Table Of ContentReference: Bio/. Bull. 200: 344-350. (June 2001)
2+
5-Hydroxytryptamine Stimulates Net Ca Flux
in the Ventricular Muscle of a Mollusc (Busycon
canaliculatum} During Cardioexcitation
LEAH DEVLIN
C.
Department ofBiology, Penn State University, Abington College, Abington, Pennsylvania 19001
+
Abstract. Noninvasive, self-referencing calcium (Ca" ) with a primary effect on long-duration chronotropic regu-
electrodes were used to study the mechanisms by which lation. Underlying this potentiation in cardiac rhythmicity is
5-hydroxytryptamine (5-HT) affects net Ca2+ flux across an increase in the amplitude and frequency ofcardiac action
the sarcolemma of myocytes from ventricular trabeculae potentials as well as the coupled systolic force (Hill et al.,
(from a marine gastropod, Busycon canaliculatum). Treat- 1992). The combined anatomical, pharmacological, and
ment of isolated trabeculae with 5-HT causes a net Ca2+ physiological studies on the gastropods Aplysia, Busycon,
efflux, which is 30% blocked by verapamil. These findings and Lymnaea have provided overwhelming evidence that
suggestthat theefflux is in part the resultofapreviousCa2+ 5-HT acts as a cardioexcitatory neurotransmitter in this
influx through L-type Ca2+ channels and is due to a rapid class of mollusc.
Ca2+ extrusion mechanism inherent to the sarcolemma of 45Ca2+ efflux studies havebeenconductedon avarietyof
these myocytes. 5-HT-induced net Ca2+ efflux is also re- molluscan cardiac and smooth muscles to determine how
duced by about40% by treatmentwith a sodium (Na+)-free, Ca2+ ions may be mobilized by 5-HT during the process of
lithium Li+(-substituted saline, which shuts down the contraction or relaxation (Bloomquist and Curtis, 1972,
Na-Ca e(xchanger during Ca2+ extrusion. Cyclopiazonic 1975; Koch and Greenberg, 1981; Sawada etal., 1984; Ishii
acid (CPA). an inhibitorofthe sarcoplasmic reticulum (SR) et til., 1989). The present study uses a newer technique to
Ca2+ ATPase, almost completely abolishes the 5-HT-in- study Ca2+ flux, noninvasive self-referencing Ca2+ elec-
duced netCa2+ efflux, suggestingthat the SR ratherthan the trodes, to determine possible mechanisms by which 5-HT
extracellular pool is the primary Ca2+ reservoir serving affects trans-sarcolemmal net Ca2+ flux in the Busycon
5-HT-induced excitation. ventricle as a clue to understanding its role during the
excitation-contraction (E-C) coupling in the gastropod
Introduction heart. An earlierstudy focusedon the action ofFMRFamide
on net Ca2+ flux in the ventricle ofBusycon canaliculatum
The rhythmicity ofmolluscan cardiac muscle is regulated (Devlin, 1997), so we may be able to compare mechanisms
by numerous neurotransmitters and neuromodulatory working at putative Ca2+ pools accessed by 5-HT and
agents, the most widely studied ofthese being the biogenic FMRFamide to achieve the same end enhanced cardiac
amine 5-hydroxytryptamine (5-HT),thetetrapeptide FMRF- performance.
amide. and acetylcholine (ACh). Pharmacological applica-
tion of 5-HT onto the isolated hearts of the gastropods Materials and Methods
Busycon canaliculatum (Hill, 1958; Huddart and Hill,
1996), Lymnaea stagnalis (Bucket! et til., 1990), and Aply- Specimens of Busycon canaliculatum, the channeled
sia californica (Liebeswar et al., 1975) causes a dose- whelk, were obtained on the day of each experiment from
dependent increase in inotropic and chronotropic activity the Marine Resources Center ofthe Marine Biological Lab-
oratory (MBL). Woods Hole, Massachusetts. All experi-
Received 18 January 2000; accepted 17 February 2001. ments were conducted at the National Institutes of Health
E-mail: [email protected] BioCurrents Research Center located at the MBL.
344
S-HT-INDUCED CA:+ FLUX IN MOLI.USCAN VENTRICLE 345
Preparation the trabecula and was positioned at two distances from the
The shell was completely cut away from the animal with muscle surface. One electrode position was 500 ju-m away
from the muscle surface. At this relatively great distance,
bpeorniecafrodricuemp.s,Tehxepopseirnigcatrhdeiuhmeawrtasanodpeintesde,nvaenldopthiengveonptraiqculee Cdeat2ec+tefdl.uxThfurso,motnhleyitohne scoonutrrcoel,(ib.ea.c,ktghreoumnudsclleev)elcsaonfnoCta2b+e
watarsiurm.emTohveedvenbtyricsleevewraisngopitesnceodnnaencdtpiionnsnedtodtohwenaoirntaadainsdh einlectthreodsealpionseitiaorne wraescoorndleyd5at/ntmhifsropmositthieonm.usTclhee ssurefcaocned,
boefcunlatauer.alAsneaiwnadtievridtuoalextproasbeectuhlea iwnnaesr ilsaotltaitceedworwkitohfftirnae- sloemnmeat.CaF2r+omflutxhecobualcdkgbreoumnedasluerveedl doifreCctal2y+atiotnhse isnarctoh-e
scissors from the inner wall of the ventricle and secured bathing saline, the Ca2+ ion concentration gradient at the
with minuten pins to a Sylgard well in a small recordin+g muscle surface, and thediffusionconstant forCa2+ ions, net
chamber. The trabecula was bathed in a nominally Ca" Ca2+ ion flux was calculated using a modification of the
MfrBeeLarftoifrimciuallas(e4a2w3atmerM(NAaS+W)9.p7rempaMredK+acc9o.r9dminMg tCoa2th+e Pick equation. The mathematical formulas used in the con-
o5H1mC.i2tOt3emdM).frMMoamggn2t+ehes, i5Cu3a8m2.+6wa-msfrMueeseCA,dlS"tW,o.r2e7pTlhmaecMentohS,meiOn94.a~9l2m,(o2Mr.3bCamac2kM+-, aavrenerdsdiaeopsnpclroiifcbavetoidlotnbasygoeSfmttiohtefhlsueextlfuan-lir.tesf(,e1r9ae9nn9dc)i.ongtheelrecttercohdneictaelchansipqeucet,s
ground) amounts ofCa2+ ions still present in the Ca2+-free
ASW were typically 100 micromolar (/o,M) or less and were
monitored continuously with a Ca2+ electrode throughout Pharmacological agents
the experiment. The neurotransmitter, 5-HT, and the L-type channel
blocker, verapamil, were obtained from Sigma Chemical
Ionflux measurement Company (St. Louis, MO). Verapamil was selected over
The self-referencing ion electrode technique is used to other Ca2"1" blockers because of its consistent antagonistic
detect netion flux generated from abiological source, in this action on other invertebrate cardiac and smooth muscle
case, the sarcolemma of myocytes from a Busycon trabec- types (Devlin 1993a, b, 1997, Devlin and Smith, 1996).
ula. The net ion flux across the membrane is the sum ofboth Diltiazem or nifedipine. which often act as agonists in
inward and outward ion movements. In the present experi- molluscan muscle preparations, were not used in this study.
ments, electrodes loaded with a Ca2+-specific ionophore The Ca2+ channel agonist, Bay K 8644, and the sarco/
(Fluka ChemikaCa2+ ionophore cocktail A with the neu- endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor,
tral carrier ETH 1001) were used to detect net Ca2+ flux (in cyclopiazonic acid (CPA), were obtained from Research
pmol cm"2 s"1) across the sarcolemma of the myocytes. Biochemicals International (Natick, MA). At the beginning
The electrode was programmed with PC-based software of each experiment each drug was diluted in the nominal
(lonprobe) to oscillate with an excursion of 10 jam and a Ca2H"-free ASW to achieve the designated experimental
slow frequency of 0.3 Hz; this minimized mixing of the concentration.
bathing saline. The difference in voltage (;u,V) between the
two ends ofthe excursion was measured and could be taken
ascorresponding to aCa2+ concentration gradient, since the Experimentalprotocol
excursion was constant.
To construct the electrodes, borosilicate micropipettes The following general protocol was used throughout the
were pulled from 1.5-mm-diameter glass capillaries (World present experiments. An initial netCa2+ efflux generated by
Precision Instruments. Inc.), then back-filled with 100 mM 10 7 M 5-HT (the control) was recorded during a 3-min
CaCl2 in 0.1% agar. The pipette was front-filled with cal- treatment; this net Ca2+ efflux immediately returns to basal
cium ionophore cocktail A containing neutral carrierETH levels when the 5-HT is washed from the trabecula by
ASW
1001 (Fluka Chemika) to produce an ionophore column of flushing with for 20 to 30 min. Next, the trabecula
40 n.m. A Ag/AgCl wire inserted into the back of the was pretreated for 10 min with verapamil or Bay K 8644
micropipette served as the coupling to the headstage. To (10 " M) or other inhibitors (CPA or Li* ASW). 5-HT
calibrate the electrode priorto the experiment, the electrode (K)"7 M) dissolved in solutions ofverapamil orBay K 8644
was tested in 0.1 mM, 1 mM, and 10 mM Ca24 solutions to (10~5 M) or other inhibitors (CPA or Li+ ASW) was
check its Nernstian properties that is, an approximate 28 rcapplied to the trabecula for a 3-min treatment period. The
mV difference per decade change in Ca2+ concentration. control, the initial response to 5-HT alone, was then com-
The return electrode was a Ag/AgCl wire inserted into a pared to the response to 5-HT in the presence ofthe agonist,
glass capillary containing 3 M KC1 agar. antagonist, or inhibitor. A r-test analysis was then per-
The electrode oscillated at right angles to the long axis of formed.
346 C. L. DEVLIN
(5-HT-induced net efflux measured prior to verapamil treat-
ment) (Fig. 2). After verapamil was washed from the prep-
aration, subsequent exposures to 5-HT are often enhanced.
Bay K 8644 has no significant effect on the 5-HT response.
The effect ofa Nti+-free, Li+-substitutedASW on 5-HT
responses
To determine whether the net Ca2+ efflux was mediated
by the Na+-Ca2+ exchanger, sodium (Na+) ions were omit-
ted from the bathing saline and replaced instead with an
equivalent concentration (423 mM) of lithium (Li"1") ions;
the rationale is that Li+ transverses the Na+ channel but
ednfiefroFeeuiccsttgluyonrfeeatt51C-.taHheT2A+iononfnleunxsxeoptuemrrCecieaam,se2un+ttrhaeeelfmfterlrnueatxcboe(rctiudanlkasep,hnmoaowwtlihanecgndmia"sttch2aeonsnc~ece'le)en.octftrAralotSediseWsonwt-a=hdasenpsepp5nlodan/ect^neamt-d c(isaLnirnpeopdtuacnbededNsbiuygbgstlthiie,tup1tr9ee9d4s)fe.onrcTeNhaeof+5L-iiHn+Tt-ihioenndsNuatco+ed5-C7nae%2t+Caex92c+%heaf(nfngleu=rx
duringtreatmentwithanartificialCa2+-freeseawater(ASW).5-HT(10~lc 3, P < 0.01) that of control (the 5-HT response in normal
Mto 10~5 M) = net Ca2+ efflux measurement taken when the electrode ASW) (Fig. 3). implicating the Na+-Ca2+ exchanger as a
wdwuaarssinsrgteilctlorrepdalteamdceenbdtetdwiwiretehecntalyr1a0ant~gsethMoeft5arn-adbHeTc1uc0lo~an7caetMntar5adt-iiHsotTna.sn.cBeMaacokfxgilremosusunmtdhnae(ntBe5Gf)f(lxu=mx Lmie"c1"htarneiastmmenotf aClao2n"e1" rexetdruucseidonthdeurbiansgalexnceittaCtai"on+beyffl5u-xHTt.o
small oscillations around the baseline that were recorded when the elec- 69% 11% (H = 3. P <0.01).
trode was placed 500 /xni from the ion source, the trabecula.
The effect ofcyclopiazonic acid (CPA) on 5-HT responses
Results Figure4 showsthe effect ofSERCA inhibitorCPA (10~5
2+
M) on 5-HT-induced net Ca efflux. The 5-HT response is
With the electrode placed 5 ju,m from the muscle surface,
a basal net Ca2+ efflux of 1.21 0.42 pmol cm"2 s~' (n =
10) isrecordedfromthe trabeculaatrest inCa"+-free ASW.
M
A range of 5-HT concentrations (10~12 to 10~5 M) was
then tested. Above a threshold of about 10"I0 M, all con-
+
centrations of 5-HT enhance basal net Ca~ efflux. The
maximal efflux is induced at 5-HT between 10~s-10~7 M;
above 10~7 M, the net Ca2+ efflux is actually smaller than
those induced by lower 5-HT concentrations (Fig. 1). Thus,
the 5-HT receptor was desensitized by exposures to higher
doses ofits ligand. Because the effect of 10~7 M5-HTdoes
not desensitize the receptor and is completely reversible, it
waschosen as the concentration to be challengedby various
Ca2+ channel antagonists or agonists. A stable net Ca2+
M
e1f.f0l1uxpmionlduccemd~b2ys~1'0~(n7 = 58)-.HT is on the order of 2.63 CaF:i+guefrfelu2x.sigAnnale(xipnerpimmoelntcalm"re2cso~r'd)sihsoiwnihnigbittheadtbtyheth5e-HLT--tiynpeduccheadnnneelt
antagonist verapamil (10~5 M). ASW = spontaneous net Ca2* efflux
Effect ofCa2+-channel antagonists or agonists on 5-HT- measurement taken when the electrode was placed directly at the ion
induced net CcT+ efflux rsoeucrocred,edthperiotrratboecaunlya,drautgatrdeiasttmaennctewohfenlestshetthraanbe5cu;luam.waTshibsathseigdnaolnlwyaisn
To test the hypothesis that the Ca2+ ions mobilized by an artificial Ca2""-free seawater (ASW). 5-HT (1st) = net Ca2+ efflux
5I-sHtuTdideudritnhgeEef-fCecctosuopflitnwgoarLe-tfyrpoemCtahe2+excthraacnenlelluldarrusgasl,inaen. mdsueurraiesnmugernettmreetanatktmeentnatwkhewneintwhhteh1en0e~tlh7eecMterloe5dc-etHrwToa.dseVpwelarasacpepadlmadicilreedc=tdliyrneeactttltCyhae~att+rtahebefefctlurulaxabemacefutale-ar
antagonist (verapamil) and an agonist (Bay K 8644), on the a 10-min treatment with verapamil (10~5 M). 5-HT in ver = net Ca2+
5-HT response. In concentration-response experiments con- efflux measurement taken when the electrode was placed directly at the
ducted with verapamil or Bay K 8644 alone, neither drug trabeculawhen treated with 5-HT (10~7M) in verapamil (10~5M). After
htraastiaonsirgannigfeicatnetsteedffe(c1t0~on12nteot1C0a~25+Mf}lu(xnootvesrhotwhne)c.onHcoewn-- wdvieasrstaapgnarcmeeaitlolfyw5aesjnhiwamnaocsrehdle.edssA.flrlBoaamcbkotghvreeomurunescdcolr(edB,iGn)tghse=aftsinmtaahllelrteorssapcbioelnclsualetaitoownes5ra-erHotTuank(de2nntdha)et
ever, verapamil inhibits the action of 5-HT. reducing the baseline that were recorded when the electrode was placed 500 /j.m from
response to 67% 16% (n = 3, P < 0.025) of the control the ion source, the trabecula.
5-HT-INDUCED CA:" FLUX IN MOLLUSCAN VENTRICLE 347
force. The remaining 80% of Ca2+ ions are probably re-
leased from the SR. as shown by experiments with SERCA
^-^ 5- rinehsipbointsoer.CLPiA+.iownhs,icwhhiaclhmoasctt tcoombplloectketlhyebNlao-cCksa tehxech5a-nHgTe
process, partially inhibit the 5-HT response, suggesting
some reliance on the Na+-Ca2+ exchanger during the Ca2+
o
extrusion process.
o. Verapamil. an L-type Ca2+ channel blocker. inhibits
5-HT-induced net Ca2+ efflux by about 30% in myocytesof
the Busycon trabeculae. Similarly, in both gastropod (Hud-
dartand Hill, 1996) and bivalve ventricles (Devlin. 1993)
U where 5-HT enhances both inotropic and chronotropic re-
sponses the effect of5-HT is dependent on the movement
ofextracellular Ca2+ ions through an L-type channel, since
5-HT responses are blocked by verapamil (Devlin. 1993b).
Although verapamil has no significant effect on basal net
Ca2+ efflux in the Busycon ventricle, it does inhibit the
5-HT response, suggesting a use-dependent mechanism of
block. This same use-dependent block by verapamil also
Figure3. The inhibitory effectofLiASWon 5-HT-induced netCa2* occurs in many other invertebrate and vertebrate muscle
efflux (in pmol cm~2 s~'l. First bar. background (BG): Net Ca2+ efflux preparations thatwereeitherchemically orelectrically stim-
measurement taken when the electrode was placed 500 j^m from the ion ulated (Lee and Tsien, 1983: Zahradnik and Zachar, 1983;
source, the trabecula. Second bar. spontaneous flux: Net Ca2* efflux Vaghy et al., 1988; Nanasi el /.. 1990).
measurement taken when the electrode was placed directly at the ion Whereas verapamil inhibits the 5-HT-induced net Ca2+
saonuracrtei,fitchiaeltsraebaewcautlear..aTthairddisbtaarn,ce5-ofHTlescsontthraonl:5N^emt Cduar:i+ngeftfrleuaxtmmeenatsuwriet-h efflux from the Busycon cardiac myocytes by about 30%, it
ment taken when the electrode was still placed directly at the trabecula
during treatment with 5-HT (10~7 Ml Fourth bar: After washing the
muscle for 30 min with artificial seawater. net Ca2* efflux measurement
was taken when the electrode was placed directly at the ion source, the
trabecula. atadistanceoflessthan5 /nmduring treatment withaNa-free.
Li-substitutedartificialseawater(LiASW).Fifthbar.5-HTinLiASW: Net
Ca2~ effluxmeasurementtakenwhentheelectrodewasstillplaceddirectly
at the muscle during treatment with 5-HT (10~7 M) in LiASW. The data
are means pooled from three muscles SD.
inhibited by CPA to 18.5% 10% (;; = 3, P < 0.005) of
CPA
the control (5-HT-induced flux prior to treatment).
CPA 10~5M) alone alsoreduces basal net efflux to 76%
(
117r (n = 3, P < 0.025) that of the control. These data
indicate that excitation by 5-HT relies more on Ca2+ ions
from the sarcoplasmic reticulum (SR) than does spontane-
ous myogenicity. The inhibitory effect of CPA on 5-HT
responses is completely reversible.
Discussion
netThefeflpurxesoefntCaex2p+eriinmethnetsvesnhtroiwceudlarthamtus5c-lHeTofstithmeulawtheeslka ascpioFdnit(gauCnrPeeAo)u4s.onflu5Tx-h:HeNTe-itinnhCdiabuic2te+odreyfnfeeltfufCxeacm-te*aosefuffrSleuEmxRe(nCitnAptmaikonelhnibciwmthoer,nceylcelc).toFrpioirdaseztobwnaairsc.
Busvcon canaliculatitm. The Ca~* signal is sensitive to placeddirectly atthe ion source, thetrabecula, atadistanceoflessthan 5
partial block by verapamil, which suggests that some ofthe p.mduringtreatment withanartificialseawater.Secondbar.5-HTcontrol:
Ca2+ movement is through sarcolemmal L-type Ca2+ chan- Net Ca2* efflux measurement taken when the electrode was still placed
Cneals2.+TmhoebsieliLz-etdypientochtahneneilnstearncacloucnotmpfaorrtambeonutt 2du0r%ingofEt-hCe ds5ti-irlHelcTptlliyancaCetdPtAdh:iereNtcretatlbyeCcaautl2ta+hedefumfnulnsuxcglmeteradesuartuimrneengmtetnwrtieattthamke5en-ntHwTwiht(eh1n05~t-h7HeMTe)l.(e1cT0thr~io7rddeAfwb)aaris.n
cCoau2p*lirnegleaasned fmraoym tphreovSiRd.e rtehseultsionugrcien fionrcrCeaas"e*d-isynsdtuocleidc CvePntAri(c1le0s~5M)S.D.Thedataaremeanspooledfromthreeexperimentsonthree
348 C. L. DEVLIN
reduces the FMRFamide response by 60% in the same intracellular stores in molluscan neurons as well
preparation (Devlin, 1997). Therefore, 5-HT relies less on (Kononecko etal., 1983). In contrast, FMRFamide does not
extracellular Ca2+ ions than does FMRFamide during the appear to work through the adenylate cyclase-cAMP signal-
process of E-C coupling in the Busycon ventricle. The ing pathway in gastropod ventricles (Drummond et al.,
results also point to two distinct Ca2+ release mechanisms 1985; Huddart and Hill, 1996) but instead may mediate
used by these respective neurotransmitters. However, both phosphoinositide hydrolysis, the process that has been im-
5-HT and FMRFamide activate L-type Ca2+ channels plicated in some bivalve molluscs (Bayakly and Deaton.
(whether directly or indirectly remains to be determined) 1992).
during cardioexcitation, since verapamil partially inhibits Both 5-HT and some cAMP analogs stimulate 45Ca2+
net Ca2+ efflux induced by either chemical. This finding efflux from Aplysia ventricular myocytes during enhanced
also indicates that the net Ca2+ efflux is in part an effect of chronotropic and inotropic activities (Sawada et al., 1984).
an earlier Ca2+ influx through L-type channels, possibly In the present experiments, 5-HT also induces a large net
carried by the HVA (high-voltage activated) current de- Ca2+ efflux (on the order of 2.6 pmol cm~2 s~') from
scribed by Yeoman el al. (1999). Busycon trabeculae that is recorded even when slow con-
5-HT (from 1(T9 to 10~6 M) depolarizes the heart cells tractions of the trabecula are visible under the microscope.
from Helix pomatia (Kiss and S.-Rosza, 1978), Aplysia This net Ca2+ efflux is sustained over many hours. Sawada
dactyiomela (Sawada et al., 1984), and Dolabella auricu- et al. (1984) also reported that excitatory drugs that stimu-
laria (Hill, 1974). This is the same concentration range in late contraction and cause simultaneous 45Ca2+ efflux do
which 5-HT stimulates a net Ca2+ efflux from the ventric- notdeplete cytoplasmic Ca2+ in the ventricularmyocytesof
ular myocytes ofB. canalicitlatum. Two depolarizing Ca2+ Aplysia. These combined Ca24" efflux data from Busycon
currents, designated the LVA (low-voltage activated), a and Aplysia ventricles suggest a substantial internal Ca2+
T-type current, and the HVA (high-voltage activated), an reserve such as the well-developed SR in the gastropod
L-type current, have been identified in the ventricular cells cardiac muscle described by Sanger (1979).
of the gastropod Lvmnaea (Yeoman et al.. 1999). The The 5-HT-induced Ca2+ efflux recorded from the Busy-
sequential activation of the LVA and HVA currents pro- con ventricle is a large, stable signal that immediately
vides the mechanism for pacemaking, AP generation, and a returns to control levels upon washing with seawater. In
Ca2+ source for E-C coupling in the gastropod ventricle contrast, in bivalve ventricles (from mussel. Geukensia de-
(Yeoman et al., 1999). Since 5-HT increases the Ca2+- missa) that are excited by 5-HT. a transient 45Ca2+ efflux
dependent (and a Na+-dependent) component of cardiac corresponds to the onset ofa 5-HT-induced contracture but
APs and coupled systolic force in bivalve ventricles (Dev- is not sustained throughout the contracture (Koch and
lin, 1993b), potentiation of an HVA-like current may be Greenberg, 1981 ). When 5-HT is washed from the Geuken-
involved given that the Ca2+-dependent component is sen- sia ventricle, a second large 45Ca2+ efflux occurs, during
sitive to verapamil, diltiazem, and Bay K 8644. 5-HT also which total tissue Ca2+ increases. Sawada et al. (1984)
induces a Ca2+ current in Aplysia RB neurons (Pellmar. similarly noted that Ca2+ efflux does not necessarily reflect
1984) and mammalian neurons (Burnashev, 1998). a reduction in cytoplasmic Ca2+, because internal reserves
The relationship between the gastropod 5-HT receptor may be available. A second application of 5-HT to the
and coupled ion channels (such as the L-type channel) mussel heart does not induce a subsequent 45Ca2+ efflux
remains unclear because the subtypes that mediate excita- (Koch and Greenberg, 1981), whereas repeated efflux sig-
tory responses in the gastropod heart are still being phar- nals are stimulated by 5-HT in the gastropod heart. These
macologically identified. However, alargebodyofevidence findings reflect the large difference in complexity between
suggests that the 5-HT receptor from both gastropod (S.- the gastropod SR and the bivalve SR as sustainable Ca2+
Rozsa and Kiss, 1976: Kebabian et al.. 1979; Mandelbaum pools.
et al., 1979; S.-Rozsa, 1984; Sawada et al., 1984; Drum- The SERCA inhibitor, CPA. blocks 5-HT responses by
mond et al., 1985; Huddart and Hill, 1996) and bivalve 80% in the Busycon heart, suggesting that the SR is the
hearts (Higgins, 1974, 1977; Higgins and Greenberg, 1974; majorCa2+ reservoirusedduring cardioexcitation by 5-HT.
Paciotti and Higgins, 1985) is associated with the adenylate CPA acts by inhibiting the binding of Ca2+ ions at high-
cyclase-cAMP signaling pathway, which when stimulated affinity binding sites on the SERCA; this shuts down both
produces an intracellular rise in cAMP. Higgins and Green- Ca2+ uptake and subsequent Ca2+ release at the SR of
berg (1974) found that cAMP increases the phosphorylation skeletal, cardiac (Balke et al., 1994), and smooth muscle
of SR proteins that mediate Ca2+ sequestration into micro- (Suzuki et al., 1992). An indirect effect of inhibiting
somes prepared from bivalve hearts. This then decreases the Ca2+sequesteration at the SR is a reduction in Ca2+-in-
length ofthe diastolic phase ofthe cardiac cycle and primes duced Ca2+ release; such a reduction is typically activated
the SR to release more Ca2+ ions during the next contrac- by the entry of extracellular Ca2+ through voltage-gated
tion. cAMP was reported to stimulate Ca2+ release from Ca2+ channels. In short, this would serve to limit the Ca2+
5-HT-INDUCED CA2+ FLUX IN MOLLUSCAN VENTRICLE
needed to activate the contractile proteins. On the other Bloomquist, E., and B. A. Curtis. 1972. The action I
hand, a reduction in Ca2+-induced Ca2+ release also de- calcium-45 efflux from the anteriorbyssal retractor muscleof/Wyimo
creases the Ca2"1"-dependent K+ current (IK.Ca>- tnus um~ Blooedmuqluiis.stJ,. GEe.n,.aPnhdysiBo.l.A5.9:Cu4r7t6i-s4.851.975. 45Ca efflux from anterior
mutely prolonging the excitability of vertebrate smooth byssusretractormuscleinphasicandcatchcontraction.Am.J.Physiol.
muscle (Suzuki et al.. 1992). How CPA is affecting excit- 229: 1237-1243.
ability in the mollusc ventricle warrants further investiga- Bucket!, K. J., G. J. Dockray, N. N. Osborne, and P. R. Benjamin.
tion, especially considering its effectiveness in blocking 1990. Pharmacology of the myogenic heart of the pond snail Lym-
CaT2h+eefnfeltuxCaan2d+theeffcloumxpltehtate rweavsersribeicloirtdyeodfditusriancgtio5n.-HT BurnnCaealselhaeCsvat,lagcnNia.ulmis1.2949J:8..3N2e5uCr-ao3lp3ch2yi.suimolp.er6m3e:ab1i4li1t3y-14of25l.igand-gated channels.
treatment was composed primarily of Ca2+ released from Devlin, C. L. 1993a. Acetylcholine-induced contractions in a holothu-
the SR and secondarilyofCa2+, fromtheextracellularfluid, rian (Isosticlmpus badionotux) smooth muscle are blocked by the
that had previously entered the myocytes via an L-type calcium antagonists, diltiazem and verapamil. Comp. Biochem.
csuarrrcenotl.emTmhaistonetrapCiad2ly+ reefdfilsutxrirbeuftleectCsa~th+e+iaobnislityoutowfartdh,e DevlPmihonyl,sliuoCsl.c. L1M.0e6r1Cc9e:9n3a5br7.i3-a57Am7ne.rcaennaalryisais. oIIf. cIoonntircolmeocfhtahneisvmesntriinclveolvoefdthien
rteheesrteafbolriesphrinegveinnttirnagcetllhueltaroxaincdefefxetcrtasceolfluClaa~r Caov2e+rlgoraadd,ieanntds Devleixnc,itCa.tiLo.n b19y975.-hydArovxiybtrraytipntgamCiane2.+-Js.elEexcpt.iveBieoll.ect1r7o9d:e6m3e-a7s5u.resCa:+
before the next successive depolarization and contraction. fluxinducedbytheneuropeptideFMRFamideinagastropodventricle.
TtlihoneedN(eLati2e+++r-mCAianS2eW+i)fetxwhcaehsanneugtseeerdf,.flTauhxNewaaL+is-+firnAeepS,arWLtidr+ue-desuutcboesstaicttthuietveibdtayssaoal-f DevlcfCilaonulm,xcpia.uCcm.r-BosisLeo.sl,cehtcheatemin.vdseaPrPhe.cvlsoeicJlo.telrm.oSmd.a1e1So6mmfAei:atash9su.3rm-eo11so90t90ha6.c.emtuysAlcclheno.olniJ-.nienC-vioanmsdpiu.vceePdhvyicsbairloalct.iiunBmg
net Ca efflux during normal, autorhythmic activity ofthe 166: 270-277.
Busycon trabeculae by only 20%-30%, whereas the 5-HT Drummond,G.I.,S.Wernham,and K. Lukowiak. 1985. Stimulation
and FMRFamide responses are inhibited during Li+ treat- of adenylate cyclase in the heart ofAplysia californica by biogenic
Ndmieafnf+te-rCbeanyc2e+40i7encxcrahenasdnpgoe7nrs3e%d,usrurigengsgepsetccsthievameilgcyrael(alDtyeevrlmiernde,liiaa1tn9ec9de7)eo.xncTihttihase- Higgba1iim9nvi0san:,levs9We.9.-mC1yJo1o.m0cp.1a.9r7d4Bi.iuomc.hIneIt.mr.AadceePlhnlyyuslliaaortle.acaCtn.ido8ng0ou:fan15y2-l9ha-yt1de3rc3oy.xcyltarsyesp.taJm.iEnxep.onBiotlh.e
+
tion than during spontaneous myogenic activity. These Li Higgins,W.J. 1977. 5-hydroxytryptamine-inducedtachyphylaxisofthe
substitution experiments on the gastropod trabeculae also molluscan heart andconcomitantdesensitization ofadenylate cyclase.
show that Na+ ions are a necessary stimulus for Ca~+ J. CyclicNudeotideRes. 3: 293-302.
tmroabciellilzualatriopnootlh,ropuegrhhavposltdaugrei-nggatNeac+h-ainnndeulcsedorCaf~ro+mraelneaisne- Higg5ti-indhsey,-ddreWopx.eyntdJr.ey,nptatapnrmdoitneMei.noknJi.ntahGsereesbeianvnbadelvrmegi.cmryo1os9co7am4r.adlicuIamnl.tcraiIcuIe.mllCuuyplctalarikcea.cntJiu.ocnElxepoo.-f
(Lipp and Niggli, 1994), and that they are also involved in Zool. 190: 305-316.
the process of Ca2+ extrusion. The data from the present Hill, R. B. 1958. The effectsofcertain neurohumors andofotherdrugs
study are in agreement with studies on the Na+-Ca~+ ex- ontheventricleandradulaprotractorofBusyconcanaliculatumandon
cluhlaanrgNera+inreodtuhecresmCyoac2y+teefsflwuhxeirneguthieneraeamtorviaallceolflsex(tRreaucteel-r Hill,othnRe.cvoBen.nttr1ra9icc7tl4ie.leoffEoSfrtfcreecotmisbnioitfshe5g-ivhgeyandstr.roicBxliyeotlr.oyfpButDlaolml.ianb1ee1l5ol:na4aa7cu1tri-io4cnu8l2pao.rtiean.tiaJ.lsEaxpn.d
and Seitz, 1968) and in internally dialyzed myocytes (Miura Biol. 61: 529-539.
and Kimura, 1989). Hill, R. B., H. Huddart, and C. L. Devlin. 1992. Activation of a
molluscan heart. Pp. 149-165 in Phylogenetic Models in Functional
Acknowledgments CouplingoftheCNSandthe CardiovascularSystem,R. B. Hilland K.
Kuwasawa, eds. S. Karger Medical and Scientific Publishers, Basel,
This work was supported by the Marine Biological Lab- Switzerland.
oratory, M. G. F. Fuortes and Lucy B. Lemann Fellowships, Huddart, H., and R. B. Hill. 1996. Modulatory mechanisms in the
the NASA Life Sciences Program, and a Pennsylvania State lisaotluamt.edGeinn.terPnhaallrymapceorlf.use21d:ve8n0t9ri-cl8e18o.fthe whelk. Busycon canalicu-
University Faculty Development Grant. Thanks are also Ishii, N., A. W. M. Simpson, and C. C. Ashley. 1989. Effects of
extended to the staff of the N. I. H. BioCurrents Research 5-hydroxytryptamine (serotonin) and forskolin on intracellular free
Center at the MBL for their support in this research. calcium in isolated and tura-2 loaded smooth-muscle cells from the
anterior byssus retractor (catch) muscle of Mytilus edulis. Pflugers
Arch. 414: 162-170.
Literature Cited
Kebabian. P. R., J. W. Kebabian, and D. O. Carpenter. 1979. Reg-
Balke, C. \V., T. M Egan, and W. G. Wier. 1994. Processes that ulation of cyclic AMP in heart and gill of Aplysia by the putative
removecalcium fromthecytoplasmduringexcitation-contractioncou- neurotransmitters dopamine and serotonin. LifeSci. 24: 1757-1764.
pling in intact rat heart cells. J. Physiol. 474: 447-462. Kiss, T., and K. S.-Rozsa. 1978. Pharmacological properties of 5-HT
Bayakly, N. A., and L. E. Deaton. 1992. The effects of FMRFamide, receptors of the Helix pomatia L. (Gastropoda) heart muscle cells.
5-hydroxytryptamine and phorbol esters on the heart of the mussel Comp. Biochem. Physio/. C. 61: 41-46.
Geukensia dernissa. J. Comp. Physiol. B. 162: 463-468. Koch,R.A.,andM.J.Greenberg. 1981. Calciumfluxesaccompanying
350 C. L. DEVLIN
the action of5-hydroxytryptamine on mussel hearts. Comp. Biochem. S.-Rozsa, K. 1984. The pharmacology of molluscan neurons. Prog.
Physiol. C. 70: 229-239. Neurobiol. 23: 79-150.
Kononecko, N. I., P. G. Kostyuk, and A. D. Sherbatko. 1983. The S.-Rozsa, K.,andT. Kiss. 1976. Roleofcyclic nucleotidesintheeffect
effect of intracellular cAMP injections on stationary membrane con- of transmitters on the heart of Helix pomatia L. Comp. Biochem.
ductance and voltage and time dependent ionic currents in identified Physiol. C53: 13-16.
snail neurons. Brain Res. 268: 321-338. Sanger, J. W. 1979. Cardiac fine structure in selected arthropods and
Lee, K. S., and R. W. Tsien. 1983. Mechanism of calcium channel molluscs. Am. Zool. 19: 9-27.
blockade by verapamil, D600, diltiazem and nitrendipine in single Sawada, M., M. Ichinose, I. Ito, T. Maeno, and D. J. McAdoo. 1984.
dialyzed heartcells. Nature 302: 790-794. Effects of 5-hydroxytryptamine on membrane potential, contractility,
Liebeswar. G., J. E. Goldman, J. Koester, and E. Mayeri. 1975. accumulation of cAMP, and Ca2+ movements in anterior aorta and
Neural control of the circulation in Aplysia. III. Neurotransmitters. ventricle ofAplysia. J. Neurophysiol. 51: 361-374.
J. Neurophysiol. 38: 767-779. Smith,P.J.S.,K.Hammar,D.M.Porterfield,R.H.Sanger,andJ.R.
Lipp, P., and E. Niggli. 1994. Sodium current-induced calcium signals Trimarchi. 1999. A self-referencing, non-invasive, ion selective
inisolatedguinea-pigventricularmyocytes.J. Physiol. 474: 439-446. electrode for single cell detection of trans-plasma membrane calcium
ManCdyecllibcauAmM,PD.meEd.i,aJt.ioKnoeosftethre,eMx.citSacthoornybeefrfegc,taonfdseKr.otRon.iWneiinssth.e1h9e7a9r.t Suzufkluix,. MM.ic,roKs.c.MuRresa.kiT,ecYh.. I46m:ai3z9u8m-i4,17a.nd M. Watanabe. 1992. Cy-
MiuroafanAd,ptcYyo.s,mipaae.tnidBtriJav.ienKbiRimensud.irna1g.77o1:f98e3x98t.8e-r3naD9l4e.pNeandaenndceCao.nJ.inGteernn.alPhCyasioaln.d9N3a: cpmluuosmpcpil,aezorcneeildclusc.aecBsird.,CJa.a2nP+h-iadnrehmpiabeicntodoler.nto1f0K7t:+he1c3su4arrr-ec1no4tp0sl.asinmicguirneetai-cpuilgumsCmoao2t+h-
1129-1145.
Nanadseip,enPd.enPt.,aAct.ioVnarorfoa,ntDi.arAr.hyLtahmtihcrodpr,ugasnidnMfr.ogDasknekloet.al19m9u0s.cleUasned Vagh1y98,8.P. LM.e,cKh.amIstamgoakfi,actKi.onMoifwcaa,lcEi.umMmcoKdeulnantao,rdarnugds.A.Pp.Sc1h7w6a-r1t8z.6
PPaeclilcomtmtaertanryiricp,,entaneGmaT.icr.naiFrea.Cd-,.ivieanancd1ntu9rdPc8iuce4lWrd.ek.iibnnyJEjc.enrfeohHarfiasisgknbeogesclrie.ininmsn.Ge.eCnmno1yt.9om8cpoP5a.fh.radBiriinmaoPwalocatchreeocdnomltn..itcrauPat2rhci1ryto:seininloi7tolt4.fy7b-ytCih7ne58s0M1e5:er.-orh3tcy2oed5nnr-iao3nr2xi9yi.-an YeoA5iHmn2caV2anC.Ada,elPC.cMmaiy."Mu.m+oS.f,cAVunSaBrctn.riaheegoLnno.utcntseiBtssier,tn.esNz:vdeeRn.ePwtnhr,PaiYacrooaumlrlneakatdc.rtioP.ml.uoasgRncy.dleBaSen.cnedljGlaCosmlvioionfnni.ict,ah1le9eL9Rd9esy..smenaNareLecwhaV,AhYeVoaaorrnltkd..
neurons ofAplysia. J. Neurophysiol. 15: 13-25. J. Neurophysiol. 82: 2428-2440.
Reuter, H.,and N.Seitz. 1968. Thedependenceofcalciumeffluxfrom Zahradnik,I.,andJ.Zachar. 1983. Inhibitoryeffectofverapamilupon
cardiac muscle on temperature and external ion composition. calcium and potassium currents in crayfish muscle membrane. Gen.
J. Phvsiol. 195: 451-470. Physiol. Biophys. 2: 181-192.
