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Settlement and metamorphosis of Capitella larvae induced by juvenile hormone-active compounds is mediated by protein kinase C and ion channels.

Introduction

The chemoreception by marine invertebrate larvae of chemical "cues" that are present in the ocean environment and induce settlement and metamorphosis is important for the recognition of habitats that favor growth and reproduction (Chia and Rice, 1978; Rittschof and Bonaventura, 1986; Scheuer, 1990). These settlement signals appear to be specific for different species, as evidenced by findings that larvae of the abalone Haliotis rufescens respond to specific chemicals in red algae (Morse et al., 1984), larvae of the nudibranch Phestilla sibogae respond to chemicals in corals (Hadfield, 1978, 1984), larvae of the polychaete annelid Phragmatopoma californica respond to chemicals present in the burrows of adult worms (Pawlik, 1988, 1990; Jensen and Morse, 1990), and larvae of the sand dollars Dendraster excentricus (Burke, 1984) and Echinarachnius parma (Pearce and Scheibling, 1990) respond to chemicals produced by adult sand dollars.

In previous studies, we have found that juvenile hormones (JH), which are known morphogens that regulate reproduction and development of insects and crustaceans (Laufer and Borst, 1983, 1988; Laufer et al., 1987), as well as chemicals with juvenile hormone activity in insect cuticle bioassays, are able to induce settlement and metamorphosis of metatrochophore larvae of the polychaete annelid Capitella sp. I (Biggers and Laufer, 1992, 1996), which is a subspecies member of the Capitella polychaete complex (Grassle and Grassle, 1976). In nature, larvae of Capitella sp. I are stimulated to settle and metamorphose [ILLUSTRATION FOR FIGURE 1 OMITTED] when they come into contact with chemical inducers present in sediments (Butman et al., 1988), although the identity of these chemicals remains in debate (Cuomo, 1985; Dubilier, 1987), they appear to have JH-activity (Biggers, 1994).

We have now investigated the signal transduction process through which the Capitella larvae respond to JH-active compounds. Our results presented in this paper indicate that JH-active compounds stimulate settlement and metamorphosis of these larvae through the activation of protein kinase C (PKC) and subsequent modulation of ion channels.

Materials and Methods

Capitella larval settlement bioassays

Stock cultures of Capitella sp. I were maintained at 18 [degrees] C in large plastic containers containing artificial seawater (Utikem Co.) and washed sea sand (Fisher Scientific) and were fed Tetramin fish food flakes. Brood tubes containing adult females along with their developing eggs and larvae were then separated from the cultures and placed into 60-mm glass petri dishes containing seawater. The dishes were checked daily for hatched, swimming metatrochophore larvae to be used for bioassays. All test chemicals, except for methyl farnesoate (MF) which was synthesized in our laboratory, were purchased from Sigma Chem. Co. Stock solutions of juvenile hormone III (JH III), MF, phorbol- 12,13 dibutyrate (PDBU), 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7), arachidonic acid, elaidic acid, verapamil, 4-aminopyridine, and nigericin were prepared in 95% ethanol. Stock solutions of KCl, Ni[Cl.sub.2], Zn[Cl.sub.2], and tetraethylammonium chloride (TEA) were prepared in distilled water. Settlement and metamorphosis bioassays were conducted at 18 [degrees] C using 60-mm glass petri dishes that were pre-baked at 250 [degrees] C to remove contaminants (Biggers and Laufer, 1996). For the assays, 10 to 100 [[micro]liter] of the test chemical stock solutions were added by micropipet into petri dishes containing 10 metatrochophore larvae less than 1 day old (1 day post-release), and 10 ml of artificial seawater. The dishes were then observed. After 1 h, the amount of settlement and metamorphosis was assessed by placing each dish under a dissecting microscope and counting the number of settled larvae crawling on the bottom of the dish. Metamorphosis after 1 hour was also more critically assessed by using a compound microscope and noting the loss of cilia, elongation, and development of capillary setae.

Protein kinase C assays

Assays for the presence of protein kinase C were carried out essentially as described by Yasuda et al. (1990), by measuring phosphorylation of an 11-residue synthetic peptide from myelin basic protein ([MBP.sub.4-14]). This method is specific for measurement of PKC, and permits selective measurement in crude tissue preparations. The PKC assay was first standardized using purified rat brain PKC (calcium and phospholipid dependent) from Calbiochem Co. Phosphorylation of [MBP.sub.4-14] was measured using 2-10 ng of enzyme, and over a time period of 30 min. Reactions were carried out in plastic Eppendorf tubes, with 50-[[micro]liter] reaction mixtures containing 20 mM Tris/HCl, pH 7.5, 5 mM magnesium acetate, 100 [[micro]molar] Ca[Cl.sub.2], 25 [[micro]molar] [MBP.sub.4-14] (Sigma Chem. Co.), 50 ng diolein and 500 ng phosphatidylserine, 2-10 ng of rat brain PKC, and 10 [[micro]molar] ATP (containing 5-6 x [10.sup.5] CPM gamma 32P-ATP). Reactions were started by addition of enzyme, and incubations were carried out at 30 [degrees] C for up to 30 min. The reactions were then stopped by placing the tubes on ice and spotting the reaction mixtures onto P-81 anion exchange paper discs (Whatman Co.), which were then immediately immersed in 75 mM [H.sub.3]P[O.sub.4], and washed eight times in 100 ml of the same solution. The filter discs were placed in Ecolume cocktail for analysis with a scintillation counter. Other PKC assays using rat brain PKC were carried out in the same manner, except with the replacement of diolein and phosphatidylserine with test chemicals as indicated.

PKC activity in Capitella larval homogenates was determined in the same manner, except with the inclusion of the larval homogenates (20-100 [[micro]gram] protein) instead of the rat brain PKC. Capitella larvae were collected within 1 day of release from the brood tubes, and were frozen at -20 [degrees] C. After thawing, the larvae were centrifuged at 5000 rpm for 1 min in a microfuge, the seawater was discarded, and larval homogenates were prepared by homogenizing 1000 larvae in 1 ml 20 mM Tris/HCL, pH 7.5, containing 0.5 mM phenylmethylsulfonyl fluoride (protease inhibitor) using small glass homogenizers. The homogenates were then centrifuged in a microfuge at 5000 rpm for 1 min to remove large cellular debris, and the PKC activity of the supernatant was assessed as previously described for studies with rat brain PKC. Protein concentrations of supernatants were determined using a microprotein assay (Sigma Chem. Co.) and a standard curve of increasing concentrations of bovine serum albumin.

Localization of PKC by RIM-1 analysis

Rhodamine-conjugated bisindolylmaleimide (RIM-1) (Calbiochem), a fluorescent PKC inhibitor (Chen and Poenie, 1993), was used to visually locate PKC in Capitella larvae and juveniles. Metatrochophore larvae less than 1 day old and 1-day-old juveniles were briefly exposed for 1 min at room temperature to 200 nm RIM-1 in seawater (100[[micro]liter]) in depression slides covered with aluminum foil. Larvae or juveniles were then fixed in 2% formaldehyde containing seawater for 15 min, transferred into methanol for 15 min to permeabilize the membranes, and then transferred three times (5 min each wash) into 1 ml fresh seawater to rinse away excess unbound RIM-1. Fixed larvae or juveniles were then transferred in 10% glycerol-seawater onto microscope slides and visualized by either light microscopy or fluorescent microscopy with a Nikon confocal fluorescent microscope equipped with a rhodamine filter.

Results

Effects of protein kinase C modulators on larval settlement and metamorphosis

We have previously found that the juvenile hormones MF, JH I, and JH III, as well as compounds with juvenile hormone activity in insect cuticle bioassays, including arachidonic acid, are able to induce settlement and metamorphosis of Capitella sp. I metatrochophore larvae (Biggers and Laufer, 1992).

The first indication that the larvae sense the presence of JH III in the seawater is the onset of excited swimming. This response, which is typical of normal settlement behavior (Butman et al., 1988; Pechenik and Cerulli, 1991), is faster than normal, with intermittent spiral-corkscrew movements, gradual body elongation, and periodic touching of the bottom of the petri dish. The larvae then settle and metamorphose into normal juvenile worms within 1 h. Both MF and JH III are very potent inducers of settlement and metamorphosis of the Capitella larvae at micromolar concentrations, whereas control larvae do not start to spontaneously settle and metamorphose until after 24 h [ILLUSTRATION FOR FIGURE 2 OMITTED].

In preliminary investigations of the signal transduction process that mediates JH-induced settlement and metamorphosis of the Capitella larvae, we found compounds that elevate intracellular cAMP concentrations to be ineffective inducers of settlement and metamorphosis, indicating that cAMP does not act as a second messenger in this signal transduction process (Biggers and Laufer, 1992).

Based upon the ability of JH I, JH III, and arachidonic acid to activate PKC in other species including insects (Yamamoto et al., 1988; Holian et al., 1989; Sevala and Davey, 1989; Shearman et al., 1989a; Shinomura et al., 1991), and on reports that PKC activation is involved in the mediation of settlement and metamorphosis of other species of marine invertebrate larvae (Muller, 1985; Baxter and Morse, 1987; Leitz and Klingman, 1990; Morse, 1990), we further investigated the involvement of PKC activation in mediating settlement and metamorphosis of the Capitella larvae by testing the effects of other known PKC modulators. The phorbol ester phorbol-12,13-dibutyrate (PDBU) is a well-studied activator of PKC, and PKC has been found to be the actual cellular receptor for PDBU in some cells (Castagna et al., 1982; Vandenbark et al., 1984). Experiments with PDBU on the Capitella larvae showed that it is also a very potent inducer of settlement and metamorphosis [ILLUSTRATION FOR FIGURE 3A OMITTED], indicating that PKC activation is involved in mediating this process.

The effect of a PKC inhibitor was next studied to determine if it could inhibit settlement and metamorphosis induced by JH. The PKC inhibitor 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine, abbreviated H-7 (Hidaka et al., 1984), caused a concentration-dependent inhibition of Capitella settlement and metamorphosis induced by 25 [[micro]molar] JH III, when the larvae were pre-exposed to H-7 for 3 h [ILLUSTRATION FOR FIGURE 3B OMITTED]. This result again indicates the involvement of PKC activation in mediating the effects of JH on settlement and metamorphosis.

Activation of a protein kinase C-like enzyme in Capitella larvae by JH-active chemicals

Although PKC is considered to be a ubiquitous enzyme present throughout the animal kingdom and has been found in marine sponges (Muller et al., 1987) and Dictyostelium discoideum (Jimenez et al., 1989; Luderus et al., 1989), its presence in polychaetes has so far not been documented. The presence of a PKC-like enzyme in Capitella larvae was therefore investigated, as was also the ability of JH-active compounds to activate this enzyme. In these investigations, we used an assay specific for PKC. Developed by Yasuda et al. (1990), this assay is based upon the specific phosphorylation by PKC of an 11-amino acid peptide sequence of myelin basic protein, which is not phosphorylated by either cAMP-dependent protein kinase (PKA), casein kinases I and II, [Ca.sup.2+]/calmodulin-dependent protein kinase II, or phosphorylase kinase. The results demonstrate that a PKC-like enzyme does exist in Capitella [ILLUSTRATION FOR FIGURE 4 OMITTED]. PKC activity was linear for up to 15 min for homogenate concentrations up to 100 [[micro]gram] [ILLUSTRATION FOR FIGURE 4A, B OMITTED]. The specific activity of PKC in the larval homogenates was calculated as being 6.7 fmoles 32P-incorporated per minute per microgram of protein. Incubations were also done without the PKC substrate [MBP.sub.4-14] to monitor endogenous larval protein phosphorylation, of which only a very small amount could be detected.

Experiments were next conducted to determine whether the PKC-like enzyme present in the Capitella larvae could be activated by JH-active compounds. Incubations were carried out using 100 [[micro]gram] of the larval homogenates for 15 min at 30 [degrees] C in the presence of either phosphatidylserine/diolein (PS/DO), 10 [[micro]molar] arachidonic acid (AA), 10 [[micro]molar] JH III, 10 [[micro]molar] MF, or 10 [[micro]molar] elaidic acid (EA). The results of this experiment indicate that JH III, MF, and AA are able to activate Capitella PKC in vitro [ILLUSTRATION FOR FIGURE 5A OMITTED]. In comparison with activation by PS/DO, arachidonic acid was the strongest activator (94% activation), followed by MF (73% activation) and JH III (51% activation). Elaidic acid, a transisomer of oleic acid that does not activate rat brain PKC (Shearman et al., 1989a) and does not induce settlement and metamorphosis of the Capitella larvae, did not activate the Capitella PKC. These results therefore again indicate the involvement of PKC activation in mediating settlement and metamorphosis of the Capitella larvae.

Since it is possible that the crude larval homogenates contain enzymes, receptors, and substrates of the diacylglycerol pathway, such as phospholipase C and guanine-binding proteins through which indirect activation of PKC may occur, we also tested whether JH could directly activate a purified preparation of rat brain PKC. Polyunsaturated fatty acids such as arachidonic acid can activate bovine brain PKC (Shearman et al., 1989a) and rat brain PKC (Holian et al., 1989; Shinomura et al., 1991); therefore, juvenile hormones might also be able to cross species lines and activate rat brain PKC. Incubations were carried out using 5 ng purified rat brain PKC (Calbiochem Co.) in the presence of either PS/DO, 10 [[micro]molar] AA, 10 [[micro]molar] JH III, 10 [[micro]molar] MF, or 10 [[micro]molar] EA, at 37 [degrees] C for 15 min. The results show that insect juvenile hormones and the crustacean juvenile hormone MF are able to directly activate rat brain PKC in the absence of phosphatidylserine and diolein [ILLUSTRATION FOR FIGURE 5B OMITTED]. Arachidonic acid was the most potent activator tested (93% activation relative to PS/DO), whereas elaidic acid was inactive, confirming earlier work reported by Shearman et al. (1989a). MF caused 59% activation of the rat brain PKC and was more active than JH III (28% activation).

Localization of PKC in Capitella by RIM-1

To visualize the location of PKC in the Capitella larvae and juveniles and to identify possible chemosensory cells that would rapidly take up external chemicals or chemicals in the environment, larvae and juveniles were briefly (1 min) exposed to a fluorescently labeled protein kinase C inhibitor, rhodamine-conjugated bisindolylmaleimide (RIM-1), which has proven useful as a fluorescent probe for PKC (Chen and Poenie, 1993). After exposure to this inhibitor, the larvae or juveniles were fixed, permeabilized, rinsed to remove excess unbound RIM-1, and viewed under a fluorescent microscope. In metatrochophore larvae, distinct cells in the ciliary bands of the prototroch and telotroch and isolated cells in the apical region of the prostomium and the rest of the body displayed RIM-1 binding, indicating that these cells possess PKC [ILLUSTRATION FOR FIGURE 6B OMITTED]. Juvenile Capitella exposed in the same manner displayed RIM-1 binding to cells in the apical region of the prostomium and scattered throughout the rest of the body [ILLUSTRATION FOR FIGURE 6D OMITTED]. These results provide more evidence for the presence of a PKC-like enzyme in Capitella larvae and juveniles, and are consistent with a chemosensory function for apical cells in regions of the prostomium as previously noted by Eckelbarger and Grassle (1987).

Effects of potassium channel modulators

In mediating cellular responses, PKC activation has in many cases been found to result in the modulation of potassium and calcium channels (Kaczmarek, 1987; Shearman et al., 1989b). The involvement of potassium channels in mediating the settlement and metamorphosis of several types of marine invertebrate larvae has also been previously demonstrated (Baloun and Morse, 1984; Yool et al., 1986; Leitz and Klingman, 1990; Carpizo-Ituarte and Hadfield, [TABULAR DATA FOR TABLE I OMITTED] 1998). Studies were therefore carried out to determine if the modulation of potassium channels may also mediate settlement and metamorphosis of the Capitella larvae. Increased external KCl concentrations in the seawater induced settlement and metamorphosis of the Capitella larvae in a concentration-dependent manner, with an added concentration of 20 mM (30 mM total including seawater) inducing 100% settlement and metamorphosis in 1 h (Table I). These effects of KCl appear to be mediated by [K.sup.+] ions and not [Cl.sup.-] since addition of NaCl had no effect on settlement and metamorphosis. The effect of tetraethylammonium chloride (TEA), a known blocker of potassium currents, was next studied to determine its effect on settlement and metamorphosis. TEA did not induce settlement and metamorphosis of the Capitella larvae, but instead inhibited the stimulatory response of the larvae to KCl, with a concentration of 100 mM TEA almost completely inhibiting settlement and metamorphosis (Table I). Settlement and metamorphosis of the Capitella larvae was, however, stimulated in a concentration-dependent manner by 4-aminopyridine (4-AP), another potassium channel blocker, which blocks outward rectifying potassium currents. A concentration of 100 [[micro]molar] 4-AP stimulated 100% settlement and metamorphosis of the larvae within 1 h (Table I). The effects of the potassium channel ionophore, nigericin, was next studied. Pre-exposure of the larvae to nigericin for 30 min inhibited the response of the larvae to JH III in a concentration-dependent manner, with 500 ng/ml nigericin causing 100% inhibition of settlement and metamorphosis in response to JH III in 1 h (Table I).

Effects of calcium channel modulators

Calcium channels are in many cases activated in response to membrane depolarization and in response to PKC activators (Kaczmarek, 1987; Shearman et al., 1989b). Calcium channel modulators were therefore studied for their effects on settlement and metamorphosis of the Capitella larvae. Pre-exposure of the larvae to the known calcium channel blockers [Ni.sup.2+] at a concentration of 10 mM, [Zn.sup.2+] at a concentration of 10 mM, and verapamil at a concentration of 17 [[micro]molar] completely inhibited the settlement- and metamorphosis-inducing effects of JH III (Table I). Next, to determine whether an influx of calcium could stimulate settlement and metamorphosis, the effect of the calcium channel ionophore A23187 was tested. A23187 proved to have a potent, concentration-dependent effect on larval settlement and metamorphosis [ILLUSTRATION FOR FIGURE 7 OMITTED], with a concentration of 400 nM stimulating 100% of the larvae to settle and metamorphose in 1 h (Table I). These results therefore suggest that JH III activation of PKC may lead to the opening of calcium channels.

Discussion

The ability of juvenile hormones and compounds with JH activity to induce settlement and metamorphosis in Capitella larvae raises the possibility that these compounds may act on the larvae through a mechanism similar to that by which they affect insect metamorphosis and reproduction. In insects, juvenile hormones have multiple mechanisms for regulating metamorphosis and reproduction. For example, they may act through nuclear receptors and transcriptional regulation (Jones et al., 1993; Palli et al., 1994; Jones and Sharp, 1997), or by affecting mRNA stability (Jones et al., 1993) and mRNA translation (Ilan et al., 1972). JH I affects follicle cell patency in Rhodnius prolixus through - another well-studied mechanism (Sevala and Davey, 1989) - a signal transduction cascade involving a membrane receptor, activation of PKC, and the subsequent activation of a Na/K-ATPase. Other studies (Yamamoto et al., 1988) have demonstrated that PKC activation and opening of calcium channels is involved in the mechanism by which JH III induces protein synthesis in Drosophila male accessory glands. Our studies indicate that PKC activation and ion channel modulation are also involved in the process of chemosensory signal transduction by which JH and compounds with JH activity induce settlement and metamorphosis of larvae of Capitella sp. I.

In addition to JH I and JH III, other known activators of PKC can also induce settlement of Capitella larvae. One example is arachidonic acid, which has shown JH activity in insect cuticle bioassays (Slama, 1962) and strongly activates PKC from bovine brain (Shearman et al., 1989a) and rat brain (Holian et al., 1989; Shinomura et al., 1991). The potency of the phorbol ester PDBU in inducing settling and metamorphosis of Capitella larvae [ILLUSTRATION FOR FIGURE 3A OMITTED] is particularly good evidence for the activation of a protein kinase C-like enzyme, since it is well established that PDBU directly activates PKC in mammalian tissues (Castagna et al., 1982; Nishizuka, 1984; Vandenbark et al., 1984; Parker et al., 1986). The PKC inhibitor H-7 is able to inhibit the settlement and metamorphosis effects of JH III [ILLUSTRATION FOR FIGURE 3B OMITTED]. Although H-7 at higher concentrations also inhibits other protein kinases such as cyclic AMP-dependent protein kinase (PKA) (Hidaka et al., 1984), the effects of H-7 on the Capitella larvae are probably due to the inhibition of PKC and not PKA, since PDBU, which induces settlement and metamorphosis, activates only PKC and not PKA (Castagna et al., 1982).

PKC is regarded as a ubiquitous enzyme present throughout the animal kingdom (Nishizuka, 1984), having been demonstrated in Dictyostelium (Luderus et al., 1989; Jimenez et al., 1989) and sponges (Muller et al., 1987) as well as in mammalian tissues. Other studies have also demonstrated that PKC activation is involved in the signal transduction processes that mediate settlement and metamorphosis of marine invertebrate larvae of the coelenterate Hydractinia (Muller, 1985; Leitz and Klingman, 1990) and the mollusc Haliotis rufescens (Baxter and Morse, 1987; Morse, 1990). It is therefore likely that Capitella also possesses a PKC-like enzyme that is involved in mediating larval settlement and metamorphosis. Our data indicate that Capitella does possess such an enzyme, since crude homogenates of the larvae show PKC activity in a selective PKC assay (Fig. 4). More studies are needed to further characterize the enzyme and determine its requirement for calcium.

JH appears to activate PKC in the Capitella larvae directly, much like the mechanism of action of phorbol esters. Our results show that MF, JH III, and arachidonic acid can, in vitro, directly activate the PKC-like enzyme in Capitella as well as purified PKC from rat brain in the complete absence of the normal membrane inducers phosphatidyl serine and diacylglycerol [ILLUSTRATION FOR FIGURE 5 OMITTED]. These data therefore indicate that juvenile hormones, as well as JH-active compounds such as arachidonic acid, are able to bind to the lipid-binding site in PKC (Parker et al., 1986; Ziesel, 1993). This is perhaps not surprising given that phorbol esters and juvenile hormones are both terpenoid compounds: phorbol esters are diterpenoids and juvenile hormones are sesquiterpenoids.

In sensing JH-active compounds in the seawater, these lipophilic compounds presumably pass through the membrane of ciliary epithelial chemosensory cells similar to those reported to transduce chemical signals for settlement and metamorphosis in larvae of the abalone Haliotis (Trapido-Rosenthal and Morse, 1986), the cnidarian Hydractinia (Schwoerer-Bohning et al., 1990), the polychaete Phragmatopoma californica (Amieva et al., 1987), and the sea star Luidia senegalensis (Komatsu et al., 1991). Our studies with the fluorescent PKC inhibitor RIM-1 provide more evidence that Capitella larvae possess a PKC-like enzyme and that PKC is present in chemosensory cells. The RIM-1 presumably was able to directly enter chemosensory cells of the larvae that allow rapid uptake of external chemicals, since the larvae were exposed to RIM-1 only briefly (1 min). The intense RIM-1 binding in isolated cells of the prostomium [ILLUSTRATION FOR FIGURE 6 OMITTED] may represent the presence of PKC in apical cilia that are thought, on the basis of evidence from electron microscopy (Eckelbarger and Grassle, 1987) to serve a chemosensory function. However, entry of RIM-1 into the larvae through other processes cannot be ruled out, since RIM-1 binding was found in cells throughout the body, especially in ciliary cells of the prototroch and telotroch, and in both metatrochophore larvae and juveniles.

It is likely that JH-active compounds, after passing through the membrane of chemosensory cells, bind with an inactive PKC present in the cytosol, which, as in other species (Nishizuka, 1984), then becomes active and is translocated to the membrane. It is evident from our studies that micromolar concentrations of JH-active compounds are able to activate PKC and thereby induce settlement and metamorphosis. Concentrations of 10 [[micro]molar] JH III, MF, and arachidonic acid activate both Capitella PKC and rat brain PKC [ILLUSTRATION FOR FIGURE 4 OMITTED], and the same concentrations of these chemicals dissolved in the seawater also induce settlement and metamorphosis of the larvae. The inability of elaidic acid to activate PKC in vitro and to induce settlement and metamorphosis is further evidence that PKC activation is involved in mediating settlement and metamorphosis of the Capitella larvae.

In Capitella larvae, PKC activation may cause several cellular events that transduce the external JH signal and lead to settlement and metamorphosis. One well-characterized effect of PKC activation is the modulation of ion transporters such as a Na+/K+ ATPase (Ilenchuk and Davey, 1987; Sevala and Davey, 1989), Na+/H+ exchangers (Berridge, 1984), and ion channels (Kaczmarek, 1987; Shearman et al., 1989b). The modulation of potassium channels, resulting in membrane depolarization and neural relay of external chemical settlement cues, has been demonstrated to be involved with the settlement and metamorphosis of several types of marine invertebrate larvae, including those of the abalone Haliotis rufescens (Baloun and Morse, 1984), the nudibranch Phestilla sibogae and the prosobranch Astraea undosa (Yool et al., 1986), the cnidarian Hydractinia (Leitz and Klingman, 1990), and the polychaetes Phragmatopoma californica (Yool et al., 1986), and Hydroides elegans (Carpizo-Ituarte and Hadfield, 1998). Pheromone reception by insects and non-insect species has also in some instances been demonstrated to be mediated by PKC activation and the concommitant modulation of ion channel activity (Zufall and Hatt, 1991; Stengl, 1993). Like those studies, our investigations suggest that ion channel modulation is also involved. We found that the addition of excess KCl to the seawater stimulated settlement and metamorphosis, whereas this effect was negated by simultaneous addition of the potassium channel blocker tetraethylammonium chloride (TEA). These results indicate that TEA blocks settlement and metamorphosis by preventing the entry of excess potassium. These results are similar to those reported by Carpizo-Ituarte and Hadfield (1998) in their investigations with larvae of the polychaete Hydroides elegans. TEA did not induce settlement and metamorphosis of the Capitella larvae at the concentrations tested; however, the larvae were induced to settle and metamophose in response to another potassium channel blocker, 4-aminopyridine (4-AP), which causes blockage of outward rectifying potassium currents (Alkon et al., 1986). These data indicate that blockage of outward rectifying potassium channels can induce settlement and metamorphosis of the Capitella larvae. Since studies by Leitz and Klingman (1990) demonstrated that potassium channel closure was involved in mediating settlement and metamorphosis of Hydractinia larvae in response to PKC-activating diacylglycerols, we tested the possibility that JH induces settlement of the Capitella larvae through closure of potassium channels in response to PKC activation. Our findings that the potassium channel ionophore nigericin inhibits settlement and metamorphosis induced by JH III and that 4-AP can directly stimulate settlement and metamorphosis provide evidence that JH stimulates settlement and metamorphosis through the closure of potassium channels.

Since potassium channel closure may cause membrane depolarization, it is possible that voltage-gated calcium channels are activated during this process and participate in mediating settlement and metamorphosis. Our studies support this idea, since the calcium channel blockers [Ni.sup.2+], [Zn.sup.2+], and verapamil inhibited settlement and metamorphosis induced by JH III, whereas the calcium channel ionophore A23187 induced settlement and metamorphosis.

Our interpretation of these data is that juvenile hormones and JH-active chemicals induce settlement and metamorphosis of Capitella larvae through activation of PKC, which causes closure of potassium channels; the reduced efflux of potassium depolarizes the membranes of the chemoreceptor cells, leading to the opening of voltage-activated calcium channels [ILLUSTRATION FOR FIGURE 8 OMITTED]. In eliciting metamorphosis, PKC activation by JH may activate transcription factors such as nuclear factor-kB (NF-kB) or stimulate the mitogen-activated protein kinase (MAP-kinase) pathway. Membrane depolarization and calcium influx in PC12 cells has been demonstrated to activate the MAP kinase pathway (Rosen et al., 1994; Rosen and Greenberg, 1996). PKC modulation also plays a role in regulating neural differentiation of Xenopus (Durston and Otte, 1991), epithelial patterning in Hydra (Shenk and Steele, 1993), and skin morphogenesis in chickens (Noveen et al., 1995). The molecular mechanisms by which PKC and calcium may be regulating settlement and metamorphosis of the Capitella larvae are now the subject of our ongoing research.

Acknowledgments

The authors thank Dr. Judith Grassle at Rutgers University for kindly supplying cultures of Capitella sp. I, Dr. David Knecht at the University of Connecticut for help and use of the fluorescent microscope, and Mrs. Mary Jane Spring of the University of Connecticut for providing illustrations. This research was supported in part by a research fellowship to WJB from the Marine Science Institute of the University of Connecticut and by the Sea Grant College Program, NOAA.

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Author:Biggers, William J.; Laufer, Hans
Publication:The Biological Bulletin
Date:Apr 1, 1999
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