). At 24 h, the highest percentage of metamorphosis (84%) was obtained with the lowest concentration tested (0.001 µM). There was no significant difference between the inductive concentrations of indolactam (0.1, 0.01, and 0.001 µM) and the control with KCl (Tukey, P > 0.05), but these concentrations were significantly different with respect to the negative control (FSW) and the control with DMSO (Tukey, P < 0.05). Indolactam was toxic to the larvae in concentrations higher than 0.1 µM (data not shown).
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Figure 2. Percentage of metamorphosis (± 1 SE) of the sea urchin Strongylocentrotus purpuratus exposed to different concentrations of indolactam as inducer (n = 3). Bars indicate the standard error. Asterisks on top of columns (*) indicate statistical differences (P < 0.05) in treatments compared to negative controls (DMSO, FSW). Treatments include indolactam concentrations as indicated on the x axis; KCl, 0.1 M (15 min, then FSW); FSW, filtered and sterilized seawater; DMSO, concentration of DMSO used in the preparation of indolactam solutions.
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Inhibitors of G proteins
Suramin inhibited metamorphosis in the presence of KCl as inducer at 24 h (Fig. 3). Inhibition was effective at suramin concentrations of 10, 50, and 100 µM, and percentages (72%, 58%, and 64% respectively) of metamorphosis were significantly different from those obtained in 1 µM (92%) and the positive control with KCl (95%) (Tukey, P < 0.05). The effect of suramin in the presence of a biofilm inducer was also tested. At 6 h after initial contact with the natural inducer and the chemical tested, the percentages of metamorphosis obtained with 50 and 100 µM suramin were significantly lower (Tukey, P < 0.05) than the ones obtained with the positive control with biofilm alone (Fig. 4A). There was no statistical difference (Tukey, P > 0.05) between these last two treatments or in comparison with the negative control with FSW (Fig. 4A). At 12 h after induction, the percentage of metamorphosis was close to 35% in the highest concentration (100 µM), but close to 60% in the lowest concentration (50 µM) (Fig. 4B). These two concentrations were significantly different from one another (Tukey, P < 0.05) and from both the positive control (with biofilm) and the negative control (with FSW) (Fig. 4B). In the same experiment, after 24 h of contact with the chemical and the biofilm, 100 and 50 µM suramin were again significantly different from one another and from the biofilm and FSW controls (Tukey, P < 0.05); in contrast, the 100 µM suramin treatment showed higher percentages of metamorphosis (close to 80%) than the treatment with 50 µM, where percentages of metamorphosis were close to 60%.
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Figure 3. Percentage of metamorphosis (± 1 SE) of the sea urchin Strongylocentrotus purpuratus exposed to different concentrations of suramin as inhibitor (n = 3). Larvae were exposed to the different concentrations of suramin for 15 min before being induced to metamorphosis with KCl in the presence of the inhibitor. After 15 min the solution was removed and replaced with a fresh solution of suramin in FSW at the concentrations indicated in the figure. Bars indicate the standard error. Asterisks on top of columns (*) indicate statistical differences (P < 0.05) in treatments compared to positive control (KCl). Treatments include suramin concentrations as indicated on the x axis; KCl, 0.1 M (15 min, then FSW); FSW, filtered and sterilized seawater.
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Figure 4. Percentage of metamorphosis (± 1 SE) of the sea urchin Strongylocentrotus purpuratus after 6 (A), 12 (B), and 24 h (C) of exposure to different concentrations of suramin as inhibitor (n = 3), and a bacterial film as inducer. Bars indicate the standard error. Asterisks on top of columns (*) indicate statistical differences (P < 0.05) in treatments compared to positive control (Biofilm). Treatments include suramin concentrations as indicated on the x axis; Biofilm, 10–12 days of bacterial film developed in a tank with adults; FSW, filtered and sterilized seawater.
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It is possible that the biofilms degraded the suramin, thus reducing the inhibitory effect at the 24-h assay point (Fig. 4C). To test this possibility, we preincubated suramin in the presence of a biofilm and found that the percentage of metamorphosis was highest (57%) when the inhibitor used was previously incubated for 24 h, and lowest of (34%) when the inhibitor was not preincubated (Fig. 5). The percentages of metamorphosis in all treatments with suramin were significantly different from those in the control with KCl (Newman-Keuls, P < 0.05), except where the inhibitor used was previously incubated for 24 h in a biofilm (P > 0.05). Mortality in the treatments with suramin was no more than 8% in all the experiments where suramin had an inhibitory effect.
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Figure 5. Percentage of metamorphosis (± 1 SE) of the sea urchin Strongylocentrotus purpuratus after 24 h of exposure to suramin as inhibitor at concentration of 100 µM (n = 3), and KCl as inducer. Bars indicate the standard error. Asterisks on top of columns (*) indicate statistical differences (P < 0.05) in treatments compared to positive control (KCl). Treatments include Biofilm + 24 h, suramin incubated 24 h in a bacterial film; 24 h, suramin incubated 24 h without bacterial film; 0 h, suramin without any incubation; KCl, 0.1 M (15 min, then FSW); FSW, filtered and sterilized seawater.
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Guanosine had a marked effect on the metamorphosis of S. purpuratus (Fig. 6). At 6 and 12 h after the initial induction with KCl, the inhibitory effect was evident at all concentrations tested (10 to 0.001 µM). The percentages of metamorphosis obtained at these concentrations were significantly lower than those obtained with KCl (Tukey, P < 0.05). The percentages of metamorphosis at 24 h were significantly different from those in the control with KCl only at 10 µM (Tukey, P < 0.05). No spontaneous metamorphosis was observed in the treatment with FSW, and mortality in the treatments with guanosine was never higher than 7%.
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Figure 6. Percentage of metamorphosis (± 1 SE) of the sea urchin Strongylocentrotus purpuratus after 6 (A), 12 (B), and 24 h (C) of exposure to different concentrations of guanosin as inhibitor (n = 3), and KCl as inducer. Larvae were exposed to the different concentrations of guanosine for 15 min before being induced to metamorphosis with KCl in the presence of the inhibitor. After 15 min the solution was removed and replaced with a fresh solution of guanosine in FSW at the concentrations indicated in the figure. Bars indicate the standard error. Asterisks on top of columns (*) indicate statistical differences (P < 0.05) in treatments compared to positive control (KCl). Treatments include guanosine concentrations as indicated on the x axis; KCl, 0.1 M (15 min, then FSW); FSW filtered and sterilized seawater.
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In the experiment where we tested the effect of guanosine in the presence of a biofilm as inducer, the percentage of metamorphosis was lowest (4%) in the 100-µM treatment after 8 h of induction, and increased with the decreasing concentration of the inhibitor in the remaining treatments. In all cases, the percentages of metamorphosis were significantly different from those in the controls with biofilm and KCl (Newman Keuls, P < 0.05) (Fig. 7). At 16 h, the percentage of metamorphosis (43%) was lowest in the highest concentration tested (100 µM), and it was different than in biofilm and KCl (Tukey, P < 0.05). The remaining concentration apparently had no inhibitory effect on the larvae, and the percentages of metamorphosis were higher than in the positive controls. After 24 h of induction, a nonselective mortality event was observed in this experiment in all treatments, including the controls.
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Figure 7. Percentage of metamorphosis (± 1 SE) of the sea urchin Strongylocentrotus purpuratus after 8 (A) and 16 (B) h of exposure to different concentrations of guanosine as inhibitor (n = 3), and a bacterial film as inducer. Bars indicate the standard error. Asterisks on top of columns (*) indicate statistical differences (P < 0.05) in treatments compared to positive control (Biofilm). Treatments include guanosine concentrations as indicated on the x axis; Biofilm, 10–12 days of bacterial film developed in a tank with adults; KCl, 0.1 M (15 min, then FSW); FSW, filtered and sterilized seawater.
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Calcium flux
Ionophore A23187. The results obtained with this compound support the theory that calcium has a role in sea urchin metamorphosis (Fig. 8). The highest percentages of metamorphosis were obtained in the 5- and 10-µM concentrations of the ionophore (48% and 49%, respectively), in which metamorphosis was significantly greater than that obtained with FSW (Tukey, P < 0.05). The percentage of metamorphosis obtained in the lower concentrations of A23187 tested (0.1 and 1 µM) were not significantly different from that obtained with FSW (Tukey, P > 0.05). However, metamorphosis in all of the concentrations of A23187 was significantly lower than obtained with KCl (Tukey, P < 0.05). Mortality never exceeded 12% in any treatment (data not shown).
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Figure 8. Percentage of metamorphosis (± 1 SE) of the sea urchin Strongylocentrotus purpuratus exposed to different concentrations of ionophore A23187 as inducer (n = 3). Bars indicate the standard error. Asterisks on top of columns (*) indicate statistical differences (P < 0.05) in treatments compared to negative control (FSW). Treatments include ionophore A23187 concentrations as indicated on the x axis; KCl, 0.1 M (15 min, then FSW); FSW, filtered and sterilized seawater. The highest amount of DMSO (50 µl) present in the treatments had no effect on the metamorphosis of the larvae.
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Thapsigargin was a more effective inducer of metamorphosis than ionophore A23187 (Fig. 9). At concentrations of 50 and 100 µM, thapsigargin induced levels of metamorphosis significantly higher than those obtained with KCl (Tukey, P < 0.05) and FSW (Tukey, P < 0.05). There was no significant difference between the treatments of 1 and 10 µM thapsigargin and the control with FSW (Tukey, P > 0.05). The control in which DMSO was used to dissolve thapsigargin had no effect on the metamorphosis of the larvae (data not shown).
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Figure 9. Percentage of metamorphosis (± 1 SE) of the sea urchin Strongylocentrotus purpuratus exposed to different concentrations of thapsigargin as inducer (n = 3). Bars indicate the standard error. Asterisks on top of columns (*) indicate statistical differences (P < 0.05) in treatments compared to negative control (FSW). Treatments include thapsigargin concentrations as indicated on the x axis; KCl, 0.1 M (15 min, then FSW); FSW, filtered and sterilized seawater. The highest amount of DMSO (50 µl) present in the treatments had no effect on the metamorphosis of the larvae.
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Discussion
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The results obtained in the present work indicate that the inositol trisphosphate (IP3)-diacylglycerol (DAG) system, G-protein-coupled receptors (GPCRs), and protein kinase C (PKC) all participate in induction of metamorphosis in the sea urchin Strongylocentrotus purpuratus. Calcium may serve as a second messenger in the transduction pathways associated with metamorphosis; however, this assumption is based on the effectiveness of only 6 of the 16 compounds tested. The external exposure of whole larvae to chemicals that affect signal-transduction pathways is an experimental limitation to elucidating the level at which the compounds may be acting. In such experiments, an absence of action of the drug tested does not necessarily mean that the signal-transduction target is not involved: we are assuming that the pharmacological compounds tested interact with the signal-transduction pathways in a way similar to their action on isolated cells of other species, but some of the compounds probably behave differently when acting in whole larvae, and their permeability could be different when tested in invertebrate cells. Future studies with isolated larval tissues or focused on regions of the larva presumed to be involved in triggering metamorphosis will help to clarify this issue.
Our results agree with those obtained for some other species of marine invertebrates where similar compounds were used to elucidate signal-transduction pathways during metamorphosis (see Freeman and Ridgway, 1990; Fleck and Bischoff, 1992; Clare, 1996). Baxter and Morse (1987) showed the existence of lysine receptors coupled to G proteins; these receptors possibly facilitate the response to a GABA receptor that stimulates metamorphosis in the larvae of red abalone Haliotis rufescens. At least one G protein that is highly similar to members of the Gq family that control the activity of phospholipase C is expressed in larval cilia in the abalone (Wodicka and Morse, 1991). GPCRs have been also indicated as being part of the signal transduction pathway for metamorphosis of the barnacle Balanus amphitrite (Clare, 1996) and the hydrozoan Hydractinia (Schneider and Leitz, 1994).
Activation of PKC during metamorphosis has also been implicated for different invertebrate larvae using phorbol esters, which are known to mimic the action of DAG during the cascade of events that activate PKC (Nishizuka, 1984). Phorbol esters have been documented as effective inducers for different species of cnidarians (see Siefker et al., 2000; Frank et al., 2001) and barnacles (Yamamoto et al., 1995). In the present study, the phorbol ester TPA was the most effective inducer of metamorphosis of larvae of sea urchins. Freeman and Ridgway (1990) reported similar results with equivalent concentrations of TPA in the hydrozoan Mitrocomella polydiademata. These same authors showed a decrease in induction to metamorphosis of M. polydiademata with concentrations of 10–7 to 10–9 M TPA. In the present study, TPA was less effective at concentrations lower than 10–9 M after 8 h of initial exposure. This result with TPA is reinforced by the inductive effect of indolactam, another activator of PKC. The counterintuitive response to indolactam, where the larvae metamorphosed higher proportions in the presence of lower concentrations of the compound, could be explained by a biphasic response of the receptors to this compound, where low concentrations potentiate the action and high concentrations become inhibitory. This type of response is known to happen in receptors that modulate the internal calcium concentration in mammalian cells (see Hajnóczky and Thomas, 1994; Clapham, 1995).
When KCl was used as the inducer, only the inhibitors of G proteins—guanosine and suramin—had an effect on metamorphosis, apparently at a site downstream of the K+ induction site. It is not known how K+ activates metamorphosis, but recent evidence by Hadfield et al. (2000) points to its action downstream of the primary sensory cells in Phestilla sibogae. However, even in this species, the locus of K+ action on metamorphosis has not been clarified.
Except for the first 6 h when guanosine shows an apparent dose response (see Fig. 6A), it appears that this compound is only delaying metamorphosis. This result could be explained by a low affinity of the compound for the corresponding G-protein motif.
On the other hand, when we compared the inductive response of S. purpuratus to a bacterial biofilm in the presence of the G-protein inhibitors suramin and guanosine, we observed an inhibitory response similar to that seen in the presence of KCl at 6 and 12 h after induction. These results open the possibility that KCl and the natural inducer contained in a biofilm activate the same transduction pathways and include G-protein-coupled receptors.
At 24 h of inhibition, the effectiveness of suramin as an inhibitor apparently diminishes. A possible explanation for this result is that bacteria degraded the compound faster than it could act in the larvae. Experiments where suramin was incubated for 24 h in the presence of a bacterial film before being put in contact with the larvae support this explanation. Guanosine may have a pattern of degradation similar to that observed with suramin, but we did not test this possibility.
The effect of G-protein inhibitors on metamorphosis during the longest periods tested is intriguing, because of their apparent potentiation of metamorphosis at the highest concentrations. One possible explanation for this effect is that a by-product of the degradation of the inhibitor by the bacteria in the biofilm enhances the metamorphic response.
With respect to the role of calcium in metamorphic signal transduction, our results confirm previous reports about the inductive effect of the calcium ionophore A23187 in the purple sea urchin S. purpuratus (Pearse and Cameron, 1991). In the present study, the calcium ionophore A23187 and an inhibitor of the calcium ATPase (thapsigargin) were effective in triggering metamorphosis. Because the ionophore transports calcium to the interior of the cell without the participation of any membrane receptor, it is possible that calcium acts as an inducer of metamorphosis by itself. On the other hand, thapsigargin, by inhibiting the calcium ATPase of the endoplasmic reticulum, allows calcium to accumulate in the cytoplasm and act as if it had increased inside the cell by other means. Although our study did not test excess calcium as an inducer, its effectiveness has been demonstrated in other species of marine invertebrates. In Balanus amphitrite, calcium concentrations 5 mM above those of normal seawater activate metamorphosis, whereas concentrations 10 mM above the normal levels inhibit metamorphosis (Clare, 1996). On the other hand, reducing the concentration of calcium in seawater inhibits metamorphosis in the hydroid Tubularia mesembryanthemum (Kawaii et al., 1999). In larvae of the sea urchin Lytechinus variegatus, excess calcium in seawater stimulates metamorphosis, and the number of metamorphosed larvae tends to decrease as calcium concentration increases (Cameron et al., 1989).
The effectiveness of calcium as a second messenger during the metamorphosis of marine invertebrates has been documented for several species (see Ilan et al., 1993; Clare et al., 1995; Clare, 1996; McCauley, 1997; Kawaii et al., 1999). In addition, the role of calcium could be related to the transduction pathways in which GPCRs and PKC participate. The cascade of events generated by the activation of the IP3/DAG system triggers calcium release from the endoplasmic reticulum and participates in the activation of PKCs (, ßI, ßII, and ) (Clapham, 1995; Cullen, 2003). This could be the case during metamorphosis of S. purpuratus, where, according to our results, these three elements, GPCRs, PKC, and calcium, have roles in signal transduction.
The model we propose for activation of metamorphosis in sea urchins via the transduction pathways referred to above is based in the phosphoinositol cycle (Nishizuka, 1984) and includes a unique route where activation of G-protein-coupled receptors stimulates release of phospholipase C (PLC). Once PLC is activated, it cleaves phosphatidyl-inositol-4,5-biphosphate (PIP2) into inositol-1,4,5-tris-phosphate (IP3) and diacylglycerol (DAG). DAG also is known to activate PKC (see Leitz and Klingmann, 1990). A second possibility is that GPCRs and PKC are activated independently during sea urchin metamorphosis, and each alone is sufficient to trigger metamorphosis. In both situations, calcium would be acting as a second messenger. Figure 10 illustrates a hypothetical model in which the signal-transduction pathways mentioned above interact and shows the target sites for the pharmacological agents we employed. In this model, we consider that GPCRs, PKC, and Ca2+ act together to modulate metamorphosis. Ca2+ and PKC are enough to trigger metamorphosis, and GPCRs act through an inhibitory mechanism, preventing metamorphosis.
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Figure 10. Hypothetical model for signal-transduction pathway during metamorphosis of Strongylocentrotus purpuratus. The model is based in the phosphoinositol cycle of Nishizuka (1984) and considers a unique route where activation of G-protein-coupled receptors stimulates release of phospholipase C (PLC). Once PLC is activated, it cleaves phosphatidyl-inositol-4,5-biphosphate (PIP2) into inositol-1,4,5-tris-phosphate (IP3) and diacylglycerol (DAG). This last element will activate protein kinase C (PKC), while IP3 activates membrane receptors in the endoplasmic reticulum that releases calcium. Calcium will contribute to activating PKC. Calcium from another source, the extracellular space, can be introduced to the cytosol via calcium channels. The diagram also shows the target sites of the pharmacological agents that had an effect on metamorphosis. MR, membrane receptor; GP, G protein; SERCA pump, smooth endoplasmic reticulum calcium ATPase.
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Recent studies by Bishop and Brandhorst (2001) on the sea urchin Lytechinus pictus indicate the participation of the NO/cGMP system as a negative regulator during metamorphosis. These authors propose that the generation of nitric oxide inhibits metamorphosis in competent larvae of the sea urchin, with the participation of the molecular chaperone HSP90. Considering that a similar mechanism could be operating in S. purpuratus, two possibilities exist to couple the routes studied in the present study with the NO/cGMP system: (1) the cascade of events that initiates the activation of GPCRs or PKC is located upstream of the NO system, and its activation ends with the cessation of production of NO by inhibition of specific genes or production of a protein that inhibits NOS or HSP90; and (2) the signal caused by the cascade of events initiated by GPCRs or PKC activation is more potent than the inhibitory effect caused by the NO system. Recently, activation of a specific type of PKC in human monocytic cells has been implicated in the regulation of iNOS (Pham et al., 2003).
Our results indicate that GPCRs, PKC, and calcium are components of the signal-transduction pathways that activate the metamorphosis of larvae of S. purpuratus. The next step is to find where and when in the larva these compounds are expressed, and how are they regulated during metamorphosis. Sequencing data from the sea urchin genome project (Cameron et al., 2000) will be of great help in elucidating the molecular regulation of metamorphosis in the larvae of sea urchins. Having sequence data for genes that could participate in the metamorphosis of sea urchins opens the possibility of documenting expression patterns of genes that are presumably regulated by the transduction pathways suggested here.
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Acknowledgments
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The authors acknowledge A. Hernández for his help in the culture of the larvae, and people in the Laboratorio de Biología del Desarrollo-IIO-UABC for their support. We also thank Dr. Michael G. Hadfield for his comments, and two anonymous reviewers whose observations helped increase the clarity of this paper. This research was supported by IIO funds (grant 4044) and UABC grants No. 4092 and 0555 to EC-I. GA-C is the recipient of a scholarship from graduate studies from CONACyT, México.
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Footnotes
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Received 30 July 2004; accepted 13 December 2005.
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Abstract
Introduction
