Fasciola hepatica: Increase of glycogen phosphorylase activity due to prostaglandins

EXPERIMENTALPARASITOLOGY

Fasciola

56,89-92

hepatica:

Increase of Glycogen Phosphorylase Due to Prostaglandins

TATJANASIMONIC~ Istituto

Policattedra

(1983)

, PAOLA SARTORELLI , AND ALBERTO

Activity

LOCATELLI

di Patologia Generale Veterinaria, Facolfd di Medicina Veterinaria, Studi di Milano, Via Celoria 10, Milan 20133, Italy

Universitd

degli

(Accepted for publication 15 March 1983) &MONK, T., SARTORELLI, P., AND LOCATELLI, A. 1983. Fasciola hepatica: Increase of glycogen phosphorylase activity due to prostaglandins. Experimental Parasitology 56, 8992. Both prostaglandin Et (PGEt) and prostaglandin Fh (PGF2,) stimulate the glycogen phosphorylase (EC 2.4.1.1.) activity of Fasciola hepatica. Whole or sliced parasites were incubated with PGE, (2.8 x IO-‘and 2.8 x IO-s&f) and PGF,,(2.1 x lo-‘and 2.1 x IO-5 M) and enzyme activity was measured in homogenates prepared immediately following the incubation. No substantially different effect was noted between the two assayed doses of prostaglandins. Prostaglandins appeared to be less effective in sliced parasites. INDEX DESCRIFTORS: Fasciola hepatica; Trematode, parasitic; Prostaglandins; Enzyme, activity, regulation; Phosphorylase (EC 2.4.1.1.); Glycogen.

fected by prostaglandins, specially by the E prostaglandins (Kuehl 1974). A stimulatory effect of prostaglandin El (PGE1) on glycogen phosphorylase activity of vertebrate cells was reported, and these actions seemed to be mediated by 3’,5’-adenosine monophosphate (3’,5’- cyclic AMP) (Satoh ef al. 1975; Drummond et al. 1977). Therefore, experiments were conducted to determine whether prostaglandins affect this important enzyme activity in F. hepatica.

INTRODUCTION

Beside its importance in medical parasitology, Fusciofa heparica is interesting from an evolutionary viewpoint “between” unicellular organisms and vertebrates. In this trematode, energy metabolism is mainly based on anaerobic utilization of monosaccharides; glycogen being the principal storage compound (Pantelouris 1971). Glycogen phosphorylase is a key enzyme of F. hepatica, the activity of which is modulated by different substances (Mansour et al. 1960; Simonic and Locatelli 1978). Nevertheless, the mechanism of its regulation has only been partially elucidated. As in mammals, the adenyl cyclase system appears to be involved. In mammals, adenyl cyclase activity is affected by various hormones (Sutherland and Robison 1966; Perkins 1973). In F. hepatica this activity is altered by 5hydroxytryptamine (5-HT) and related compounds (Mansour 1979). It is well known that adenyl cyclase activity of some mammalian tissues is also af-

MATERIALS

AND METHODS

Fasciola hepatica were extracted from cattle bile ducts and maintained at 37 C for 20 hr in Tyrode solution, with added horse serum (10%) and antibiotics (penicillin 100,090 U and streptomycin 0.25 g/100 ml). The washed parasites were then incubated at 30 C for 30 min in flasks containing incubation medium (20 ml/ five flukes) under nitrogen, as described by Mansour et al. (1960). Three flasks were incubated at the same time, one of which was the control, while the other two contained 100 ng and 10 &ml of PGE, (corresponding to 2.8 x lo-‘and 2.8 x 10-5M concentrations). Using the same experimental design, prostaglandin F,, (100 ng and 10 &ml, corresponding to 2.1 x IO-‘and 2.1 x 10mSMconcentrations) was also assayed. In a separate set of flasks, prepared as above, the same substances (PGE, and PGF,) at the same concentrations were assayed on sliced parasites (five par-

’ Present address: Istituto di Fisiologia Veterinaria e Biochimica, Universita degli Studi di Milano, Via Celoria, 10 I-20133 Milano, Italy. 89

0014-4894/83 $3.00 Copyright@ 1983 by Academic Press,Inc. rights of reproduction in any form resewed.

AU

90

SIMONIC,

SARTORELLI,

asitesiflask). The prostaglandins were supplied by Upjohn Diagnostics, Kalamazoo, Michigan, and used without further purification. At the end of the incubation, the parasites were blotted on filter paper, weighed, and placed in 10 vol of a solution containing 0.035 M potassium glycerophosphate, 0.03 M L-cysteine, and 0.1 M sodium chloride, which was adjusted to pH 6.5 with HCl. The F. hepntica were then homogenized with a Teflon-pestle motor-driven homogenizer at 0 C. Glycogen phosphorylase (EC 2.4.1.1) activity was measured in the homogenate, as described by Mansour et al. (l%O), but sodium fluoride was substituted for a sodium chloride solution of the same molarity because a possible effect of prostaglandins could be masked by fluoride (Simonic et al., 1980).The reaction mixture contained at final concentration: glucose lphosphate, 0.0375 M; glycogen, 2.5%; and NaCl, 0.125 M, dissolved in the above-described solution at pH 6.5. Total volume of the assay mixture was 0.8 ml. The reaction, at 30 C, was started by the addition of 0.2 ml of the homogenate; aliquots of the reaction mixture (0.2 ml) were transferred to 0.2 ml of 5% trichloracetic acid at different times (0, 5, 15, and 30 min). The samples were then centrifuged at 4000 rpm for 10 min and the supematants assayed for inorganic phosphorus by the method of Fiske and Subbarow (1925). Protein contents of the F. hepatica homogenates were determined by the method of Lowry et al. (1951). Statistical evaluations were performed by the two-way analysis of variance.

AND LOCATELLI

patica both in whole and in sliced parasites.

In whole parasites, the stimulation of glycogen phosphorylase was more apparent with PGEl (Table I) than PGF2, (Table II); however, with both prostaglandins, the effect became more evident with increased incubation time, the differences from the controls being highly significant after 30 min. The effects of the two concentrations of PGE, and PGF2, were not statistically different. In sliced parasites, the treatment with the prostaglandins produced increases of glycogen phosphorylase activity quantitatively smaller than those obtained with whole parasites (Tables I, II). DISCUSSION

Regulation of metabolic pathways is already recognizable at the early stage of the animal evolution. Often such regulation occurs by similar mechanisms; cyclic nucleotides appear to have a regulatory role in bacteria (Peterkofsky 1976) and more complex organisms. An endogenous synthesis of prostaglandins was recently demonstrated in bacteria and clams (Gulbis et al. 1979; Nomura et al. 1979). Prostaglandins constitute another ubiquitous system, at least in mammals, and appear to be important in the synthesis of

RESULTS

Prostaglandin El and FZuincreased the glycogen phosphorylase activity of F. he-

TABLE I Glycogen Phosphorylase Activity of Whole and Sliced Fasciola hepatica, Untreated and Treated with Two Different Doses of PGE, Added substances

Whole Fasciola

Sliced Fusciola hepatica

hepatica

5 min

IS min

30 min

5 min

15 min

30 min

None

1.30 f 0.18

3.42 f 0.44

5.91 5 0.82

1.35 2 0.16

3.60 f 0.35

7.11 2 0.72

PGE, (2.8 x lo-‘M)

2.24* -r 0.54

4.81* 2 0.59

8.79** k 0.98

1.36 t 0.26

4.49** ‘: 0.43

8.19* + 0.61

PGE, (2.8 x lo-5M)

1.82* f 0.19

3.92% k 0.52

7.96** + 0.99

2.40** k 0.20

4.70** f 0.37

9.25* k 0.78

Note. The values of the enzyme activity (ug P/mg protein), measured in the homogenates after 5, 15, and 30 min of reaction, are the mean values + SE of eight replications. * P < 0.05. ** P < 0.01.

Fusciolu hepatica: GLYCOGEN PHOSPHORYLASE AND PROSTAGLANDINS

91

TABLE II Glycogen Phosphorylase Activity of Whole and Sliced Fasciolu hepanca, Untreated and Treated with Two Different Doses of PGF*. Added substances

Whole Fasciola hepatica

Sliced Fasciola hepatica

5 min

15 min

30 min

5 min

15 min

30 min

None

1.11 2 0.16

2.35 f 0.27

3.73 2 0.59

1.05 k 0.10

2.83 f 0.25

4.93 + 0.50

PC% (2.1 x lo-‘A!!)

1.13 f 0.16

2.88* + 0.27

5.11** f 0.55

1.40* k 0.13

2.96 f 0.26

5.67* * 0.45

PC%. (2.1 x lo-JM)

1.16 f 0.14

2.66* f 0.30

5.00** f 0.67

1.39* 2 0.07

2.96 f 0.22

5.43* * 0.47

Note. The values of the enzyme activity (up P/mg protein), measured in the homogenates after 5, 15, and 30 min of reaction, are the mean values f SE of 13 replications. * P < 0.05. ** P < 0.01.

regulatory substances. This is suggested by the ability of prostaglandins to modulate the activities of enzymes responsible for cyclic nucleotides synthesis (Kuehl 1974), and is thought to play a homeostatic role at the cellular level (Markelonis and Garbus 1975). The present investigation showed that glycogen phosphorylase of Fusciolu heputicu is sensitive to prostaglandins (PGE1 and PGF2,). A stimulating effect of PGEl was also observed on this enzyme in human placenta (Satoh et al. 1975) and C-6 astrocytoma cells (Drummond et al. 1977). In addition, the latter authors found that the action of PGEl was mediated by cyclic AMP. Such a mechanism may also function in F. heputicu (Mansour et al. 1960; Mansour 1979). Satoh et al. (1975) reported no effect of PGF*, on human placenta phosphorylase, whereas our results show an activating effect on the trematode’s enzyme. No statistically different effects of prostaglandins between the two doses assayed were noted in whole worms. Therefore, maximal effects were shown with concentrations of PGEl and PGF2, as low as 2.8 x 10-7and 2.1 x 10-7M. It is possible that a transport process of prostaglandins through the absorbing tegument of F. heputicu (Pantelouris, 1971) was involved. Bito (1975) provided evidence demonstrating the exis-

tence for prostaglandins of facilitated and active transport processes, operating across the biological membranes. The finding that F. heputicu is sensitive to prostaglandins at an important step of metabolic regulation must be emphasized. A possible physiological role of these substances in trematodes can be only hypothesized, because evidence of endogenous prostaglandin biosynthesis is still lacking. However, a prostaglandin synthetase was detected in lower organisms as bacteria (Gulbis et al. 1979), and some precursor fatty acids of prostaglandins were found in Fusciolu heputicu (Oldenborg et al. 1976). Moreover, this trematode, despite its poor oxygen environment, was able to carry out some other oxygen-requiring process, like hardening of egg shell (Mansour 1958; Smyth and Clegg 1959), and a limited oxydative phosphorylation (Van Vugt et al. 1976). ACKNOWLEDGMENT This investigation was supported by Grant CT 73.00623.04from the CNR. REFERENCES BITO, L. Z. 1975. The role of transport processes in

the distribution and disposition of prostaglandins. In “International Conference on Prostaglandins,” Florence, Italy, May 26-30.

SIMONIC, SARTORELLI,

92

DRUMMOND,A. H., BAGULEY, B. C., AND STAEHELIN, M. 1977. Beta adrenergic regulation of glycogen phosphorylase activity and adenosine cyclic 3’S’monophosphate accumulation in control and desensitized C-6 astrocytoma cells. Molecular Pharmacology 13, 1159-1169. FISKE,~. H., ANDSUBBAROW, Y. 1925.Determinationof inorganic phosphorus in serum and plasma. Journal of Biological

Chemistry

66, 375-400.

GULBIS, E., MARION, A. M., DUMONT,J. E., AND SCHELL-FREDERICK,E. 1979. Prostaglandin formation in bacteria. Prostaglandins 18, 397-400. LOWRY,0. N., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265-275. KUEHL, F. A., JR. 1974. Prostaglandins, cyclic nucleotides and cell function. Prostaglandins 5, 325-340. MANSOUR,T. E. 1958. Effect of serotonin on phenol oxidase from the liver fluke Fasciola hepatica and from other sources. Biochimica et Biophysics Acta 30, 492-500. MANSOUR, T. E. 1979. Chemotherapy of parasitic worms: New biochemical strategies. Science 205, 462-469. MANSOUR,T. E., SUTHERLAND,E. W., RALL, T. W., AND BUEDING, E. 1960. The effect of serotonin (5hydroxytryptamine) on the formation of adenosine 3’,5’-phosphate by tissue particles from the liver fluke, Fasciola hepatica. Journal of Biological Chemistry

235, 466-470.

MARKELONIS,G., AND GARBUS,J. 1975. Alterations of intracellular oxidative metabolism as stimuli evoking prostaglandin biosynthesis. Prostaglandins 10, 10871106. NOMURA,T., OGATA, H., AND LUCAS, A. 1979. Mise en evidence de prostaglandines chez des mollusques bivalves. Revue Internationale d’Oce’anographie Me’dicale

53-54, 87-93.

OLDENBORG, V., VAN VUGT, F., VAN GOLDE,L. M. G., AND VAN DEN BERGH,S. G. 1976. Synthesis of fatty

AND LOCATELLI

acids and phospholipids in Fasciula hepatica. In “Biochemistry of Parasites and Host-Parasite Relationships” (H. Van den Bossche, ed.), pp. 159166. North-Holland, Amsterdam/New York/Oxford. PANTELOURIS, E. M. 1971.PhysiologyofFasciola hepatica. In “Second International Liverfluke Colloquium,” pp. 126-147, Wageningen, The Netherlands, 2-6 October 1967. Merck Sharpe & Dohme. PERKINS,J. P. 1973. Adenyl cyclase. In “Advances in Cyclic Nucleotide Research” (P. Greengard and G. A. Robison, eds.), Vol. 3, pp. l-64. Raven Press, New York. PETERKOFSKY,A. 1976. Cyclic Nucleotides in Bacteria. In “Advances in Cyclic Nucleotide Research” (l? Greengard and G. A. Robison, eds.), Vol. 7, pp. l-48. Raven Press, New York. SATOH,K., KINOSHITA, K., AND SAKAMOTO,S. 1975. In vitro activation of the placental phosphorylase. In “International

Conference

on Prostaglandins,”

Florence, Italy, May 26-30. SIMONIC,T.,ANDLOCATELLI,A. 1978.Effectof3’,5’-cyclic GMP on Fasciola hepatica phosphorylase. Archivio Veterinario Italiano 29, 101-103. SIMONIC, T., SARTORELLI,P., GENCHI, C., AND LoCATELLI,A. 1980. Liver glycogen-phosphorylase activity in rats after intraperitoneal implantation of Fasciola hepatica. Archivio Veterinario Italian0 31, 69-72. SMYTH, J. D., AND CLEGG, J. A. 1959. Egg-shell formation in Trematodes and Cestodes. Experimental Parasitology

8, 286-323.

SUTHERLAND,E. W., AND ROBISON,G. A. 1966. The role of cyclic 3’,5’-AMP in responses to catecholamines and other hormones. Pharmacological Reviews 18, 145-162. VAN VUGT, F., KALAYCIOGLU, L., AND VAN DEN BERGH,S. G. 1976. ATP production in Fasciola hepatica mitochondria. In “Biochemistry of Parasites and Host-Parasite Relationships” (H. Van den Bossche, ed.), pp. 151-158. North-Holland, Amsterdam/New York/Oxford.