1~ - . . ~ • ~,, trnal f o r Parasitology VoL 13, No. I, pp. 10t-10~, 1983. 002o-75191~3/0101f11..06503 00/0 ~, .s Britain. I~¢rgarat~n t~'r~s Ltd.

i:-: 1983 Au~lraltan ~ociely lop I~tra.silolo~y

THE SYNTHESIS OF 5-HYDROXYTRYPTAMINE FROM TRYPTOPHAN AND 5-HYDROXYTRYPTOPHAN IN THE CESTODE HYMENOLEPIS

DIMINUTA

PAULA RIBEIRO a n d RODNEY A. WEan

Department of Biology, York University, Downsview. Ontario. Canada, M3J I P3

(Received 17 March t 9821

Abstract--RlaEIRO P. and WEan R. A. 1983. The synthesis of 5-hydroxytryptamine from tryptophan and 5-hydroxytryptophan in the cestode tlymenolepis diminuta. International Journal for Parasitology ! 3:101 - 106. The synthesis of 5-HT in Hymenolepisdirninuta was investigated. Homogenates of/-/, diminuta incubated in 3H-5-HTP or 3H-tryptophan resulted in the formation of a substantial radioactive product which comigrated on several TLC systems with 5-HT. Furthermore, considerable 3H-5-HTP was produced on incubation of homogenates of H. diminuta with 3H-tryptophan. Boiled homogenates w'ere not effective in the synthesis of these products from 3H-trS"ptophan or 3H-5-HTP. The formation of 3H-5-HT from precursor 3H.5-HTP was stimulated by addition of pyridoxal phosphate. On the other hand, addition of iproniazid phosphate increased the amount of extractable JH-5-blT indicating the presence of a mammalian-like monoamine oxidase. Incubation of intact H. diminuta in saline containing 3H-tryptophan for 3 and 12 h resulted in substantial radioactivity in 5-HT. The present study clearly demonstrates the enzymatic capacity of H. dimim,ta for the synthesis of 5-HT from both tryptophan and 5-HTP and suggests that this pathway is of significance to the parasite in its natural environment.

INDEX KEY WORDS: Serotonin; 5-hydroxytry'0tamine; 5-hydroxytryptophan; synthesis; iproniazid phos- phate; Hyn)enolepis dintinuta.

INTRODUCTION

SEROTONtN (5-hydroxytryptamine: 5-HT) has been demonstrated in numerous free-living and parasitic platyhelminths (Welsh & Moorhead, 1960; Mansour & Stone, 1970; Chou, Bennett & Bueding, 1972). Substantial quantities of 5-HT have been shown in the cestodes Hymenolepis dirninuta, Diphyllo- l~othrium dendriticum, Mesocestoides corti, Hymen. olepis nana, Dipylidium caninum and Spirometra ntansonoldes, by fluorescence histochemistry and biochemical separation methodologies (Chou et aL, 1972; Gustafsson & Wikgren, 1981; Harir i , 1974; Lee, Bueding & Schiller, t978; Shield, 1971; Tomosky, Jardine, Mueller & Bueding, 1977). Furthermore, while acetylcholine has been found to inhibit muscular activity in a number o f platyhel- n in ths (Wilson & Shiller, 1969; Barker, Bueding & Timms, 1966), exogenously applied 5-HT was found to stimulate the rhythmical movement o f several platyhelminths (Mansour, 1957; Tomosky, Bennett & Bueding, 1974). Such observations collectively suggest that 5-HT functions as an excitatory neuro- transmitter and that acetylcholine functions as an inhibitory neurotransmit ter in the platyhelminths. Moreover, 5-I--IT has been detected in the nerves supplying the subintegument of 19. dendriticum and H. nana which suggests a role for 5-HT in the sensory

nerves (Gustafsson & Wikgren, 1981; Lee et aL, 1978). Thus 5-biT may play a central role in the nervous system of platyhelminths.

Serotonin has been found to be an important regu- lator o f platyhelminth metabolism. The evidence (see Mansour, 1979) suggests that , through the activation of certain key enzymes, 5-HT will stimulate glycolysis in Fasciola hepatica and Schistosoma mansonL Furthermore 5-HT appears to increase glycogen breakdown in starved worms and to activate glycogen phosphorylase and adenylate cyctase (see Mansour, 1979). Thus the essential stages of a 5-HT modulated cyclic AMP-dependent protein kinase system, similar to that seen in the vertebrates, has been found in platyhelminths.

While the previous experimental data clearly support 5-FIT as an important metabolic modula tor and putative neurotransmitter in parasitic platy- helminths, the origins of the endogenous stores of 5-HT have not received much attention. Surprisingly there is no direct evidence to show the synthesis o f 5-HT from tryptophan although the ability of F. hepatica to synthesize 5-HT from 5-hydroxytrypto- phan (5-HTP) and the presence of the enzyme 5-HTP decarboxylase have been demonstrated (Mansour, 1964; Mansour & Stone, 1970), Similarly S. rnansoni possesses the enzymatic capacity to decarboxylate

101

102 I)~.ULA ~IBEIRO and RODNEY A. WEIIFI ~.J,~. voL. 13. 1983

5 -HTP but appears to lack the abil i ty to. hydroxylate t ryp tophan (Cairo, 1981). On the o ther hand both S. rnansoni and M. corti show uptake o f 5 -HT by a carrier mediated process (Hariri , 1975; Bennett & Bueding, 1973), and thus endogenous 5 -HT may be derived from exogenous sources. More recently, Mettrick & Cho (1981) postulated that the 5 .HT found in cestodes may result from the absorpt ion of this m o n o a m i n e f rom the intestine of the m a m m a l i a n host,

The present study, however, clearly demonstra tes the abil i ty of both intact and homogenized H. ditninuta to synthesize 5-FIT from exogenously sup- plied t ryp tophan and 5 -HTP in a m a n n e r analogous to that found previously in m a m m a l i a n tisst~es (Green & Sawyer, 1966; Lovenberg, Weissbach & Udenfr iend , 1962; Lovenberg, Jecluier & Sjoerdsma, t 96B). Moreover , the effects o f iproniazi~ phosphate on the radiolabelled 5 - H T content o f these worms suggest that 5 -HT is turned over in the tissues by a mammal ian- l ike m o n o a m i n e oxidase.

M A T E R I A L S A N D M E T H O D S

tqryr~tophan, DL-5-hydroxytryptophan, 5-hydroxylryp- tamable hy~* chloride, pyrldoxal-5-phosphat¢ and ipronlazld ,~h~pha~e ~,ere purchased from Sigma Chemical

',~mpao:. Radioisotopes t-[G:Hl-tryotophan (6"9 ( ' i/m), 'Jl) and r~l.-5-hydroxy[G-3 H]-tryptophan (3"0 Ci. m-r:~,'.) were obtained from Amcrsham Corp,, Toronto. At[ ,~-~er compounds used were of reagent grade. Cellulose TLC P'-:~'.c s were purchased from Eastman Kodak. The Bio- Rad Protein Assay Kit was used for protein determinations.

Collection of worms and preparation for incubation. 1-1. diminuta was maintained in" mate Wistar rats. Rats (125-175 g, Canadian Breeding Farms) were infected with a group of 20 cysticercoids and the worms were removed 18-20 days post-infection. The worms were rinsed in three changes of warm safine {CaCI2, 0"9froM; NaCI, 102raM; NazHPO4/NaH2PO4 buffer, 25raM, pH 7"2; and glucose, 5"5tara) containing a mixture of antibiotics (penicillin, 10,000 units/ml; streptomycin, 10,000 mcg/ml; fungizone, 25 mcg/ml; Gibed) to remove bacteria and intestinal debris. The radiochemical parity of the two isotopes 3H-tryptophan and 3H-5-hydroxytryptophan was assayed in this laboratory. Aliquots were chromatographed on cellulose TLC plates using tert-amyl alcohol : 14°70 methylamine {4 : I v/v) and n-butanol : water : acetic acid (120 : 50 : 30 v/v) solvent systems which completely separate :5-HT from tryptophan and 5-HTP. 1'4o det~-'table 3H-5-HT contamination was found in 3H-5-HTP or 3H-5-HTP in 3H- tryptophan. Hence, radioisotopes were used directly as supplied.

Incubations. TOe ~ y procedures consisted of incubating whole worms or worm homogenates with a tritrlated substrate (3H-tryptophan or 3H-5-HTP) followed by separation, and radloassay of possible products by thin layer chromatography. The procedure for the deteml~nation of tryptophan hydroxyla- don in homogenates was as follows: the anterior third of gravid worms were homogenized in ice cold Tris-HC1 (50raM, pH7"5; 800/.d/worm). 3H-tryptophan (5 x IO"6M) was added to 100 ~1 of homogenate and 100/A of homogenate which had been pre,,iously boiled for 20 rain (blank). Unlabelled 5-HTP (10":3M) was added to both sample and blank. The presence of

excess 5-HTP in the reaction mixture minimized metabolic loss of newly fun'ned 3H-5-HTP (Lovenberg etaL, 1968). Blanks and samples were incubaled at 37°C for 1-5 h in a shaking water bath and subsequently centrifuged at 12,000g for 20 rain at 0°C. The supernatants were evaporated to dryness under a stream of nitrogen and the dry extracts were redissolved in 40~I of the following mixture of standards (mg/ml): tryptophan (1"0) 5.FITP (0"3) and 5-HT (0"3) in water and melhanoI (l : I v/v). Twenty microliter aliquots were spotted and chromatographed on cellulose TLC plates using a two- solvent system. The plates were first developed with n-butanol : acetic acid : water (120 : 50 : 30 v/v) for 3 h. They were then dried and developed in the same dimension with tert- amyl alcohol : 140/0 methylamine (4 : I v/v) for approx. 4 h. This procedure was found to be necessary for the complete separation of 5-HTP from both tryptophau and 5-HT. Following chromatography the spots were detected by spraying with diazotized-p.nitroaniline. The plates were then divided into 0'5-1-0 cm lanes and each lane radio- assayed in ACS (Amersham Corp,). The determination of 5-HTP decarboxylation in homogenates was carried out as follows: Whole worms were homogenized in ice cold Tris- t-ICI buffer (50tara pHT"5; 800 pl/worm). One ht, ndred microliters of crude homogenate was added to microconical tubes containing { 1) 3H-5-HTP (10-sr~) with 5-HT (10raM), and pyridoxal-5-phosphate (10"4M}, (2) 3H-5-HTP (10-5~i) and 5-HT (10-'try), (3) :;H-5-HTP (10-SM) and iproniazid phosphate (10-3M). (4.))H-5-HTP tl0-S~t) only. The presence of exce~ 5-HT diluted the newly synthesized ~ H-5- V1T and therefore minimized loss of 3H-5-HT through :~ubsequent metabolism. The effect of added 5-HT on the :~H-5-HT levels was compared to that of the monoamine oxidase inhibitor iproniazid pltosphate. The blanks contained t00 el of crude bomogenate which had been previously boiled for 20 rain, ~H-5-HTP (10-'~M) and 5-HT (10-4M). Samples and blanks were ~hen incubated, centrifuged and prepared for chromatography as above. Twenty microliter aliquots were chromatographed in cellu- lose TLC plates for 3"5 h using n-butanol : acetic acid : water (120 : 50 : 30 v/v), and the spots were visualized with diazotized p-nitroaniline. The cellulose was cut into 0 '5 - 1 : cm bands and radioassayed in ACS. Live, intact worms were incubated for 3 and t2 h at 37°C in saline, as above, containing 3H-tryptophan (10-6M) and a mixture of anti- biotics (200 o/ml) to prevent bacterial growth. After incubation the worms were washed three times in cold saline, transferred to tubes containing 0"01 t~ formic acid and stored at -20°C. The frozen worms were subsequently thawed and homogenized in 0'01 r~ formic acid (0"5ml/3 worms), centrifuged at 12,000g at 0*C for 20 rain and the clear supernatant evaporated to dryness under a stream of dry nitrogen. The dry extracts were then redissolved in a mixture of standards, as above, and spotted on TLC plates. Following chromatography in tert-amyl alcohol: 14% metbylamine (4 : I v/v) the individual spots were visualized with dlazotized p-nitroaniline, and the cellulose was radioassayed in ACS. Samples of the incubation media, taken both at the beginning and at the end of each incubation, were chromatographed in tert-amyl alcohol : 14% methylamine {4 : i v/v) to determine whether there was any ~H-5-HT present in the media.

RESULTS

FoIlowing incuba t ion o f homogenates of H. dirninuta in 3H-labelled t ryp tophan the extracts were separated by thin- layer chromatography. The results

x

¢,., c~

13. 1983

7 2 . ~ n ' r p

4 8 "

7

Synthesis of 5-HT in Hymenolepis dimimaa

TRV ,~HT

IIr

FIG. 1. Chromatography of the ~H-labelled extracted products following incubation of homogenates of H. diminuta in aH-tryptophan. The data were determined by subtracting the radioactivity in boiled homogenates (blanks) from that in unboiled homogenates. Standards consisting of unlabelled tryptophan, 5-HTP, and 5-HT were chromatographed with the extracts in n-butanol : acetic acid : water and tert-amyl alcohol : 14e70 methylamine as described in Materials and Methods. The error bars represent the J7,4-S.D. (N = 6). or, origin; fr, solvent

front.

103

shown in Fig. 1 were determined by subtracting the activity found in the boiled sample (blank) from that in the unboiled sample, Initially, equal amounts of aH-tryptophan were added to both blank and sample. However, after a 1"5 h incubation the tissue levels o f free 3H-tryptophan in the unboiled sample dropped well below that in the boiled sample, whereas the ~H-5-HTP and 3H-5-HT levels were considerably increased. The data indicate that the added radiolabelled t ryptophan was metabolized in

the unboiled tissues to produce, at least in part,3H-5 - HTP and 3H-5-HT.

Incubation o f whole worm homogenates with ~H- 5-HTP for !.5 h resulted in an increase in labelled 5-HT (see Table 1). The results presented in Table I were corrected by subtracting the boiled tissue level of ~H-5-HT (blank value) from the unboilcd tissue levels o f -aH-5-HT (sample values). The effects o f added pyridoxal-5-phosphate, cold 5-HT, and iproniazid phosphate were determined by comparison with a control sample of unboiled tissue which was incubated in the presence of aH-5-HTP only. Flooding the incubation mixture with 5-HT resuhed in a 99% increase over the control level o f )H-5-HT. When the coenzyme pyridoxal-5- phosphate was added to unboiled tissue in the presence of excess 5-HT a 368°7o increase in ~H-5- HT was observed. Finally, the addit ion of the monoaminc oxidase inhibitor iproniazid phosphate to unboiled tissue increased the control level of~H-5 - HT by 260o70.

The ability o f H. dlmffnuta to synthesize 5-HT was also investigated in live intact worms. Following incubation o f intact H. diminuta with ~H-labelled t ryptophan ' for varying periods of time the tissues were extracted and the extracts were separated by thin-layer chromatography. The results are shown in Table 2. Initially 3H-5-HT was not detected in the medium, However, after both 3 and 12 h incubations o f the tissues in 31-I-tryptophan, substantial radio- activity associated with 5-HT was prescnt in the tissue extracts. After 3 h approx. 6"20,b o f the total radiolabel in the extract was found to eomigrate with authentic 5-HT (see Fig. 2). By 12 h this propor t ion had increased to 11"3~o. Only traces of radio- activity were found on the chromatogram that migrated between 5-HT and t ryptophan. After a 12 h incubation lower levels o f ~H-tryptophan were detected than after a 3 h incubation. Analysis of the medium at the end of each incubation found no detectable amounts of ~H-5-HT.

I) ISCUSSION The present s tudy is the first to demonstra te the

enzymatic capacity of H. diminuta to synthesize 5-HT in vitro. Exogenously supplied t ryptophan was rapidly metabolized in unboiled homogenates to

TABt.E I~METABOLISM OF 3H-5-HTP By HOMOGENATES OF H. diminuta AF'rEa itl V[tlro INCUBATION

eT0 increase DPM x 10"~/mg protein from

Treatment* Art (X ± S.D.) the control

Control 6 547 ± 159 - 5-HT 6 1040 ± 424 99% ;proniazid phosphate 6 1972 _ 134 260~0 5-HT + pyridoxal phosphaze 6 2558 ± 243 368°70

*The data were determined by subtracting the radioactivity in the boiled samples (blanks) from that in the unboiled samples.

tNumber of determinations.

104 I..).F. VOL. 13. 1983 PALrLA R]r&E~Roand R O D N E Y A . W E B B

TABLE 2 ~ M E T A B O L I S M OF 3H - T RY PT O PH A N BY INTACT / ' i t. tliminttla FOLLC)"WING 3 .~NID 12 h in vitro INCUnATtONS

Time of N* 5-HT Tryplophan incubation DPM/mg protein 0/0('t) DPM/mg pro~tcin 070

(h i (,V ± S.D.) (,~ ± S.D.)

3 3 3858 ± 607 6-2 56,56fl z "/~28 91-0 12 3 2314+-411 11"3 16.823 _ 967 82-3

*Number of determinations. "lPercentage of Iota] radioactivity

T~ Y . . . . . . . ~ - H T 31-

29-

: '7.

c :

¢J 2 s .

o.

23- )o

x 21,, G.

3~

J, ! d i s t a n c e t crl l )

Fro. 2. Typical histogram of the 3H-labelled extracted products following a 3 h incubation of intact 1"1. dim]aura in 3H-tryptophan. Standards consisting of unlabelled tryptophan, 5-HTP. and 5-HT were chromatographed with the extracts in tert-amyi alcohol : 14% methylamine as described in Materials and Methods. Although the location of 5-HTP is not shown in this Figure, previous experiments have indicated that 5-HTP runs in the vicinity of the origin. The percentages of the total radioactivity found in the tryptophan and 5-HT areas are marked. The values were corrected for quenching and background activity, or,

origin; fr, solvent front.

produce substantial amounts o f 5-HTP and 5-HT. Similarly, exposure to 5-HTP under similar conditions resulted in considerable 5-HT formation. However, boiled homogenates were not effective in the synthesis o f these products. Collectively, these observations indicate the presence o f two heat- sensitive enzymes in the tissues of H. diminuta which synthesize 5-HT through the hydroxylat ion o f t ryptophan and the deearboxylat ion o f 5-HTP. The pathway of serotonin synthesis in this animal is then similar to that previously described for mammals (Lovenberg et aL, 1962, 1968; Green & Sawyer, t966)

recovered.

and other invertebrates (Osborne & Neuhoff, 1974a, b). Furthermore, the 5-HTP decarboxylase found in H. dimhn,ta was markedly activated by the addition of pyridoxal phosphate, suggesting that this enzyme has the same cofactor requirements as its mammalian pyridoxal-phosphate-dependent analog (Lovenberg et aL, 1968).

The occurrence o f 5-HT turnover in homogenates of t f . diminuta was also demonstrated in this study. Considerable 5-HT accumulation was observed when iproniazid phosphate, a potent inhibitor of mamma- lian monoanaine oxidase, was supplied to the incubation medium. The data suggest that at least one o f the mechanisms o f serotonin catabolism in this animal involves inactivation by a monoamine oxidase. In mammals, serotonin is metabolized either by a monoamine oxidase in the brain to form 5-hydroxyindole acetic acid or by an N-acetylase and a 5-hydroxyindole-o-methyhransferase in the pineal g land to form mela tonin . Unfor tuna te ly , the mechanism(s) o f 5-HT inactivation in eestodes have not yet been investigated. A membrane-bound monoamine oxidase was demonstrated in homogenates o f H. dim#ruin but it did not appear to be active towards 5-HT (Moreno & Barrett, 1979). However, Moreno & Barrett (1979) suggested that another monoamine oxidase may be present in the nervous tissue which is capable o f breaking down 5-hydroxytryptamine, The present studies give credence to that suggestion.

Hariri (1975) found that, following incubation of whole tetrathyridia of Mesocestoidea corti in unlabelled t ryptophan, in the presence or absence o f various metabolic inhibitors, he could not detect an increase in endogenous 5-HT levels. Using a similar approach, Mansour & Stone (1970) also did not find 5-HT synthesized front t ryptophan in Fasciola hepatica. However, the sensitivity o f this approach may not have allowed for the detection o f small amounts o f newly synthesized 5-HT.

Exogenous 5-HTP and 5-HT were added to samples being assayed for t ryptophan hydroxylation and 5-1--ITP decarboxylation respectively, in order to minimize the metabolic loss o f the newly formed radioactive products. Despite the presence of excess 5-HTP, however, some of the newly formed -)H-5- HTP was still decarboxylated to produce 3H-5-HT. It seems that in H. diminuta, as in mammals (Lovenberg et aL, 1962), 5-HTP is a short-lived

:~ 13. 1983 Synthesis ofS-HTin 14ymenolepisdimimtta 105

~mc~mcdiatc which is rapidly metabolized and converted to serotonin, in facl, the high rate at which 5-HTP is decarboxylated may be the reason why some workers have been unable to detect 5-HTP formation following incubation with Iryptophan. Ca/to (1981) reported that while the addition of t~C-5-HTp to both aduh and larval Schistoyonta mansoni resulted in an increase in radioactive serotonin, incubation in ~aC-tryptophan caused no detectable formation of ~4C-5-HTP. However, this work did no~ indicate any precautions being taken against 5-HTP breakdown, either by adding a 5-HTP decarboxylase inhibitor or exogenous 5-HTP. it is possible that 5-HTP was synthesized but because it was rapidly decarboxylated, never accumulated up to detectable tissue levels.

Exposure of live intact worms to 3H-tryptophan for 3 and 12 h resulted in substantial 5-HT forma- tion. The data revealed that although the proportion of~H-5-HT appeared to increase over the 12 h period (6'2°70 at 3 It and 11,3% at 12 h) there was less radioactivity associated with 5-HT at 12 It than at 3 h. The higher percentage found at 12 It can be explained by the fact that while the levels of free 3H- tryptophan decreased by 70% from 3 to 12 h of incubation, the levels of ~H-5-HT decreased only by 40070, The 70°/'0 decrease of tryptophan is not sur- prising since this ana~no acid is an important precursor in the variety of metabolic pathways, in particular protein synthesis, and therefore must be rapidly metabolized by the animal. On the other hand, the decreased levels o f 5-HT suggest that this monoamine is being turned over in the tissues of 1f. dirninutu, and gives additional support to our previous finding that these animals can catabolize as well as synthesize 5-HT.

It has been previously suggested that the serotonin found in cestodes is due to absorption of this amine, from mammalian intestinal tissue (see Me/trick & Cho, 1981 for review), However, the results reported here revealed that while ~here may indeed be some absorption occurring in viva, the serotonin levels present in 14. diminuta cannot be entirely exogenous in origin. Clearly, the endogenously synthesized 5-HT must contribute to some extent to the serotonin pool in this animal, and is probably of significance to the parasite in its natural environment. Considerable information remains to I~e gathered before a clear understanding of the function(s) of 5-HT in the cestode nervous system can be ascertained. However, the finding that 5-HT can be synthesized and turned over by 1-1, diminuta lends support to the hypothesis that 5-HT may serve as a neurotransmitter in the cestode nervous system.

Acknowleclgemeats~We wish to express our appreciation to Eva Budziako~ski for providing us with infected animals. This work was supported by a Natural Science and Engineering Research Council of Canada grant in aid of Research A6508 to R. A. Wcbb.

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106 PAULA RIBEII~o and RODNEY A. WEBB t.J.P, voL. 13. 1983

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