5-Hydroxytryptamine, glucose uptake, glycogen utilization and carbon dioxide fixation in Hymenolepsi microstoma (Cestoda)

Comp. Biochem. Physiol. Vol. 73B, No. 4, pp. 901 to 906, I982 Printed in Great Britain.

0305-0491/82/120901-06503.00/0 © 1982 Pergamon Press Ltd

5-HYDROXYTRYPTAMINE, GLUCOSE UPTAKE, GLYCOGEN UTILIZATION AND CARBON DIOXIDE FIXATION IN H Y M E N O L E P I S M I C R O S T O M A (CESTODA) M. SAIDURRAHMAN,* D. F. METTRICK* and R. B. PODESTA'~ *Department of Zoology, University of Toronto, Toronto. Canada M5S I AI and +Department of Zoology, University of Western Ontario, London, Canada N6A 5BL (Received 2 April 1982)

Abstract I. In citro glucose uptake and glycogen utilization by Hvmenolepis microstoma decreased under high oxygen concentrations. 2. 5-Hydroxytryptamine did not stimulate in z:itro glucose uptake but did increase glycogen utilizations by H. mierostoma. 3. The reduced glucose uptake under high oxygen concentrations (21 and 95",i) resulted in a reduction in excretory products. 4. l'*CO2-incorporation studies confirmed that, under both 95"~, 02:5?o CO/ and air-minus-CO2 (=- 21¢'{;O2), COx-fixation by phosphoenolpyruvate carboxykinase (EC 4.1.1.32) was inhibited. 5. The specific activity of hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40) was not stimulated by 5-HT. 6. The concentration of ATP required for optimal stimulation of phosphofructokinase activity was 0.67 raM. Activity was further significantly increased by the addition of cAMP and even greater by AMP.

INTRODUCTION The mouse bile-duct tapeworm, Hymenolepis microstoma, possesses branched pathways for carbohydrate metabolism, characteristic of many parasitic helminths (Behm & Bryant, 1975a, 1975b; Cornish & Bryant, 1976; R a h m a n & Bryant, 1977; R a h m a n & Mettrick, 1982). In vitro the rate and direction of carbon flow through the pathways is significantly influenced by the concentration of oxygen, irrespective of the presence or absence of COz in the ambient medium (Rahman & Mettrick, 1982). High oxygen concentration resulted in a diminution in the end-products of CO2-fixation. In the absence of CO2 lactate production increased while that of succinate, acetate and proprionate decreased; the tissue revels of A T P and total adenine nucleotides of H. microstoma were also significantly reduced. These results led to the suggestion that the actions of Oz and C O / on the energy metabolic pathways of this cestode are independent of each other (Rahman & Mettrick, 1982). The amine 5-hydroxytryptamine (5-HT, Serotonin), enhances glucose uptake and, in the absence of external glucose, increases internal glycogen utilization in Fasciola hepatica (Mansour, 1959, 1962; M a n s o u r et al., 1960) and in Hymenolepis diminuta (Mettrick et al., 1981). In H. diminuta 5-HT also increased the specific activity of phosphofructokinase but did not affect hexokinase or pyruvate kinase. These three enzymes appear to play a regulatory role in the glucose metabolism of a n u m b e r of helminths including Moniliformis dubius (Cornish et al., 1981) and H. microstoma (Rahman & Mettrick, 1982). The present studies on H. microstoma were therefore directed towards confirming the inhibitory action of Oz on COz-fixation and to determine the effect of 901

5-HT on glucose uptake, glycogen utilization and the specific activity of the three regulatory enzymes identified in the previous studies on H. diminuta and M. dubius. The importance of adenylate nucleotides in relation to enzyme activity, in the absence and presence of 5-HT, was also investigated, MATERIALS

AND

METHODS

Experimental or,qanisms

Mice were infected with 25 cysticercoids of H. microstoma, and sacrificed when the worms were 5-6 weeks old. Recovery rates were around 65°o Worms were collected from the bile duct and washed in ice-cold Krebs Ringer phosphate buffer (KRP). Glucose uptake and glycogen utilization For the determination of in vivo glycogen content worms were rapidly homogenised within 10-12 min, following sacrifice of the host. The homogenates were digested for 60 min in KOH using a boiling water-bath. Glycogen was determined following a modification of the method described by Pfleiferer (1963), in which glycogen was precipitated in the presence of 96y~, ethanol, and then hydrolyzed to glucose with 2N H2SO4 for subsequent determination of the glucose present. For further details see Mettrick et al. (1981). In the studies on glucose uptake, worms were maintained in ice-cold KRP containing 5 mM glucose, and randomly assigned into groups of 12 before incubation at 37°C under the appropriate experimental conditions. The length of time from sacrifice to incubation was less than 30 rain. The glucose concentration in the incubation media was either 2, 5 or 10 mM. In concurrent experiments 1 mM 5-HT was added to the incubation media, using the 95% N2:5% CO2 gas phase. The procedures follow those of Mettrick et al. (1981). Glucose uptake was also studied by adding 5#Ci D-[U-igC]glucose (sp. act. 3mCi/mmol) to 10ml KRP

M. SA1DUR RAHMAN el aL

902

containing 10 m M cold-glucose, pregased with the appropriate gas mix, 955; O2:5";; CO2 or 95'I, N,:51~; CO2. After 60 min incubation at 37 C the worms were removed from the medium, washed in three quick changes of K R P and homogenized in 80% ethanol (5 ml:g wet weight). The homogenate was centrifuged and the ethanolic extract (supernatant) frozen and stored for later scintillation counting. Two miflilitres of the incubation medium was filtered through a column of D O W E X l-X8 formate-form ion exchange resin (BIO-RAD Camloque, April 1979, pp. 15-18) packed in a 10-ml syringe and equilibrated with the eluting buffer. Two millilitres of incubation medium was layered on the column and eluted with 2 x 2 ml of 50 m M Triethanolamine HC1 buffer, pH 7.2. The column was washed several times with the buffer and the elute collected separately until no more glucose was coming out of the column. Glucose was assayed enzymatically, following Si(Jma Chemical Co., Technical Bulletin No. 510. The elutes were pooled together and aliquotes counted for radioactivity as [~4C]glucose. Aliquotes of incubation medium were counted directly for total radioactivity and the result of the former count subtracted to determine the radioactivity due to excretory end-products. Fifteen millilitres of PCS (Amersham) were added to 200 itl of the column elute and 50/21 of the straight incubation medium prior to counting using a Packard Tricarb Liquid Scintillation Spectrometer. C O 2 incorporation Carbon dioxide incorporation was determined by adding 0.5 ~Ci/ml NaH~'~CO3 (sp. act. 0.1 mCi/mmoll to the pregased incubation medium. Incubation was carried out for 60 min at 37°C in tightly stoppered Erlynmeyer flask. Ethanolle extract of the worms was counted with PCS for incorporation of radiocarbon. After incubation 100 I~1 of the medium was placed in a scintillation vial containing 200 #1 2N HCI and evaporated to near dryness. Fifteen millilitres of PCS was added, and the sample counted to determine the label in the excretory end-products. Prior to scintillation counting all samples were darkadapted for 18hr at 4 ' C to reduce chemiluminescence. Samples were counted using an Automatic External Stan-

dard for quench correction: no further correction for counting efficiency was made.

Enzyme act±city Nongravid portions {= anterior two thirds of strobilal of mature H. microstnmu were homogenized in 3 vol of the following buffers: (al for hexokinase and pyruvatc kinasc. 50raM Triethanolaminc HCI containing 10 m M KF. pH 7.2; ib) for phosphofructokinase, 20 mM Tris. 4 mM MgSO,,, 3 0 m M KF and 0.1 mM EGTA. pHS.0, t t o m ogcnates were centrifuged in a Sorval RC-5 centrifuge at 25000 for 5min, the supernatant tiltered through three layers of surgical gauze and reccntrifuged m a Beckman Model k centrifuge at 100,000 0 for 60min. The floating lipid-scum wits carefully removed with a pasteur pipette and clear supernatant was sampled by filtering through two layers of presoaked lens-tissue. All procedures were performed at 0 4 ( " and the enzyme preparations were kept on ice. Enzyme activities were assayed spectrophotometricall~ at 30 C following the method of Bchm & Bryant {1975a.b) with some modifications as outlined in Table 4. Protein was assayed by the Bio-Rad method using bovine serum albumin as standard. All substrates, cofactors and enzymes were very high grade preparations from Sigma Chemical Co., St Louis, Missouri.

RESt LTN

5 - H T , qluco.~e uptake and .qlvcoqen corttent H i g h (95!~, O z : 5 " ; C O 2 ) in l,itro o x y g e n c o n c e n t r a t i o n h a d no effect o n t h e internal g l y c o g e n p o o l s of the w o r m s , irrespective of t h e presence o r a b s e n c e of external g l u c o s e (Table ]). H o w e v e r , in the a b s e n c e of external g l u c o s e t h e internal g l y c o g e n p o o l s were greatly r e d u c e d u n d e r b o t h a e r o b i c a n d a n a e r o b i c c o n d i t i o n s . A c o m p a r i s o n of t h e in vitro a n d in i'ico g l y c o g e n p o o l s indicated that in the p r e s e n c e of 1 0 r a M glucose, the w o r m s i n c r e a s e d their g l y c o g e n reserves by 31% a n d that, in the a b s e n c e of external

Table 1. Effect of 5-HT on in vitro glucose uptake and gb.cogen utilization by It. microstoma

In citro 95./; O 2 : 5 . , CO2 10raM glucose No glucose 9 5 ,"o l,,l t ' 2 . J .~,,o CO2 10raM glucose I0 m M glucose + 1 m M 5-HT 5 m M glucose 5 m M glucose + I m M 5-HT 2 m M glucose 2 m M glucose + 1 m M 5-HT No glucose No glucose + I m M 5-HT In civo

Glucose uptake (ktmol, g wet weight)

Glycogen content* (mg:g wet weight)

51.10 + 8.61~.

68.40 + 13.31 35.21 ~ 1.01

72.80 83.22 5/).00 51.23 19.11 19.73

74.00 ± 55.67 ± 58.23 ± 47.,";6 ± 52.77 ± 44.51 + 39.70 + 33.00 4 52.31/ ~

+ 3.62 + 4.20+ + 2.00 +_ 4.24 + 2.00 m 1.00

10.55 3.42t 3.42 2.20+ 4.1)3 2.64t 4.11 1.66+ 7.40

W o r m s were incubated at 3 7 1 for 60 min and the incubation media were gassed for 60 rain prior to and during incubation with the appropriate gas mix. Glucose uptake was calculated from the glucose remaining in the media after incubation. In rico glycogen was determined in unincubated worms as described in the text. * Glycogen values are expressed in glucosyl unit. t P < 0.02, significantly different from corresponding non-5-HT group. + P < 0.001, significantly different from corresponding anaerobic value. The results are means + SD (n = 3 7).

Carbohydrate utilization in H. microstoma

903

Table 2. In vitro [14C]glucose uptake by H. microstoma (cpm x 105/g wet weight) Incubation (n = 3) {.Air minus CO2 95~/o O2:5~o CO2 95% N2:53o CO2

Radioactivity in worm

[14C]glucose uptake

4.87 ± 0.24* 4.14 ± 0.37t 5.34 ± 0.04

54.91 ___ 10.04-t 76.02 ± 2.75t 91.20 ± 1.26

Excretion in medium 4.19 ± 1.19t 7.34 + 0.70t" 10.55 ± 1.00

Worms were incubated in KRP containing 10mM glucose plus 0.5pCi/ml act. 3 mCi/mmol) for 60 min at 37°C. The incubation medium was gassed for 60 rain prior to and during incubation with the appropriate gas mix. [~4C]glucose uptake was calculated by subtracting the radioactivity remaining as glucose after incubation, from the initial cpm (means ± SD). * P < 0.05; + P < 0.01, indicating degree of significance from anaerobic (95% N 2:5°,, CO2 ) incubation. ++Prcssurized laboratory air (from tap) contained about 21°,i O:

D-[U-14C]glucose (sp.

glucose, the worms' glycogen reserves decreased by 33%. U n d e r the high 0 2 concentration glucose uptake was significantly reduced as compared with the anaerobic (95'~i N2:5'~;~ C O z) conditions. Glucose uptake and glycogen utilization by H, microstoma was also studied at various concentrations of external glucose in the presence or absence of I m M 5-HT. 5-HT did not significantly increase glucose uptake irrespective of the external glucose concentration. In the presence of 5-HT internal glycogen reserves of the worms were depleted 16 19~/; at all concentrations of external glucose, and also in the absence of glucose. The results of ~4C-lahelled glucose uptake by H. microstoma show that incorporation of radiocarbon into the ethanol-soluble intermediates of the worms was significantly reduced when incubated under aerobic conditions (21 and 95~0 O:), Radioactivities that remained in the media after incubation, as glucose, largely corresponded to the above results. U n d e r aerobic conditions excretion of end-products was significantly reduced; in the absence of CO2 (Air-minus-COz) this reduction was even more pronounced (Table 2). C02-lixation

CO2-fixation by H. microstoma was investigated using N a i l ~4CO3 in the incubation media as a source of C O > The results demonstrated that, under 95~,,; O2:53f, CO2, incorporation of ~4CO2 was significantly decreased as compared with the 95% N 2:5~o CO2 incubation. Excretion of labelled end products was also significantly reduced under high 0 2 concentrations. Although the incubation medium was gased with CO2-free air, the addition of N a H I 4 C O 3 to the incubation nullified the air-minus-CO 2 conditions, and the results were therefore not significantly different from those under a 95% N2:5~o C O , gas phase (Table 3). Glycolytic e n z y m e s

5-HT had no significant effect on the activity of the glycolytic enzymes hexokinase, phosphofructokinase and pyruvate kinase from H. microstoma (Table 4). Further investigations revealed that the activity of P F K was significantly influenced by the concentration of A T P in the reaction system; the addition of 5-HT had no effect on the enzyme's activity at any

Table 3. hi citro 14CO2 incorporation from NaHI4CO3 by H. microstomas (cpm x 105/g wet weight of worm) Incubation (n = 3)

Radioactivity in worm

Excretion in medium

Air minus CO2 95'~',, O2 + 5~, CO 2 95%; N 2 + 5% CO2

2.30 ± 0.22 1.14 ± 0.21" 2.52 ± 0.32

7.58 ± 1.05 3.82 ± 0.96* 8.00 _+ 0.80

Worms were incubated in pregassed medium containing 10raM glucose and 0.5/*Ci/ml NaH14CO3 (sp. act. 0.1 mCi/mmol) in tightly stoppered conical flasks for 60rain at 37°C. See text for details. The results are means + SD. * P < 0.01, indicates values significantly different from anaerobic incubation. concentration of A T P tested (Fig. 1). Cyclic-AMP, in the presence or absence of 5-HT, stimulated P F K activity significantly (P < 0.02); similarly, with or without 5-HT, A M P was found to be even more effective (P < 0.001) in stimulating P F K activity (Fig. 1). Table 4. Effect of 5-HT on specific activity of selected glycolytic enzymes of H. microstoma nmol/min per mg protein Control 0.5 mM 5-HT Hexokinase Phosphofructokinase Pyruvate kinase

64 _+ 10 165 ± 10 47 _+ 12

65 ± 7 170 ± 14 37 + 12

Enzyme activities were assayed in duplicate and the results are presented as means + SD of at least 5 experiments. Reaction system for HK contained 90 mM Triethanolamine-HC! buffer, pH 7.4; 5 mM MgCI2; 8 mM glucose; 0.2 mM NADP: 20/*g glucose-6, phosphate dehydrogenase; 50/.1 enzyme preparation (50-60,ug protein) and 0.67 mM ATP, in a final volume of 3 ml. The PK assay system contained 90 mM triethanolamine buffer, pH 6.8; 66mM KC1; 0.33mM ADP; 0.1raM NADH; 5 m M MgCI 2 ; 20/*g LDH ; 50/*1 enzyme preparation (50-60/*g protein) and 1.67 mM PEP, in a final volume of 3 ml. The PFK system contained 90mM Triethanolamine-HCl, 90mM (NH4)2SO4, 0.1 mM EGTA, pH 8.0; 2 m M F-6, P; 0.67mM ATP; 0.1 mM NADH; 5 U c~-glycerophosphate dehydrogenase; 3 U trisephosphate isomerase; 3 U aldolase; 20/,1 (20-30/*g) sample in a final volume of 3ml. (n = 5 6.)

904

M. SAIDURRAHMANet al.

/

~8 o

S. 0 . 2

F

>,

e- . . . . . . . . . . . . o

0.YmM

~* 0 . 5 r a M

a.

o

0.1

015

o

¢.0

O.5mM

5-HT

cAMP* AMP*

¢.5

210

ATP (raM)

Fig. 1. Effect of 5-HT. cAMP and AMP on the activity of phosphofructokinase from H. microstoma tit increasing concentrations of ATP (n = 3-6). PFK activity was assayed as previously' described {Rahmand & Mettrick, 1982). * The addition of 5-HT did not further significantly increase PFK activity in the presence of either ATP, cAMP or AMP.

DISCUSSION

The present study confirms that H. microstoma is capable of active in zitro glucose uptake from an external pool and that, in the presence of high ambient 0 2 concentration, glucose uptake is significantly inhibited. A comparison of the size of the in vitro glycogen pool with that in vivo (Table 1) suggests that, in the presence of external glucose, the worms are capable of glyconeogenesis under both aerobic and anaerobic conditions. Therefore, the increased glucose uptake under anaerobic conditions may have been utilized for increased flux through the pathways. Our previous investigations (Rahman & Mettrick, 1982) showed that under anaerobic conditions (95°,i, N2:5% CO2) production and excretion of total end products was greatly increased. Although lactate production increased under aerobic conditions, production of succinate, acetate and propionate was decreased: this decrease was more pronounced in the absence of CO2 in the incubation media. The results of the experiments with radio-labelled glucose (Table 2) show that under aerobic conditions less 14C was incorporated into the ethanol-soluble intermediates of the worms as compared to the level of incorporations under anaerobic conditions. This leads to the conclusion that high ambient 02 concentration inhibits glucose uptake by H. microstoma. The absence of CO2 also produced the same effect. Analysis of the media

after incubation revealed that more [~4Clglucose remained in the media under aerobic than under anaerobic conditions. The increased radiocarbon in the worm extract following incubations under Air-minus-CO2, as compared to incubation under 95<~,~ 02:5,°0 CO2, may be explained b~ the fact that. in the absence of CO2, one of the vital branches of the pathways from phosphoenol pyruvate to malate may be partially or completely inhibited leading to the accumulation of labelled intermediate(s) in the glycolytic sequence. The increased glucose remaining in the incubation medium under the Air-minus-CO2 gas phase could also be explained in the same manner. Reduction of glucose uptake under a 95~}i; O2:Y},, CO2 gas phase, as evidenced by the significantly diminished ~4C incorporation in the worms and the larger amount of glucose remaining in the incubation medium (Table 2), may have been due to an inhibitory effect of high 02 concentration on the glycolytic sequence. Excretion of end-products was reduced despite the presence of CO2 in the incubation, suggesting an inhibition by the high 02 concentration of CO2-fixation by PEPCK in the transformation from phosphoenol pyruvate to oxaloacetate. It is possible that the oxidation of membrane phospholipid by the high 02 tension may also have affected glucose uptake. The experiments with NaHI4CO3 confirmed that under the 95% 02:5°'~, CO2 gas phase, incorporation

Carbohydrate utilization in H. microstoma of ~4CO2 was significantly decreased to only 45~ of the incorporation under a 95% N2:5°J~ CO2 gas phase. Incubation under high oxygen tension also resulted in a corresponding decrease in the excretion of end products (Table 3). In the present study 5 ~mol/ml NaH~'~CO3 was added to the incubation after gasing with CO2-free air. Therefore, the incubation gas phase designated "Air-minus-CO2" was not actually CO2-free as was evident by both ~4CO2 fixation and the high levels of excreted end-products. The results from the "Air-minus-CO2" gas phase were similar to those under anaerobic (95°~ N 2 .5,,, °/ CO2} conditions. We therefore conclude that high oxygen concentration inhibits CO2-fixation in H. microstoma, whereas atomospheric concentration of 02 (21~) had no detrimental effect on this vital metabolic function in H. microstoma. The ability of many parasitic helminths to fix CO2 through the enzyme PEPCK, and the subsequent utilization of carbon from this source for energy metabolism, has been well established (see Bryant, 1975, 1978). The amine 5-HT stimulates glucose uptake and, in the absence of glucose, increases the rate of glycogen breakdown in F. hepatica (Mansour, 1959, 1962: Mansour et al., 1960), H. diminuta (Mettrick et al., 1981) and in Schistosoma mansoni (Rahman & Mettrick, unpublished results). However, the results of the present study show that 5-HT has no significant stimulatory effect on glucose uptake by H. microstoma. Further, in both the presence and absence of external glucose, there was a 16-197/o decrease in glycogen content when 5-HT was present (see Table 1), indicating increased glycogen breakdown, possibly through stimulation of the glycogen phosphorylase system. In Ascaris suum 5-HT has been shown to activate phosphorylase with a concomitant rise in cAMP levels (Donahue et al., 1981). In H. diminuta 5-HT does not reduce glycogen levels when external glucose is present. The differences in the action of 5-HT on glucose uptake and glycogen utilization in the two congeners, H. diminuta and H. microstoma, are surprising. The most notable ecological difference between the two species is that H. diminuta is an active worm, whose migratory behaviour in the small intestine may be correlated with the circadian luminal 5-HT levels in the intestinal lumen (Mettrick & Cho, 1981, 1982; Cho & Mettrick, 1982). Adult H. microstoma lie in the bile duct of the mouse (Dvorak et al., 1961; Litchford, 1963), and even in heavy infections only the terminal part of the strobila reaches into the duodenal lumen. The physiological implications in the difference between habits do not appear to affect carbohydrate metabolism, as both H. diminuta and H. microstoma posses complex metabolic pathways involving COx-incorporation, resulting in at least three major end-products and the generation of ATP by both substrate-level and electron transport-mediated phosphorylation (Bryant, 1975, 1978; Rahman & Mettrick, 1982). Mettrick & Cho (1982) provided evidence that, in the case of carbohydrate uptake and metabolism by H. diminuta, 5-HT has a number of roles. First, 5-HT acts as a neurotransmitter stimulating worm motility; secondly 5-HT mobilizes glycogen reserves to provide energy for migration; thirdly, the luminal 5-HT

905

gradient may act as a directional guide to migrating worms, which results in H. diminuta moving up the luminal glucose gradient and activating 5-HT-stimulated glucose uptake by the worms. 5-HT is obviously important in the migrational responses of H. diminuta, and the lack of such response to 5-HT by H. microstoma may simply be related to the fact that H. microstoma is not a migrant. Further. the importance of the circadian variation in blood 5-HT levels, matching similar changes in blood glucose levels, explains why in the blood-feeders F. hepatica and S. mansoni glucose uptake is enhanced in the presence of 5-HT (Mansour, 1959; Bennett & Bueding, 1973). Comparison of observed mass action ratios and published apparent equilibrium constants (K') for glycolytic enzymes indicated that in Monil(formis duhius (Cornish et al., 1981), H. microstoma (Rahman & Mettrick, 1982), and H. diminuta (Rahman & Mettrick, unpublished results), the three enzymes hexokinase, phosphofructokinase and pyruvate kinase are potential regulating enzymes whose specific activities control the glycolytic pathways. Therefore, any effect on these enzymes would lead to marked changes in the metabolic process. In the case of H. diminuta the activity of PFK is significantly enhanced (P < 0.05) in the presence of 5-HT, which results in an increased rate'of carbon flux through the pathway. However, in the present study on H. microstoma 5-HT did not stimulate any of the three enzymes examined. In F. hepatica 5-HT has been shown to activate glycogen phosphorylase and adenylate cyclase leading to an increase in the formation of cAMP (Mansour, 1959, 1962; Mansour et al., 1960). It was further suggested that the generation of endogenous cAMP in the presence of 5-HT increased the activity of PFK (Mansour, 1967). Activation of adenylate cyclase by 5-HT has also been demonstrated in Schistosoma japonicum (Higashi et al., 1973), but 5-HT-stimulated cAMP production could not be confirmed. In A. suum 5-HT increases the level of cAMP three-fold; glycogen synthase and phosphorylase activity also increased (Donahue et al., 1981). In H. microstoma the specific activity of PFK increased over an ATP concentration range of 0q3.67 raM, before an asymptote was reached. The addition of 0.5 mM 5-HT had no significant effect on PFK activity. Phosphofructokinase activity was significantly enhanced by the addition of cAMP, in the presence of ATP; activity was further enhanced by the addition of AMP. In both cases the presence of 5-HT neither stimulated nor inhibited enzyme activity, ATP is apparently the most important adenylate involved in the regulation of PFK activity; the addition of AMP increased the activity of PFK by 50%. Our studies on H. microstoma do not support the suggestion that 5-HT increases endogenous cAMP, nor that 5-HT, in the presence of ATP, AMP or cAMP, enhances PFK activity (Mansour, 1959, 1962, 1967; Mansour et al., 1960). We conclude that there are significant differences in the action of 5-HT on glucose uptake and carbohydrate metabolism in H. microstoma and in H. diminuta; as these two cestodes are the only cases in which the action of 5-HT has been followed from glucose

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906

uptake, through intermediary metabolism and enzyme activity to excretory products, it would be premature to generalize on the role of host endogenous 5-HT in helminth biology. To date no helminth has been shown to be capable of complete de novo synthesis of 5-HT, leading both Bennett & Bueding (1973) and Mansour (1979) to conclude that any 5-HT present in helminths is of host origin. The role(s) of host 5-HT in helminth carbohydrate metabolism is therefore intriguing, adding a new dimension to the complexity of the host parasite interaction at the physiological biochemical level. The present study also raises the question of the closeness of the taxonomic relationship of H. diminuta and H. microstoma. Acknowledgement--This work was supported by the Natural Sciences and Engineering Research Council of Canada through grant No. A4667 to DFM.

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