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Secretory cholinesterase of Ancylostoma ceylanicum: Effect of tubulin binding agents and benzimidazole anthelmintics
Life Sciences, %'ol. 50, pp. 747-752 Printed in the USA
Pergamon Press
SECRETORY CHOLINESTERASE OF Ancylostoma ceylanicum :EFFECT OF TUBULIN BINDING AGENTS AND BENZIMIDAZOLE ANTHELMINTICS
Babu L. Tekwani* Division of Biochemistry, Central Drug Research Institute Lucknow-226001 (India) (Received in final form January 7, 1992)
Summarv
Ancylostoma ceylanicum, the human hookworm parasite, exhibited significant secretion of cholinesterase when maintained in vitro in RPMI-1640 medium. Secretion of the enzyme was linear up-to 4 hours of incubation. About 40 percent of the total cholinesterase activity was localized in the soluble fraction, while remaining activity was associated with the particulate fraction of the nematode. Exposure of the hookworms to colchicine in vitro caused significant inhibition in secretion of the enzyme by the parasite with concomitant accumulation of cholinesterase within the adult worms. Vinblastin did not show noticeable effect on the enzyme secretion as well as activity within the parasite. Incubation of hookworms with some benzimidazole anthelmintics viz., mebendazole or albendazole significantly reduced the capacity of the worms to secrete cholinesterase and increase in enzyme activity within the parasite. Adult worms recovered from mebendazole treated hamsters exhibited about 3 fold greater activity of cholinesterase as well as significantly lower capacity to secrete cholinesterase in vitro as compared to the worms recovered from untreated animals. These observations indicate role of microtubules in the secretion of cholinesterase by hookworms and as a target for the action of benzimidazole anthelmintics. Interest in the cholinesterases of parasitic nematodes was initially centered on their physiological role in the nematode nervous system (1). Several species of nematode parasites also secrete cholinesterase during incubation in vitro and compelling immunological evidences indicate that the enzyme is also secreted by the parasites within their hosts (2-4). The nematode secretory cholinesterases are present either singly (2) or in multiple molecular forms (5) and differ from the classical mammalian acetylcholinesterase in that they show substantial hydrolysis of butyrylcholine, are cytosolic in nature, and are inhibited by higher substrate concentrations (3). Secretory cholinesterases provide an adaptive mechanism through which certain parasites regulate the intra-host physiological and immunological environment (3). In gastrointestinal nematodes secretion of cholinesterase provides a "biochemical holdfast" by reducing the intestinal peristaltic movements (6) mucus secretion and immunological intervention (7). Analysis of this function of nematodes has been *Present address- Department of Cellular and Molecular physiology, Milton S Hershey Medical Center, College of Medicine, Pennsylvania State University, Hershey, PA 17033 USA.
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press plc All rights reserved.
748
C h o l i n e s t e r a s e S e c r e t i o n by Hookworms
Vol. 50, No. 10, 1992
suggested as a parameter to screen anthelmintics in vitro (8,9). In view of the importance of microtubules in secretory functions (10) and site for the action ol benzimidazole anthelmintics (11) effect of tubulin binding agents viz., colchicine and vinblastin as well as some broad spectrum anthelmintics viz., mebendazole (MBZ) and albendazole (ALBZ) was studied on the secretion of cholinesterase by a human hookworm parasite, Ancylostoma ceylanicum. Materials and Methods parasite and the laboratory hosts-Infection of A n c y / o s t o m a ceylanicu~ (Nematoda: strongyloidea) was maintained in golden hamsters (Mesocricetus auratus). hamsters weighing about 50-60 g were orally inoculated with 100+10 infective larvae suspended in normal saline. The larvae after passing through the oesophagus, stomach and duodenum reach the jejunum and develop into adult worms within 18-+3 days posl inoculation. Infected hamsters were sacrificed by decapitation, jejunum was excised oul and opened longitudinally. Adult hookworms were picked by the forceps, suspended in RPMI-1640 medium and immediately used for the analysis of cholinesterase secretion. In vitro drug treatment- For analyzing the effects of tubulin binding agents and benzimidazole anthelmintics in vitro , adult parasites were incubated with desired concentration of the drug at 37oc in RPMI-1640 medium for 60 min. The worms were then transfered to drug free medium and subjected to analysis of cholinesterase secretion and assay. In vivo drug treatment- Infected hamsters were treated with a single oral dose ol MBZ ( 6 mg/kg body weight) (therapeutic dose) and the animals were sacrificed 18 h post drug treatment. Adult worms were mostly recovered in the distal part of the intestine and subjected to analysis of enzyme secretion and assay. Acetylcholinesterase assay and secretion in vitro-Untreated or drug treated adult A. ceylanicum were incubated in RPMI-1640 medium at 37oc and aliquots from the media were drawn after every 60 min for the assay of acetylcholinesterase activity. For analysis of enzyme activity in the adult worms, the parasites were homogenized (10 % w/v) in 0.15M KCI. Acetylcholinesterase was assayed by the spectrophotometric method of EIIman et aL (12). Protein was determined by the method of Lowry et al. (13) using BSA as the standard. All the results are presented as mean+S.D..The results were analyzed by Students t test and p values less than 0.05 were considered statistically significant. Results TABLE I Sub-cellular Distribution of Cholinesterase in Ancylostoma ceylanicum worms Fraction Homogenate 12,000 g pellet 105,000g pellet 105,000 g supt.
U/g tissue 692.3+103.3 353.0+ 94.3 13.8+ 3.9 290.7+ 63.4
U/mg protein 15.21 +6.10 6.92+1.93 3.14_+0.39 16.11+4.67
U-enzyme units (~mol acetylcholine hydrolyzed / hour) values are mean+S.D, of six observations
% activity 100 51 2 42
Cholinesterase Secretion by Hookworms
Vol. 50, No. i0, 1992
749
Ancylostoma ceylanicum. Analysis of subcellular distribution of acetylcholinesterase in A. ceylanicum homogenate showed 51% activity associated with the particulate fraction and 42 % of the enzyme was recovered in the cytosolic/soluble fraction (Table I) Secretion of acetylcholinesterase from
Hookworms exhibited significant secretion of cholinesterase in the culture medium when maintained in vitro. Quantitatively secretion of the enzyme was linear upto 4 hours of the incubation (Fig.l). 100
E o
80
J
60 40 20 0
. . . .
0
I
1
. . . .
I
. . . .
I
. . . .
I
2 3 4 time (hour)
'
'
'
'
I
5
. . . .
6
Fig.1 Secretion of cholinesterase by Ancy/ostoma cey/anicum worms maintained in vitro in RPMI 1640 medium. Each point represents mean___S.D, of four observations. Unit- nmol acetylcholine hydrolyzed/h.
VBLsoo CLCsoo 1oo 5o lO
Contr
0
20
40
60
80 100 Percent
Of o
50 100 control
150
200
Fig.2 Effect of in vitro treatment by tubulin binding agents viz., colchicine (CLC) and vinblastin (VBL) on (A) the secretion of cholinesterase by A. ceylanicum and (B) the enzyme activity within the adult worms. Each bar represents mean+S.D. of four observations. * indicates significant difference over control value.
750
Cholinesterase
Secretion by Hookworms
Vol. 50, No. i0, 1992
Effect of tubulin binding agents-Adult A. ceylanicum worms incubated in vitro with colchicine showed significantly lower capacity to secrete acetylcholinesterase as compared to untreated worms. The effect was not significant at 10 ~M level, while the effect was statistically significant at 50 and 100 liM concentrations. Secretion of cholinesterase was completely abolished in the worms treated with 500 ~M colchicine (Fig.2A). Adult A. ceylanicum worms exposed to 50 and 100 llM coIchicine were significantly less motile than the controls. Colchicine also caused concentration dependent accumulation of acetylcholinesterase within the adult worms (Fig.2B). However, another tubulin binding agent, vinblastin, did not show noticeable effect on the enzyme secretion capacity as well as activity within the adult hookworms even upto to 500 llM concentration (Fig.2A &2B). Effect of benzimidazole anthelmintics- The worms treated in vitro with MBZ and ALBZ exhibited significantly decreased capacity to secrete cholinesterase (Fig.3A). Concomitant accumulation of cholinesterase was also observed in the worms treated with MBZ and ALBZ (Fig.3B). The effect of both the anthelmitics on the enzyme secretion and accumulation was concentration dependant. However, the effect of MBZ was more prominent than ALBZ. Exposure of worms to these benzimidazole anthelmintics also decreased motility of the worms, Adult A. ceylanicum worms recovered from the hamsters treated with therapeutic dose of MBZ (single oral dose 6 mg/kg body weight) showed markedly reduced capacity of cholinesterase secretion than untreated worms (Fig.4A). Concomitantly the worms obtained from drug treated animals exhibited about 3 fold higher activity of cholinesterase as compared to those recovered from untreated infected animals (Fig.4B).
A L B Z 0.1o mM 0.05
mM
MBZ O.lO
mM
0.05
mM
Control
0
20
40
60
80
100
percent
0
of
50
100
150
control
Fig.3 Effect of in vitro treatment with benzimidazole anthelmintics viz., mebendazole (MBZ) and albendazole (ALBZ) on (A) secretion of cholinesterase by A. ceylanicum and (B) the enzyme activity within the adult worms . Each bar represents the mean+S.D, of four observations. * indicates significant difference over control values.
200
Vol. 50, No. 10, 1992
Cholinesterase Secretion by Hookworms
751
0 o
o tO
P
c o n t r o l MBZ treated
control
MBZ treated
Fig.4 Effect of in vivo treatment by mebendazole (MBZ)(single oral dose, 6 mg/kg body weight) on (A) secretion of cholinesterase by A. ceylenicum and (B) the enzyme activity within the adult worms (B). Each bar represents mean+S.D, of four observations. * indicates significant difference over control values. Discussion
Microtubules constitute an integral part of cellular cytoskeleton and perform several vital functions including cellular secretion (10). Recently Kitazumi etal (14) have shown the involvement of microtubular system in the secretion of endothelin 1, a vasoactive peptide by aortic endothelial cells. Microtubular-microfilamentous system plays an essential role in the intracellular transport of secretory granules and golgi derived vesicles. Results presented indicate that considerable proportion of cholinesterase activity of hookworms resides in the soluble fraction which represents the enzyme present extracellular fluids as well as the cellular cytoplasm. This fraction of the enzyme is secreted by the parasites and play important role in the host-parasite interactions (3). Microtubules and the their constituent proteins, tubulins, have been isolated and characterized in nematodes and have been indicated as the site of action of various benzimidazole anthelmintics (11, 15-17). Benzimidazoles bind with tubulins and inhibit their polymerization into microtubules (11). Inhibition of secretion of cholinesterase from A. ceylanicum by colchicine indicate the role of microtubules in enzyme secretion. Colchicine has also been shown to inhibit translocation of proline through tegument of Hymenolepis diminuta, an intestinal cestode, indicating inhibition of intracytoplasmic movement of peptides (18). A dense accumulation of secretory vesicles in the area of tegument proximal to internuncial processes has also been observed in the helminth exposed to colchicine (18). Benzimidazoles inhibit the polymerization of tubulins through binding at the colchicine site (19). However, vinblastin or ligands active at vinblastin site do not bind with the nematodal tubulins (11). This explains the unaffectiveness of vinblastin on cholinesterase secretion by hookworms. These properties of nematodal microtubules distinguish it from the mammalian microtubules and provide a specific target for the chemotherapy. MBZ and ALBZ bind with the mammalian tubulins with similar affinity while incase of nematodal tubulins binding affinity of MBZ is greater than ALBZ (11). This also correlates with the differential effects of MBZ and ALBZ on cholinesterase secretion by hookworms where
752
Cholinesterase Secretion by Hookworms
Vol. 50, No. 10, 1992
MBZ caused greater effect than ALBZ. In vitro effects of MBZ on cholinesterase secretion by hookworms were also confirmed in vivo. Studies thus reveal the role of microtubules in the secretion of cholinesterase by hookworms and also as a target for the action of benzimidazole anthelmintics. Acknowledaements
The author is grateful to Prof. B.N.Dhawan, Director, CDRI, Lucknow, for providing necessary facilities and also to Drs L.M.Tripathi and Sonjoy Mukerjee for their help. Parasitic material was provided by Dr J.C.Katiyar, Scientist Incharge, Division of Parasitology,CDRI, Lucknow. This Paper bears a communication No. 4659 from Central Drug Research Institute, Lucknow. References
1. 2. 3. 4.
S.L. GOH and K.G. DAVEY. Canadian J. Biochem. 54 752-771 (1976). M.L. RHOADS, J. Biol. Chem. 256 9316-9323 (1981). M.L. RHOADS, Trop. Vet. 2 3-10 (1984). S. RATHAUR, B.D. ROBERTSON, M.E. SELKIRK and R.M. MAIZELS, Mol. Bichem. Parasitol. 26 257-265 (1987) 5. A.J. EDWARDS, J.S. BURT and B.M. OGLVIE, Parasitology 62 339-347 (1971) 6. D.L. Lee, Tissue & Cell 2 225-231 (1970). 7. M. PHILIPP, Trans. Roy. Soc. Trop. Med Hyg. 78 138-139 (1984) 8. E.B. RAPSON, A.S. CHILWAN and D.C. JENKINS, Parasitology 92 425-430 (1986) 9. S.D.M. WATTS, E.B. RAPSON, A.M. ATKINS and D.L. LEE, Biochem Pharmacol. 31 3035-3040 (1982). 10. S.L. HOWELL, Dibetologia 26 319-327 (1984). 11. E. LACEY, Int. J. Parasitol. 18 885-936 (1985). 12. G. L. ELLMAN, K. D. COURTNEY, V. ANDERS and R. M. FEATHERSTONE, Biochem. Pharmacol. 7 88-95 (1961). 13. O.H. LOWRY, N.J. ROSEGROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). 14. K. KITAZUMI, M. MIO and K. TASAKA, Biochem. Pharmacol. 42 1079-1085 (1991) 15. C. DAVIS and K. GULL, J. Parasitol. 69 1090-1094 (1983). 16. P.I. DAWSON, W.E. GUETTERIDGE and K. GULL, Mol. Biochem. Parasitol. 7 267-277 (1983). 17. R.H. FETI'ERRER, J. Vet. Pharmacol. Ther. 9 49-54 (1986). 18. D.J. ETGAS and B.J. BOGTISH, J. Parasitol. 71 290-296 (1985). 19. E. LACEY and T.R. WATSON, Biochem. Pharmacol. 34 3603-3605 (1985).
Pergamon Press
SECRETORY CHOLINESTERASE OF Ancylostoma ceylanicum :EFFECT OF TUBULIN BINDING AGENTS AND BENZIMIDAZOLE ANTHELMINTICS
Babu L. Tekwani* Division of Biochemistry, Central Drug Research Institute Lucknow-226001 (India) (Received in final form January 7, 1992)
Summarv
Ancylostoma ceylanicum, the human hookworm parasite, exhibited significant secretion of cholinesterase when maintained in vitro in RPMI-1640 medium. Secretion of the enzyme was linear up-to 4 hours of incubation. About 40 percent of the total cholinesterase activity was localized in the soluble fraction, while remaining activity was associated with the particulate fraction of the nematode. Exposure of the hookworms to colchicine in vitro caused significant inhibition in secretion of the enzyme by the parasite with concomitant accumulation of cholinesterase within the adult worms. Vinblastin did not show noticeable effect on the enzyme secretion as well as activity within the parasite. Incubation of hookworms with some benzimidazole anthelmintics viz., mebendazole or albendazole significantly reduced the capacity of the worms to secrete cholinesterase and increase in enzyme activity within the parasite. Adult worms recovered from mebendazole treated hamsters exhibited about 3 fold greater activity of cholinesterase as well as significantly lower capacity to secrete cholinesterase in vitro as compared to the worms recovered from untreated animals. These observations indicate role of microtubules in the secretion of cholinesterase by hookworms and as a target for the action of benzimidazole anthelmintics. Interest in the cholinesterases of parasitic nematodes was initially centered on their physiological role in the nematode nervous system (1). Several species of nematode parasites also secrete cholinesterase during incubation in vitro and compelling immunological evidences indicate that the enzyme is also secreted by the parasites within their hosts (2-4). The nematode secretory cholinesterases are present either singly (2) or in multiple molecular forms (5) and differ from the classical mammalian acetylcholinesterase in that they show substantial hydrolysis of butyrylcholine, are cytosolic in nature, and are inhibited by higher substrate concentrations (3). Secretory cholinesterases provide an adaptive mechanism through which certain parasites regulate the intra-host physiological and immunological environment (3). In gastrointestinal nematodes secretion of cholinesterase provides a "biochemical holdfast" by reducing the intestinal peristaltic movements (6) mucus secretion and immunological intervention (7). Analysis of this function of nematodes has been *Present address- Department of Cellular and Molecular physiology, Milton S Hershey Medical Center, College of Medicine, Pennsylvania State University, Hershey, PA 17033 USA.
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press plc All rights reserved.
748
C h o l i n e s t e r a s e S e c r e t i o n by Hookworms
Vol. 50, No. 10, 1992
suggested as a parameter to screen anthelmintics in vitro (8,9). In view of the importance of microtubules in secretory functions (10) and site for the action ol benzimidazole anthelmintics (11) effect of tubulin binding agents viz., colchicine and vinblastin as well as some broad spectrum anthelmintics viz., mebendazole (MBZ) and albendazole (ALBZ) was studied on the secretion of cholinesterase by a human hookworm parasite, Ancylostoma ceylanicum. Materials and Methods parasite and the laboratory hosts-Infection of A n c y / o s t o m a ceylanicu~ (Nematoda: strongyloidea) was maintained in golden hamsters (Mesocricetus auratus). hamsters weighing about 50-60 g were orally inoculated with 100+10 infective larvae suspended in normal saline. The larvae after passing through the oesophagus, stomach and duodenum reach the jejunum and develop into adult worms within 18-+3 days posl inoculation. Infected hamsters were sacrificed by decapitation, jejunum was excised oul and opened longitudinally. Adult hookworms were picked by the forceps, suspended in RPMI-1640 medium and immediately used for the analysis of cholinesterase secretion. In vitro drug treatment- For analyzing the effects of tubulin binding agents and benzimidazole anthelmintics in vitro , adult parasites were incubated with desired concentration of the drug at 37oc in RPMI-1640 medium for 60 min. The worms were then transfered to drug free medium and subjected to analysis of cholinesterase secretion and assay. In vivo drug treatment- Infected hamsters were treated with a single oral dose ol MBZ ( 6 mg/kg body weight) (therapeutic dose) and the animals were sacrificed 18 h post drug treatment. Adult worms were mostly recovered in the distal part of the intestine and subjected to analysis of enzyme secretion and assay. Acetylcholinesterase assay and secretion in vitro-Untreated or drug treated adult A. ceylanicum were incubated in RPMI-1640 medium at 37oc and aliquots from the media were drawn after every 60 min for the assay of acetylcholinesterase activity. For analysis of enzyme activity in the adult worms, the parasites were homogenized (10 % w/v) in 0.15M KCI. Acetylcholinesterase was assayed by the spectrophotometric method of EIIman et aL (12). Protein was determined by the method of Lowry et al. (13) using BSA as the standard. All the results are presented as mean+S.D..The results were analyzed by Students t test and p values less than 0.05 were considered statistically significant. Results TABLE I Sub-cellular Distribution of Cholinesterase in Ancylostoma ceylanicum worms Fraction Homogenate 12,000 g pellet 105,000g pellet 105,000 g supt.
U/g tissue 692.3+103.3 353.0+ 94.3 13.8+ 3.9 290.7+ 63.4
U/mg protein 15.21 +6.10 6.92+1.93 3.14_+0.39 16.11+4.67
U-enzyme units (~mol acetylcholine hydrolyzed / hour) values are mean+S.D, of six observations
% activity 100 51 2 42
Cholinesterase Secretion by Hookworms
Vol. 50, No. i0, 1992
749
Ancylostoma ceylanicum. Analysis of subcellular distribution of acetylcholinesterase in A. ceylanicum homogenate showed 51% activity associated with the particulate fraction and 42 % of the enzyme was recovered in the cytosolic/soluble fraction (Table I) Secretion of acetylcholinesterase from
Hookworms exhibited significant secretion of cholinesterase in the culture medium when maintained in vitro. Quantitatively secretion of the enzyme was linear upto 4 hours of the incubation (Fig.l). 100
E o
80
J
60 40 20 0
. . . .
0
I
1
. . . .
I
. . . .
I
. . . .
I
2 3 4 time (hour)
'
'
'
'
I
5
. . . .
6
Fig.1 Secretion of cholinesterase by Ancy/ostoma cey/anicum worms maintained in vitro in RPMI 1640 medium. Each point represents mean___S.D, of four observations. Unit- nmol acetylcholine hydrolyzed/h.
VBLsoo CLCsoo 1oo 5o lO
Contr
0
20
40
60
80 100 Percent
Of o
50 100 control
150
200
Fig.2 Effect of in vitro treatment by tubulin binding agents viz., colchicine (CLC) and vinblastin (VBL) on (A) the secretion of cholinesterase by A. ceylanicum and (B) the enzyme activity within the adult worms. Each bar represents mean+S.D. of four observations. * indicates significant difference over control value.
750
Cholinesterase
Secretion by Hookworms
Vol. 50, No. i0, 1992
Effect of tubulin binding agents-Adult A. ceylanicum worms incubated in vitro with colchicine showed significantly lower capacity to secrete acetylcholinesterase as compared to untreated worms. The effect was not significant at 10 ~M level, while the effect was statistically significant at 50 and 100 liM concentrations. Secretion of cholinesterase was completely abolished in the worms treated with 500 ~M colchicine (Fig.2A). Adult A. ceylanicum worms exposed to 50 and 100 llM coIchicine were significantly less motile than the controls. Colchicine also caused concentration dependent accumulation of acetylcholinesterase within the adult worms (Fig.2B). However, another tubulin binding agent, vinblastin, did not show noticeable effect on the enzyme secretion capacity as well as activity within the adult hookworms even upto to 500 llM concentration (Fig.2A &2B). Effect of benzimidazole anthelmintics- The worms treated in vitro with MBZ and ALBZ exhibited significantly decreased capacity to secrete cholinesterase (Fig.3A). Concomitant accumulation of cholinesterase was also observed in the worms treated with MBZ and ALBZ (Fig.3B). The effect of both the anthelmitics on the enzyme secretion and accumulation was concentration dependant. However, the effect of MBZ was more prominent than ALBZ. Exposure of worms to these benzimidazole anthelmintics also decreased motility of the worms, Adult A. ceylanicum worms recovered from the hamsters treated with therapeutic dose of MBZ (single oral dose 6 mg/kg body weight) showed markedly reduced capacity of cholinesterase secretion than untreated worms (Fig.4A). Concomitantly the worms obtained from drug treated animals exhibited about 3 fold higher activity of cholinesterase as compared to those recovered from untreated infected animals (Fig.4B).
A L B Z 0.1o mM 0.05
mM
MBZ O.lO
mM
0.05
mM
Control
0
20
40
60
80
100
percent
0
of
50
100
150
control
Fig.3 Effect of in vitro treatment with benzimidazole anthelmintics viz., mebendazole (MBZ) and albendazole (ALBZ) on (A) secretion of cholinesterase by A. ceylanicum and (B) the enzyme activity within the adult worms . Each bar represents the mean+S.D, of four observations. * indicates significant difference over control values.
200
Vol. 50, No. 10, 1992
Cholinesterase Secretion by Hookworms
751
0 o
o tO
P
c o n t r o l MBZ treated
control
MBZ treated
Fig.4 Effect of in vivo treatment by mebendazole (MBZ)(single oral dose, 6 mg/kg body weight) on (A) secretion of cholinesterase by A. ceylenicum and (B) the enzyme activity within the adult worms (B). Each bar represents mean+S.D, of four observations. * indicates significant difference over control values. Discussion
Microtubules constitute an integral part of cellular cytoskeleton and perform several vital functions including cellular secretion (10). Recently Kitazumi etal (14) have shown the involvement of microtubular system in the secretion of endothelin 1, a vasoactive peptide by aortic endothelial cells. Microtubular-microfilamentous system plays an essential role in the intracellular transport of secretory granules and golgi derived vesicles. Results presented indicate that considerable proportion of cholinesterase activity of hookworms resides in the soluble fraction which represents the enzyme present extracellular fluids as well as the cellular cytoplasm. This fraction of the enzyme is secreted by the parasites and play important role in the host-parasite interactions (3). Microtubules and the their constituent proteins, tubulins, have been isolated and characterized in nematodes and have been indicated as the site of action of various benzimidazole anthelmintics (11, 15-17). Benzimidazoles bind with tubulins and inhibit their polymerization into microtubules (11). Inhibition of secretion of cholinesterase from A. ceylanicum by colchicine indicate the role of microtubules in enzyme secretion. Colchicine has also been shown to inhibit translocation of proline through tegument of Hymenolepis diminuta, an intestinal cestode, indicating inhibition of intracytoplasmic movement of peptides (18). A dense accumulation of secretory vesicles in the area of tegument proximal to internuncial processes has also been observed in the helminth exposed to colchicine (18). Benzimidazoles inhibit the polymerization of tubulins through binding at the colchicine site (19). However, vinblastin or ligands active at vinblastin site do not bind with the nematodal tubulins (11). This explains the unaffectiveness of vinblastin on cholinesterase secretion by hookworms. These properties of nematodal microtubules distinguish it from the mammalian microtubules and provide a specific target for the chemotherapy. MBZ and ALBZ bind with the mammalian tubulins with similar affinity while incase of nematodal tubulins binding affinity of MBZ is greater than ALBZ (11). This also correlates with the differential effects of MBZ and ALBZ on cholinesterase secretion by hookworms where
752
Cholinesterase Secretion by Hookworms
Vol. 50, No. 10, 1992
MBZ caused greater effect than ALBZ. In vitro effects of MBZ on cholinesterase secretion by hookworms were also confirmed in vivo. Studies thus reveal the role of microtubules in the secretion of cholinesterase by hookworms and also as a target for the action of benzimidazole anthelmintics. Acknowledaements
The author is grateful to Prof. B.N.Dhawan, Director, CDRI, Lucknow, for providing necessary facilities and also to Drs L.M.Tripathi and Sonjoy Mukerjee for their help. Parasitic material was provided by Dr J.C.Katiyar, Scientist Incharge, Division of Parasitology,CDRI, Lucknow. This Paper bears a communication No. 4659 from Central Drug Research Institute, Lucknow. References
1. 2. 3. 4.
S.L. GOH and K.G. DAVEY. Canadian J. Biochem. 54 752-771 (1976). M.L. RHOADS, J. Biol. Chem. 256 9316-9323 (1981). M.L. RHOADS, Trop. Vet. 2 3-10 (1984). S. RATHAUR, B.D. ROBERTSON, M.E. SELKIRK and R.M. MAIZELS, Mol. Bichem. Parasitol. 26 257-265 (1987) 5. A.J. EDWARDS, J.S. BURT and B.M. OGLVIE, Parasitology 62 339-347 (1971) 6. D.L. Lee, Tissue & Cell 2 225-231 (1970). 7. M. PHILIPP, Trans. Roy. Soc. Trop. Med Hyg. 78 138-139 (1984) 8. E.B. RAPSON, A.S. CHILWAN and D.C. JENKINS, Parasitology 92 425-430 (1986) 9. S.D.M. WATTS, E.B. RAPSON, A.M. ATKINS and D.L. LEE, Biochem Pharmacol. 31 3035-3040 (1982). 10. S.L. HOWELL, Dibetologia 26 319-327 (1984). 11. E. LACEY, Int. J. Parasitol. 18 885-936 (1985). 12. G. L. ELLMAN, K. D. COURTNEY, V. ANDERS and R. M. FEATHERSTONE, Biochem. Pharmacol. 7 88-95 (1961). 13. O.H. LOWRY, N.J. ROSEGROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). 14. K. KITAZUMI, M. MIO and K. TASAKA, Biochem. Pharmacol. 42 1079-1085 (1991) 15. C. DAVIS and K. GULL, J. Parasitol. 69 1090-1094 (1983). 16. P.I. DAWSON, W.E. GUETTERIDGE and K. GULL, Mol. Biochem. Parasitol. 7 267-277 (1983). 17. R.H. FETI'ERRER, J. Vet. Pharmacol. Ther. 9 49-54 (1986). 18. D.J. ETGAS and B.J. BOGTISH, J. Parasitol. 71 290-296 (1985). 19. E. LACEY and T.R. WATSON, Biochem. Pharmacol. 34 3603-3605 (1985).