Settlement and Metamorphosis of Capitella Larvae Induced by Juvenile Hormone-Active Compounds Is Mediated by Protein Kinase C and Ion Channels

12 Pages·1999·5.2 MB·English

by  W J Biggers

Reference: Binl. Bull. 196: IS7-I9S. (April
Settlement and Metamorphosis of Capitella Larvae
Induced by Juvenile Hormone-Active Compounds Is
Mediated by Protein Kinase C and Ion Channels
WILLIAM BIGGERS' AND HANS LAUFER12 *
J.
^Department ofMolecular ami Cell Biolag\, The University ofConnecticut, Starrs, Connecticut 06268;
and 'The Marine Biological Laborator\, Woods Hole, Massachusetts 02543
Abstract. The signal transduction pathway by which ju- and induce settlement and metamorphosis is important for
venile hormone-active compounds induce settlement and the recognition of habitats that favor growth and reproduc-
metamorphosis ofmetatrochophore larvae ofthe polychaete tion (Chia and Rice. 1978; Rittschof and Bonaventura.
annelid Capitella sp. 1 was investigated. The known protein 1986; Scheuer. 1990). These settlement signals appearto be
kinase C (PKC) activator phorbol-12.13-dibutyrate was an specific for different species, as evidenced by findings that
active inducer of settlement and metamorphosis, whereas larvae ofthe abalone Haliotis nifescens respond to specific
H-7. an inhibitor of PKC. inhibited settlement and meta- chemicals in red algae (Morse el al. 1984). larvae of the
morphosis in response tojuvenile hormone III (JH III). JH nudibranch Phestilla sibogae respond to chemicals in corals
III and methyl farnesoate (MF) also directly activated, in (Hadfield. 1978. 1984). larvae of the polychaete annelid
vitro, both a PKC-like enzyme present in Capitella homog- Phragmatopoma californica respond to chemicals present
enates and PKC purified from rat brain. In addition, binding in the burrows ofadult worms (Pawlik, 1988, 1990; Jensen
studies using the fluorescent PKC inhibitor RIM-1 revealed and Morse. 1990), and larvae ofthe sand dollars Dendraster
the presence of a PKC-like enzyme in intact Capitella excentricus (Burke. 1984) and Echinarachniits parma
larvae and juveniles. Settlement and metamorphosis of the (Pearce and Scheibling, 1990) respond to chemicals pro-
larvae was also stimulated by membrane-depolarizing con- duced by adult sand dollars.
centrations of KC1. This response to KC1 was inhibited In previous studies, we have found that juvenile hor-
by tetraethylammonium. The potassium channel blocker mones (JH). which are known morphogens that regulate
4-aminopyridine induced settlement and metamorphosis, reproduction and development of insects and crustaceans
whereas settlement and metamorphosis in response toJH III (Lauferand Borst, 1983, 1988; Laufer et al.. 1987), as well
was inhibited by the potassiumchannel ionophore nigericin. as chemicals withjuvenile hormone activity in insectcuticle
Settlement and metamorphosis induced by JH III was in- bioassays, are able to induce settlement and metamorphosis
hibited by the calcium channel blockers Ni2+. Zn2+, and of metatrochophore larvae of the polychaete annelid Capi-
verapamil. whereas settlement and metamorphosis was in- tella sp. I (Biggers and Laufer. 1992. 1996). which is a
duced by the calcium ionophore A23187. These results subspecies member of the Capitella polychaete complex
suggest that in mediating this response, juvenile hormones (Grassle and Grassle. 1976). In nature, larvae of Capitella
may cause activation of PKC. leading to subsequent mod- sp. I are stimulated to settle and metamorphose (Fig. 1)
ulation of potassium and calcium channels. when they come intocontactwith chemical inducers present
in sediments (Butman et al., 1988). although the identity of
Introduction
these chemicals remains in debate (Cuomo. 1985; Dubilier.
The chemoreception by marine invertebrate larvae of 1987). they appear to have JH-activity (Biggers. 1994).
chemical "cues" that are present in the ocean environment We have now investigated the signal transduction process
through which the Capitella larvae respond to JH-active
Received 6 August 1998; accepted 29 December 1998. compounds. Our results presented in this paper indicate that
*To whom correspondence should be addressed. JH-active compounds stimulate settlement and metamor-
187
188 W. J. BIGGERS AND H, LAUFER
LifeCycleofthe PolychaeteAnnelidCapitellasp. I petri dishes containing 10 metatrochophore larvae less than
prostomium 1 day old ( 1 day post-release), and 10 ml of artificial
prototroch "N Freeswimming seawater. The dishes were then observed. After 1 h, the
Metatrochophorelarva amount of settlement and metamorphosis was assessed by
placing each dish under a dissecting microscope and count-
telotroch
ing the number of settled larvae crawling on the bottom of
the dish. Metamorphosis after 1 hour was also more criti-
cally assessed by using a compound microscope and noting
the loss of cilia, elongation, and development of capillary
setae.
Settlementoflarvaintosediments,
metamorphosisandgrowth Protein kinase C assavs
Assays for the presence ofprotein kinase C were carried
out essentially as described by Yasuda et a/. (1990), by
measuring phosphorylation of an 1 1-residue synthetic pep-
Formationofbroodtube, tide from myelin basic protein (MBP4_U). This method is
Incubationanddevelopmentofeggsandlarvae specific for measurement of PKC, and permits selective
measurement in crude tissue preparations. The PKC assay
sp.FiIgulrareva1e.. IDniapagrreanmtalofbtrhoeodlifetucbyecs,letarnodchompehtoarmeorlpahrovsaiesdoefveClaoppiteilnltao was first standardized using purified rat brain PKC (calcium
segmented metatrochophore larvae. After hatching and release from the and phospholipid dependent) from Calbiochem Co. Phos-
brood tubes, in response to chemical settlement cues, swimming meta- phorylation of MBP4_,4 was measured using 2-10 ng of
trochophorelarvaesettleandmetamorphoseintojuvenilewormsby losing enzyme, and over a time period of 30 min. Reactions were
hthoeoikrscilnieacenseseadreydffoorrcsrwaiwmlimnigngtharnodugdhevseeldoipmienntgsc.apillarysetaeandhooded carried out in plastic EpmpeMndorf tubes, with 50-ju.ml rMeaction
mixtures containing 20 Tris/HCl. pH 7.5, 5 mag-
nesium acetate, 100 /iA/ CaCl:, 25 juM MBP4 I4 (Sigma
phosis of these larvae through the activation of protein Chem. Co.), 50 ng diolein and 500 ng phosphatidylserine,
kinaseC (PKC) and subsequent modulation ofion channels. 2-10 ng of rat brain PKC, and 10 H.M ATP (containing
5-6 X 10s CPM gamma 32P-ATP). Reactions were started
Materials and Methods by addition ofenzyme, and incubations were carried out at
30C for up to 30 min. The reactions were then stopped by
Capitella larval settlement bioassays
placing the tubes on ice and spotting the reaction mixtures
Stock cultures ofCapitella sp. I were maintained at 18C onto P-81 anion exchange paper discs (Whatman Co.),
mM
in large plastic containers containing artificial seawater which were then immediately immersed in 75 H,PO4,
(Utikem Co.) and washed sea sand (Fisher Scientific) and and washed eight times in 100 ml ofthe same solution. The
were fed Tetramin fish food flakes. Brood tubes containing filter discs were placed in Ecolume cocktail for analysis
adult females along with their developing eggs and larvae with a scintillation counter. Other PKC assays using rat
were then separated from the cultures and placed into brain PKC were carriedout in the same manner, except with
60-mm glass petri dishes containing seawater. The dishes the replacement ofdiolein and phosphatidylserine with test
were checked daily for hatched, swimming metatrocho- chemicals as indicated.
phore larvae to be used for bioassays. All test chemicals, PKC activity in Capitella larval homogenates was deter-
except formethyl farnesoate (MF) which was synthesized in mined in the same manner, except with the inclusion ofthe
our laboratory, were purchased from Sigma Chem. Co. larval homogenates (20-100 jug protein) instead of the rat
Stock solutions ofjuvenile hormone III (JH III), MF, phor- brain PKC. Capitella larvae were collected within 1 day of
bol-12,13 dibutyrate (PDBU), l-(5-isoquinolinyl-sulfonyl)- release from the brood tubes, and were frozen at 20C.
2-methylpiperazine (H-7), arachidonic acid, elaidic acid, After thawing, the larvae were centrifuged at 5000 rpm for
verapamil, 4-aminopyridine, and nigericin were prepared in 1 min in a microfuge, the seawaterwasdiscarded, and larval
95% ethanol. Stock solutions of KC1. NiCl2, ZnCK. and homogenatesmweMre prepared by homogenizing 1000 larmvaMe
tetraethylammonium chloride (TEA) were prepared in dis- in 1 ml 20 Tris/HCL, pH 7.5, containing 0.5
tilled water. Settlement and metamorphosis bioassays were phenylmethylsulfonyl fluoride (protease inhibitor) using
conducted at 18C using 60-mm glass petri dishes that were small glass homogenizers. The homogenates were then cen-
pre-baked at 250C to remove contaminants (Biggers and trifuged in a microfuge at 5000 rpm for 1 min to remove
Laufer, 1996). For the assays, 10 to 100 ;ul of the test largecellulardebris, andthe PKC activity ofthe supernatant
chemical stock solutions were added by micropipet into was assessed as previously described for studies with rat
SIGNAL TRANSDUCTION IN CAPITELIA BY PKC 189
brain PKC. Protein concentrations of supernatants were 120 -/
determined using a microprotein assay (Sigma Chem. Co.)
and a standard curve of increasing concentrations ofbovine
o 100 -
serum albumin. .c
Q.
6
Localisation ofPKC by KIM-1 analysis (E0 80-
Rhodamine-conjugated bisindolylmaleimide (RIM-1) (Cal- 60-
biochem). a fluorescent PKC inhibitor (Chen and Poenie,
1993), was usedto visually locate PKC in Capitellalarvae and 40-
juveniles. Metatrochophore larvae less than 1 day old and
1-day-old juveniles were briefly exposed for 1 min at room 20-
temperature to 200 nm RIM-1 in seawater(100 ^il) in depres-
sion slides covered with aluminum foil. Larvae or juveniles
were then fixed in 2% formaldehyde containing seawater for uM 1 uM 10 uM 25 uM
15 min, transferred into methanol for 15 min to permeabilize Figure2. Concentration-dependentstimulatoryeffectsofjuvenilehor-
the membranes, and then transferred three times (5 min each mone III (JH III) and methyl farnesoate (MF) on Capitella larval settle-
wash) into 1 ml fresh seawater to rinse away excess unbound ment. Metatrochophore larvae, less than I day afterrelease from parental
RIM-1. Fixed larvae orjuveniles were then transferred in 10% brood tubes, were exposed to several concentrations ofJH III or MF in
egiltyhceerrollig-hsteamwiactreorsconotpoy omricfrlousocreospceenstlimdiecsroasncdopvyisuwailtihzeadNbiy- sisseetastwhlaoetmewernn,tfroaarnngdeianmcgehtfcaromonocmrepn1httorosati2is5onf/jo,arMf.teeaPrcehrIccheo.nntEcaesncetthrtalbteaimroennrte(parmneedsaenmntestatmShoeErMpp,ehrocnsein=st
kon confocal fluorescent microscope equipped with a rhoda- 6. representing 60 larvae/concentration). Controls (0 p.M) received 50 /nl
mine filter. 95%ethanol.Asterisksindicatesignificantdifferences(P<0.001,F>50)
between control and experimental values at each concentration tested by
ANOVA
one-way with a Bonferroni correction for multiple comparisons
Results where a = 0.017.
Effects ofprotein kinase C modulators on Ian-til
settlement and metamorphosis
to activate PKC in other species including insects (Yamamoto
We have previously found that the juvenile hormones et al.. 1988; Holian et al. 1989; Sevala and Davey, 1989;
MF. JH I, and JH III, as well as compounds with juvenile Shearmanetai, 1989a; Shinomura<>?/.. 1991),andonreports
hormone activity in insect cuticle bioassays. including ara- that PKC activation is involved in the mediation ofsettlement
chidonic acid, are able to induce settlement and metamor- and metamorphosis of other species of marine invertebrate
phosis of Capitella sp. I metatrochophore larvae (Biggers larvae (Muller, 1985; Baxter and Morse, 1987; Leitz and
and Laufer, 1992). Klingman, 1990; Morse, 1990), we further investigated the
The first indication that the larvae sense the presence of involvement of PKC activation in mediating settlement and
JH III in the seawateristheonsetofexcited swimming. This metamorphosis ofthe Capitella larvae by testing the effects of
response, which is typical of normal settlement behavior other known PKC modulators. The phorbol ester phorbol-
(Butman et ai. 1988; Pechenik and Cerulli. 1991), is faster 12.13-dibutyrate (PDBU) is a well-studied activator of PKC,
than normal, with intermittent spiral-corkscrew movements, and PKC has been found to be the actual cellular receptor for
gradual body elongation, and periodic touching of the bot- PDBU in some cells (Castagnaetal, 1982; Vandenbarketal..
tom of the petri dish. The larvae then settle and metamor- 1984). Experiments with PDBU on the Capitella larvae
phose into normaljuvenile worms within 1 h. Both MF and showed that it is also a very potent inducer of settlement and
JH III are very potent inducers ofsettlement and metamor- metamorphosis (Fig. 3A). indicating that PKC activation is
phosis ofthe Capitella larvae at micromolarconcentrations, involved in mediating this process.
whereas control larvae do not start to spontaneously settle The effect of a PKC inhibitor was next studied to deter-
and metamorphose until after 24 h (Fig. 2). mine if it could inhibit settlement and metamorphosis in-
In preliminary investigations of the signal transduction duced by JH. The PKC inhibitor l-(5-isoquinolinyl-sulfo-
process that mediates JH-induced settlement and metamor- nyl)-2-methylpiperazine, abbreviated H-7 (Hidaka et al..
phosis of the Capitella larvae, we found compounds that 1984), caused a concentration-dependent inhibition of Cap-
elevate intracellular cAMP concentrations to be ineffective itella settlement and metamorphosis induced by 25 /u,A/ JH
inducers of settlement and metamorphosis, indicating that III, when the larvae were pre-exposed to H-7 for 3 h (Fig.
cAMP does not act as a second messenger in this signal 3B). This result again indicates the involvement of PKC
transduction process (Biggers and Laufer. 1992). activation in mediating the effects ofJH on settlement and
Based upon the ability ofJH I, JH III, and arachidonic acid metamorphosis.
190 W. J. BIGGERS AND H. LAUFER
120- discoideum (Jimenez et a/., 1989; Luderus et al., 1989). its
presence in polychaetes has so far not been documented.
The presence ofa 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 Yasudaet
til. ( 1990), this assay is based upon the specific phosphor-
A ylation 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"+/calmodulin-dependent protein kinase II, or
phosphorylase kinase. The results demonstrate that a PKC-
like enzyme does exist in Capitella (Fig. 4). PKC activity
was linear for up to 15 min for homogenate concentrations
up to 100 ;ag (Fig. 4A, B). The specific activity of PKC in
the larval homogenates was calculated as being 6.7 fmoles
P-incorporated per minute per microgram of protein. In-
cubations were also done without the PKC substrate
MBP
4_|4 to monitor endogenous larval protein phosphory-
lation, ofwhich only a very small amountcould be detected.
Experiments were next conducted to determine whether
B
14000
12000
10000
CQL
8000
A
100 6000
= 4000
Figure3. EffectsofproteinkinaseC(PKC) modulatorson larval settle-
mentand metamorphosis. (A) Inductionofsettlementand metamorphosisby
12.13 dibutyrate (PDBLI): Cupilella metatrochophore larvae, less than 1 day
afterrelease,wereexposedtothe PKCactivatorphorhol PDBLI added tothe 20 40 60 80 100
seawater at concentrations ranging from 1 to 25 jiM, and settlement and
metamorphosiswasnotedafter 1 h.Controlsreceived50/al95%ethanol.Bars Larvalprotein(ug)
represent the percent settlement and metamorphosis for each concentration
(mean SEM. n = 6. representing (SO larvae/concentration). Asterisks indi-
cate significant differences (P < 0.001. F> 25)betweencontrol and exper-
imental values at each concentration tested by one-way ANOVA with a
Bonterronicorrectionformultiplecomparisonswhere a = 0.017. (Bl Inhibi-
tion ofjuvenile hormone (JH) Ill-induced settlement and metamorphosis by QQ.
H-7. Cn/iitclUi metatrochophore larvae, less than 1 day after release, were
pre-exposed lor 3 h to concentrations of H-7. a PKC inhibitor, in seawater Bo
1
ranging from 1 to 100 /xA/. before being exposed toJH III (20/jjV/) lor I h.
Controls (0 fj.A'7) received 100 Ml 45<7r ethanol followed by JH III. Bars
represent the percent settlement and metamorphosis for each concentration
after the 4-h time period (mean SEM, n = 6. representing 60 larvae/
concentration). Asterisks same as in (A).
10 15 20 25
Activation ofu protein kimiae C-like enzyme in Capitella Time(minutes)
larvae b\ JH-active chemicals Figure 4. Protein kinase C (PKC) activity in Capitella larvae. PKC
preAsletnhtotuhgrhouPghKoCutisthceonasniidmearledkitnogbdeomaaunbdiqhuaistobuesenenfzoyumned aticintdciyrvleisatesyriinwngeasparndoedtteeidcnitocelodeinnci.nenlt'a;rrPav-tailinohcnoosrmprooagrneagntiianotgnesfirnaotfmote2rM0aBctPtoi4v1a0t]04ionw^xagwsi(tAlh)inpeahanordspwwhiaats-h
in marine sponges (Muller ct a/.. 19H7) and Dictwsti'Iimu also linear for 15 min using 100 |U,g protein (B).
SIGNAL TRANSDUCTION IN CAP1TELH BY PKC 191
the PKC-like enzyme present in the Capitella lar\rae could 120-f
he activated by JH-active compounds. Incubations were
carried out using 100 ^g of the larval homogenates for IS 100 -
inin at 30"C in the presence of either phosphatidylserine/
diolein (PS/DO). 10 ^M arachidonic acid (AA), 10 ^M JH
tIIhIi,s e10xpjeurAi/mMeFnt.ionrdi1c0atMeAt/haetlJaiHdiIcII,acMiFd,(EaAn)d.ATAhearreesaubltlseotof A i
activate CapiteUa PKC //; \-itn> (Fig. 5A). In comparison
with activation by PS/DO, arachidonic acid was the stron-
W<
MF
gest activator activation), followed by (73% ac-
tivation) and JH III (51% activation). Elaidic acid, a trans-
isomer of oleic acid that does not activate rat brain PKC
(Shearman ctai. 1989a) and does not induce settlement and
metamorphosis of the Capitellci larvae, did not activate the
Capitella PKC. These results therefore again indicate the
involvement ofPKC 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 diacyl-
glycerol pathway, such as phospholipase C and guanine- B
binding proteins through which indirect activation of PKC
may occur, we alsotestedwhetherJH coulddirectly activate
a purified preparation of rat brain PKC. Polyunsaturated
fatty acids such as arachidonic acid can activate bovine
brain PKC (Shearman et til.. 1989a) and rat brain PKC
(Holian et al., 1989; Shinomura et ai, 1991); therefore, PS/DO
juvenile hormones might also be able to cross species lines
and activate rat brain PKC. Incubations were carried out Figure5. ActivationofanenzymeresemblingproteinkinaseC(PKC)
using 5 ng purified rat brain PKC (Calbiochem Co.) in the cinheCmtiicpailtse.Ha(Al)arAvcaltihvaotmioognenofattehse PaKnCd-lpuirkiefieednzryatmebrpariensePnKtCinblyarJvHae-.acPtiKvCe
presenceofeitherPS/DO, 10/uAfAA, lO^A/JHIII. \() ^M assaysusingCapitellalarvalhomogenateswerecarriedoutinthepresence
MF. or 10JU.A-/EA, at 37C for 15 min. The results show that of either phosphatidylserine/diolein (PS/DO). 10 \j.M arachidonic acid
insect juvenile hormones and the crustacean juvenile hor- (AA). 10 /nA/ juvenile hormone (JH) III. 10 juiW trans, trans methyl
ambosneencMeFofarpehoasbplheattioddyilrseecrtilnyeacatnidvadteiorlaetinbr(aFiing.P5KBC).inArtah-e iMfanBrcnoPersp4oo_arIta4etfioo(rnMAFrA)e.l.atJoirHveI1tI0Io.tpMhaFMt.feaolnuadinddiEcfAoaricstihdseh(coEowAnnt)r.eoxlIpn(rcPeosSrs/peDodOr)aat.sio(tnhBe)opAfecrt3ci2evPnattiaingoteno
chidonic acid was the most potent activator tested (93% ofpurified rat brain PKC. PKC assays using 5 ng purified rat brain PKC
activation relative to PS/DO), whereas elaidic acid was were carried out in the presence ofeither phosphatidylserine/diolein (PS/
inactive, confirming earlier work reported by Shearman et DO). 10 fj-M arachidonic acid (AA). 10 /xM JH III. 10 ^M trans, tram,
cailn.d( 1w9a8s9am).orMeFacctaiuvseetdh5an9%JHacItIiIva(t2i8on%oafcttihveatriaotnb)r.ain PKC mpineetrtocheyMnltBafPgaer4n_ie,ns4ocaoftroepro(rAaMAtFi,)o,nJoHrrel1IaI0tIi,vpe.MMFto,eltahaiandtdicfoEaucAniddis(foEsrAh)t.ohweInnccooenrtxprpoorlreas(tsiPeodSn/aoDsfO1tI.2hPe
Locali-ution ofPKC in Capitella />v RIM-1
To visualize the location ofPKC in the CapiteUa larvae and
juveniles and to identify possible chemosensory cells that and isolated cells in the apical region ofthe prostomium and
winoutlhde reanpviidlryonmteaknet,uplarevxateernaanld cjhuevmeincialless owrercehebmriiceafllsy tthhaetrtehsetseofcetlhles bpoosdsyesdsisPpKlaCye(dFiRg.IM6-B1).bJiunvdeinnigl,eiCnadpiictaetlilnag
( 1 min) exposed to a fluorescently labeled protein kinase exposed in the same manner displayed RIM-1 binding to
C inhibitor, rhodamine-conjugated bisindolylmaleimide cells in the apical region of the prostomium and scattered
(RIM-1), which has proven useful as a fluorescent probe for throughout the rest of the body (Fig. 6D). These results
PKC (Chen and Poenie. 1993). After exposure to this in- provide more evidence for the presence of a PKC-like
hibitor, the larvae or juveniles were fixed, permeabilized. enzyme in CupitelUi larvae andjuveniles, and are consistent
rinsed to remove excess unbound RIM-1. and viewed under with a chemosensory function for apical cells in regions of
a fluorescent microscope. In metatrochophore larvae, dis- the prostomium as previously noted by Eckelbarger and
tinct cells in theciliary bands ofthe prototroch and telotroch Grassle (1987).
192 W. J. BIGGERS AND H. LAUFER
B
Figure6. Localization ofprotein kinaseC (PKC) in Ctipitellu by RIM-1 analysis. Metatrochophore larvae
lessthan 1 dayafterreleasefromparentalbrc>odtubesand 1-day-oldjuvenileswereexposedfor 1 mintoRIM-1.
a rhodamine-conjugated PKC inhibitor. (A) Light microscopy ofmetatrochophore larva (400X). (B) Fluores-
cenee microscopy ofmetatrochophore larva showing binding ofRIM-1 to ciliary cells ofthe prototroch (p) as
well as isolated apical cells located in the prostomium (arrow) (800X). (C) Light microscopy of juvenile
Cupiti'llnprostomium(400X). (D)FluoresceneemicroscopyofjuvenileCapilellaprostomiumshowingbinding
of RIM-1 to apical cells (arrow) (400X).
Effects ofpotassium chiinnel modulators man et ai, 1989b). The involvement ofpotassium channels
in mediating the settlement and metamorphosis of several
In mediating cellular responses. PKC activation has in types ofmarine invertebrate larvae has also been previously
many cases been found to result in the modulation of demonstrated (Baloun and Morse, 1984; Yool et ai, 1986;
potassium and calcium channels (Kaczmarek, 1987; Shear- Leitz and Klingman. 1990; Carpizo-Ituarte and Hadfield,
SIGNAL TRANSDUCT1ON IN CAPITELLA BY PKC 193
Table I vators (Kaczmarek. 1987; Shearman etu/.. 1989b). Calcium
Effects ofion channel modulators on settlement uiii/metamorphosis of channel modulators were therefore studied for their effects
Capitclla .s/>. / lamu on settlement and metamorphosis of the Capitella larvae.
Pre-exposure of the larvae to the known calcium channel
% Larvae settled blockers Nr+ at a concentration of 10 mM, 7.n2+ at a
and metamorphosed concentrationof 10mA/. and verapamil ataconcentrationof
Chemical (after I h) 17 JU.A/ completely inhibited the settlement- and metamor-
Potassium channel modulators phosis-inducing effects of JH III (Table I). Next, to deter-
KCI (20 mA/) 100 mine whether an influx of calcium could stimulate settle-
KCI (20 mA/) + TEA (KM) mA/) 5 ment and metamorphosis, the effect of the calcium channel
4J-HAmIIiIno(p38yrvidMi)ne (100 fj.A/1 110010) ionophore A23187 was tested. A23187 proved to have a
JH III (38 /iA/> + nigericin (500 ng/ml) (I potent, concentration-dependent effect on larval settlement
Calcium channel modulators and metamorphosis (Fig. 7), with aconcentration of400 nM
A23187 (400 nM) 101) stimulating 100% ofthe larvae to settle and metamorphose
JJJHHH II1II1II1 (((333888 /i|UMj.AMA//))) ++ ZNinCClI,,((1100mmAA//)) 100 aincti1vahti(oTnabloef IP).KTChesmeayresluelatds tthoertehfeoreopseungignegstotfhatcaJlHciuHmI
JH 111 (38 /aA/l + Verapamil (17 /j.M) channels.
JH = juvenile hormone. Discussion
The ability ofjuvenile hormones and compounds with JH
1998). Studies were therefore carried out to determine ifthe activity to induce settlement and metamorphosis in Capi-
modulation ofpotassium channels may also mediate settle- tella larvae raises the possibility that these compounds may
ment and metamorphosis ofthe Capitella larvae. Increased act on the larvae through a mechanism similar to that by
external KCI concentrations in the seawater induced settle- which they affect insect metamorphosis and reproduction.
ment and metamorphosis of the Capitella larvae in a con- In insects,juvenile hormones have multiple mechanisms for
centration-dependent manner, with an added concentration regulating metamorphosis and reproduction. For example,
mM mM
of20 (30 total including seawater) inducing 100% they may act through nuclear receptors and transcriptional
settlement and metamorphosis in 1 h (Table I). Theseeffects regulation (Jones et al.. 1993: Palli et al., 1994; Jones and
ofKCI appearto be mediated by K+ ions and not Cl~ since
addition ofNaCl had no effect on settlement and metamor-
phosis. The effectoftetraethylammoniumchloride (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 Capi-
tella larvae, but instead inhibited the stimulatory response
ofthe larvae to KCI, with a concentration of 100 mM TEA
almost completely inhibiting settlement and metamorphosis
(Table 11. Settlement and metamorphosis of the Capitella
larvae was, however, stimulated in a concentration-depen-
dent mannerby 4-aminopyridine (4-AP), another potassium
channel blocker. which blocks outward rectifying potassium
currents. A concentration of 100 ju,jW4-AP stimulated 100%
settlement and metamorphosis of the larvae within 1 h
(Table 1). 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<7r inhibition of settlement and meta- 50 100 150 200 250 300 350 400
morphosis in response to JH III in 1 h (Table I). A23187(nM)
Figure 7. Effect ofthe calcium ionophore A23187 on settlement and
Effects ofcalcium channel modulators metamorphosisofCapitellalarvae.Metatrochophorelarvaelessthan I day
Calcium channels are in manycases activated in response taofte4r0r0elneaMse,inexsepaowsaetderf.orwe1rhetostciomnucleantterdattioonssettolfeAa2n3d1m8e7traamnogripnhgofsreomin25a
to membrane depolarization and in response to PKC acti- concentration-dependent manner.
194 W. J. BIGGERS AND H. LAUFER
Sharp. 1997). or by affecting mRNA stability (Jones et al.. rectly, much like the mechanism ofaction ofphorbol esters.
1993) and mRNA translation Ulan el til., 1972). JH 1 affects Our results show that MF, JH III, and arachidonic acid can,
follicle cell patency in Rliodniitsprolixus through another /'// vitro, directly activate the PKC-like enzyme in Capitella
well-studied mechanism (Sevala and Davey, 1989) a sig- as well as purified PKC from rat brain in the complete
nal transduction cascade involving a membrane receptor, absence of the normal membrane inducers phosphatidyl
activation of PKC, and the subsequent activation of a Na/ serine and diacylglycerol (Fig. 5). These data therefore
K-ATPase. Other studies (Yamamoto et nl.. 1988) have indicate that juvenile hormones, as well as JH-active com-
demonstrated that PKC activation and opening of calcium pounds such as arachidonic acid, are able to bind to the
channels is involved in the mechanism by which JH III lipid-binding site in PKC (Parkeretai. 1986; Ziesel, 1993).
induces protein synthesis in Drosophilu male accessory This is perhaps not surprising given that phorbol esters and
glands. Our studies indicate that PKC activation and ion juvenile hormones are both terpenoid compounds; phorbol
channel modulation are also involved in the process of esters are diterpenoids and juvenile hormones are sesqui-
chemosensory signal transduction by which JH and com- terpenoids.
pounds with JH activity induce settlement and metamorpho- In sensing JH-active compounds in the seawater. these
sis of larvae of Capitella sp. I. lipophilic compounds presumably pass through the mem-
In addition to JH I and JH III. other known activators of brane of ciliary epithelial chemosensory cells similar to
PKC can also induce settlement of Capitella larvae. One those reported to transduce chemical signals for settlement
example is arachidonic acid, which has shown JH activity in and metamorphosis in larvae of the abalone Haliotis
insect cuticle bioassays (Slamu, 1962) and strongly acti- (Trapido-Rosenthal and Morse. 1986), the cnidarian Hy-
vates PKC from bovine brain (Shearman ct ai. 1989a) and dractinia (Schwoerer-Bohning et ai, 1990). the polychaete
rat brain (Holian et ul.. 1989; Shinomura et at., 1991 ). The Phragmatopoma culifornica (Amieva et ul., 1987), and the
potency ofthe phorbol esterPDBU in inducing settling and sea star Lnidia senegalensis (Komatsu et al., 1991). Our
metamorphosis of Capitella larvae (Fig. 3A) is particularly studies with the fluorescent PKC inhibitor RIM-1 provide
good evidence for the activation of a protein kinase C-like more evidence that Capitella larvae possess a PKC-like
enzyme, since it is well established that PDBU directly enzyme and that PKC is present in chemosensory cells. The
activates PKC in mammalian tissues (Castagna etai. 1982; RIM-1 presumably was able to directly enterchemosensory
Nishizuka. 1984; Vandenbark et al.. 1984; Parker et ai. cells ofthe larvae that allow rapid uptake ofexternal chem-
1986). The PKC inhibitor H-7 is able to inhibit the settle- icals, since the larvae wereexposedto RIM-1 only briefly (1
ment and metamorphosis effects of JH III (Fig. 3B). Al- min). The intense RIM-1 binding in isolated cells of the
though H-7 at higher concentrations also inhibits other prostomium (Fig. 6) may represent the presence of PKC in
protein kinases such as cyclic AMP-dependent protein ki- apical cilia that are thought, on the basis of evidence from
nase (PKA) (Hidaka et ul., 1984). the effects of H-7 on the electron microscopy (Eckelbarger and Grassle, 1987) to
Capitella larvae are probably due to the inhibition of PKC serve a chemosensory function. However, entry of RIM-1
and not PKA. since PDBU. which induces settlement and into the larvae through other processes cannot be ruled out,
metamorphosis, activates only PKC and not PKA (Castagna since RIM-1 binding was found in cells throughout the
et al., 1982). body, especially in ciliary cells of the prototroch and
PKC is regarded as aubiquitous enzyme present through- telotroch, and in both metatrochophore larvae andjuveniles.
out the animal kingdom (Nishizuka. 1984). having been It is likely that JH-active compounds, after passing
demonstrated in Dictyostelium (Luderus et ai. 1989; Jime- through the membrane ofchemosensory cells, bind with an
nez etui., 1989) and sponges (Mullere? /., 1987) as well as inactive PKC present in the cytosol, which, as in other
in mammalian tissues. Otherstudieshave alsodemonstrated species (Nishizuka. 1984). then becomes active and is trans-
that PKC activation is involved in the signal transduction located to the membrane. It is evident from our studies that
processes that mediate settlement and metamorphosis of micromolarconcentrationsofJH-activecompounds are able
marine invertebrate larvae of the coelenterate Hydractinia to activate PKC and thereby induce settlement and meta-
(Muller. 1985; Leitz and Klingman, 1990) and the mollusc morphosis. Concentrations of 10 p.M JH III. MF, and ara-
Haliotis nifescens (Baxter and Morse. 1987; Morse. 1990). chidonic acid activate both Capitella PKC and rat brain
It is therefore likely that Capitella also possesses a PKC- PKC (Fig. 4), and the same concentrations of these chem-
like enzyme that is involved in mediating larval settlement icals dissolved in the seawater also induce settlement and
and metamorphosis. Our data indicate that Capitella does metamorphosis ofthe larvae. The inability ofelaidic acid to
possess such an enzyme, since crude homogenates of the activate PKC in vitro and to induce settlement and meta-
larvae show PKC activity in a selective PKC assay (Fig. 4). morphosis is further evidence that PKC activation is in-
More studies are needed to further characterize the enzyme volved in mediating settlement and metamorphosis of the
and determine its requirement for calcium. Capitella larvae.
JH appears to activate PKC in the Capitella larvae di- In Capitella larvae. PKC activation may cause several
SIGNAL TRANSDLICT1ON IN CAPITELLA BY PKC 195
Initial State
Voltage gated
Ca++ channel
Illfl
JJJJJ
JHA/Vw,
.- ..-..,,.,... - - . -,
JHA-activation
!
K+ channel blockage
;
Protein Jttft*****
Kinase C
(inactive)
Protein
KinaseC
(active)
f
Ca++ channel activation
Membrane
depolarization;
closed open
Figure 8. Model for thejuvenile hormone (JH) signal-transduction pathway in Capitella sp. I larvae. In this
proposed model, JH induces settlement and metamorphosis by directly interacting with and activating cytosolic
proteinkinaseC(PKC), whichactsasthecellularreceptorinthelarvalchemosensorycells.ThisactivationofPKC
then causes the closure of potassium channels by phosphorylation. invoking membrane depolarization, and the
subsequent opening of voltage-activated calcium channels. PKC and calcium may in subsequent steps activate
transcriptionfactorsandproteinkinases, leadingtochangesingeneactivity,andalsocauseneurotransmitterrelease.
cellularevents that transduce the external JH signal and lead ers such as a Na+/K+ ATPase (Ilenchuk and Davey. 1987;
to settlement and metamorphosis. One well-characterized Sevala and Davey. 1989), Na+/H+ exchangers (Berridge,
effect of PKC activation is the modulation of ion transport- 1984), and ion channels (Kaczmarek, 1987; Shearman etai,
196 W. J. BIGGERS AND H. LAUFER
1989b). The modulation ofpotassium channels, resulting in and JH-active chemicals induce settlement and metamor-
membranedepolarizationand neural relayofexternalchem- phosis ofCapitella larvae through activation ofPKC, which
ical settlement cues, has been demonstrated to be involved causes closure ofpotassium channels; the reduced efflux of
with the settlement and metamorphosis of several types of potassium depolarizes the membranes ofthe chemoreceptor
marine invertebrate larvae, including those of the abalone cells, leading to the opening of voltage-activated calcium
Haliotis rufescens (Baloun and Morse. 1984), the nudi- channels (Fig. 8). In eliciting metamorphosis, PKC activa-
branch Phestilla sibogae and the prosobranch Astraea un- tion by JH may activate transcription factors such as nuclear
dosa (Yool et /., 1986). the cnidarian Hydractinia (Leitz factor-kB (NF-kB) or stimulate the mitogen-activated pro-
and Klingman. 1990), and the polychaetes Phragmatopoma tein kinase (MAP-kinase) pathway. Membrane depolariza-
califomica (Yool et <;/., 1986), and Hydroides elegans tion andcalcium influx in PC12 cells hasbeen demonstrated
MAP
(Carpizo-Ituarte and Hadfield, 1998). Pheromone reception to activate the kinase pathway (Rosen et al., 1994;
by insects and non-insect species has also in some instances Rosen and Greenberg, 1996). PKC modulation also plays a
been demonstrated to be mediated by PKC activation and role in regulating neural differentiationofXenopus(Durston
the concommitant modulation of ion channel activity (Zu- and Otte, 1991), epithelial patterning in Hydra (Shenk and
fall and Hatt, 1991; Stengl, 1993). Like those studies, our Steele, 1993), and skin morphogenesis in chickens (Noveen
investigations suggest that ion channel modulation is also ctnl., 1995). The molecularmechanisms by which PKC and
involved. We found that the addition of excess KC1 to the calcium may be regulating settlement and metamorphosis of
seawaterstimulated settlement and metamorphosis, whereas the Capitella larvae are now the subject of our ongoing
this effect was negated by simultaneous addition of the research.
potassium channel blocker tetraethylammonium chloride
(TEA). These results indicate that TEA blocks settlement Acknowledgments
and metamorphosis by preventing the entry of excess po-
The authors thank Dr. Judith Grassle at Rutgers Univer-
tassium. These results are similar to those reported by
Carpizo-Ituarte and Hadfield (1998) in their invesTtiEgaAtions sDiatvyidforKnkeicnhdtlyatstuhpeplUyniinvgersciutltyuroefsCoonfneCcatpiicteultlaforsph.elIp, aDnrd.
with larvae of the polychaete Hydroides elegans. did
use of the fluorescent microscope, and Mrs. Mary Jane
not induce settlement and metamorphosis of the Capitella
larvae attheconcentrations tested; however, the larvae were Spring ofthe University ofConnecticut for providing illus-
trations. This research was supported in part by a research
piontdaucsesdiutmocsheatntlneelanbdlocmkeetra,m4o-pahmoisnoepyinrirdeisnpeon(s4e-AtPo).anwohtihcehr fellowship to WJB from the Marine Science Institute ofthe
University of Connecticut and by the Sea Grant College
causes blockage of outward rectifying potassium currents NOAA.
Program,
(Alkon et at.. 1986). These data indicate that blockage of
outward rectifying potassium channels can induce settle-
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