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A biochemical and ultrastructural study of the mode of action of bunamidine against Hymenolepis nana
International Jormai for Parasifology.
1977. Vol. 7. pp. 129-134. Pergamon Press. Printed in Great Britain.
A BIOCHEMICAL AND ULTRASTRUCTURAL STUDY OF THE MODE OF ACTION OF BUNAMIDINE AGAINST HYMEiVULEPIS AJANA R. J. HART,” *Wellcome Research Laboratories,
R. TURNERS and
R. G.
WILSON*
Berkhamsted, Herts., U.K. and ?Rothamsted Harpenden, Herts., U.K.
Experimental
Station
(Received 13 J&y 1976) AbS&a&-HART
R. J., TURNERR. and WILSONR. G. 1977. A biochemical and ultrastructural study of the mode of action of bunamidine against Hymenolepis nana. International Journal for Parasitology 7: 329-134. In the presence of bunamidine the cestode Hymenolepis nana shows a decrease in the rate of glucose uptake and an increase in the rate of glucose efflux. The surface phosphatase activity is stimulated many fold and much of the enzyme activity is released into the incubation medium. It is suggested that disruption of the integument by bunamidine is the cause of these effects and the mode of action bv which death of the worm is caused. These biochemical indications of integumental damage are confirmed by ultrastructural studies which show that there is complete loss of the surface tissue down to the level of the fibrous basal lamina. 1NDEX KEY WORDS: bunamidine; cestode; glucose integument; mode of action; phosphatase; ultrastructure.
INTRODUCTION THE ANTHELMINTIC, bunamidine, N,iV-di-n-butyl4-hexyloxy-l-naphthamidine (Fig. l), has a broad spectrum of activity against tapeworms in a range of domestic animals (Keeling, 1969). NH
transport;
Hymenolepis
nana;
section and the intestines removed. The worms were flushed from the intestines using Krebs-Ringer saline (Krebs, 1933) modified to contain 0.1 M-tris/HCl buffer pH 7.4 in place of 0.1 M-phosphate buffer pH 7.4. The saline was modified to avoid any precipitation of bunamidine phosphate at later stages in the experiments. The worms were washed twice and kept in saiine at 37°C. Materials. Bunamidine was used in the form of bunamidine hydrochloride obtained from the Wellcome Research Laboratories, Beckenham.
FIG. 1. Structure of bunamidine. Cestodes lack a mouth and gut and all nutrients must be absorbed through the integumental surface. Glucose, an important nutrient, can be taken up against a concentration gradient (Phifer, 196On, 1960b) and surface phosphatases are thought to be important in the transport mechanism (Dike & Read, 1971). This paper describes a series of experiments to determine the effect of bunamidine on glucose transport and the phosphatase of this absorptive surface using Hya&epis nana as the test organism. hfATERLALS AND METHODS Collection of worms. Mice, infested 21 days previously with Hymenolepis nuna eggs, were killed by cervical
Uptake of &cose by H. nana. One or two worms in each experiment were incubated in 10 ml saline containing 5 x lo-3M IU-@+Cl alucosewith anactivitvof O-1 !&i/ml. They were removedafter various time intervals up to 30 min and washed twice for 30 s with 75 ml of saline. They were then blotted on filter paper, placed in 5 ml of 50% aqueous ethanol, shaken for 1 h and left overnight at room temperature. One ml of extract was added to 4 ml of Brav’s scintillation fluid (Bray. 1960) and the radioactivity- measured in an I~tert~hniqu~ SL 30 scintillation counter. The worms were removed from the extraction mixture, blotted dry on filter paper and homogenised with 0.25 ml water. The protein content of the homogenate was determined by the method of Lowry, Rosebrough, Farr & Randall (1951). Similar experiments were carried out using incubation solutions containing bunamidine hydrochloride. Release of glucose by H, nana. Worms were incubated for 15 min in 10 ml saline containing 5 X lo-3M[U-‘4C] glucose (1 pa/ml) at 37°C. After three half-minute washes in glucose-free saline they were transferred to a perfusion chamber maintained at 37°C and perfused with glucose-
129
R. J. HART, R.
130
TURNER
free saline at a rate of 2-3 ml/min. 1 ml fractions of perfusate were collected and added to 4 ml of Bray’s scintillation fluid and the radioactivity measured as described earlier. In certain experiments the perfusate contained various concentrations of bunamidine hydrochloride. Assay of integumental phosphatase. 0.1 ml of 0.5~n-nitronhenvl disodium ohosvhate was added to 10 ml bf s&e containing one t-0 four worms, depending on the size, and the incubation carried out at 37% for up to 30 min with occasional shaking. At the end of the incubation period the worms were removed with forceps and the optical density of an aliquot of the incubation medium immediately measured at 410 nm. Further measurements of the optical density of the incubation medium were made at various times, the interval depending on the activity observed. In certain experiments the incubation medium contained various concentrations of bunamidine hydrochloride. Protein content of the worms and also of the remaining incubation medium, after removing the worms, was measured by the method described earlier. Electron microscopy. Worms were incubated for 15 min in saline containing various concentrations(5 X 10msM, 1 x 10-4~) of bunamidine hydrochloride and washed twice for half a minute in fresh saline. For scanning electron microscopy specimens were prepared either by freeze-drying or dehydrating with acetone then air drying. Dry worms were mounted on sticky stubs, coated with 25-30 nm gold and examined in a Cambridge Instruments ‘Stereoscan” 2A scanning electron microscope. For transmission electron microscopy, worms were cut into about 5 mm lengths and fixed at 0°C in 6% glut~raIdehyde in 0.05 M-cacodylate buffer at pH 7.4 for about I h. Smali pieces of worm (approximately 05 mm33 were then embedded in 1% Agar using the method of Wright & Jones (1965). The smali agar blocks containing the specimens were then returned to the fixative for another 2 h after which they were rinsed six times in buffer and post-fixed in 1% osmium tetroxide in 0.05 M-veronal acetate buffer at pH 7.4 for 3 h at 0°C. The tissue was rinsed in buffer and dehydrated in an alcohol series before embedding in the low-viscosity epoxy resin ernb~d~~~ medium of Spurr (19691. Ultrathin sections of the worm were mounted on large slot grids coated with pyroxylen film, or on uncoated 300/75 mesh grids. They were stained in 0.9% potassium permanganate in 0.1 M-phosphate buffer at pH 6.5 (Soloff, 1973) for 1 min., rinsed in buffer, then in distilled water and stained in 3% aqueous uranyl acetate for 45 min and in Bevnolds’ 09631 lead citrate for IO min, all at room temperature. The s&ions were examined b$ transmission microscopy using a Phillips 201 microscope. RESULTS E+ect of bunamidine on glucose uptake and release
The increasing ~~~e~t~ti~ns of ~u~amid~n~ increasingly inhibit the uptake of gIucose by the worms, as indicated by the radiocarbon content of the worms shown in Fig. 2.88 % inhibition of glucose uptake is produced by a bunamidine concentration of 3 s 10-5~. During the experiments some glucose taken up by the worms will be converted to respiratory end products and excreted into the medium. Thus the radiocarbon content, expressed as m-mol of
and R. G. Wnsohi
I.J.P. VOL.7. 1977
[U -14C] glucose equivalents, will, in fact, be a low estimate of the amount of glucose actually processed by the worms. Figure 3 illustrates that bunamidine also reduces the ability of the worms to retain
f E
I
i
1
I
m-6
3x10-6
6 x IO-6
10-5
Bunclmidine
concentration,
td
Fra.
2. Effect of various concentrations of bunamidine on the in vitro uptake of glucose by intact W. FWZCZ, indicated
by the radiocarbon content of the worms. glucose and its metabolites against a ~on~en~rat~o~ gradient. After commencement of perfusion with bunamidine there is an initial decrease followed by a increase in the amounts of glucose andmeta bolites released. The results are expressed in terms of the relative rate of radiocarbon release. This is defined as the rate of radiocarbon release during any particular time interval divided by the average rate of radiocarbon release during the period before the addition of bunamidine. E$Pct of bunamidine
on integumentary
phosphatase
The activity of the ~ntegument~y phosphatase of worms treated with bun~~d~ne is much greater than that of control worms as shown in Fig. 4. At a bunamidine concentration of 3 x 10-h the integumentary phosphatase activity is 15 times that of control worms. After worms were removed from an incubation medium containing bunamidine, the optical density of these solutions at 410 nm continued to increase. Of the total phosphatase activity measured, the amount released into the medium following a 20 min treatment with various concentrations of bunamidine is shown in Fig. 4. At the bunamidine concentration of 3 x lo-so, about 85 % of the total phosphatase activity is released into the medium after 20 min. Scanning electron microscopy
Untreated worms showed a dense covering of spa&late microtriches on a deeply folded surface (Fig. 5), whereas worms which had been treated with 5 x 10-5~ bunamidine showed almost no microtriches (Fig. 6)=
I.J.P.
VOL.7.1977
131
The mode of action of bunamidine
Bunamidine v
Perfusion
time,
min
FIG. 3. Effect of bunamidine on the rate of release of glucose and its metabolites into perfusing saline by intact H. nanu (after prior incubation of worms in [U-%1 glucose for 15 min). completely removed lamina (Fig. 10).
DPhosphotose oP-Nitrophenol
Bunomidine
concentrotton,
M
FIG. 4. Active phosphatase released by H. nana into
incubation medium when treated with bunamidine, and effect of bunamidine on total in vitro phosphatase activity of intact H. nuna. Transmission electron microscopy The integument of H. nana consists of a syncytial
epidermis made up of an outer anucleate cytoplasmic region and an inner nucleated region. The two are separated by an internal plasma membrane and a fibrous basal lamina, but connected by pore canals. A typical section is shown in Fig. 7. The worms treated with 1 x lo-%f-bunamidine showed a varying degree of damage to the integument. First there was an intense vacuolisation of the outer anucleate cytoplasmic region (Fig. 8), followed by varying degrees of sloughing off of this outer layer (Fig. 9). Finally the complete outer layer was
down
to the fibrous
basa
DISCUSSION The integument of H. nana conforms to the generalised structure for cestode integuments described by Lee (1972) and Smyth (1972). The electron micrographs of untreated H. nana confirm the results obtained with the same species by Berger & Mettrick (1971) using scanning electron microscopy and by Ciofi Luzzatto & Ferretti (1966) using transmission electron microscopy. Inhibition of glucose uptake and failure of the worms to retain glucose against a concentration gradient in the presence of bunamidine (Figs. 2 & 3) suggest that the integument of the worms has been disrupted in some way. During the early stages of perfusion with IO-%i-bunamidine when the concentration of bunamidine at the worms would be low, there is an apparent stabilisation of the integument as indicated by the decreased efflux of radioactivity. Disruption of the membrane occurs as the perfusion progresses and the concentration of bunamidine increases to that in the perfusing solution, as indicated by the increase in the efflux of radioactivity. The stabilisation of membranes at low concentrations and disruption at high concentrations is characteristic of the effect of many monovalent cationic detergent-like molecules (Seeman & Weinstein, 1966). At neutral pH bunamidine (pKa 10.6) is largely in the protonated form and has a hydrophilic cationic end and a lipophilic hydrocarbon chain and may be considered as a cationic detergent. Cytochemical studies with the related species H. diminuta (Lumsden, 1972, 1973) have shown that the glycocalyx of the microtriches is made up from polyanionic macromolecules. In view of the
R. J.
132
HART,
R. TURNERand R. G. WILSON
FIG. 5. Scanning electron micrograph of the integument of untreated H. nana showing dense covering of spatulate microtriches
on a deeply folded surface.
very similar integumental ultrastructure of H. nanLa it is possible that its surface has similar anionic centres which would form excellent binding sites for the bunamidine cation. Thus when worms are exposed to bunamidine it would accumulate in the outer integumental layer and apparently this is disrupted when the concentration reaches a certain level. Both acid and alkaline phosphatase activity have been demonstrated on the surface of IX nnna (Waitz & Schardein, 1964). The surface phosphatases of H. diminuta are thought to be important in the sugar transport mechanism of the worm (Dike & Read, 1971) so that glucose can be taken up against a concentration gradient (Phifer, 196Ou, 1960b.) Surface phosphatases could be similarly involved in H. na~a. Stimulation of this surface phosphatase activity and the release of a high proportion of the enzyme activity into the medium in the presence of
I.J.P. VOL. 7. 1977
FIG. 6. Scanning electron micrograph of the integument of N.nanu treated with 5 x IO-%-bunamidinefor 15min showing almost complete absence of microtriches.
bunamidine provide further evidence that bunamidine is adversely affecting integument function and causing disruption of the surface tissues. This is confirmed in the ultrastructural observations which show that the outer surface layer of the worm is removed down to the fibrous basal iamina. We suggest, therefore, that bunamidine causes the death of the cestode by disruption of the outer layers of the integument. Acknowledgement-We
thank Mrs. S. A. Clark and Dr. A. M, Shepherd of Rothamsted Experimental Station, ~arpenden, for advice on t~hniques for transmission electron microscopy. Our thanks also to Parasitology Department, Wellcome Research Laboratories, Beckenham for supplying the tapeworm infested mice, and to the Wellcome Pharmaceutical Development Laboratory, Dartford, for determining the pKa of bunamidine.
.Legends to fisures on p. 133
FIG. 7. Transmission electron micrograph of the integument of untreated H. nanfa showing outer anucleate cytoplasmic regions and inner region separated by the fibrous basal lamina and muscle layers.
FIG. 9. Transmission electron micrograph of the integument of H. nurru treated with I x 10 -4M-bunamidine for 15 min showing the sloughing off of the outer cytoplasmic layer.
FIG. 8. Transmission electron micrograph of the integument of H. nanu treated with 1 x 10 -%f-bunamidine for 1.5 min showing intense vacuolisation of the outer cytopiasmic region.
10. Transmission electron micrograph of the integument of H. nana treated with 1 x 10 -%r-bunamidine for 15 min showing that the outer cytoplasmic region has been completely removed down to the fibrous basal lamina. FIG.
I.J.P. VOL. 7. 1977
The mode of action of bunamidine
133
134
R. J. HART, R. TURNI3~ and R, G. WILSON REFERENCES
BERGIZRJ. & METTRICK D. F. 1971. Microtri~hiai polymorphism among hymenolepid tapeworms as seen by scanning electron microscopy. Transactions of the American microscopical Society PO: 393-403. BRAY6. A. 1960. A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter, Analytical Biochemistry 1: 279-285. CIOFI LUZZATTOA. & FERRETTIG. 1966. Osservazioni con il microscopio elettronico sulla superficie di rivestimento di alcuni platelminti parasitti. Am&i dell ‘Zstituto Superiore San& 2: 675-686. DIKE S. C. & READ C. P. 1971. Tegumenta~ phosphohydrolases of Nymenolepis diminuta. Journal of Parasitology 57: 81-87. KEELING J. E. D. 1969. Activity of some dialkylnaphthamidines against tapeworms. In Veterinary Pesticides. Society of Chemical Industry Monograph No. 33, pp. 56-73. Society of Chemical Industry, London. KREBS H. A. 1933. Untersuchungen fiber den Stoffwechsei der Aminos~uren im TierkCirper. HoppeSeylers Zeitschrifi fir Physiolonische Chemie 217: 191-227. ” _ LEED. L. 1972. The structure of the Helminth cuticle. Advances in Par~~fo~o~~ 10: 347-379. LOWRY 0. H., Rossnn&o~ N. J., FARR A. L. & RANDALL R. J. 1951. Protein measurement with the protein phenol reagent. Journal of ~iu~og~calChemistry 193:265-275. LUMSDENR. D. 1972. Cytological studies on the absorptive surfaces of cestodes-VI. Cytochemical evaluation of electrostatic charge. Journal of Parasitology 58: 229-234. LUMSDENR. D. 1973. Cytological studies on the absorp-
I&P. VOL. 7. 1977
tive surfaces of cestodes VII. Evidence for the function of the tegument glycocalyx in cation binding by Hymenolep~s d~minata. Journal of ParasitoIogy 59: 1021-1030. PH~FERK. 196Oa. Permeation and membrane transport in animal parasites. The absorption of glucose by Hymenolepis diminuta. Journal of Paras~io~ogy 46: 51-62. PHIFER K. 196Ob. Permeation and membrane transport in animal parasites. On the mechanism of glucose uptake by ~yrneno~e~~s d~m~nuta. Joar~l of Parasitology 46: 145-l 53. REYNOLDS E. S. 1963. Lead citrate at high pH as an el~tron-opaque stain in electron microscopy. Joarna~ of Cell BioIogy 17: 208-212. SEEMANP. & WEINSTEINJ. 1966. Erythrocyte membrane stabilisation by tranquilisers and antihistamines. ~ioc~ern~ca~Pharmacology 15: 1737-1752. SMYTR J. D. 1972. Changes in the dig~tiv~absorptive surface of cestodes during larval/adult differentaition. In ~unct~onai Aspects of Parasite Surfaces (Edited by TAYLOR A. E. R. & MULLER R.) p. 41-70. Blackwell Scientific Publications, Oxford. SOLOFPB. L. 1973. Buffered potassium permanganateuranyl acetate-lead citrate staining sequence for ultrathin sections. Stain Techno~agy 48: 159-165. SPURRA. R. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructure Research 21: 3143. WAXTZ J. A. & SCHARIXIN J. L. 1964. Histochemical studies of four cyclophyllidean cestodes. Journal of Parasitology SO: 271-277. WRIGHTK. A. & JONESN. 0.1965. Some techniques for the orientation and embedding of nematodes for electron microscopy. Nematologica 11: 125-130.
1977. Vol. 7. pp. 129-134. Pergamon Press. Printed in Great Britain.
A BIOCHEMICAL AND ULTRASTRUCTURAL STUDY OF THE MODE OF ACTION OF BUNAMIDINE AGAINST HYMEiVULEPIS AJANA R. J. HART,” *Wellcome Research Laboratories,
R. TURNERS and
R. G.
WILSON*
Berkhamsted, Herts., U.K. and ?Rothamsted Harpenden, Herts., U.K.
Experimental
Station
(Received 13 J&y 1976) AbS&a&-HART
R. J., TURNERR. and WILSONR. G. 1977. A biochemical and ultrastructural study of the mode of action of bunamidine against Hymenolepis nana. International Journal for Parasitology 7: 329-134. In the presence of bunamidine the cestode Hymenolepis nana shows a decrease in the rate of glucose uptake and an increase in the rate of glucose efflux. The surface phosphatase activity is stimulated many fold and much of the enzyme activity is released into the incubation medium. It is suggested that disruption of the integument by bunamidine is the cause of these effects and the mode of action bv which death of the worm is caused. These biochemical indications of integumental damage are confirmed by ultrastructural studies which show that there is complete loss of the surface tissue down to the level of the fibrous basal lamina. 1NDEX KEY WORDS: bunamidine; cestode; glucose integument; mode of action; phosphatase; ultrastructure.
INTRODUCTION THE ANTHELMINTIC, bunamidine, N,iV-di-n-butyl4-hexyloxy-l-naphthamidine (Fig. l), has a broad spectrum of activity against tapeworms in a range of domestic animals (Keeling, 1969). NH
transport;
Hymenolepis
nana;
section and the intestines removed. The worms were flushed from the intestines using Krebs-Ringer saline (Krebs, 1933) modified to contain 0.1 M-tris/HCl buffer pH 7.4 in place of 0.1 M-phosphate buffer pH 7.4. The saline was modified to avoid any precipitation of bunamidine phosphate at later stages in the experiments. The worms were washed twice and kept in saiine at 37°C. Materials. Bunamidine was used in the form of bunamidine hydrochloride obtained from the Wellcome Research Laboratories, Beckenham.
FIG. 1. Structure of bunamidine. Cestodes lack a mouth and gut and all nutrients must be absorbed through the integumental surface. Glucose, an important nutrient, can be taken up against a concentration gradient (Phifer, 196On, 1960b) and surface phosphatases are thought to be important in the transport mechanism (Dike & Read, 1971). This paper describes a series of experiments to determine the effect of bunamidine on glucose transport and the phosphatase of this absorptive surface using Hya&epis nana as the test organism. hfATERLALS AND METHODS Collection of worms. Mice, infested 21 days previously with Hymenolepis nuna eggs, were killed by cervical
Uptake of &cose by H. nana. One or two worms in each experiment were incubated in 10 ml saline containing 5 x lo-3M IU-@+Cl alucosewith anactivitvof O-1 !&i/ml. They were removedafter various time intervals up to 30 min and washed twice for 30 s with 75 ml of saline. They were then blotted on filter paper, placed in 5 ml of 50% aqueous ethanol, shaken for 1 h and left overnight at room temperature. One ml of extract was added to 4 ml of Brav’s scintillation fluid (Bray. 1960) and the radioactivity- measured in an I~tert~hniqu~ SL 30 scintillation counter. The worms were removed from the extraction mixture, blotted dry on filter paper and homogenised with 0.25 ml water. The protein content of the homogenate was determined by the method of Lowry, Rosebrough, Farr & Randall (1951). Similar experiments were carried out using incubation solutions containing bunamidine hydrochloride. Release of glucose by H, nana. Worms were incubated for 15 min in 10 ml saline containing 5 X lo-3M[U-‘4C] glucose (1 pa/ml) at 37°C. After three half-minute washes in glucose-free saline they were transferred to a perfusion chamber maintained at 37°C and perfused with glucose-
129
R. J. HART, R.
130
TURNER
free saline at a rate of 2-3 ml/min. 1 ml fractions of perfusate were collected and added to 4 ml of Bray’s scintillation fluid and the radioactivity measured as described earlier. In certain experiments the perfusate contained various concentrations of bunamidine hydrochloride. Assay of integumental phosphatase. 0.1 ml of 0.5~n-nitronhenvl disodium ohosvhate was added to 10 ml bf s&e containing one t-0 four worms, depending on the size, and the incubation carried out at 37% for up to 30 min with occasional shaking. At the end of the incubation period the worms were removed with forceps and the optical density of an aliquot of the incubation medium immediately measured at 410 nm. Further measurements of the optical density of the incubation medium were made at various times, the interval depending on the activity observed. In certain experiments the incubation medium contained various concentrations of bunamidine hydrochloride. Protein content of the worms and also of the remaining incubation medium, after removing the worms, was measured by the method described earlier. Electron microscopy. Worms were incubated for 15 min in saline containing various concentrations(5 X 10msM, 1 x 10-4~) of bunamidine hydrochloride and washed twice for half a minute in fresh saline. For scanning electron microscopy specimens were prepared either by freeze-drying or dehydrating with acetone then air drying. Dry worms were mounted on sticky stubs, coated with 25-30 nm gold and examined in a Cambridge Instruments ‘Stereoscan” 2A scanning electron microscope. For transmission electron microscopy, worms were cut into about 5 mm lengths and fixed at 0°C in 6% glut~raIdehyde in 0.05 M-cacodylate buffer at pH 7.4 for about I h. Smali pieces of worm (approximately 05 mm33 were then embedded in 1% Agar using the method of Wright & Jones (1965). The smali agar blocks containing the specimens were then returned to the fixative for another 2 h after which they were rinsed six times in buffer and post-fixed in 1% osmium tetroxide in 0.05 M-veronal acetate buffer at pH 7.4 for 3 h at 0°C. The tissue was rinsed in buffer and dehydrated in an alcohol series before embedding in the low-viscosity epoxy resin ernb~d~~~ medium of Spurr (19691. Ultrathin sections of the worm were mounted on large slot grids coated with pyroxylen film, or on uncoated 300/75 mesh grids. They were stained in 0.9% potassium permanganate in 0.1 M-phosphate buffer at pH 6.5 (Soloff, 1973) for 1 min., rinsed in buffer, then in distilled water and stained in 3% aqueous uranyl acetate for 45 min and in Bevnolds’ 09631 lead citrate for IO min, all at room temperature. The s&ions were examined b$ transmission microscopy using a Phillips 201 microscope. RESULTS E+ect of bunamidine on glucose uptake and release
The increasing ~~~e~t~ti~ns of ~u~amid~n~ increasingly inhibit the uptake of gIucose by the worms, as indicated by the radiocarbon content of the worms shown in Fig. 2.88 % inhibition of glucose uptake is produced by a bunamidine concentration of 3 s 10-5~. During the experiments some glucose taken up by the worms will be converted to respiratory end products and excreted into the medium. Thus the radiocarbon content, expressed as m-mol of
and R. G. Wnsohi
I.J.P. VOL.7. 1977
[U -14C] glucose equivalents, will, in fact, be a low estimate of the amount of glucose actually processed by the worms. Figure 3 illustrates that bunamidine also reduces the ability of the worms to retain
f E
I
i
1
I
m-6
3x10-6
6 x IO-6
10-5
Bunclmidine
concentration,
td
Fra.
2. Effect of various concentrations of bunamidine on the in vitro uptake of glucose by intact W. FWZCZ, indicated
by the radiocarbon content of the worms. glucose and its metabolites against a ~on~en~rat~o~ gradient. After commencement of perfusion with bunamidine there is an initial decrease followed by a increase in the amounts of glucose andmeta bolites released. The results are expressed in terms of the relative rate of radiocarbon release. This is defined as the rate of radiocarbon release during any particular time interval divided by the average rate of radiocarbon release during the period before the addition of bunamidine. E$Pct of bunamidine
on integumentary
phosphatase
The activity of the ~ntegument~y phosphatase of worms treated with bun~~d~ne is much greater than that of control worms as shown in Fig. 4. At a bunamidine concentration of 3 x 10-h the integumentary phosphatase activity is 15 times that of control worms. After worms were removed from an incubation medium containing bunamidine, the optical density of these solutions at 410 nm continued to increase. Of the total phosphatase activity measured, the amount released into the medium following a 20 min treatment with various concentrations of bunamidine is shown in Fig. 4. At the bunamidine concentration of 3 x lo-so, about 85 % of the total phosphatase activity is released into the medium after 20 min. Scanning electron microscopy
Untreated worms showed a dense covering of spa&late microtriches on a deeply folded surface (Fig. 5), whereas worms which had been treated with 5 x 10-5~ bunamidine showed almost no microtriches (Fig. 6)=
I.J.P.
VOL.7.1977
131
The mode of action of bunamidine
Bunamidine v
Perfusion
time,
min
FIG. 3. Effect of bunamidine on the rate of release of glucose and its metabolites into perfusing saline by intact H. nanu (after prior incubation of worms in [U-%1 glucose for 15 min). completely removed lamina (Fig. 10).
DPhosphotose oP-Nitrophenol
Bunomidine
concentrotton,
M
FIG. 4. Active phosphatase released by H. nana into
incubation medium when treated with bunamidine, and effect of bunamidine on total in vitro phosphatase activity of intact H. nuna. Transmission electron microscopy The integument of H. nana consists of a syncytial
epidermis made up of an outer anucleate cytoplasmic region and an inner nucleated region. The two are separated by an internal plasma membrane and a fibrous basal lamina, but connected by pore canals. A typical section is shown in Fig. 7. The worms treated with 1 x lo-%f-bunamidine showed a varying degree of damage to the integument. First there was an intense vacuolisation of the outer anucleate cytoplasmic region (Fig. 8), followed by varying degrees of sloughing off of this outer layer (Fig. 9). Finally the complete outer layer was
down
to the fibrous
basa
DISCUSSION The integument of H. nana conforms to the generalised structure for cestode integuments described by Lee (1972) and Smyth (1972). The electron micrographs of untreated H. nana confirm the results obtained with the same species by Berger & Mettrick (1971) using scanning electron microscopy and by Ciofi Luzzatto & Ferretti (1966) using transmission electron microscopy. Inhibition of glucose uptake and failure of the worms to retain glucose against a concentration gradient in the presence of bunamidine (Figs. 2 & 3) suggest that the integument of the worms has been disrupted in some way. During the early stages of perfusion with IO-%i-bunamidine when the concentration of bunamidine at the worms would be low, there is an apparent stabilisation of the integument as indicated by the decreased efflux of radioactivity. Disruption of the membrane occurs as the perfusion progresses and the concentration of bunamidine increases to that in the perfusing solution, as indicated by the increase in the efflux of radioactivity. The stabilisation of membranes at low concentrations and disruption at high concentrations is characteristic of the effect of many monovalent cationic detergent-like molecules (Seeman & Weinstein, 1966). At neutral pH bunamidine (pKa 10.6) is largely in the protonated form and has a hydrophilic cationic end and a lipophilic hydrocarbon chain and may be considered as a cationic detergent. Cytochemical studies with the related species H. diminuta (Lumsden, 1972, 1973) have shown that the glycocalyx of the microtriches is made up from polyanionic macromolecules. In view of the
R. J.
132
HART,
R. TURNERand R. G. WILSON
FIG. 5. Scanning electron micrograph of the integument of untreated H. nana showing dense covering of spatulate microtriches
on a deeply folded surface.
very similar integumental ultrastructure of H. nanLa it is possible that its surface has similar anionic centres which would form excellent binding sites for the bunamidine cation. Thus when worms are exposed to bunamidine it would accumulate in the outer integumental layer and apparently this is disrupted when the concentration reaches a certain level. Both acid and alkaline phosphatase activity have been demonstrated on the surface of IX nnna (Waitz & Schardein, 1964). The surface phosphatases of H. diminuta are thought to be important in the sugar transport mechanism of the worm (Dike & Read, 1971) so that glucose can be taken up against a concentration gradient (Phifer, 196Ou, 1960b.) Surface phosphatases could be similarly involved in H. na~a. Stimulation of this surface phosphatase activity and the release of a high proportion of the enzyme activity into the medium in the presence of
I.J.P. VOL. 7. 1977
FIG. 6. Scanning electron micrograph of the integument of N.nanu treated with 5 x IO-%-bunamidinefor 15min showing almost complete absence of microtriches.
bunamidine provide further evidence that bunamidine is adversely affecting integument function and causing disruption of the surface tissues. This is confirmed in the ultrastructural observations which show that the outer surface layer of the worm is removed down to the fibrous basal iamina. We suggest, therefore, that bunamidine causes the death of the cestode by disruption of the outer layers of the integument. Acknowledgement-We
thank Mrs. S. A. Clark and Dr. A. M, Shepherd of Rothamsted Experimental Station, ~arpenden, for advice on t~hniques for transmission electron microscopy. Our thanks also to Parasitology Department, Wellcome Research Laboratories, Beckenham for supplying the tapeworm infested mice, and to the Wellcome Pharmaceutical Development Laboratory, Dartford, for determining the pKa of bunamidine.
.Legends to fisures on p. 133
FIG. 7. Transmission electron micrograph of the integument of untreated H. nanfa showing outer anucleate cytoplasmic regions and inner region separated by the fibrous basal lamina and muscle layers.
FIG. 9. Transmission electron micrograph of the integument of H. nurru treated with I x 10 -4M-bunamidine for 15 min showing the sloughing off of the outer cytoplasmic layer.
FIG. 8. Transmission electron micrograph of the integument of H. nanu treated with 1 x 10 -%f-bunamidine for 1.5 min showing intense vacuolisation of the outer cytopiasmic region.
10. Transmission electron micrograph of the integument of H. nana treated with 1 x 10 -%r-bunamidine for 15 min showing that the outer cytoplasmic region has been completely removed down to the fibrous basal lamina. FIG.
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The mode of action of bunamidine
133
134
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