ISSN 1063�0740, Russian Journal of Marine Biology, 2014, Vol. 40, No. 1, pp. 53–58. © Pleiades Publishing, Ltd., 2014.Original Russian Text © A.V. Chernyshev, E.P. Kotsyuba, 2014, published in Biologiya Morya.

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INTRODUCTION

Hydrogen sulfide (H2S) acts as a gaseous neu�rotransmitter that is involved in many physiologicaland pathological processes in vertebrates and humans[6, 7, 24]. Endogenous H2S is synthesized from L�cys�teine mostly by pyridoxal 5'�phosphate�dependentenzymes: cystathionine β�synthase (CBS, EC4.2.1.22) and cystathionine γ�lyase (CSE, EC 4.4.1.1).

The first evidence of the existence of the hydrogensulfide signaling system in invertebrates was obtainedwhen high concentrations of H2S and sulphides werediscovered in the organs and tissues of the marineinvertebrates Ruditapes philippinarum, Arenicolamarina [14] and Mercenaria mercenaria [11]. Rela�tively recently, the data were obtained on the presenceof endogenous H2S synthesis in the nervous system ofsome arthropods [3, 25]. Although the role of H2S ininvertebrate animals has been poorly investigated, thethus�far available information indicates the involve�ment of hydrogen sulfide in the mechanisms of learn�ing and memory [25] and in protective reactions [8]. Itwas also experimentally found that the impact of exog�enous H2S increases the longevity of the nematodeCaenorhabditis elegans [17].

However, it is not clear thus far how phylogeneti�cally ancient the hydrogen sulfide signaling system isand whether the system is actually important for lowerinvertebrates. The phylum Nemertea belongs to thelarge invertebrate group Trochozoa (or Lophotro�chozoa) and it is morphologically separated fromeither flatworms or annelids. The study of a new classof regulatory systems, the so�called gaseous transmit�

ters, has been launched in nemerteans relativelyrecently. Currently, active investigations are being car�ried out on the distribution and the functional signifi�cance of nitric oxide [2], but no information is avail�able in the literature on the presence of H2S�synthe�sizing structures in the invertebrates of this group.

In this connection, the present study is aimed atinvestigation of the CBS and CSE distribution in thetissues of the nemertean Cerebratulus marginatusRenier, 1804.

MATERIALS AND METHODS

The study was conducted on the nemertean Cere�bratulus marginatus (Heteronemertea: Lineidae); theanimals were collected at a depth of 5–8 m in VostokBay (Sea of Japan) in September 2012.

For immunochemical detection of H2S�synthesiz�ing structures, whole nemerteans were fixed for 2 h in4% paraformaldehyde solution in 0.1 M phosphatebuffer (pH 7.2) at 4°C. After washing in phosphate�buffer solution (PBS), the material was placed for1 day in a cold 30% sucrose solution in 0.1 M phos�phate buffer.

Cystathionine β�synthase was detected by the stan�dard methods in 30� to 35�micron�thick sections pre�pared on a freezing microtome and mounted on slides.After inhibition of endogenous peroxidase in 1% H2O2solution and at suppression of nonspecific binding ofantibodies in 1% normal serum, the sections wereincubated with monoclonal CBS antibodies (Abcam,United Kingdom) (dilution 1 : 500) for 18 h at 4°C.The sections were then washed in several changes of

MORPHOLOGY

Cystathionine β�Synthase and Cystathionine γ�Lyase in Tissuesof the Nemertean Cerebratulus marginatus Renier, 1804 (Nemertea)

A. V. Chernysheva, b and E. P. Kotsyubab

aZhirmunsky Institute of Marine Biology, Far East Branch, Russian Academy of Sciences,ul. Pal'chevskogo 17, Vladivostok, 690041 Russia

bFar Eastern Federal University, ul. Sukhanova 8, Vladivostok, 690600 Russiae�mail: nemertea@fromru.com

Received June 6, 2013

Abstract—Using immunohistochemistry, we examined the distribution of the hydrogen sulfide (H2S) syn�thesizing enzymes cystathionine β�synthase (CBS) and cystathionine γ�lyase (CSE) in tissues of the nem�ertean Cerebratulus marginatus Renier, 1804 (Heteronemertea: Lineidae). The expression of both CBS andCSE was found in the body wall and in the receptor cells of the canals of the cerebral organs; the expressionof CBS was found in the foregut and intestine; and CSE was found in the brain, lateral nerve cords, and cere�bral organs. The peculiarities of the distribution and the possible functional role of H2S�synthesizing ele�ments in nemerteans are discussed.

Keywords: hydrogen sulfide, cystathionine β�synthase, cystathionine γ�lyase, nemerteans

DOI: 10.1134/S1063074014010039

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CHERNYSHEV, KOTSYUBA

0.1 M phosphate�buffered saline (PBS) (pH 7.2)and incubated for 2 h in a solution of secondary bioti�nylated antibodies (Vector Labs, United States) at adilution of 1 : 200. After washing, the sections wereincubated with an avidin–biotin–peroxidase complex(Vectastain Elite ABC Kit, Vector Labs, United States)for 1 h at room temperature in the dark, then washedthree times in PBS. The reaction products were visual�ized United States the substrate (VIP Substrate Kit,Vector Labs, United States) to control the stainingprocess under the microscope. Thereafter, the sectionswere dehydrated according to standard proceduresand embedded in Canada balsam.

For identification of structures containing cys�tathionine γ�lyase, the sections were incubated withpolyclonal antibodies against CSE (Santa Cruz Bio�technology, United States) (dilution 1 : 500) for 18 h at4°C. After washing in three changes of PBS, the sec�tions were incubated in a solution of secondary anti�bodies labeled with fluorescein isothiocyanate (FITC,1 : 400) (Santa Cruz Biotechnology, United States) for2 hours in the dark at 4°C. They were then washed withPBS and embedded in a 1% glycerol solution. In thecontrol experiments, the primary antibodies werereplaced with an equivalent amount of 1% non�immune serum. In all the control experiments, noimmuno positive reactions were detected. The prepa�rations were photographed on an inverted microscopewith an Axiovert 200 M fluorescence module.

RESULTS

Cystathionine β�Synthase

In the studied nemertean Cerebratulus marginatus,CBS immunoreactivity was detected in the body wall,the foregut, and midgut; the lining cells of the canal ofthe cerebral organ were labeled as well.

In the body wall, a majority of the CBS�immu�noreactive cells were found in the epidermis. Theylocalized singly or in small groups, mainly in the upperpart of the epithelial layer (Figs. 1a and 1c). Many ofthe cells belonging to the open type were elongated�oval or spindle shaped. The cells were from 7 to 20 μmlong and 3 to 6 μm wide. The number of CBS�positivecells in different parts of the epidermis differed signif�icantly in the nemerteans. The greatest number of thecells were labeled in the antero�lateral and mid�lateralparts of the body (Fig. 1a); they were somewhat lesslabeled in the head region (Fig. 1b) and the smallestnumber of the cells were detected in the postero�lat�eral part of the body (Fig. 1c).

In the cutis, numerous CBS�immunoreactivefibers formed a plexus; these were most intenselystained. Basically, they are situated anteriorly, mainlyin the head region (Fig. 1d). Some labeled fibersseemed to be the processes of CBS�positive cells of theepidermis and/or cutis. In addition to the fibers, wefound some single round or pear�shaped cells (10–18 μm in size) in the cutis under the epidermis

(Fig. 1e). Their processes were sometimes traced tothe distal part of the cutis.

In the digestive system, CBS�immunoreactive cellswere detected mainly in the foregut (stomach)(Fig. 1f). CBS�immunoreactive elements were signifi�cantly more numerous in the anterior part than in theposterior part of the stomach (Fig. 1g). Only singlecells were labeled in the midgut. In the stomach andthe midgut, round or oval CBS�immunoreactive cells,8 to 12 μm in size, were found predominantly in thegastric epithelium (Fig. 1h). In a majority of thesecells, the thin process directed to the lumen of the gas�trointestinal tract reached the surface of the epithe�lium. No CBS�immunoreactive elements weredetected under the epithelium.

The CBS�positive response was also expressed inthe cells lining the canals of the cerebral organs(Fig. 1g).

Cystathionine γ�Lyase

CSE�immunoreactive cells and fibers were foundin the brain, the lateral nerve cords, the cerebralorgans, and the body wall of C. marginatus.

In the brain, CSE�immunoreactivity was recordedmainly in the ventral ganglia (Fig. 2a). The mostintense fluorescence in neurons and nerve fibers of thelateral nerve cords was observed immediately behindthe ventral ganglia; the fluorescence decreased gradu�ally along the nerve cords to the level of the stomach�midgut junction (Fig. 2b). In the distal part of thenerve trunks, the specific fluorescence was absent. Inthe lateral�nerve neuropils we detected very intensefluorescence of the fibers with a relatively small num�ber of tiny (about 10 μm in size) CSE�immunoreactiveneurons in the area of the cell bodies (Figs. 2a and 2b).In the simultaneous fluorescence in the fibers of thelateral nerve cords, specific fluorescence was alsoobserved in the intramuscular nerve layer that is con�nected with the lateral nerve cords and surrounds theouter circular muscles of the body wall (Fig. 2c).

Intensive CSE�immunoreactivity was also revealedin the cerebral organs and in the epithelium of the lat�eral cephalic slits through which these organs open tothe outside (Figs. 2a and 2d). In the cerebral organs,the strongest fluorescence was observed in the cellslining the inner channel and also in the rear cluster ofneuro�glandular cells (Fig. 2a, gco). In the body wall,the numerous oval or polygonal cells in the cutis werethe most intensely labeled (Figs. 2e and 2f). Theirlengths varied between 10 and 25 μm; their width var�ied from 3 to 7 μm. These cells were arranged relativelyuniformly along the length and circumference of thebody wall, with some predominance in the anteriorand the lateral parts of the body.

In the epidermis, CSE�immunoreactive cells werefound only in the anterior� and the postero�lateralareas of the body. They have an elongated spindle andpear shape; the cell sizes ranged from 10 to 20 μm(Fig. 2f).

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CYSTATHIONINE β�SYNTHASE AND CYSTATHIONINE γ�LYASE 55

(a) (b) (c)

(d) (e)

(f)

(h) (g)

ep

ctct

epep

ct

ctct

ep

1cs

ct

1cs

ep

ep

cco

cco

br

gep

stis

n

n

n

nln

gep

st

st

Fig. 1. Cystathionine β�synthase in the body wall of Cerebratulus marginatus (a–c), in the brain (g), in the anterior part of thestomach (f), in the anterior part of the body (d, e), and in the posterior part of the stomach (h). br, brain; cco, cerebral organ canal;ct, cutis; ep, epidermis; gep, gastral epithelium; is, intramuscular neural layer; lcs, lateral cephalic slits; n, neurilemma; nln, neu�ropil of the lateral nerve cord; st, stomach. Arrowheads indicate chemoreceptor cells lining the canal of the cerebral organs;arrows show the internal neurolemma. Scale: 50 μm.

DISCUSSIONOur study, which was conducted on the nemertine

Cerebratulus marginatus, showed the presence of

enzymes of hydrogen sulfide synthesis, CBS and CSE.However, these enzymes had significantly differentdistributions. Expression of both CBS and CSE was

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CHERNYSHEV, KOTSYUBA

(a) (d)

(c)

(b)

(e) (f)

gco

ln

nln

cnlbr

is

st

ct

ln

co

lcs

ct

epep

ct

Fig. 2. Cystathionine γ�lyase in Cerebratulus marginatus: the anterior part of the body (a), in the lateral neural trunk (b), in theintramuscular layer (c), in the cerebral organ (d) and in the body wall (e, f). cnl, cells of the lateral nerve cord; co, cerebral organ;gco, glandular cells of the cerebral organ; is, intramuscular neural layer; ln, lateral nerve cord; other designations as in Fig. 1. Thearrow points to the canal of the cerebral organ. Scale: 50 μm.

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CYSTATHIONINE β�SYNTHASE AND CYSTATHIONINE γ�LYASE 57

detected in the body wall and in the receptor cells ofthe channels of the cerebral organs, CBS was found inthe foregut and the midgut, while CSE was found inthe brain, in the lateral nerve cords, and in the cerebralorgans.

In the body wall, CBS� and CSE�immunoreactivecells and fibers were labeled in the surface epitheliumand in the cutis. It is known that heteronemerteans,like other nemerteans, possess in their epidermis basalintraepithelial and subepithelial nerve plexuses, aswell as scattered sensory cells [22]. According to Zai�tseva et al. [1], the epidermis and the cutis of heter�onemerteans contain catecholamine� and serotonin�containing cells; among them the authors identifiedthe cells of the open and the closed types according totheir morphology and position in relation to the epi�thelial layer.

A comparison of our results with literature datashowed that a majority of CBS�positive cells that wedetected in the cutis of C. marginatus are similar intheir size and location to the basic types of subepithe�lial cells of the closed type that have been describedearlier for other nemertean species [1, 4]. According toZaitseva et al. (2007), such cells are associative andeffector neurons that communicate within the periph�eral nervous system and are involved in the innervationof the skin glands, ciliated epidermal cells, and mus�cles of the body wall. The distribution of the intraepi�thelial CBS� and CSE�positive cells of the open typethat we identified in our study was identical to that ofthe receptor�effector cells that have been described forthe White Sea nemerteans Lineus viridis, L. ruber, Cer�ebratulus sp., Cephalothrix linearis, and Amphiporuslactifloreus [1]. There is evidence that similar cells inthe Hydra, sea anemones, and the mollusk Aplysiacarry a receptor�motor function and participate in theinnervation of the muscular elements of the body wallor some organs [5].

The presence of the numerous CBS� and CSE�immunoreactive fibers and cells that we detectedamong muscular and glandular elements of the cutisnear the body�wall musculature suggests that the mainfunctional targets of hydrogen sulphide are musclecells. This assumption is also supported by a signifi�cantly greater density of H2S�synthesizing elements inthe cephalic section of C. marginatus, where the longi�tudinal muscles of the body wall are developed moststrongly. Most species of the genus Cerebratulus live insilt and silt�sandy bottoms and move there by means ofbowel movements, with the greatest effort being exe�cuted entirely in the head region.

Another confirmation of the influence of H2S onthe locomotor activity of some invertebrates is pro�vided by the results of reported experimental studies[15], which have shown that low doses of sodiumhydrosulfide (NaHS, an H2S donor) cause the musclecontraction of the body wall in the echiuran wormUrechis caupo. In vertebrates, hydrogen sulphide mayalso have a modulating effect on muscle motility. It hasbeen experimentally proven in mammalians that H2S

is involved in the relaxation of the aortic smooth mus�cle, the ileum, the portal vein, and the vas deferens[13, 21, 26], as well as reducing the contractile activityof the uterine [20] and the cardiac muscle [12].

The role of H2S in invertebrate animals, unlikemammalians, is poorly investigated. There is evidencethat in nematodes hydrogen sulfide synthesized by theCBS can cause a relaxation of smooth muscles of thepharynx and participates in the regulation of theexpression of HIF�1 (hypoxia�inducible factor 1) inthe hypodermis [8]. Moreover, the authors suggestedthat the presence of CBS in the hypodermis and inmuscle cells is necessary for the synthesis of cysteine,which plays an important role in the formation of thecuticle [23].

The studied nemertine C. marginatus had high CBSimmunoreactivity in the digestive system with thedensity of distribution of CBS�immunoreactive ele�ments changing along the digestive tract. The numberof labeled cells in the foregut was significantly higherthan that in the midgut. The presence of subepithelialCBS�immunoreactive components in the stomach,which is characterized by the secretion of digestiveenzymes and high metabolic activity, supports theinvolvement of hydrogen sulfide in the regulation ofdigestive functions in C. marginatus. It is known thatH2S�synthesizing neurons are arranged along theentire length of the digestive tract in mammalians [16,21]. In the digestive system of vertebrate animals andhumans, hydrogen sulfide is recognized as a neuro�modulator of the motor activity of the digestive tractmuscles and the secretory activities of epithelial cells[19]. Furthermore, it has been shown that H2S acts inthe gastric mucosa as a protective factor against dam�age [10]. In invertebrate animals, the distribution ofCBS has been investigated by Vozdek et al. only in thedigestive system of the nematode Caenorhabditis ele�gans [23]. The authors assume that, as in mammals[9], high CBS activity in the gut plays an importantprotective role in C. elegans [23].

Interestingly, we did not detect CBS�containingneurons in the central nervous system of C. margina�tus, although CBS is the main enzyme involved in thesynthesis of hydrogen sulfide in the brain of mamma�lians [6] and some invertebrates [3, 25]. Similar datawere obtained for the nematode C. elegans [23].

At the same time, CSE expression in neurons andnerve fibers of the cerebral ganglia and lateral nervecords indicates an endogenous synthesis of hydrogensulfide in the CNS of C. marginatus with the participa�tion of the CSE. This seems to be a reflection of theevolutionary peculiarities of the formation of the cen�tral regulatory systems of the central nervous system innemerteans. In addition to neurons and nerve fibers,high activity of this enzyme was observed in the cere�bral organs, i.e., specialized chemosensory structuresinvolved in the perception of food stimuli and chemi�cal changes in the quality of the medium. Consideringthe importance of H2S in the regulation of adaptivemechanisms in the formation of a compensatory

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response to habitat change [18], we can suggest that inthe brain of nemerteans hydrogen sulfide has animportant modulatory role in the transmission andprocessing of chemosensory information and the reg�ulation of adaptive mechanisms in the formation ofthe compensatory response to changes in the condi�tions of the environment.

Thus, our findings have shown that the nemertineC. marginatus has a well�developed H2S�producingsystem, which involves neurons and nerve fibers thatare included in the central and peripheral nervous sys�tem.

ACKNOWLEDGMENTS

This work was partially funded by the Governmentof the Russian Federation to provide the state supportfor research conducted under the supervision of lead�ing scientists at Russian institutions of higher educa�tion (contract no. 11.G 34.31.0010) and by grants ofthe Far Eastern Branch, Russian Academy of Sciencesno. 12�III�B�06�090 and the Russian Foundation forBasic Research no. 13�04�01664.

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Translated by I. Barsegova