Comparative Biochemistry and Physiology Part A 124 (1999) 561–567

Serotonergic control of the heart and pericardium in the chitonAcanthopleura japonica

Shinji Matsumura a,1, Makoto Kurokawa a, Kiyoaki Kuwasawa a,*, Robert B. Hill b,Kenji Ohsuga a

a Department of Biological Sciences, Tokyo Metropolitan Uni6ersity, Minami-Ohsawa 1-1, Hachioji-shi, Tokyo 192-0397, Japanb Department of Biological Sciences, Uni6ersity of Rhode Island, Kingston, RI 02881, USA

Received 21 September 1998; received in revised form 11 January 1999; accepted 27 January 1999

Abstract

Serotonergic control of the heart and pericardium of the chiton, Acanthopleura japonica, was examined during this combinedhistological and physiological study. Glyoxylic acid (2%) induced fluorescence in neural elements of fibers and varicosities in theheart and pericardium. Serotonin exerted positive chronotropic and inotropic actions on the heart and pericardium in adose-dependent manner. The threshold dose of serotonin that stimulated excitatory effects on the heart and pericardium was lessthan 10−9 M. The histological and pharmacological results suggest that serotonin may be a neurotransmitter present in excitatorynerves innervating the heart and pericardium. We conclude that serotonergic excitatory control of the heart appeared early duringmolluscan phylogeny with the evolution of the chitons. The pericardium of the chiton may receive serotonergic excitatory neuralcontrol, which has not been reported in higher molluscan taxa. © 1999 Elsevier Science Inc. All rights reserved.

Keywords: Chiton; Heart; Pericardium; Serotonin; Chronotropic effect; Inotropic effect; Excitatory innervation; Methysergide; Neural control;Glyoxylic acid

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1. Introduction

In three molluscan classes, gastropods, bivalves andcephalopods, physiological and neurochemical evidenceindicates that serotonin (5-hydroxytryptamine, 5-HT)has an important physiological role in excitatory con-trol of the heart (reviewed by [16,19,38]). Serotoninacted like an excitatory neurotransmitter on ventricularmyocardial cells of the systemic heart in thecephalopod, Octopus hummelincki, increasing the rateof rise of prepotentials and thus heart rate [14]. InMercenaria mercenaria, Greenberg [8] found that sero-tonin (around 10−9 M) increased the amplitude ofcontractions of the heart. In 50 bivalve species, sero-tonin predominantly caused cardioexcitation, although

serotonin at some doses inhibited heart beat in somespecies [32]. Excitatory junctional potentials (EJPs) inthe auriculo-ventricular valve of Mercenaria appearedto be serotonergic since they were reduced by treatmentwith methysergide (UML) [14,26]. In gastropods, sero-tonin had profound inotropic and chronotropic effectson ventricles of gastropods [13,15,17,18,21,25]. Sero-tonin applied directly to Helix pomatia heart muscleeither depolarized or hyperpolarized the membrane po-tential of the muscle cells depending on the treatment[22]. Concentrations between 10−8 and 10−6 M depo-larized, and between 10−6 and 10−4 M hyperpolarizedthe membrane potential. Other evidence supporting therole of serotonin as an excitatory neurotransmitter wasin the molluscan models, Aplysia californica [27,31] andLymnaea stagnalis [4,5].

Few physiological studies have been carried out withpolyplacophora. Greenberg [9] stated that activities ofthe heart of Cryptochiton stelleri were increased byserotonin. Greenberg et al. [10], however, showed amechanical record in which serotonin (10−6 M) rarelyinduced rhythmical activity in the quiescent isolated

* Corresponding author. Tel.: +81-426-772578; fax +81-426-772559.

E-mail address: kuwasawa-kiyoaki@c.metro-u.ac.jp (K.Kuwasawa)

1 Present address: Department of Medical Chemistry, Kansai Med-ical University, Moriguchi, Osaka 570-8506, Japan.

1095-6433/99/$ - see front matter © 1999 Elsevier Science Inc. All rights reserved.PII: S 1 0 9 5 -6433 (99 )00150 -6

S. Matsumura et al. / Comparati6e Biochemistry and Physiology, Part A 124 (1999) 561–567562

heart of Mopallina muscosa and its predominant actionon the beating heart was inhibitory.

In Cryptochiton, serotonin increased the tone of thequiescent pericardium and increased pericardial rhyth-micity [10]. Tissue extracts from the cerebral commis-sure and heart of Cryptochiton were shown to containserotonin, as shown by gel-column chromatographicseparation [1,10].

The previous experiments have found that both theheart and pericardium of Acanthopleura was myogenic,and that both the heart and pericardium may receivedual innervation from each of the lateral and ventralnerve cords [29].

Using electrophysiological and histochemicaltechniques in this study, we examined whether or notthe heart and pericardium receive innervationfrom serotonergic nerves. Results showed the ex-istence of serotonergic excitatory innervation ofboth the heart and pericardium in the chiton. A prelim-inary report has appeared elsewhere in abstract form[30].

2. Materials and methods

More than 62 specimens of the polyplacophoranmollusc Acanthopleura japonica (45–80 mm in bodylength) were used in this study. They were collected atthe seashore in the Tokai area and kept as describedpreviously [29].

2.1. Fluorescent histochemistry with glyoxylic acid

For detection of biogenic monoamines by glyoxylicacid-induced fluorescence, we used the method de-scribed by Kurokawa et al. [24] with some modifica-tions for the whole mount preparations of the heart orpericardium. Freshly dissected preparations of the heartand pericardium were incubated on a slide glass, in 0.1M phosphate buffer solution containing 2% glyoxylicacid and 30% sucrose (pH 7.4), for 10–30 min. Duringthe incubation the tissues were gently stretched.Then, specimens were dried with a fan at room temper-ature and heated at 98°C for 3–5 min. These wereembedded in liquid paraffin and covered with a coverslip. Slides were observed under epi-illumination usinga Nikon Optiphoto fluorescent microscope equippedwith blue excitation and 515 nm emission filters,and photographed on Fujichrome Provia 100 (ASA,100).

2.2. Pharmacological methods

The semi-intact and isolated preparations were madeas described previously [29]. To study serotonin effectson the heart and pericardium in semi-intact animals,

the heart was perfused with SW or serotonin containingSW from cannulae inserted into a pair of efferentbranchial vessels, while the whole specimen was super-fused with SW. The middle of the ventricle on theventral side was connected to straingauge mechano-transducers via a fine suture on the heart, and a fineneedle on the pericardium, to record the mechanogram.Stock solutions of serotonin-creatinine sulfate (WakoPure Chemical), methysergide (UML, a gift from San-doz) or other antagonists (Research Biochemical Inter-national) dissolved in distilled water (10−2 or 10−3 M)were stored at 4°C and diluted with sea water (SW) toprepare given concentrations to apply to specimens justprior to experiments.

3. Results

3.1. Fluorescent histochemistry with glyoxylic acidtreatment

The freshly isolated heart used for the wholemount preparation was treated with glyoxylic acid.Yellowish green fluorescent fibers were observed on theinner surface of the treated whole mount heart (Fig.1A).

A portion of the pericardium covering the ventralside of the heart was treated with glyoxylic acid. Yel-lowish green fluorescent fibers and varicosities rangingfrom 2.0 to 7.8 mm in diameter were observed on theouter surface (opposite to the side facing the pericardialcavity) of the pericardium preparation (Fig. 1B). Theprocesses appeared denser in the anterior part of thepericardium than in the posterior part.

3.2. Effects of serotonin on the heart

Effects of serotonin on the heart were investigated bymeans of mechanical recording of the heartbeat. Sero-tonin dose-dependently increased both chronotropicand inotropic responses of the chiton heart over therange of concentrations tested (10−9–10−7 M) (Fig.2A) and up to 10−6 M (Fig. 2B). The threshold dosefor excitation was less than 10−9 M.

The effects of UML on serotonin-induced cardiacexcitation were examined, as shown in Fig. 2C. Sero-tonin at concentration of 10−7 M increased systolicforce of the heart to 277% (Fig. 2C, the 1st trace). After20 min from the serotonin application, UML (10−6 M)increased systolic force to 215% (Fig. 2C, the 2ndtrace). After 3 min from the UML application, 10−7 Mserotonin didn’t increase systolic force (Fig. 2C, the 3rdtrace). After wash, 10−7 M serotonin increased systolicforce to 230% (Fig. 2C, the 4th trace). Thus, UML,which itself augmented systolic force, had antagonisticeffect against serotonin in the chiton heart.

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3.3. Effects of serotonin on the pericardium

Effects of serotonin on the pericardium were exam-ined by means of mechanical recording of the pericar-dial beat (Fig. 3). Serotonin (10−8 M) slightly increasedrate of pericardial beating (Fig. 3A, top trace). Sero-tonin (10−7 M) increased both rate and force of thepericardial beat more markedly (Fig. 3A, middle trace).At 10−6 M serotonin, the excitatory responses were

more apparent (Fig. 3A, bottom trace). Thus, positivechronotropic and inotropic effects of serotonin ap-peared in a dose-dependent manner over the range upto 10−6 M (Fig. 3B). The threshold concentration ofserotonin for excitatory effects of the pericardium ap-peared to be at the order of 10−9 M. Excitationinduced by serotonin (10−7 M) was clearly blocked byUML (10−6 M), which itself slightly augmented peri-cardial beat (data not shown).

Fig. 1. Micrographs showing glyoxylic acid-induced fluorescence of monoamine-containing neural elements. Fluorescence of monoamine-contain-ing neural elements in the whole mount preparations of the auricle (A) and pericardium (B). Arrowheads show the fluorescent fibers. Bars, A, 10mm; B, 50 mm.

S. Matsumura et al. / Comparati6e Biochemistry and Physiology, Part A 124 (1999) 561–567564

Fig. 2.

Fig. 3. Excitatory responses of the pericardium to serotonin. (A)While mechanical records were obtained from a ventral part of thepericardium in a semi-intact preparation, serotonin was applied to thespecimen by addition to the bath. Underlining beneath each traceshows the period of serotonin application. (B) Dose-response curvesfor effects of serotonin on rate and force of pericardial beating. S.E.is shown on one side of each mean value. n, number of experiments.

Fig. 2. (A) Mechanical records showing excitatory effects of serotoninon the whole heart. Serotonin was applied to the heart in a semi-in-tact preparation via a pair of efferent branchial vessels. Underliningbeneath each trace shows the period of drug application. (B) Dose-re-sponse curves for effects of serotonin on heart rate and systolic force.S.E. is shown on one side of each mean value. n, number ofexperiments. (C) Methysergide (UML) blocked serotonin-inducedcardiac excitation. Bars under the traces show a period of serotonin(10−7 M) or UML (10−6 M) application. Mechanical records wereobtained from the heart in a semi-intact preparation while drugs wereapplied to the heart via a pair of efferent branchial vessels.

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Other serotonin antagonists, ergonovine (10−6 M),metergoline (10−5 M), propranorol (10−6 M), spiper-one (10−5 M) and sulpiride (10−5 M), augmentedpericardial beat (data not shown).

4. Discussion

4.1. Serotonergic excitatory control of the heart

Serotonin produced positive inotropic andchronotropic effects on the heart in the polypla-cophoran Acanthopleura. The threshold concentrationwas in the order of 10−9 M. In a variety of species ofother molluscan taxa such as the Gastropoda, Bivalviaand Cephalopoda, serotonin is generally accepted to acton the heart as an excitatory agent (reviewed by[16,19]). In the gastropod, Aplysia, treatment with sero-tonin mimicked the effect of electrical stimulation ofthe cardiac excitatory nerve. Stimulation of this nerveincreased heart rate more than the bath application of10−9 M serotonin [27]. In Busycon canaliculatum, sero-tonin affected heart rate and systolic force, showingpositive chronotropic and inotropic responses at athreshold of around 10−9 M [25,35]. In the bivalveMercenaria, serotonin produced positive inotropic andchronotropic effects on the whole heart at concentra-tions of more than about 10−9 M [28]. Hill andKuwasawa [14] revealed that diastolic depolarizationand cardiac rate were increased by serotonin (10−6 M)in Octopus. The threshold of serotonin for excitatoryeffects on the heart of Acanthopleura obtained in thisstudy was within the threshold concentrations obtainedfrom other molluscan species.

UML was a highly effective antagonist of serotoninin Mercenaria [11,28], in Tapes watlingi [33], in Strom-bus gigas [12,25], in Rapana thomasiana [21] and inHelix aspersa [3]. UML had an antagonistic effect onthe action of the cardio-accelerator nerves to the heartin Mercenaria [26,28] and in Tapes [33]. Indeed, it wasshown that EJPs in the auriculo-ventricular valve ofMercenaria were reduced by treatment with UML[14,26].

In Chiton tuberculatus, serotonin was identified innerve tissue by its fluorescence [39]. In Cryptochiton, atissue extract from the heart and the cerebral commis-sure contained serotonin [1,10]. Histological resultsshowing glyoxylic acid-induced fluorescent fibers in theheart may support our pharmacological data obtainedduring experiments with UML (Fig. 1). In our prelimi-nary study, neural processes showing serotonin-like im-munoreactivity were observed in the heart [30].

The chiton belongs to one of three classes, Polypla-cophora, in the lowest molluscan subphylum Aculifera[34]. We may conclude that serotonergic excitatorycontrol of the heart, a feature of more complex mollus-

can cardiovasular systems, appeared early during mol-luscan evolution with the chitons, the most primitive ofthe molluscan taxa.

4.2. Serotonergic excitatory control of the pericardium

The pericardium of chitons receives both excitatoryand inhibitory innervation from both the lateral andventral nerve cords [29]. The pericardium was contrac-tile in the gastropod species, Aplysia [7], Busycon [20]and Lymnaea [4]. Neurons which induced the contrac-tion of the pericardium were identified in both Aplysia[23] and Lymnaea [4]. However, in these animals onlythe excitatory innervation was found, though the exci-tatory neurotransmitter was not chemically identified.Serotonin produced positive inotropic and chronotropiceffects on the Acanthopleura pericardium. There is noreport of a serotonin effect on the pericardium in avariety of species of other molluscan taxa. Thethreshold concentrations of serotonin for cardiac exci-tation ranged from about 10−10 to 10−7 M in a varietyof molluscan species and, in the Acanthopleura heart,serotonin also produced positive inotropic andchronotropic effects at the threshold concentration of10−9 M. The threshold concentration of serotonin forexcitatory effects on the Acanthopleura pericardium ob-tained in this study was less than 10−9 M. Glyoxylicacid-induced fluorescent processes were visualized inthe pericardium which had neural processes showingserotonin-like immunoreactivity in our preliminarystudy [30]. This supports a belief that the pericardiumof the chiton may receive serotonergic excitatory inner-vation, which has not been reported in higher mollus-can taxa.

4.3. Neural plexus in the heart and pericardium

A histochemical technique using glyoxylic acid wasapplied to molluscan specimens to detect neural ele-ments containing monoamines [2,24,36]. Glyoxylic acid-induced fluorescence in neural processes formed areticular structure accompanied with varicosities of theheart and pericardium of Acanthopleura. Large varicosestructures in this study were often observed at junctionsbetween processes, while the varicosities ranged from2.0 to 7.8 mm in diameter. In sections of the Acantho-pleura central nervous system (the cerebral commissure,lateral and ventral nerve cords and buccal ganglion)which were stained with hematoxylin and eosin, the cellbodies were approximately 3.0–8.0 mm in diameter(unpublished observation). In other polyplacophoranspecies, Chiton oli6aceus and Acanthochitona discrepans,neurons in the nerve cords and in the cerebral commis-sure were 3.5–4.5 mm in diameter [37], so the range ofthe values was rather narrower than that of Acantho-pleura. The size of the large varicose structures (up to

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7.8 mm) in the present heart and pericardium wascomparable to the largest neuronal cell bodies in thecentral nervous system. The existence of nerve cells organglia in the heart of some molluscs has been reported(reviews of [15,23]). In Helix, Phillis [33] reported that anumber of uni- and bipolar nerve cells were observed inthe ventricular wall by means of silver staining meth-ods. Cottrell and Osborne [6] suggested the presence ofcell bodies in the dense network of nerve fibers in theHelix heart as shown by methylene blue stainingmethod. We do not exclude the possibility that theneural plexus existing on the inner surface of the heartand pericardium might contain the cell bodies of intrin-sic neurons.

Acknowledgements

This study was partly supported by a Grant-in-Aidfor Scientific Research No. 0854271 from the Ministryof Education, Science and Culture of Japan. This studysupported in part by the Sumitomo Foundation. Con-tribution from the Shimoda Marine Reseach Center,No. 636.

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