Cholinergic mechanisms in hydra

Comp, B~ochem Physiol. 1978. Vol. 59C. pp, 39 to 43 PergamonPre.~ Printed m Great Britain

CHOLINERGIC MECHANISMS IN HYDRA IDA ER~EN and MIRO BRZIN Institutes of Anatomy and Institute of Pathophysiology, Medical Faculty, University of Ljubljana, 61105 Ljubljana. Yugoslavia (Received 10 M a y 1977)

Abstract-HI. In the homogenate from hydra tissue an enzyme was demonstrated which hydrolyses acetylcholine and butyrylcholine. 2. By the usual criteria this enzyme does not belong to the cholinesterase group since its activity is not influenced by cholinesterase inhibitors except TEPP. 3. The localization of this enzyme is not reliably proved by cytochemical and histochemical methods, 4. No ChAT activity can be detected above 4.16 × 10 -3 pmoles/,ug wet wt per hr. 5. Pharmacological experiments suggest the presence of cholinergic and adrcncrgic clcments which influence the movements of hydra.

INTRODUCTION

MATERIALS AND METHODS

In the cnidarians the function of neurotransmitters is not clear, but W o o d & Lentz (1964) consider the cnidarian nervous system to be highly specialized and to be provided with cholinergic (Lentz & Barrnett, 1961) and adrenergic mechanisms as well as with 5-hydroxytryptamine, which plays here a more important role than it does in vertebrate nervous system (Wood & Lentz, 1964). In the literature two contradictory views concerning the cholinergic system in cnidarians are presented: mostly older evidence suggests that this system is not present in cnidarians (Bacq & N a c h m a n s o h n , 1937; Bacq & Oury, 1937; Augustinsson. 1946) however, evidence to the contrary was presented later (Mitropolitanskaya, 1941; Bullock & N a c h m a n s o h n , 1942; Lentz & Barrnett, 1961; Wood & Lentz, 1964). This contradiction aroused our interest in the cnidarians, especially because an insight into primitive nervous systems promised to shed light on the phylogenetic origin of cholinergic transmission mechanisms typical of all higher animals. The purpose of the present work was: to determine the activity and fhe type of the enzyme which hydrolyses acetylcholine (ACh): to localize the enzyme on the cellular or subcellular level; and, finally, to visualize the pharmacological effect of some cholinergic and adrenergic drugs on the movement of the hydra, In comparison with similar experiments on the hydra published in the literature, in the present work the methods with specific substrates (Koelle & Friedenwald, 1949; Karnovsky, 1964) were used for the localization of the enzyme activity, and a highly sensitive and specific radiometric method ( M c C a m a n et al., 1968) for the enzyme assay was employed. The radiometric method permits the use of selective inhibitors for the determination of the specificity of the enzyme in spite of the extremely low rate of ACh-hydrolysis by the tissues of hydra. If this enzyme signifies a part of functional cholinergic system in the hydra, another enzyme is also postulated for this animal, i,e. an enzyme for acetylcholine synthesis. Therefore in our work we try to determine acetylcholinetransferase (CHAT).

The experiments were carried out on two species of hydrozoans, Hydra viridis and Pelmatohydra oli(tactis, from the marshes in the neighborhood of Ljubljana. The animals were kept under conditions described previously (Loomis & Lenhoff, 1956; Bode et al., 1973). Since the green hydras thrived best, many more experiments were carried out on them than on the other species. The procedures used in our experiments were the following: the radiometric assays (McCaman et al.. 1968; Fonnum, 1975). time-lapse photography, and histochemical and cytochemical methods for localizing cholinesterase activity (Koelle & Friedenwald, 1949: Karnovsky. 1964; Brzin & Pucihar, 1976). (a) The radiometric assay/or determination of cholinesterase activity (McCaman et al., 1968). With the use of a glass

homogenizer whole freeze-dried animals were homogenized in phosphate buffer to which 5 mM of MgCI2 was added. The homogenate concentration was I mg of dry tissue per 40/A of buffer. As substratcs, 3 mM and 10raM of ACh iodide (Serva) and 3 mM and 10mM of butyrylcholine iodide (BuCh) (Sigma) were used. The specific radioactivity of acetyl-[a*C]choline chloride was 0.53 mCi/ mmole and that of butyryl-[t'*C]cholinc chloride. 0.25 mCi/mmole. Both chlorides were obtained from Amersham. Incubation was carried out in a shaking water bath at 30"C for 4hr. Each sample was run in three parallel assays. The following inhibitors --added to the homogenate I hr before the substrate--were tested: general inhibitors of ChE-s escrine 1 raM, 0.1 mM, 0.01 mM (BDH), tetraethylpyrophosphate (TEPP)0.1 mM (Bios Lab.)and prostigmine 0.1 mM (Roche), selective inhibitor of AChE 1,5-bis-4-allyl dimethylammonium phenyl pentan-3-one dibromide (BW 284C51)0.1 mM IWelcome Research), and selective inhibitor of BuChE tetra-isopropylpyrophosphoramide (iso-OMPA) 0.2 mM, 0.1 mM (Koch Light Lab.). (b) Quantitative determination of cholineacetyltransferase activity (Fonnum, 19751. The cholineacetyltransferase ac-

tivity was measured by radiometric a s h y by Fonnum (1975). The homogcnate concentration was 2.5 mg of fresh tissue in 10/A of Ringer solution, to which 0.1 mM TEPP was added. (c) Time-lapse photography. In order to determine the effect of some pharmacological agents on the movement of the animals the following drugs were added to the normal medium in which the animals were kept: acetylcholine iodide I mM (Serva), d-tubocurarinc I mM (Nutritional Biochemicals Corporation), atropine sulphate I mM, 0.1 mM. 0.001 mM (Kemika). tetramethylammonium 39

40

IDA ER~.I:N and MIRO BRZIN

iodide I mM (BDH). tctracth.,,lammonium iodide I mM (BDH). hexamethonium bromide I mM (Sigma). amcchole (acctyl-fl-mcth)l-choline chloride) I mM (Savor,, and Moore Ltd. LondonL dccamethonit, m iodide I mM (Koch Light Lab.). l-adrenaline I mM. 0.1 mM. 0.01 mM (Eastman organic chemicals). Hydra were observed on special test slides with hollows. I.'or easier examination the bottom was covered with a thin n.~lon net. The animals were photographed through the microscope I min after the addition of the drt, g and subsequently every 30 scc. Each series of experiments was begun with the normal medium, which was then replaced with the medium containing the drug [irst at a low concentration. In the subsequent replacements of the medium the concentration of the drug was progrcssivcl) raised. The last pictures were taken 2 hr after the addition of the drug The exposure was 1 scc in order that the blurred picture of the tentacles could serve as an indicator of the animal's movement. (d) tti.stochcmical and c)'tochcmical localization o/ the enzy.w. The animals were starved for at least I week and then fixed in a mixture of 2",, glutaraldehyde and 2°. paratormaldehyde. For histochemical examination they wcrc fro~'en in liquid nitrogen or ('O_, and ct, t with a cryotome whereas for cytochcmical studies the); were postfixed in I",, OsO4. dehydrated and embedded in epon. The samples wcrc examined under a Jcol T 8 electron microscope. The incubation media ~ere prepared according to Koellc & Fricdenwald 119491. Karnovsk.~ 119641 and Brzin & Pucihar (1976); as substrates, acetylthiocholinc (AThCh) and butyrylthiocholine (BuThCh) were used: and as inhibitors. eserinc. BW 284(.51 and iso-OMPA were cmplo)cd. RESt LTS

A. Quantitatirc anal)'scs A(i) Radiometrical examination revealed that the tissue homogcnatcs contained an enzyme which hydrolysed ACh and, to a smaller degree, BuCh. It was found that I ,ug of dry tissue hydrolysed between 2.5 to 3.0 × 10-"l~moles of A ( ' h : h r and 0.3 × 10 - " umoles of BuCh/hr. If the substrate concentration was raised to 10mM. the enzyme activity increased to 10-51tmoles of ACh/tag dry weight per hr and "~ × 10 - " ,umoles of BuCh.',ug dry weight per hr. Of the tested inhibitors only T E P P (0.1 mM) inhibited the enzyme activity by 40 50°.: eserine was without effect even at a concentration of 1 mM as were prostigmine, BW284C51 and i s o - O M P A (Table 1). A(ii) The cholineacctyltransfcrase activity ,,'.as determined with an average value of 18 measurements. In I hr 4.16 + 0.94 × 10 s pmoles of acctylcholine per ,ug of fresh tissue weight was synthesized. This result proved statistically non-sig,fificant in comparison with spontaneous synthesis of acetylcholinc. One of the

reasons for this negative rcsuh was the incomplete inhibitory effect of T E P P t, pon the activity of the enzyme which splits acetylcholinc. B. The effect t?f some dru,q.s on the animal,s" mot'ement The effect of the drugs on the animals' movement was especially pronotinced during the first 10rain after application: later, however, the animals seemed to have adapted themselves to at least some of the noxious agents. The animals died after protracted exposure to atropine (1 mM and 0.1 mM}. adrenaline (0.1 mM and 0.01 mM) and hexamethonium (I mM) whereas they behaved quite normally in the remaining agents. ACh (1 mM) and d-tubocurarine (I mM) caused no effect whatsoever. Atropine (1 mM, 0.1 mM. 0.01 mM. 0.001 mM). t c t r a m e t h y l a m m o n i u m (1 mM) and tctracthylammonium (1 mM) caused the animals to relax whereas hexamethonium (1 mM). amecholc (I mM) and dccamcthonium (I mM) made them contract (Table 2). C. The histochemical and cvtochemical localization o f the ¢'H.Tyl?l[" No reliable positive results concerning the localization of the enzyme activity wcrc obtained, either on the histochemical or cytochemical level, even though a wide range of incubation times, temperatures, substratc concentrations and three different incubation media were used. Only two experiments carried out on the brown hydra yielded a reaction product in a few cells, primarily in nematocysts and growing nerve cells. The reaction product was membranebound and similar to that obtained by the hydrolysis of AThCh in higher animals. However, eserine, BW284C51 and i s o - O M P A did not inhibit the formation of the precipitate. DISCI

"KSION

The hydra possesses an enzyme which hydrolyses ACh more readily than it does BuCh. The activity of the enzyme increases with the increasing concentration of the substratc but is. for example. 1000 times lower than that recast, red in the nervous tissue of the frog (Majccn & Brzin. 1975). The selective inhibitots for the mammalian ChE-s are without effect. Since. in hydra even at 1 mM concentration of escrinc. no inhibition of the enzyme was observed, the results obtained by Lcntz & Barrnctt (1961} arc difficult to t, nderstand. For the determination of the activity the radiomctric method ( M c C a m a n et al.. 1968).

Table 1. The rate of hydrolysis of acctylcholine and the influence of some inhibitors

Specimen

t~moles × 10-5 of hydrolysed ACh:iig dry weight per hr (in parentheses ",, of inhibition1 Substrate Eserine TEPP Prostigm. conch Control 11 mM) (0.1mM)(0.01mM) (0.1mM) (0.1mM) (mM)

P. oli(4actis

3

0.23

It. ciridis

3

0.25

0.23 (5.!)

0.19 (17.8) 0.22 (11.2)

0.32

0.16 [27.8) 0.22 (11.2) 0.17 (48.8)

H. riridi~

10

0.94 1.14

0.99

(13.01

0.70 138.5)

(I.84 (10.61

41

Cholincrgic mechanisms in hydra Table 2. The influence of atropine, hexamethonium and adrenaline on hydra's movement After

addition

Drug added in

concent,.

Before

4 mlnul"es

add i?lon

4 rninuCes

2 hours

o.i

o.I

(raM)

o.oJ

J

c o +-

c o .c

E

o

¢,....

f

"l

\/

,

,A-

*r

T

I m t~

I ¸ "~i

)" f

i!f~

"x

;!

All three added drugs cause a slower movement of tentacles, and slow also the spontaneous movement of the animals. After a longer action they cause even exitus. ments, as it does adrenergic, which is in keeping with a very sensitive and specific method for the quantitathc findings published by Wood & Lcntz (1964). ACh tive determination of ACh and BuCh hydrolysis was used. Thcrc arc fcw data in the literature on the and d-tubocurarinc have no effect on the animals' amount of the enzyme which, in thc cnidarians, hydmovcmcnt. Thc qucstion arises whether this is due rolyses ACh. but the results obtained on the species to the poor permeation of these drugs through the llydra fi~sca by Mitropolitanskaya (1941) using the tissue. It is known that in the molluscs ACh can bc biological ACh assay, arc in good agreement with our demonstrated in the cells but if applied from the outown results. In like manner the order of magnitudc side no effect can bc recorded (Bryant & Brzin, 1966; of the results obtaincd by Bullock & Nachmansohn Webb et aL, 1966). Diffcrcnt drugs, at any ratc. affect (1942). who used Warburg's manometric mcthod on the animals' movemcnts diffcrently. It is unknown othcr cnidarians, is in keeping with our results. The which tissue structurcs are affected and how thcy arc low enzyme activity demonstrated in the tissue homoaffected by thc tested drugs. It is possible that differgcnatcs of the hydra seems to indieatc that the ent receptors or different structures arc involved, hut enzyme is either diffused throughout the entire tissue that some agents cause inhibition whereas others mass or clsc localized in only a few special cells. again cause excitation. Wc did not succced in reliably localizing the Our expcriments do not permit the conclusion that cnzymc either on the histochcmical or cytochcmical cholincrgic mechanisms arc involved in the transmislevel, though more specific substrates (AThCh and sion of nerve impulscs in hydra• Pharmacological exBuThCh) than thioacctic acid uscd by Lcntz & periments, especially thc influencc of atropine and Barrnctt (1961) wcrc employed. Thus. the well- hexamethonium, suggest their presence and there dcfincd localization suggested by Lcntz & Barrnctt exists an enzyme which splits acetylcholine but chomight be intcrpreted in terms of non-specitic csterascs. lineacctyltransfcrasc if present would bc below the whereas inhibition with cscrinc remains uncxplaincd. sensitivity of the mcthod. Both the histochcmical and the cytochemical localization of thc enzyme arc greatly hampered by the St ~,IMAR~ enzyme's low activity, which very likely creates conditions unfavorable for the formation of prccipitates of With the aid of cytochemical, histochemical and the reaction product. Moreover. the presence in the • radiometric methods and time-lapse photography cells of granulated glycogcn or secrctory cellular further information has been obtained regarding the products may obscure small deposits of the precipipossible existence of a cholinergic systcm in two hydtate, even if present. rozoans, i.e. Hydra riridis and Pehnatohydra oligactis. The rcsults of our pharmacological cxpcriments in- In thc hydra tissue an enzyme which hydrolyses ACh dicate that the hydra may possess cholinergic clc- and BuCh has bccn demonstrated. At a substrate con-

42

IDA ER]~EN AND MtRO BRZIN

centration of 3 mM, 1/zg of dry tissue hydrolyses from 2.5 to 3.0 x 10-6/amoles of ACh and 0.3 x 10-6/amoles of BuCh in 1 hr; at a substrate concentration of 10mM again, the same amount of dry tissue hydrolyscs 10-s #moles of ACh and 2 x 10 -6/amolcs of BuCh. T E P P (0.I mM) inhibits the enzyme activity by 40 50~o whereas eserine, prostigmine, BW284C51 and i s o - O M P A have no inhibitory effect. No reliable localization of the enzyme activity on the cellular and subcellular level was obtained, possibly as a consequence of the extremely low rate of hydrolysis so that the precipitate cannot be formed. No ChAT activity can be detected above 4.16 × 10-3pmoles/~ug w/w per hr. Some cholinergic and adrenergic drugs, primarily L-adrenaline (0.01 mM), atropine (0.01 mM), hexamethonium (0.01 mM) impede the hydra's movement whereas ACh (10 mM) and d-tubocurarine (I mM) are without effect. Their mode of action is not precisely known. The assumption that in hydra nervous excitation is transmitted via a cholinergic system can not be confirmed. This may bc due either to the actual lack of an effect of the latter drugs or a consequence of poor penetration into cell.

REFERENCES AUGUS~NSSON K. B. (1946) Choline esterases in some marine invertebrates. Acta physiol, scand. 11, 141-150. BACQ Z. M. & NACHMANSOHN O. (1937) Cholinesterase in invertebrate muscles. J. Physiol., Lond. 89, 368-371. BACQ Z. M. & OURY A. 0937) Note sur le repartition de la cholinesterase chez les 6tres vivants. Bull. Acad. r. Belg. CI. Sci. 23, 891 893. BODE H.. BERKING S., DAVIS C. N., GIERER A., SCHALLER H. & TRENKNER E. (1973) Quantitative analysis of cell types during growth and morphogenesis in hydra. Wilhelm Rous Arch. EntwMech. 171. 269-285. BRYANT S. H. & BRZtN M. {1966) Cholinesterase activity of isolated giant synapses. J. Cell Physiol. 68, 107-108. BRZIN M. & Pt:CmAR S. (1976) Iodide, thiocyanate and cyanide ions as capturing reagents in one-step copper thiocholinc method for cytochemical localization of cholinesterase activity. Histochemistry 48. 283-292. BULLOCK T. H. & HORRIDGE G. A. (1962) Structure and Function of Nert~ous Systems t~f lnL'ertehrates. Freeman. San Francisco. BULLOCK T. H. & NACHMANSOHND. (1942) Choline esterase in primitivc nervous systems. J. cell. comp. Physiol. 20, 239 242. BLRNrTT A. L. & DIEHL N. A. (1964) The nervous system of hydra. J. exp. Zool. 157. 217-226. Bt;RNE'Iq" A. L., DIEHL N. A. & DIEHL F. (1964) The nervous system of hydra. I1. Control of growth and regeneration by neurosecretory cells. J. exp. Zool. 157. 227-236. BURSZTAJN S. & DAVISL. E. (1974)The role of the nervous system in regeneration, growth and cell differentiation in hydra, I. Distribution of nerve eloments during hypostomal regeneration. Cell Tissue Res. 150, 213-229. COHrN J. E. (1975) The control of foot formation in transplantation experiments with Hydra viridis. J. Theor. Biol. 50. 87-105. DAHL E.. FALK B. & VON MECKLENBERG C. (1963) An adrenergic nervous system in sea anemones. Q. JI microse. Sci. 104, 531-.534. DAvis L. E. (1971) Differentiation of ganglionic cells in hydra. J. exp. Zool. 176, 107-128. DAWS L. E. (1972) Ultrastructural evidence for the pres-

ence of nerve cells in the gastrodermis of hydra. Z. Zellforsch, mikrosk. Anat. 123, 1-17. DAVIS L. E. (1973) Histological and ultrastructural studies of the basal disk of hydra, I. The glandulomuscular cell. Z. Zellforsch. mikrosk. Anat. 139. 1-27. DAVIS L. E. & BURSZT^m S. (1973) Histological and ultrastructural studies of the basal disk of hydra, II. Nerve cells and other epithelial cells. Z. Zellforsch. mikrosk. Anat. 139, 29-45. DAVIS L. E & BURSZTAJNS. (1974)The role of the nervous system in regeneration, growth and cell differentiation in hydra, It. Ultrastructural study of nerve ccll elements during hypostomal regeneration. Cell Tissue Res. 150, 231- 247. DAVIS L. E., BURNETTA. L. & HAYNESJ. F. 0968) Histological and ultrastructural study of the muscular and nervous systems in hydra, ll. Nervous system. J. exp. Zool. 167, 295-33 I. FONNUM F. (1975) A rapid radiochemical method for the determination of cholineacetyltransferase. J. Neurochem. 24, 407-409. HAND A. R. & GOBELS. (1972) The structural organization of the septate and gap junctions of hydra. J. Cell Biol. 52. 397 408. HORRIDGE G. A. & MACKAYB. (1962) Nacked axons and symmetrical synapses in coelenterates. Q. JI microsc. Sci. 103, 531-541. JHA R. K. & MACKtEG. O. (1967) The recognition, distribution and ultrastructure of hydrozoan nerve elements. J. Morph. 123, 43-61. KARNOVSKYM. J. (1964) The localization of cholinesterase activity in rat cardiac muscle by electron microscopy. J. Cell Biol. 23, 217. KOrLLE G. B. & FRtEDENWALDJ. S. (1949) A histochermcal method for localizing cholinesterase activity. Proc. Soc. exp. Biol. Med. 70, 617-622. LENTZ T. L. & BARRNETTR. J. (1961) Enzyme histochemistry of hydra. J. exp. Zool. 147, 125-149. LENTZ T. L. & BARRNE'rr R. J. (1962) The effect of enzyme substrates and pharmacological agents on nematocyst discharge. J. exp. Zool. 149, 33-38. LENTZ T. L. & BARRNETT R. J. (1965) Fine structure of the nervous system of hydra. Am. Zool. 5, 341-356. LOOMlSW. F. & LENHOFFH. M. 0956) Growth and sexual differentiation of hydra in mass culture. J. exp. Zool. 132, 555- 574. MAJCFN Z. & BRZIN M. (1975) The inhibition of frog tissue cholinesterases and the influence of eserine and prostigmine on the action of acetylcholine on the frog heart. Biochem Pharmac. 24, 1599-1602. McCaman M. W., Tomey L. R. & McCaman R. E. (1968) Radiometric assay of acetylcholinesterase activity in submicrogram amounts of tissue. Life Sci. 7, 233. McCONNELL C. (1932)The development of the ectodermal nerve net in the buds of hydra. Q. Jl microsc. Sci. 75, 495-513. MITROPOLITANSKAYA R. L. (1941) On the presence of acetylcholine and cholinesterase in the Protozoa, Spongia and Coelenterata. Acad. Sci. URSS 31, 717-718. PHtLPOTT D. E., CHART A. B. & BURNET'r A. L. (1966) A study of the secretory granules of the basal disk of hydra. J. UItrastruct. Res. 14, 74-84. SLAU'r'rERBACKD. B. (1967) The cnidoblast musculoepithelial cell complex in the tentacles of hydra. Z. Zellforsch. mikrosk. Anat. 79. 296- 318. SLAU'r'n!RBACKD. B. & FAWCErr D. W. (1959) The development of the cnidohlasts of hydra. An electron microscope study of cell differentiation. J. biophys, biochem Cytol. 5, 441-453. WEBB G. D., DE'rrBARN W. D. & BRZtN M. (1966) Biochemical and pharmacological aspects of the synapses of the squid stellate ganglion. Biochem. Pharmac. 15, 1813--1819.

Cholinergic mechanisms in hydra WLS'I'F^LL J. A. (1970) Ultrastructure of synapses in a primitive Coelenterate. J. UItrastruct. Res. 32, 237--246. WESTr'ALL J. A. (1973) Ultrastructural evidence for a granule containing sensory--motor-interneuron in Hydra litoralis. J. Ultrastruct. Res. 42, 268-282. WESTFALLJ. A. Y^MATAK^S. & ENOS P. (1971a) Scanning and transmission microscopy of nematocyst batteries in epitheliomuscular cells of hydra. 29th Annual Proceedings of the Electron Microscopy Society of America, (edited by ARC~:NEAt:SC. J.). Boston, Mass.

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WESTFALLJ. A., YAMATAKAS. & ENOS P. D. (1971b) Ultrastructural evidence of polarized synapses in the nerve net of hydra. J, Cell Biol. 51, 318-323. WOOD J. G. & LENTZ T. L. (1964) Histochemical localization of amines in hydra and in the sea anemone. Nature. Lond. 201. 88. 90. WOOD R. L. 0959) Intercellular attachment in the epithelium of hydra as revealed by electron microcopy. J. hiophy.~, hiochem. Cytol. 6. 343-353.