Putative neurotransmitters in three experimental filariasis models

Life Sciences, Vol. 39, pp. 1539-1542 Printed in the U.S.A.

Pergamon Journals

PUTATIVE NEI3~C~T RANSMITTE RS IN TH REE EXPE RIMENTAL FILA RIASIS MODELS C.' 3ACQUOT, H. BARRETEAU, 3.H. TROUVIN, P. GAYRAL, and 5.P. LEROY Lab. Pharmacol. and Parasitol. Fac. Pharmacie. F-92290 Chatenay-Malabry Rhone-Poulenc. C e n t r e Nicolas Grillet F-9tt4OO Vitry sur Seine

(Received in final form August I, 1986)

Summary Some p u t a t i v e n e u r o t r a n s m i t t e r s in three e x p e r i m e n t a l f i l a r i a s i s models were investigated by a new relevant chromatographic method, sensitive and specific. No catecholaminergic compounds have been detected, but serotonin was found in Dipetalonema vitae. However, f u r t h e r i n v e s t i g a t i o n s revealed very high levels of gammaamino butyric acid (GABA) in the macro-filariae. These data allow us to foresee new fields in filariasis therapeutics.

Filariasis is an endemic disease which affects 250 millions people in the world and is registered as one of the top priorities at the World Health Organization. The active drugs, filaricides, are often toxic for men. These substances are selected from animal filariasis models with a good predictive value; but this research is d i f f i c u l t for, at present, there exists no method of culture of the macro-filariae, i_nn vitro. This, perhaps, explains the lack of knowledge of these parasites biochemistry, p a r t i c u l a r l y for the n e u r o t r a n s m i t t e r s of these Nematodes, e x c e p t for Acetylcholine (I,2). The discovery of putative neurotransmitters would allow one to foresee the existence of various receptor units. This interesting approach has already found an a p p l i c a t i o n for the t r e a t m e n t of O n c h o c e r c i a s i s , thanks to A v e r m e c t m , a GABA-ergic drug (3,4). We propose determining the levels of putative neurotransmitters (catecholamines, indoleamines and GABA) in three experimental filariasis models. Materials and Methods Parasites • the macrofilariae (Ig. per sample) were removed thus : - from the pleural cavity of the Cotton Rat (Sigmodon hispidus) for Litomosoi'des carinii (L.c), - from the peritoneal cavity of Proechymis oris for Dipetalonema dessetae (D.d)(5), - and beneath the skin of the jird (Meriones inguiculatus) for Dipetalonema vitae

(D.v). 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Journals Ltd.

1540

Putative

Neurotransmitters

in Filariae

Vol.

39, No.

17,

The fdartae of both sexes were rinsed in physiological serum, then homogenized in perchloric acid O.# N with anttoxidant (cysteine 0,1%, sodium metabisulfite 0,1% and EDTANa2 0,1%). After centrifugatlon, determinations were performed on the perchloric layer. In order to concentrate the biological samples, we performed a lyophhzation of the f i l a r i a e b e f o r e the h o m o g e n i z a t i o n step. L y o p h i l i z a t l o n did not a l t e r the neurotransm~tters, and allowed detection of lower quantities. Separation and quantitation of biogemc amines : Chromatograpkc analysis with electrochemical detection (LC-ECD) was performed according MORIER and RIPS (6). The chromatographtc system consists of a BECKMAN 112 pump with a constant flow rate set at lml/mm., a rheodyne injecnon valve with a 201Jl loop, a reversed phase column (Ultrasphere ODS, 5pm, 150x4 mm, BECKMAN) and an electrochemical detector METROHM 641 VA with a glassy carbon electrode set to a potential of 0.g Volt versus an Ag/AgCI reference electrode. The mobile phase was prepared with potasstum phosphate buffer 0.1M (KH2PO4), heptane sulfonic acid 5mM and methanol 10% (v/v). pH was adjusted to 3,g with KOH 3M. By modifying the composition of the mobile phase (pH, polarity or methanol concentration), we established the optimal conditions for the separation of 13 compounds, total elution was achieved within 30 minutes (fig. l) and the sensitivity of the LC-ECD technique enabled the detection of amines to be made to the extent of lng/g of wet tissues.

13

10

1

J 5

10

15

20

30

nt~n.

FIG. 1

Chromatogram of a standard m i x t u r e (0.5 n g / i n j e c t i o n of each compound, e x c e p t gng f o r T r p ) . C h r o m a t o g r a p h i c c o n d i t i o n s and d e t e c t i o n were performed as described in the t e x t . 1= 3,~-methoxyhydroxyphenylglycol (M-FG), 2 n o r e p i n e p h r i n e (BE), 3- e p i n e p h r i n e (E), 4= 5-hydroxytryptophan (5-HTP), 5 n o r m e t a n e p h r i n e (NVtq), 6= 3 , 4 - d i h y d r o x y p h e n y l a c e t l c a c i d (DOPAC), 7- m e t a n e p h r i n e (/V¢4), g_ dopamine (DA), 9- ~ - h y d r o x y i n d o l a c e t i c acid (5-HIA&), 10- honovanillic acid (I-NA), 11- tryptophan (Trp), 12- 3-rrethoxytyra~nine (3-MY) and 13 s e r o t o n i n (5-HT).

1986

Vol. 39, No. 17, 1986

Putative Neurotransmitters in Filariae

We also h a v e m e a s u r e d o c t o p a m i n e (Oc) and t y r a m i n e (Ty) with t h e s a m e sensitivity range but with an higher potential (1 Volt). By analyzing some filariae samples previously spiked with known amounts of standard c a t e c h o l and indole derivatives, we had a positive control demonstrating that no a l t e r a t i o n occurred during preparation steps. Radioenzymatic assay of norepmephrine and dopamine : This method has been described by DA PRADA and Z U R C H E R (7) and by PEULER and J O H N S O N (8). It is s e n s i t i v e ( 1 0 0 p g / g w e t t i s s u e s ) and s p e c i f i c for c a t e c h o l a m i n e s . The p a r a s i t e s w e r e h o m o g e m z e d in a p e r c h l o r i c a n t i o x i d i z e r mixture (200rag of parasites in 1,5ml). The e n z y m a t i c methoxylation was assured by the c a t e c h o l - O - m e t h y l transferase (COMT), removed from r a t ' s liver (9), in the presence of a t r i t i a t e d m e t h y l donor ( J H - S - a d e n o s y l m e t h i o n i n e ) ( A M E R S H A M ) . 3H-methoxylated derivatLves were then measured a f t e r separation by thin layer chromatography (TLC) and counted by liquid scintillation. G a m m a - a m i n o - b u t y r i c acid (GABA) assay : The levels of GABA have been assayed according SEILER's method (10). A f t er h o m o g e n i z a t i o n and p e r c h l o r i c d e f e c a t i o n of t h e f i l a r i a e s u s p e n s i o n , GABA undergoes a dansylation by the dansyl chloride in excess. The dansylated derivate was next e x t r a c t e d in a toluene phase, then separated by TLC. A f t er elution, dansyl-GABA was measured by s p e c t r o f l u o r i m e t r y . Results C a t e c h o l a m i n e levels : In our e x p e r i m e n t a l conditions, we have not d e t e c t e d the presence of DA, NE and E. These negative results were confirmed by the more sensitive radioenzymatic m e t h o d . In h k e m a n n e r , no c a t e c h o l a m i n e r g i c m e t a b o l i t e s w e r e found too.

TABLE I

L e v e l s of G A B A , Trp,

GABA

pg/g

5-HTP

and

5-HT

Trp

pg/g

in t h r e e

5-HTP ng/g

macrofilariae.

5-HT ng/g

D.vitae mead

+ SEM

n-10

D.dessetae mean n=3 + SEM

L.carinii mean n:10 + SEM

13.00 + 2.52

8.01 + 0.8#

190.50 + 26.90

210.80

25.87 + 1.37

7.48 + 0.82

25.57 + 1.17

N.D.

25.36 + 2.83

5.78 + 1.32

28.76 +2.70

N.D.

+ 20.70

N.D. - no d e t e c t a b l e Mean of n determinations,

each performed

on I g o f p a r a s i t e s .

1541

1542

Putative Neurotransmitters in Filariae

Vol. 39, No. 17, 1986

Indolamine levels : The results (Table I) show d i f f e r e n t contents varying w l t h the type of macrofHaHae one considers. Serotomn was only d e t e c t e d in D.vitae, associated w i t h related levels of its precursor 5-HTP. The a m i n o - a c i d precursor for this l a t t e r substance (Trp) was at a ten t i m e s g r e a t e r level than that of 5-HT. In the other types, Trp was present at the same level but t h e r e existed very weak levels of 5-HTP and an absence of 5-HT. Nor 5 - H I A A was d e t e c t e d for a l i m i t value of I n g / g of f l l a H a e (wet tissue). G a m m a - a m i n o - b u t y r i c acid levels • The ]evels of GABA were very high in the m a c r o f i l a r l a e and depending on the type, varying f r o m I0 to 25 pg/g (Table I). Discussion

Our results show for the three species of macrofilariae, very high levels of GABA, but u n d e t e c t a b l e l e v e l s of NE, E, D A , Oc and t h e i r p r e c u r s o r s and m a j o r c a t a b o l i t e s . Serotonin is also u n d e t e c t a b l e m all, except in D.vitae. F u r t h e r investigations should be p e r f o r m e d in order to d e t e r m i n e whether the GABA is located in neurons or in o t h e r tissues. Among the available methods, we can propose p h o t o a f f i n i t y labelling to observe more precisely GABA d l s t r i b u t ] o n m the worms. By specific and sensitive methods (i.e. L C - E C D and r a d i o e n z y m a t i c assay) we have not d e t e c t e d the presence of c a t e c h o l a m i n e r g l c compounds in these t h r e e studied species. These results are not in a g r e e m e n t w i t h the hypothesis of a p u t a t i v e role of o c t o p a m i n e in neurotransmission (11) and are also in opposition w i t h previous q u a n t i t a t i v e r e s u l t s of S A X E N A and al. (12) w h o f o u n d c a t e c h o l a m m e s and serotonin in L.c. at s i g n i f i c a n t levels. However, the f l u o r i m e t r i c method used by Saxena had weak s p e c i f i c i t y and s e n s i t i v i t y (e.g. t r y p t o p h a n has been described as i n t e r f e r r i n g compound in 5-HT d e t e c t i o n ) (13). We do not neglect that presence of c a t e c h o l a m i n e s at a r e a l l y weak level, below our d e t e c t i o n h m i t , is possible. T h s could be c o n f i r m e d by using another a n a l y t i c a l method (e.g. f o r m a l d e h y d e - i n d u c e d fluorescence). Presence of a s i g n i f i c a n t level of serotonm in D . v i t a e w l t h no t r a c e of 5-HIAA, could be explained e i t h e r by a d i f f e r e n t m e t a b o l i c pathway or by a very low t u r n - o v e r rate of this n e u r o t r a n s m i t t e r . In conclusion, this study provides arguments for GABA as a second putatLve neurot r a n s m i t t e r , in addition w i t h a c e t y l c h o l i n e (2). These findings could posslbly open new fields for a n t i f i l a r i a s i s drug research. References

1. 2. 3.

H. MELLANBY, Parasitology 45 287-294 (1955). D.3. Mc LAREN, Parasitology 655 507-524 (1972). M.A. AZIZ, S. DIALLO, I.N. DIOL, M. LARIVIERE and M. PORTA, Lancet 24 1 71-1 73 (1982). 4. A. ROUGEMONT, Lancet, 20 1182 (1982). 5. P. GAYRAL, G. DREYFUSS and 3.C. GANTIER, 3. Pharmacol (PARIS), 13 49-63 (1982). 6. E. MORIER and R. RIPS, IRCS Medical Science 9 454-455 (1981). 7. M. DA PRADA and G. ZURCHER, Life Sci. 19 H61-1174 (1976). 8. 3.D. PEULER and G.A. 3OHNSON, Life Sci. 21 625-636 (1977). 9. 3. AXELROD and R. TOMCHIK, 3. Biol. Chem__ 233 702-705 (1958). 10. N. SELLER, Research Method in Neurochemistry Eds N. MARKS and R. RODNIGHT, 3 419-4t~3 Plenum Press, New York (1975). 11. C.A. BREEN and-D.H.L. ATWOOD, Nature 303 716-718 (1983). 12. 3.K. SAXENA, S.K. BOSE, R. SEN, R.K. CHATTER~]EE, A.B. SEN and S.GHATAK, Exp. Parasitol. 43 239-2t~3 (1977). 13. G.M. GAL, R.B. YOUNG and A.D. SHERMAN, 3. Neurochem. 31 237-244 (1978).