A Myomodulin-CARP-related Peptide Isolated from a Polychaete Annelid, Perinereis vancaurica

6 Pages·1994·2.5 MB·English

by  Toshio Takahashi

ZOOLOGICAL SCIENCE 11: 33-38 (1994) © 1994ZoologicalSocietyofJapan
A Myomodulin-CARP-related Peptide Isolated from a Polychaete
Annelid, Perinereis vancaurica
Toshio Takahashi1 Osamu Matsushima2*, Fumihiro Morishita2
, ,
Masaaki Fujimoto2 Tetsuya Ikeda3 Hiroyuki Minakata3
, ,
and Kyosuke Nomoto3
1Faculty ofIntegrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 724,
2Department ofBiological Science, Faculty ofScience, Hiroshima University,
Higashi-Hiroshima 724, and 3Suntory Institutefor
Bioorganic Research, Osaka 618, Japan
—
ABSTRACT MyomoduIin-CARP-family peptides have been isolated only from molluscs. In the present study, a
heptapeptide, Ala-Met-Gly-Met-Leu-Arg-Met-NH2, termed Pev-myomodulin, was isolated from a polychaete annelid,
Perinereis vancauricausingthe esophagusofthe animal as the bioassay system. The sequenceofthe annelid peptide is
highly homologous with those of the myomodulin-CARP-family peptides found in molluscs. The annelid peptide is
regarded as a member of the myomodulin-CARP family, though all the molluscan peptides have a Leu-NH2 at their
C-termini. Theannelidpeptideshowedapotnetcontractileactionontheesophagusoftheannelid. Thepeptidemaybe
an excitatory neuromediator involved in the regulation of the esophagus. Among various myomodulin-CARP-family
peptides and their analogues, the annelid peptide showed the most potent contractile action on the esophagus.
Replacementofthe C-terminal Met-NH2ofthe annelidpeptidewith aLeu-NH2decreaseditscontractilepotency, while
replacement of the C-terminal Leu-NH2 of myomodulin and CARP with a Met-NH2 increased their potency. The
C-terminalMet-NH2oftheannelidpeptideseemstobeimportant, butnotessential, forexhibitingitscontractileactivity
on the esophagus. On the anterior byssus retractor muscle of the bivalve mollusc Mytilus edulis, the annelid peptide
showed catch-relaxing and contraction-modulating effects qualitatively similar to those ofthe authentic peptide CARP,
though the annelid peptide was less potent than CARP.
sequence of the peptide is highly homologous to myomodu-
INTRODUCTION lin, catch-relaxing peptide (CARP) and their related pep-
tides, all ofwhich have been isolated from molluscs ([2, 3, 8,
Over the past two decades, a large number of bioactive
10, 11], See also Table 1). That is, the annelid peptide is
peptides have been isolated from invertebrates, especially regarded asamemberofmyomodulin-CARPfamily. Here,
from arthropods and molluscs [5, 9, 12, 18, 20. 21]. As to we report purification, structure determination and pharma-
annelids, however, only several FMRFamide-related pep-
cologicalcharacterizationofthe annelidmyomodulin-CARP-
tides have been identifiedusingradioimmunoassayfordetec-
family peptide.
tionofthepeptides. Krajniak andPrice [13] foundFMRFa-
mide in the polychaete Nereis virens. Baratte et al. [1]
isolated FMRFamide and its analogue from Nereis diversico- MATERIALS AND METHODS
lor. Evans et al. [4] identified FMRFamide and four analo- Animals
gues in the medicinal leech Hirudo medicinalis. The marine polychaete annelid worms, Perinereis vancaurica,
In the previous study, we isolated two S-Iamide-family werepurchasedfromafishing-baitstoreandtheseamussels,Mytilus
peptides, AKSGFVRIamide and VSSFVRIamide, from the edulis, were collected in the Hiroshima Bay. These animals were
polychaete annelid, Perinereis vancaurica [16]. The pep- kept in laboratory tanks filled with aerated seawater at 15°C.
tides show apotentcontractile effect on theesophagus ofthe
annelid [16], while FMRFamide shows an inhibitory effect Extraction andpurification
[14]. This was the first finding of annelid peptides which Extraction procedures for bioactive peptides in P. vancaurica
were not considered to be FMRFamide-family peptides. wereessentiallythesameasthosereportedpreviously[16]. Briefly,
Since this finding, we have continued to search for bioactive 0.5kgofthe animals were boiled for 10min at 100°Cin 21 ofwater
containing 4% acetic acid and homogenized with a Waring blender
peptides in the annelid P. vancaurica using its esophagus as
and then with a Polytron homogenizer. The homogenate was
the bioassay system. In the presentstudy, we found anovel
centrifuged at 15,000Xg for 40min at 4°C. The supernatant was
heptapeptide, Ala-Met-Gly-Met-Leu-Arg-Met-NH2, that concentratedwith arotaryevaporator, and 1/10volume of 1 N HC1
showed a potent excitatory action on the esophagus. The wasaddedtotheconcentratedsolutionwithconstantagitation. The
concentrated solution was centrifuged again. The supernatant was
Accepted October27, 1993 applied to two C-18 cartridges (Mega Bond Elut, Varian) in series.
Received October4, 1993 After the cartridges were washed with 0.1% trifluoroacetic acid
To whom correspondence should be addressed. (TFA), the retained material was eluted with 50% methanol. The
34 T. Takahashi, O. Matsushima et al.
eluate was applied to four steps of reversed-phase and cation- ABRM in response to repetitive electrical stimulation at lower
exchange high performance liquid chromatography (HPLC). The concentrations and inhibits the contraction at higher concentrations
eluting substances were monitored with an UV detector at 220nm. [6, 7, 18]. Therefore, the actions of the annelid peptide on catch
Each of the fractions obtained at each HPLC step was bioassayed tension andphasiccontraction ofthe ABRM were also examinedto
using the isolated esophagus ofP. vancaurica. compare them with those of CARP. Catch contraction was pro-
At the first HPLC-purification step, the eluate from the C-18 duced by applying 10~4M acetylcholine (ACh) to the ABRM for2
cartridges was applied to aCapcell-Pak C-18reversed-phasecolumn min at 20min intervals and phasic contractions was elicited by
(Shiseido, 10x250mm). The column was eluted with a 120-min stimulatingthe musclewithrepetitiveelectricalpulses(15V,3msec,
lineargradientof0-60% acetonitrile (ACN) in0.1%TFA(pH2.2). 10Hz, 50 pulses) at 10min intervals. This procedure was basically
The flow rate was 1 ml/min. An aliquot (1/250) of each 2-ml the same as that ofMuneoka and Twarog [19].
fraction was evaporated to dryness, dissolved in artificial seawater
(ASW) and subjected to bioassay. A bioactive peak eluted at Saline
around 20% ACN was then subjected to the second step of HPLC The saline used for the esophagus of P. vancaurica and the
purification. At this step, another C-18 reversed-phase column ABRMofM. eduliswasASWofthefollowingcomposition:445mM
e(lOuDteSd-8wi0tThM,a5T0o-smoihn,li4ne.a6rxg1r5a0dimenmt)ofwa1s5-u2s5e%d.ACTNheinc0o.l1u%mnTFwAa.s NHaCC1l,(p1H07.m6)M. KC1, 10mM CaCl2, 55mM MgCl2 and 10mM Tris-
A bioactive peak was observed at around 16% ACN. At the third
step, the bioactive substance was applied to a cation-exchange
RESULTS
column (SP-5PW, Tosoh, 7.5x75mm), and the column was eluted
M mM
with a 70-min linear gradient of0-0.7 NaCl in 10 phosphate After the three steps of HPLC purification, the final
buffer (pH7.1) at a flow rate of 0.5ml/min. The bioactive sub-
M purification was performed with the reversed-phase column
stance was eluted at around 0.22 NaCl. At the fourth step (final
step), the bioactive substance was applied to the reversed-phase (Fig. 1). The single absorbance peak obtained was found to
column (ODS-80TM), and the column was eluted with a 25-min coincide with an active peak. At the first step of HPLC
linear gradient of 15-25% ACN in 0.1% TFA at a flow rate of0.5 purification on a Capcell-Pak C-18 reversed-phase column,
ml/min. The substancewaseluted at around20% ACN asasingle the active substance was recovered in the same fraction (No.
absorbance peak (Fig. 1). 34) as a previously reported S-Iamide peptide,
The purified bioactive substance was subjected to peptide se- VSSFVRIamide [16]. At the second step of HPLC using
quence analysis (Shimadzu PSQ-1 Protein Sequencer) and fast atom another C-18 reversed-phase column (ODS-80TM), these
bombardment mass spectrometric (FAB-MS) analysis (JEOL JMS- two substances were separated from each other:
HX 110/110A). These analyses suggested that the substance is a VSSFVRIamide was recovered in fractions 7, 8, and 9, while
pentapeptidewithanamidatedC-terminus. Thepeptidehavingthe
the present substance was in fractions 12 and 13.
suggested structure was then synthesized by a solid-phase peptide
synthesizer(AppliedBiosystems430A)andpurifiedbyHPLC. The
structure of the synthesized peptide was confirmed by amino acid
sequenceanalysisandFAB-MSanalysis. Thesyntheticpeptidewas
compared with the native one in the behavioron HPLC. 0.4r
Bioassay R 0.2
The anterior half of the body was cut open ventrally and the L±J|
esophagus (about 15 mm long) was excised. Both ends of the
20 40
isolated esophagus were ligated with cotton threads. One of the
thread was tied to a fixed support of a trough (2ml) filled with Time (min)
aerated ASW, and the other was connected to a force-displacement
transducer attached to a manipulator. Changes in the esophagus 1min
tension in response tobioactive substanceswere recordedon achart 03g
recorder through an amplifier.
Aliquots (1/250-1/100) ofthe fractionsobtainedateach HPLC
pAuSriWficaatnidoninsjteecptewderientoevtahpeoraaetreadtedtotdrroyungehss,indwihsiscolhvetdheinis0o.l1atmeld Fig.s1u.bstHaPncLeCopnroftihleeatC-t1h8e fOinDalSs-t8ep0TofMpucroifliucmatnion(uopfpetrhe)baionadcttihvee
esophagus was mounted. Aftereach recordingofthe effectsofthe activity of the purified substance on the esophagus of P. van-
fractions, the esophagus was washed with ASW. The next test was caurica (lower). The elution was performed with a linear
started 10min after the recording. gradient of 15-25% ACN (0.1% TFA) at a flow rate of0.5ml/
min. The absorbance was monitored at 220nm. The broken
Pharmacology line shows the concentration of ACN. An aliquot (1/100) of
The contractile actions of 10 myomodulin-CARP-family pep- the purified substance was applied at the time indicated by an
arrow head.
tides including the annelid peptide isolated in the present study and
ninesyntheticanalogueswereexaminedon the isolatedesophagusof
P. vancaurica. It has been shown that CARP powerfully relaxes The determined sequence and detected amount (pico-
catch tension in the anterior byssus retractor muscle (ABRM) ofM. moles) ofeach aminoacid in the aminoacidsequenceanalysis
edulis and that the peptide potentiates phasic contraction of the of the purified substance were as follows: Ala (820.6)-Met
Myomodulin-CARP-related Annelid Peptide 35
(892.2)-Gly (508.6)-Met (808.2)-Leu (729.2)-Arg (114.3)-
Met (293.1). In the FAB-MS spectrum of the purified 10"6M -V*V*J\
substance, a molecular ion peak was observed at 810.1 m/z
(M+H)+ The results of these analyses suggested that the
.
purified substance is an amidated heptapeptide having the
following primary structure: Ala-Met-Gly-Met-Leu-Arg-
Met-NH2. The yieldofthispeptide from500goftheworms
wasroughlyestimatedtobe3nmolesusingthedataonamino
acid sequence analysis. The value was comparable to the 10~8 M oi
yield of an S-Iamide peptide, AKSGFVRIamide, and larger
than the yield of another S-Iamide peptide, VSSFVRIamide
2 min
[16].
A mixture of the synthetic peptide with the suggested Fig. 3. Actionsofthesyntheticpeptideontheisolatedesophagusat
structure and the native peptide (purified substance) showed different concentrations. The peptidewas applied atthe times
indicated by arrow heads.
a single absorbance peak when applied to a C-18 reversed-
phase column and a cation-exchange column (Fig. 2). We
were not able tocompare the bioactivitybetween the synthe- The sequence of the annelid peptide is highly homolo-
tic and native peptides, because the quantity of the native goustothose ofmyomodulin-CARP-familypeptidesfoundin
peptide obtained was not enough for the comparison. several molluscs (Table 1). We examined the effects of
However, we confirmed contraction-eliciting activity of the these molluscan peptides and several synthetic analogues on
syntheticpeptide at concentrations of 10~8M or higher (Fig. the esophagus of P. vancaurica, and compared the effects
3). with those of the annelid peptide which we designated
Pev-myomodulin (Table2).
Pev-myomodulin showed the most potent contractile
S-06r effect on the esophagus, and was approximately 10 times
CM more potent than myomodulin and CARP. In Figure4, the
effects of 10"6M Pev-myomodulin and 10"6M CARP on a
.025 preparation of the esophagus are shown. Met7-CARP and
L Met7-myomodulin were also more potent than the respective
< 20 40 20 30 authentic molluscan peptides, though they were slightly less
potent than Pev-myomodulin. Phe7-CARP and Phe7-
Time (min)
myomodulin were far less potent than Pev-myomodulin.
Fig.p2.eptiHdPesLConparoCf-i1l8esODofS-m8ix0tTuMrescolofumnthewitnhatainveisoacnrdatiscynetlhuettiionc Leu7-Pev-myomodulin was also far less potent than the
of21%ACN(0.1%TFA) ataflowrateof0.3ml/min(left)and authentic annelid peptide. C-terminal-free CARP did not
oofn0t.h2eMcatNiaoCnl-e(x1c0hamngMeSphPo-s5pPhWatceolbuufmfnerw,ipthHa7n.1i)soactraatifcleolwutriaoten cshoomwmoannyCe-ftfeercmtienvaelnfratag1m0e^n5tMo.ftheMemto-sLtemuo-lAlrugsc-aLneum-yNoHm2o,-a
of0.5ml/min (right). dulin-CARP-family peptides, showed a considerable effect,
Table 1. Myomodulin-CARP-family peptides
Phyla Animals Structures References
Annelida Perinereis AMGMLRMamide this study
Mollusca Mytilus AMPMLRLamide Hirata et al. (1987)
Aplysia PMSMLRLamide Cropper et al. (1987)
GSYRMMRLamide Cropper et al. (1991)
Fusinus PMSMLRLamide Kanda et al. (1990)
PMNMLRLamide Kanda et al. (1990)
Helix PMSMLRLamide Ikeda et al. (1993)
SLGMLRLamide Ikeda et al. (1993)
GLNMLRLamide Ikeda et al. (1993)
pQLSMLRLamide Ikeda et al. (1993)
pQLPMLRLamide Ikeda et al. (1993)
Achatina SLGMLRLamide Ikeda et al. (unpublished)
GLHMLRLamide Ikeda et al. (unpublished)
pQ, pyroglutamic acid.
36 T. Takahashi, O. Matsushima et al.
Table2. Contractile effects of myomodulin-CARP-family
peptidesandsyntheticanaloguesonthe isolatedesophagusof
Perinereis vancaurica
Concentrations (M)
Peptides 10_8 10_7 10-6 10-5 10"6M AMGMLRMa
Myomodulin-CARP-family peptides
AMGMLRMamide + ++ +++ NT
AMPMLRLamide NT - ++ +++ 0.5 g
PMSMLRLamide - + +++ NT
GSYRMMRLamide - + ++ +++ 10"6M AMPMLRLa 2 min
PMNMLRLamide NT - ++ +++ Fig.4. Actions of 10~6M AMGMLRMamide (Pev-myomodulin)
SLGMLRLamide - + ++ +++ and 10-6MAMPMLRLamide (CARP)ontheisolatedesopha-
GLNMLRLamide - + ++ +++ gus ofP. vancaurica. A, Pev-myomodulin. B, CARP.
pQLSMLRLamide + ++ +++
pQLPMLRLamide - +++
GLHMLRLamide + ++ +++ phasic contraction of the ABRM were also examined, and
compared with those of CARP. The effects of 10-6M of
Analogue peptides ABRM
AMPMLRMamide ++ +++ NT these peptides on a preparation of the are shown in
PMSMLRMamide - ++ +++ NT Figure 5. The actions ofCARP and Pev-myomodulin (10~6
AMGMLRLamide NT - - ++ M) on the phasic contractions were apparently opposite.
AMPMLRFamide NT NT - + However, those actions were found to be dose-dependent
PMSMLRFamide - - + ++ (Fig. 6), indicating that actions of these peptides on the
AMPMLRL NT NT NT - phasic contractions were not qualitatively different. Dose-
MLRLamide NT + ++ +++ response relationships of these peptides showed that CARP
WLRLamide NT - - +++ wasmuchmorepotentthanPev-myomodulininexertingboth
LRLamide NT - - - modulatory effects on the phasic contraction and relaxing
effects on the catch tension (Fig. 6).
—+,+n+o,esftfreoctn.gefNfeTc,t.not++tes,tmedo.derpaQt,eepfyfercotg.lut+a,miwceaackide.ffect.
DISCUSSION
-5M
but Leu-Arg-Leu-NH2 did not show any effect at 10 or The novel heptapeptide Pev-myomodulin isolated from
lower. Replacement of the Met residue of Met-Leu-Arg- the annelid, P. vancaurica, in the current study is apparently
Leu-NH2with amore hydrophobicresidueTrpdecreasedthe a member of myomodulin-CARP family. The members of
contractile potency. the myomodulin-CARP family have so far been identified
The effects of Pev-myomodulin on catch tension and only in molluscs (Table 1). Thus, this is the first report on
I L 5g
^_ L_
1CT6M AMPMLRLa 1(T6M AMGMLRMa
2 min
2.5 g
10 4M Ach 5M AMPMLRLa Ach 1CT6M AMGMLRMa
Fig. 5. EffectsofAMPMLRLamide (CARP) and AMGMLRMamide (Pev-myomodulin) on phasiccontractions (A,. A:) in
r1e0s"p4onMseAtCohr.epetitiveelectricalstimulation (15V,3msec, 10Hz,50pulses)andoncatchcontractions(B,. B:)inducedby
Myomodulin-CARP-related Annelid Peptide 37
100
80
200
60
*-> 150
o
O
0X3
c
~CD 100 Tas) 40
cc
03
0)
Q_
20
10" 10" io- 10" 10" 10'
Concentration (M)
Fig. 6. Dose-responserelationshipsofAMPMLRLamide (CARP) and AMGMLRMamide (Pev-myomodulin) formodulat-
ingactionsonthephasiccontractions(left)andforrelaxingactionsonthecatchcontractions(right)oftheMytilusABRM.
the occurrence of a member of the family in the Annelida. Leu-OH, does not show anycontractile effecton the esopha-
Since Pev-myomodulin showed a potent contractile effect on gus, suggesting that the C-terminal amide is essential for
theesophagus ofP. vancaurica, this peptidemaybe involved exertion ofthe contractile effects ofthe myomodulin-CARP-
in the regulation of the esophagus of the animal. We have family peptides. It has been reported that the C-terminal
isolated previously two S-Iamide peptides from P. vancauri- amide is essential for catch-relaxing action of CARP on the
ABRM
ca, both of which elicit spontaneous contraction of the ofM. edulis[6]. Anotherexampleforessentialityof
esophagus of the same animal as did Pev-myomodulin [16]. C-terminal amide for bioactivity is the RPCH-related pep-
It is probable that some other peptides, which affect gut tide, APGWamide, which has been isolated from ganglia of
motility, maybefoundinP. vancaurica, andinotherannelids the gastropod mollusc Fusinusferrugineus [15]. On electri-
as well, in future. cally-elicited phasic contractions of the Mytilus ABRM, the
A conspicuous difference in structure betweenthe anne- C-terminal dipeptide fragment of APGWamide, GWamide,
lid peptide and the molluscan peptides is at their C-termini. shows a comparable activity to the native tetrapeptide, while
The annelid peptide has a Met-NH2 at its C-terminus, while C-terminal-free dipeptide, GW, does not show any activity
all the molluscan peptides have a Leu-NH2. Among the [17]. Therefore, the C-terminal amide of small bioactive
myomodulin-CARP-family peptides including Pev- peptides having amidated C-termini may be generally essen-
myomodulin, the annelid peptide shows the most potent tial for exertion of their bioactivity.
contractile effect on the esophagus of P. vancaurica. The The tetrapeptide fragment, Met-Leu-Arg-Leu-NH2,
C-terminal Met-NH2ofthe peptide seems to be important in shows a considerable effect on the esophagus, but the
eliciting the potent effect. This notion is supported by the tripeptide fragment, Leu-Arg-Leu-NH2, does not show any
facts that Leu7-Pev-myomodulin is far less potent than Pev- effect. These facts suggest that the C-terminal tetrapeptide
myomodulinandthatMet7-CARPandMet7-myomodulinare sequence istheminimum structure requiredforexpression of
more potent than CARP and myomodulin, respectively. the contractile effect of the myomodulin-CARP-family pep-
The synthetic analogues Phe7-CARP and Phe7- tides on the esophagus, though the Met residue can be
myomodulin show less potent effects than CARP and substituted at least with Trp.
myomodulin, respectively. This may be partly due to IthasbeenshownthatCARPpotentiatesphasiccontrac-
FMRFamide-likeactionsofthe analoguepeptides, becauseit tion of the ABRM of M. edulis in response to repetitive
hasbeenknownthatFMRFamidehasaninhibitoryactionon electrical stimulation at lower doses and inhibits at higher
the esophagus of a polychaete annelid, N. virens [14]. doses [7, 18]. The annelid peptide Pev-myomodulin also
FMRFamide has been shown to be present in some annelids shows a potentiating effect at lower doses and an inhibitory
[1, 4, 13]. effect at higher doses on phasic contraction of the ABRM.
C-terminus-free CARP, Ala-Met-Pro-Met-Leu-Arg- However,CARPexertstheseeffectsatabout 100timeslower
38 T. Takahashi, O. Matsushima et al.
concentrations than Pev-myomodulin. As shown in Figure Effects of the catch-relaxing peptide on molluscan muscles.
5A, therefore, 10~6M CARP inhibits the phasic contraction Comp Biochem Physiol 92C: 283-288
while 1CT6M Pev-myomodulin potentiates it. The reason 8 HMiurnaetaokTa, KYub(1o9t8a7)I,TCaaktacbha-trealkaexiIn,gKpaewpathiadreaisAo,laStehdifmraommotMoytNi,-
why the myomodulin-CARP-family peptides show such dual
lus pedal ganglia. Brain Res 422: 374-376
actionsisobscure atpresent. The annelidpeptide, aswellas 9 Holman GR, Nachman RJ, Wright MS, Schoofs L, Hayes TK,
CARP, relaxes catch tension of the ABRM. However, the DeLoof A (1991) Insect myotropic peptides. Isolation,
annelid peptide is approximately 10 times less potent than structuralcharacterization, and biological activities. In "Insect
CARP. In the ABRM, therefore, the authentic peptide Neuropeptides. Chemistry, Biology and Action" Ed by JJ
CARP is more potent than the foreigh peptide Pev- WMaesnhni,ngtToJn,KeDlCl.y,ppEP40-M5a0sler, American Chemical Society,
myomodulin.
10 Ikeda T, Minakata H, Fujita T, Muneoka Y, KissT, Hiripi L.
In conclusion, we isolated anovel memberofmyomodu- NomotoK(1993) NeuropeptidesisolatedfromHelixpomatia.
lin-CARPfamily from the marine polychaete, P. vancaurica. I. Peptides related to MIP, buccalin, myomodulin-CARP and
It has been shown that S-Iamide-family peptides [16] and SCP. In "Peptide Chemistry 1992" Ed by N Yanaihara,
mFoMlRlFuasmcsidbeu-tfaalmsioliyn paenpnetliiddess.[1,A4n,ne13l]idasrespereemsetnothnaovteonmlayniyn 11 EKaSnCdOaMT,SKcuireonkceiYPu,bKliusbhoetras BI,VM,unppeo5k7a6-Y5,78KobayashiM(1990)
Neuropeptides isolated from the ganglia ofa prosobranch mol-
neuropeptides closely related to molluscan neuropeptides. lusc, Fusinusferrugineus. In "Peptide Chemistry 1989" Ed by
N Yanaihara, Protein Research Foundation, Osaka, pp39-44
ACKNOWLEDGMENTS 12 Keller R(1992) Crustacean neuropeptides: structure, function
and comparative aspects. Experientia48: 439-448
The authorswish to expresstheirsincerethanksto ProfessorY. 13 Krajniak KG, Price DA (1990) Authentic FMRFamide is
Muneoka,HiroshimaUniversity,forhisvaluablecommentsthrough- present in the polychaete Nereis virens. Peptides 11: 75-77
out the present study and critical review ofthe manuscript. 14 Krajniak KG, Greenberg MJ (1992) The localization of
FMRFamide in the nervous and somatic tissuesofNereis virens
and its effects upon the isolated esophagus. Comp Biochem
REFERENCES Physiol 101C: 93-100
15 Kuroki Y, KandaT, Kubota I, Fujisawa Y, Ikeda T, Miura A.
1 Baratt B, Gras-Masse H, Ricart G, BuletP, Dhainaut-Courtois Minamitake Y, Muneoka Y (1990) A molluscan neuropeptide
N (1991) Isolation and characterization ofauthentic Phe-Met- related to the crustacean hormone, RPCH. Biochem Biophys
Arg-Phe-NH2 and the novel Phe-Thr-Arg-Phe-NH2 peptide Res Commun 167: 273-279
from Nereis diversicolor. Eur J Biochem 198: 627-633 16 MatsushimaO,TakahashiT,MorishitaF,FujimotoM,IkedaT.
2 CKrRop(p1e9r87E)C,MTyeonmeondbualuinm:R,aKboilokacstMivAeGn,euKrouppefpetrimdaenpnreI,seWnetisisn AKuKbSoGtFaVRI,IaNomsiedeT,anMdikViSSWFVR(I19a9m3i)deT,wiosolSa-tIeadmfirdoempaenptaindnees-,
an identifiedcholinergic buccalmotorneuron ofAplysia. Proc lid, Perinereis vancaurica. Biol Bull 184: 216-222
Natl Acad Sci USA 84: 5483-5486 17 Minakata H, Kuroki Y, Ikeda T, Fujisawa Y, Nomoto K,
3 CMrAoGpp,erRoEsCe,n SV,ilKiumpfFeSr,maAnlenviIz,oWseiAs,s TKeRne(n19b9a1u)m SRt,rucKtoulrkes, AKuPbGoWtaamIi,dMeunaendokraelYated(19c9o1m)pouEfnfdesctsonofmotlhleusnceaunrompuespctliedse
bioactivity, and cellular localization of myomodulin B: a novel —GWamideshowspotentmodulatoryeffects. CompBiochem
Aplysia peptide. Peptides 12: 683-690 Physiol 100C: 565-571
4 Evans BD, Pohl J, Kartsonis NA, Calabrese RL (1991) Iden- 18 Muneoka Y, Kobayashi M (1992) Comparative aspects of
tification of RFamide neuropeptides in the medicinal leech. structure and action of molluscan neuropeptides. Experientia
Peptides 12: 897-908 48: 448-456
5 Greenberg MJ, Price DA (1992) Relationships among the 19 Muneoka Y, TwarogBM (1977) Lanthanum blockofcontrac-
9F2M"RFEadmibdye-JliJkooesep,eptRidMes.BuijIins,"PFrJogHreTsisldienrsB,raEilnseRveiseerarScchieVnocle tmioolnluasncdanofcraetlcahxamtuisocnlei.n reJspPohnasremtaocsEerxoptoTnhienra2n0d2:do6p0a1m-i6n0e9in
Publishers BV, pp25-37 20 Penzlin H (1989) Neuropeptides—occurrence and function in
6 HirataT,KubotaI, ImadaM,MuneokaY(1989) Pharmacolo- insects. Naturwissenschaften 76: 243-252
gy of relaxing response of Mytilus smooth muscle to the catch- 21 Walker RJ (1992) Neuroactive peptides with an RFamide or
relaxing peptide. Comp Biochem Physiol 92C: 289-295 Famide carboxyl terminal. Comp Biochem Physiol 102C: 213-
7 HirataT, KubotaI, ImadaM, MuneokaY, KobayashiM (1989) 222