Modulation of Crayfish Hearts by FMRFamide-related Peptides

8 Pages·1992·3.5 MB·English

by  A J Mercier

Reference: Biol. Bull 182: 333-340. (June. 1992)
Modulation of Crayfish Hearts
FMRFamide-related
by Peptides
A. JOFFRE MERCIER1 AND RUNE T. RUSSENES
Department oj Biological Sciences. Brock University. St. Catharines. Ontario L2S 3A1 Canada
Abstract. Thepresent studyexamineseffectsofFMRF- 1987; Li and Calabrese. 1987; Elphick el al.. 1989: Robb
amide-related peptides(FaRPs)oncrayfish heart. Lobster et al.. 1989; Krajniak and Price, 1990). It is now recog-
peptides F, (TNRNFLRFamide) and F2 (SDRNFLRF- nized that FaRPs represent membersofaverylarge family
amide) increase the rate and amplitude of heart beat in ofneuropeptidesthatiswidelydistributed throughoutthe
hearts isolated from Procambarus clarkii. Thresholds for animal kingdom (Greenberg and Price, 1983; Price and
M
these effects were betweenM10~' and 1CT9 for F2 and Greenberg. 1989).
between 1(T9 and 10~8 for F,. FMRFamide and In crustaceans. FMRFamide-like immunoreactivity
FLRFamide elicited similar cardioexcitatory effects, but (FLI) has been found throughout most of the nervous
at thresholds ofapproximately 1CT7M. Thus, the amino- system, but the highest amounts are present in the peri-
terminal extensions "TNRN" and "SDRN" enhance the cardial organs (POs) (Koberski etal., 1987; Marder etal.,
excitatory actions of FMRFamide and FLRFamide. 1987; Krajniak, 1991: Mercierel al.. 199Ib). Becausethe
SchistoFLRFamide (PDVDHVFLRFamide) and leu- POs are located in the pericardia! sinus just outside the
comyosuppressin (pQDVDHVFLRFamide) markedly heart (Maynard, 1960). and because they release car-
dMecrease the rate ofcardiac contractions at 1CT9 to 10~8 dioactive hormones (e.g.. Cooke and Sullivan. 1982;
and can suppress the cardiac rhythm for one minute Kravitz elal.. 1980), the heart islikelytobean important
or more at 1CT7 M. The amino-terminal extensions of target forcrustacean FaRPs. So far, onlytwo FaRPs have
these two peptides, therefore, are necessary for inhibition been sequenced and identified in extracts ofthe lobster
of heart rate. Both ofthese peptides cause an initial re- POs, although other FMRFamide-like immunoreactive
duction in contraction amplitude, but contractions sub- material ispresent(Trimmeretal.. 1987). Thesetwopep-
sequently increase in the presence ofSchistoFLRFamide. tides have the sequences TNRNFLRFamide (F,) and
Thus, crayfish heartsare sensitiveto several FMRFamide- SDRNFLRFamide (F:). Both ofthese peptidesexciteiso-
related peptides, but the sites and mechanisms ofaction lated hearts of lobsters (Kravitz et al.. 1987) and blue
remain to be determined. crabs (Krajniak. 1991). However, the effects ofthese and
other FaRPs on crustacean hearts have not been thor-
Introduction oughly investigated.
The primary aim ofthe present study was to examine
Since the discovery ofthe neuropeptide FMRFamide in greater detail the cardio-regulatory effects of lobster
(caPlhlies-tMaenti-mAbrogs-aP(hPer-iNcHe:a)nidnGtrheeenbbievragl.ve19m7o7l)l.uFskMRMFacarmo-- spteupdtiieddesoFn,iasnodlatFe:d. Tcrhaeyfeifsfhechtesaortfst.heTsoe tewxoampienpetidtehsewreelrae-
ide-related peptides (FaRPs) have been reported in nu- tionship between amino acid sequence and biological ac-
merous invertebrate and vertebrate species (e.g.. Boer et tivity, the effects ofF, and F2 were compared with those
ai, 1980; Dockray et al, 1983; Watson el al., 1984: of FMRFamide. FLRFamide. and two FaRPs with sig-
Grimmelikhuijzen and Graff, 1985; Lehman and Price. nificantly different amino-terminal extensions: leuco-
myosuppressin (LMS). with the sequence pQDVDHV-
R1eTcoeiwvhedom16cSoerrpetsepmobnedren1c9e91s;hoauclcdepbteedad2d6reMsaserdc.h 1992. aFmLiRdFeam(iScdhe). (wHiothlmtahne seleqauie.nc1e98P6D)VaDnHdVSFcLhRisFtaomFLiRdFe-
333
334 A J. MERCIER AND R. T RUSSENES
(Robb el al.. 1989). The crayfish heart was sensitive to all effectseach time. The results reported herewere obtained
ofthe compounds tested. from atotal of37 preparations. In all butsix experiments,
each peptide was tested on one preparation only. The
Materials and Methods number of preparations used for testing each peptide is
indicated in the figure legends.
Crayfish were obtained commercially and were main- SchistoFLRFamide and leucomyosuppressin were ob-
tained in aerated freshwater tanks at 14.5C on a mixed tained from Peninsula Laboratories Ltd. (Belmont, Cal-
vegetable diet. ifornia). Peptides F[ and F2 were synthesized by Dr. D.
Synthetic peptideswere applied to spontaneously active McCormack (Rochester, Minnesota) and wereagift from
crayfish hearts. Thedorsal carapace, containing the heart Dr. M. Schiebe. FMRFamide, FLRFamide, and all other
and pericardium, was dissected from crayfish weighing chemicals were obtained from Sigma Chemical Co. (St.
approximately 3 gand waspinned toa Sylgard-lined dish Louis, Missouri).
with the ventral side up. The pericardial membrane was In each case, the error value reported is the standard
severed and pinned at each side to allow thebathing fluid error of the mean. Statistical significance of differences
access to the heart. The recording chamber, which had a between mean values was determined using a Student's t
volume of0.5 ml, was perfused with crayfish saline (van Test (Furguson, 1971).
Harraveld, 1936). which had the following constituents
(in mM): Na+, 205; Cr, 232; K+, 5.3; Ca++, 13.5; Mg++, Results
2.5; HEPES, 5.0 (pH 7.4). Saline was added to the cham-
berata rate of3.0 ml min^1 usinga peristaltic pump and Ofthe six neuropeptides tested, fourelicited responses
was removed at the otherend ofthechamber by suction. that were exclusively cardio-excitatory. FMRFamide,
The entire preparation was superfused continuously in FLRFamide, F, , and F: increased both the rate and am-
this manner. The temperature was maintained at 14- plitude of contractions of isolated Procambarus hearts.
16C, butdid not vary by more than 1Cduringany one Figure 1 shows representative examples ofthe effects of
experiment. Heart preparations were viable for up to 8 h. these four peptides. at doses that elicited approximately
Contractions were recorded by connecting the sternal equivalent responses. Theonset ofsuch responses usually
artery to a tension transducer using two insect pins that
were hooked at one end and glued to the transducer at
the other end. The artery and heart were stretched until
the maximum contraction amplitudewasobtained. Con-
tractionsweredisplayed on aGrass Model 7B Polygraph.
The rate and amplitude ofcontractions were measured 10-9 M F2
manually over intervals ofeither 30 s or 1 min.
Peptides were applied by changing the perfusate to a
solution containing a selected peptide concentration.
Peptides were present in the bathing chamber for 8-10
min; 10 min waschosen arbitrarily asthe maximum time 108 M
ofexposure. In a few cases, it was obvious that the max-
imal effect ofthepeptide hadalreadyoccurred, andwash-
out was begun after 8 min. The maximal effect of the
peptides generally occurred well before this time, except
forthe increased amplitude caused by SchistoFLRFamide
107M FLRFa
(see Results).
During washout, the effects of the peptides began to
subside within 5 min and had completelyworn offwithin
20-30 min. In many cases (e.g.. Fig. 7), the effects wore
offmore rapidly. Each peptide wastested by startingwith
the lowest concentration (10~': M) and subsequently in- 10-6M FMRFa
creasing the dosage in 10-fold increments until the entire Figure 1. EffectsofexcitatoryFaRPsoncrayfishheartcontractions.
response range had been tested. Successive doses were Eachpanelshowschartrecordingsofspontaneouscontractions. Peptides.
always given after the previous dose was completelMy adturtihnegctohencpeenrtiroadtsioinnsdiciantdeidcabtyedt,hewethrieckprheosreinztontinaltbhaersb.aTthhiengrecsoorldutiinogns
washed out. Three successive applications of 1 X 10~9 were obtained from different preparations. (Abbreviations: FLRFa for
F: onto the same preparation yielded virtually identical FLRFamide, FMRFa forFMRFamide, F, and F2asin text.)
FARPS MODULATE CRAYFISH HEART 335
occurred within 60-90 s after the peptide entered the
bathing chamber, and the lag-time was shorter at higher
peptideconcentrations. Theeffectson heartrateandcon-
traction amplitude were always completely reversed by
20-30 min ofwashing in normal saline (data not shown).
Noneofthese fourpeptideselicitedany inhibitoryeffects.
Log dose-response curves, constructed for the fourex-
citatory peptides (Fig. 2), were based on the responses of
five to six preparations in each case. Responses were ex-
pressed as the percentage change in contraction rate and -12 -11 -10 -9 -8 -7 -6 -5
the percentage change in amplitude by comparing the log [peptide cone (M)]
maximal effect ofeach peptide with the average contrac-
tion rate, orcontraction amplitude, during the 3 min pe- B 350l
300
riod immediately precedingpeptide application. Peptides
F, and F2 caused a more pronounced increase in the am- 250"
200-
plitude of the contractions than on their rate. For
FLRFamide and FMRFamide, however, the percentage 150-
100-
change in amplitude was similar to the change in rate in
50-
each case.
o-
Differencesin the relativepotenciesofthepeptideswere
-50
more prominent for the effect on contraction amplitude -13 -12 -11 -10 -9 -8 -7 -6 -5 -4
(Fig. 2A) than for the effect on rate (Fig. 2B). A compar- log [peptide cone (M)]
ison ofthe effects on contraction amplitude gave the fol- Figure2. Log-dose versusresponsecurvesfortheeffectsofexcitatory
lowing results. F2 was the most potent peptide, with a FaRPs on the amplitude ofcardiac contractions (A) and on heart rate
threshold concentration between 10"M' and 10~9 M. MA (B). The percentage change in rate or in amplitude wasdetermined by
comparison of the effects of 10~9 F2 and 10~5 comparingthe maximum valueobtained in the presenceofthepeptide
FMRFamide suggeststhat F2wasupto 10,000times more cfartoimon:theaverage valueduringthe three minutespriorto peptideappli-
potent than FMRFamide. F, was the next most potent
peptide, with a threshold between 10~9 and 10~8 M, and % change = [(peak value - initial valuel/initial value] x 100.
was approximately 1000 times more potent than Each point represents the mean value for five preparations in the case
MFMRFamide (based on the effects of 10~8 MF, and 10~5 ofF3and forsixpreparationsinallothercases. Errorbarsdepictstandard
FMRFamide). FLRFamide was about 10 times more errorsofthe means. (Abbreviationsareasin Fig. 1.)
M
potent than FMRFamide (based on the effects of 10~7
M
FLRFamideand 10~6 FMRFamide), butthethreshold
concentrations forboth appeared to be between 10 8 and contractions resumed at a rate that was lowerthan before
10~7M. FMRFamidegavearelatively broadlog-dose ver- peptide application (Figs. 3, 4, and 6A). Dose-response
sus response curve, which rose steadily over the concen- curves, based on the maximal reduction in rate, were
tration range of 10~8 to 10~5 M, while the other peptides markedly similar for these two peptides (Fig. 5A). The
appeared to reach saturation within slightly narrower threshold for inhibition of heart rate was between 10~'
concentration ranges. and 10~9 M.
Differences in relative potency were not as marked Effects ofSch and LMS on the amplitude ofcontrac-
when comparing the effects ofthe excitatory peptides on tionswere more complex. Formost preparations, such as
contraction rate (Fig. 2B). F2 and F, had approximately theone represented in Figure4,thecontractionsthat per-
equivalent effects on heart rate, butboth compoundswere sisted in the presence ofeither Sch or LMS were initially
about 100 times more potent than FLRFamide and reduced in amplitude. This type ofeffect was observed in
FMRFamide. The log-dose versus response curves for 4 of6 preparations exposed to Sch and in 6 of8 prepa-
FLRFamide and FMRFamide were very similar. rations exposed to LMS. Figure 3 is an example of re-
SchistoFLRFamide (Sell) and leucomyosuppressin cordings from a preparation in which no substantial re-
(LMS) had inhibitory effects oMn cardiac contractions at duction in contraction amplitude occurred during expo-
concentrations of 10~9 to 10~7 (Figs. 3-5). The rate of sure to LMS. When observed, reductions in contraction
spontaneous contractions was reduced consistently by size were usually transient. Approximately 2-4 min after
M
both peptides. At 10 7 M. contractions were completely peptide exposure began. 10 7 Sch caused a substantial
suppressed foraperiod lasting 1 min orlonger, afterwhich increase in contraction sizeabovethelevel observed prior
336 A. J. MERCIER AND R. T. RUSSENES
The increase in contraction amplitude caused by Sch
developed more slowly than did the reduction in con-
10-9 M LMS tractionMrate. Figure6Ashowsthetimecourseoftheeffects
of 1(T8 Sch foran individual preparation. In thiscase,
heart rate began to decline within 2 min ofpeptide ex-
posure and reached its lowest value 6 min later. Contrac-
tion amplitude, however, did notbegin torise until 6 min
after the peptide entered the bath and required an addi-
10-8 M LMS wtiiotnhalsi8x mprienpatroatrieoancsheixtspomsaexdimtoal10l~ev7elM. ISnche,xptehreimmeenatns
time forthe maximal inhibition ofheart rate (1.3 0.36
min) was shorter than the mean time for the maximal
increase in amplitude(10.9 2.0 min), andthedifference
in means was statistically significant (t = 5.56; P< 0.01).
In contrast, the increased amplitude caused by F2 oc-
curred rapidly and usually coincided with the maximal
increase in rate, as in the example shown in Figure 6B.
Mean times for the maximal rate (2.7 0.68 min) and
10-7 M LMS animfpilcianttuldyed(i3f.fe1rent0.(9t9=) f0o.r93f;ivPe>pre0p.a4)r.atAisonisnwtehreeenxoatmpslige-
JJJjJJjJJJJJJJJM shown in Figure 6B, the heart rate often declined rapidly
from the peak value, even though F2 was still present in
the bathing solution. Contraction amplitude, however,
1 1 rtiN remained elevated until the peptide was removed.
20 s As a first step toward examining potential interactions
chaFritgurreeco3r.dingEsffoefctsspoofntLaMneSouosncahredaritacccoonnttrraaccttiioonnss..ELacMhSpwaanselprsehsoewnst bsteutdwieede.n WFaeRPwse,rethepaerftfieccutlaorflaymiinxtetruerseteodfiFni daentdeSrmcihnwiansg
inthebathingsolutionduringtheperiodsindicatedbythethickhorizontal
bars at the concentrations indicated. The recordings were all from the
same preparation.
10-9 M Sch
to peptide exposure (Fig. 4). Thus, the effect of Sch on
contraction amplitude appeared to be biphasic.
Log-dose versus response curves forthe initial effect of
LMS and Sch on contraction amplitude were obtained
by comparing responses 1 min after the peptide entered
the bath with the average amplitude overa 2-min period
prior to peptide exposure (Fig. 5B). (The change in con- 10-8 M Sch
traction size was expressed as a percentage ofthe ampli-
tude during the period prior to peptide application.) The
M
dramatic reduction in contraction size at 1(T7 wasdue
mainly to the fact that this dose completely suppressed
contractions in most preparations. Log-dose versus re-
sponse curvesforthe increase in amplitude that occurred
laterwereobtainedbycomparingtheaveragecontraction
amplitude during the 2-min period before peptide appli- 10-7 M Sch
|2mN
cation with the highest average amplitude over a 1-min
20 s
period during exposure to the peptide or during the first
ti5nramc1it(niTo7onfMatmhpSelcwiha.tsuIhdn-eoscuodtnotprueabsrltie,odd1(i0Fnig71.0M~5C8)LM.MOSSncchaavueasrneaddgeto,rniclpoylne-ad cbinhaartFrshitegautbrreatecthohe4ri.dncigonnsgEcosflefunoettcfirtosasntpoidofounnrtSsiacnnhigenotdohiunecsapthceeeardari.rdotidTashccoiennctdorrineactccarottariecdodtinibnsoy.gnsst.EhwaeeSctrchhehicpawklalanhseoflrrpiorzsmeohsnoettwanhslte
25% increase in contraction size. same preparation.
FARPS MODULATE CRAYFISH HEART 337
A 3- 1 50
I 25=-
-20 1
30-
-40"
20- 075"
-60-
CC 050 a.
-80 E
025<
I
-100 Sch 000
-13 -12 -11 -10
log [peptide cone (M)) 10 Time20(min) 30 40
B 20-
0- B -200
-20- -1 75
-40- -1 50
-60- -1 25
0> 40-
-80- ra 1.00
CC
ci. -100 c 30- 0.75
< -13 -12 -11 -10 -9 -8 -7
-0,50
log [peptide cone (M)]
025
300-1 10 15 20 25 30 35
Time (min)
200- Figure 6. Time course forthe effects ofSch (A) and F: (B) on the
rate and amplitude ofcardiac contractions. The pMeptides were present
a. in the bathingsolution atconcentrations of 1CT8 duringthe periods
< o- indicatedbythehorizontalbars.Thedatafor(A)and(B)wereobtained
from thesameexperimental preparation.
S -100
-12 -11 -10 -9 -8 -7
log [peplide cone (M)]
in each case. Sch always antagonized the chronotropic
pSecphFtiiogdnuereheexa5p.rotsurLaroteeg-((dBA)o)s,aeonndvetrohsneustahrmeepslmpiaotnxusdieemcuoufmrvceoasnmtprfloairctteuifdofenecstosaffotcfeornLtIrMamScitniaonnodsf cefhfreocntootfroFp2icbaultlynoatnditsiniontortorpoipciaclleyffeexctc.itTahtoursy,,wahnidleScFh: iiss
duringpeptideexposureorinthefirst 5 minofthewash-outperiod(C). chronotropically inhibitory, a mixture ofthese FaRPs can
The change in rate or in amplitude was determined by comparing the produce a response that is predominantly inotropic.
value obtained in response to the peptide with the average value prior
to peptide application and was expressed as a percentage ofthe initial
valueasin Figure2. Effectsonheartrate(A)weredeterminedusingthe
minimum heartrateduringpeptideexposure. Eachpointrepresentsthe 30
meanvalueobtainedfromeightpreparationsexposedtoLMSandfrom
sixpreparationsexposedtoSch. Errorbarsdepictstandarderrorsofthe
whether the chronotropic action of one peptide might
predominate or, alternatively, whether the two substances Sch
might produce an "additive" response. Figure 7 illustrates 20n
the responses ofan individual preparation in which 10 s
M
M FSc:hcaduescerdeaaserdaphiedar6t0%r.atiencbryeaasbeouinth5e0a%r.t rAatemiaxntdur1e0of8
thetwopeptidesproduced aheart ratethatincreased only
slightly and was comparatively stable. Subsequent appli-
cation of F showed that the heart was capable of re-
: Sch
sponding to this peptide as before. Thus, these two pep-
50 100
tides exert chronotropic actions that are mutually antag- Time (min)
SoncihstdiicdannodttaenntdagtoonciaznecetlheeaefcfhecottohferFo:uotnwchoenntrcacotmiboinnesidz.e panFeilg)uraend7.ampTlhietuedffeec(tloofwearmpiaxnteulr)eooffcFar2daiancdcSocnhtroanctitohnes.ratTehe(updpaetra
and mayevenhavepotentiated it (Fig. 7). Thisexperiment were obtained from a single preparation. Peptides were present in the
M
was performed six times with qualitatively similar results bathingsolutionat IO"8 atthetimesindicatedbythehorizontalbars.
338 A. J. MERCIER AND R. T. RUSSENES
Discussion tide. Similar structure-activity relationships have been
reported forthelocustextensortibiae nerve-muscle prep-
FaRPs have been reported to modulate the activity of aration (Cuthbert and Evans, 1989) and for neuromus-
hearts from several types ofinvertebrates, including mol- cular synapses on crayfish abdominal extensor muscles
lusks (e.g.. Painter and Greenberg, 1982; Price et al, (Mercierrffl/.. 1990).
1990), insects (Cuthbert and Evatis, 1989; Robb et al.. The sensitivity ofhearts from Homarus(Kravitz et al.,
1989), leeches (Li and Calabrese, 1987), and crustaceans 1987), Callinectes (Krajniak, 1991), and Procambarnsto
(Kravitz et al., 1987; Krajniak, 1991). Some FaRPs elicit F, suggeststhatF, orclosely relatedpeptidesarecommon
responsesthataremainlyexcitatory, otherselicit responses cardioexcitatory hormones in decapods. F, and F2 were
that are primarily inhibitory, and others are reported to originally isolated from lobster POs(Trimmeretal.. 1987)
elicit mixed or biphasic responses (Cuthbert and Evans, and have not been positively identified in anyothertissues
1989). The type of response depends on the chemical todate. The POs ofProcambarnsclarkii. however, appear
structure of the peptide and on the invertebrate species to contain cardioexcitatory FaRPs that are very similar
(Painter and Greenberg, 1982). to F, and F2 (Mercier et al.. 1991a, b).
Thepresent studydid not investigate thesitesormech- The crayfish heart was also sensitive to Sch and LMS,
anisms ofaction ofthe various FaRPs tested. Changes in which have markedlydifferent N-terminal extensionsthan
rate are most likely to result from effects on the cardiac the other FaRPs studied. The primary effect ofSch and
ganglion, which sets the overall rhythm for cardiac con- of LMS appears to be a reduction in heart rate, which
tractions (Maynard, 1960). Changes in the amplitude of occurs at a threshold concentration between 10"I0 and
contractions could be due to changes in the number or 10~9 M for both peptides. Similar thresholds have been
frequency of nerve impulses per burst produced in the reported forinhibition oflocust hearts(Robbetal.. 1989)
cardiac motor neurons (Maynard, 1960), or they could andoviducts(Lange etal., 1991)bySch,andforinhibition
result from direct effects on the cardiac muscle cells, as ofcockroach hindguts (Holman et a/.. 1986) and locust
in Limulus (Watson el al., 1985). hearts (Cuthbert and Evans, 1989) by LMS. Because
It should be noted, however, that the POs, which con- FLRFamide excites the crayfish heart, the inhibitory ef-
tain several cardioexcitatory agents (e.g., Cooke and Sul- fects observed in the present study must be due to the
livan, 1983), were still present in the preparations used presence of the N-terminal extensions PDVDHV- and
for study. We cannot exclude the possibility that the ap- pQDVDHV- on Sch and LMS, respectively.
plied peptides act by inducing the release ofother agents In contrast, Krajniak (1991) has reported that LMS
from thePOs. Such hormone-induced releaseofhormones causes cardioexcitation in Callinectes. The effect was ob-
fromthesame neurosecretory organ hasnotbeen reported served in a single preparation and has a threshold 10,000
fIonraadndiytioofnt,hethpeeriinchairbdiitao!rysuebffsetcatnscoefsSacnhdasnedemosfLunMliSkelayr.e treifmleescthsipgehceirest-hdaenpethnadtenotfdFiIf.feSruecnhcesaninobtsheertvaartgieotntimsisugehst,
qualitatively similar to those reported in insect hearts, but this possibility requires further study.
which lack POs (Cuthbert and Evans, 1989; Robb et al.. Reduction in the amplitude of contractions was not
1989). observed asconsistently with crayfish hearts as with insect
F| and F: exert purely excitatory effects on hearts of preparations (Cuthbert and Evans, 1989; Lange el al.,
Procambarns, increasing both the rate and amplitude of 1991). The large increasein contraction sizeat higherSch
spontaneous contractions. F, and F2, respectively, are up concentrations was distinct from the inhibitory effect in
to 1,000 and 10,000 times more potent than FMRFamide that itdeveloped much more slowly. In addition, the large
when comparing inotropic responses. Similar results have increase in amplitude wasonly produced bySch, whereas
been obtained with isolated hearts ofthe blue crab, Cal- both Sch and LMScould inhibit heart rate. Thisindicates
linecles sapidus (Krajniak, 1991) and of the lobster, that Sch activatesa receptorthatisnotactivated by LMS.
Homarusamericanus(Kravitz et al., 1987). F, isalso 1000 This might seem surprising, in view ofthe high degree of
times more potent than FMRFamide in excitingthe semi- homology between thetwo peptides, which differby only
isolated heart ofthe locust. Schistocerca gregaria (Cuth- the N-terminal amino acid. It is possible, however, that
bert and Evans, 1989). Thus, arthropod hearts appear to atthe lowconcentrations, both peptidesactivatethesame
be more sensitive to N-terminally extended analogues of receptor or receptor class (one responsible for reducing
FLRFamide than to FMRFamide. FMRFamide and heartrate), andthatadifferentclassofreceptorisactivated
FLRFamide are both excitatory, but the addition ofthe at higher concentrations of Sch (causing the increase in
amino acid sequence thr-asn-arg-asn- or of ser-asp-arg- amplitude). This hypothesis is consistent with the high
asn- to the amino terminal of the tetrapeptide-phe-leu- similaritybetween the log-dose versusresponsecurves for
arg-phe-NH2 effectively increases the potency ofthe pep- the effects ofSch and LMS on contraction rate (Fig. 5A).
\RPS MODULATE CRAYFISH HEART 339
I
The reason for the delayed inotropic effect of Sch is Burnett, L. 1979. The respiratoryfunctionofhemocyanin inthespider
not clear. One possibility may be that the peptide is hy- crab Lihinia emarginala and the ghost crab Ocypode auadrala in
drolyzed beginning at the N-terminal end. This would Cookneo,rmI.oxMi.,aaannddRh.ypKo.xSiual.liJv.anE.xp1.98Z2o.ol.H2o1r0m:o2n8e9s-3a0n0d.neurosecretion.
gradually produce a peptide more closely resembling Pp.205-290in TheBiologyofCrustacea. D. E. Bliss, H. L.Atwood,
FLRFamide, which, at 10 7 M, caused a substantial in- and D. C. Sandeman.eds. Academic Press, NewYork.
crease in contraction size (Figs. 1, 2). The combination Cuthbert, B. A., and P. D. Evans. 1989. A comparison ofthe effects
of Sch with an excitatory FaRP (F2) was capable of in- ofFMRFamide-like peptideson locust heartandskeletal muscle.J.
creasingtheamplitudeofcontractionswithoutincreasing Exp. Biol 144: 395-415.
heart rate (Fig. 7). Thus, if hydrolysis were to cause an DockBraaryn,arGd.. J1.9,8J3..R.AReneovvee,lJarc.,tiJv.eSpcehnitvaepleyp,tiRd.eJ.frGoamytcohni,ckaennd bCr.aiSn.
accumulation ofan excitatory FaRP, the combination of identified byantibodiesto FMRFamide. Nature305: 328-330.
Sch anditsbreakdown productwould beexpectedtopro- Klphick, M. R., R. H. Emson, and M. C. Thorndyke. 1989. FMRF-
ducearesponsesimilartothatobserved afterseveral min- amide-like immunoreactivity in the nervous system ofthe starfish
utesofexposure to Sch (Figs. 4, 6A). Such an explanation, Asteriasnibens. Biol. Bull. Ill: 141-145.
howReevsepro,nsisesspetcoulmaitxivteu,reasndofotehxecristaatroeryposasnibdlei.nhibitory GFrueregMuncsboGenrr,ga,Gw.MA.Hi.lJl1.,,97Na1en.dwDYS.toarAtk.i.stPirPcipac.leA.1n4a16l9-8y13s5.i9s.inInPvseyrctheoblroatgeyanenudrEodpuecpattiidoens,:
agentsmaybeofsomephysiological significance. Increases nativeand naturalized. .-Imi. Rev. PhysioL 45: 271-288.
in cardiac output (the fluid volume ejected by the heart Grimmelikhuijzen, C. J. P., and D. Graff. 1986. Isolation of <Glu-
per minute) during physical activity (e.g., McMahon et Gly-Arg-Phe-NH: (AnthoRFamide), a neuropeptide from sea ane-
ul.. 1^79) and hypoxia (e.g.. Burnett, 1979; McMahon mones. Proc. Null. ACM/. Sa. USA 83:9817-9821.
and Wilkens. 1975) generally involve changes in both Ilolm.m, G. M., B.J.Cook, and R.J. Nachman. 1986. Isolation,pri-
heart rateand stroke volume (theamount offluidejected mroapreyptsitdreuctthuarteainnhdibsitysnthsepsoinstaofneloeuuscocmoynotsruapcptrieosnssionf,tahneicnoseccktronaecuh-
perbeat). In somecases, however, stroke volume increases hindgut. Comp. Bioehem. Physiol 85C: 324-333.
independentlyofheart rate(Taylor, 1976; McMahonand kobierski, L. A., B. S. Beltz, B. A. Trimmer, and E. A. Kravitz.
Wilkens, 1977; Taylor and Butler, 1978), and the mech- 1987. FMRFamide-like peptides ofHomarus americanus: distri-
anisms that underly such an effect are not known bution,immunocytochemical mapping,andultrastructurallocaliza-
(McMahon and Burnett, 1990). It istemptingtospeculate tion in terminal varicosities.J. Camp. Neural. 266: 1-15.
thata mixtureofpeptideslike F2and Sch, whichiscapable krajonfiaak,caKr.diGo.ac1t9i9v1e. FMTRhFeamiidednet-ifrieclataitoendanpdepsttirducetufrre-oamctitvhietyblrueleatciroanbs
ofincreasing the amplitude ofcontractions independently Callinectes sapidtix. Peptides 12: 1295-1302.
ofrate, might be involved. Tension recordings, however, krajniak,k.G.,andD.A.Price.1990. AuthenticFMRFamideispres-
do not provide a direct measure ofstroke volume or of entinthe polychaeteNereis virens. Peptides 11: 75-78.
cardiac output. In addition, crayfish POs do not contain kravitz, E. A., S. Glusman, R. M. Harris-Warrick, M. S. Livingstone,
Sch or LMS (Mercier et al. 1991b), and none ofthe neu- Tr.ohSocrhmwoanrze,sianndlobMs.terFs.:Gaocyti.on1s98o0n.neAurmoimnuesscualnadraprpeeppatriadteioanssnaenud-
rohormones isolated from POs to date decreases heart preliminary behavioural studies.J E\p. Biol. 89: 159-175.
rate. More research aimed at identifying neuropeptides kravitz, E. A., L. kobierski, B. A. Trimmer, and M. F. Goy.
and examining their effects may provide a better under- 1987. Peptide F,: a myoactive lobster peptide related to FMRF-
standing of how stroke volume and cardiac output are amide. Sot Xeurosci. Abslr 13: 1257.
Lange, A. B., 1.Orchard,and V.A.TeBrugge. 1991. Evidenceforthe
regulated.
involvementofaSchistoFLRF-amide-likepeptideintheneuralcon-
troloflocustoviduct.J. Comp. Physiol. A 168: 383-391.
Acknowledgments Lehman, H. k.,and D. A. Price. 1987. Localization ofFMRFamide-
like peptidesin thesnail Helixaspersa. J Exp. Biol 131: 37-53.
This work was supported by a grant to AJM from the Li,C.,and R. L.Calabrese. 1987. FMRFamide-likesubstancesinthe
Natural Sciences and Engineering Research Council of leech. III. Biochemical characterization and physiological effects.J.
Canada. Wethank PatQuigleyforassistanceinanalyzing Neurosa. 1: 595-603.
data for some of the experiments and Dr. Ian Orchard Marder, E., R. L. Calabrese, M. P. Nusbaum, and B. Trimmer.
for commenting on a draft of the manuscript. Dr. R. p1e9p8t7i.desDiinsttrhiebusttioomnataongdasptarritcianlecrhvaoruacstseyrisztaetmisonofotfhFeMrRoFcakmcirdabe.-lCiakne-
Baines also provided some helpful suggestions. cerborealis. and the spiny lobster, Paiui/iriis interruptus. J Comp.
Neural. 259: 150-163.
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