Department of Biomedical Sciences, Iowa State University, Ames,
Iowa (A.P.R., C.L.C., T.A.B., S.M.T., R.J.M.); and Pharmacia Animal
Health, Kalamazoo, Michigan (D.P.T., T.G.G.)
Paraherquamide is a novel natural anthelmintic product with a mode of
action that is incompletely characterized. Nicotine and
cholinergic-anthelmintic agonists of different chemical classes were
used to produce contraction in Ascaris muscle strips.
Paraherquamide and a semisynthetic derivative, 2-deoxy-paraherquamide,
antagonized these responses. Analysis of the actions of the antagonists
was made using the simple competitive model and nonlinear regression to
estimate the pKB values of the antagonists.
The analysis was tested using Clark plots. The
pKB values for paraherquamide were: nicotine, 5.86 ± 0.14; levamisole, 6.61 ± 0.19; pyrantel,
6.50 ± 0.11; and bephenium, 6.75 ± 0.15. The
pKB of nicotine was significantly different
from the pKB values for levamisole,
pyrantel, and bephenium, showing that paraherquamide can distinguish a
subtype of cholinergic receptors sensitive to nicotine and a subtype of
cholinergic receptors sensitive to levamisole, pyrantel, and bephenium.
The pKB values for 2-deoxy-paraherquamide
were: levamisole, 5.31 ± 0.13; pyrantel, 5.63 ± 0.10; and
bephenium, 6.07 ± 0.13. The Clark plots of the antagonism
illustrated the degree of fit to the competitive model for
2-deoxy-paraherquamide. 2-Deoxy-paraherquamide selectively antagonized
the effects of bephenium; the pKB values of
levamisole and pyrantel were significantly different from the
pKB of bephenium. Paraherquamide and
2-deoxy-paraherquamide are selective competitive cholinergic
antagonists that distinguish subtypes of cholinergic receptor in
Ascaris muscle corresponding to nicotine-, levamisole-, and bephenium-sensitive receptors.
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Introduction |
Nematode
parasite infections of humans and animals cause disease with loss of
productivity, debility, and occasionally death. Ascariasis and hookworm
infections are carried by 1.6 billion people throughout the world and
in 2% of cases cause loss of life. The use of therapeutic compounds
forms a major component of control, and the development of novel
therapeutic agents is required to deal with the increasing levels of
resistance to existing drugs.
Paraherquamide (Fig. 1) is a novel
anthelmintic (Yamazaki et al., 1981
) that is an alkaloid fermentation
product originally isolated from Penicillium paraherquii.
The anthelmintic property of paraherquamide was first identified using
jirds infected with Trichostrongylus colubriformis (Ostlind
et al., 1990). Paraherquamide produces paralysis of parasitic nematodes
in culture, without an effect on ATP, suggesting that it does not act
as a metabolic poison (Thompson et al., 1996). Interestingly, one of
the toxic effects of paraherquamide in the dog (Shoop et al., 1990) is
a prolapsed nictitating membrane, an effect that suggests antagonism of
neuronal nicotinic receptors (nAChRs). Recently, it has been reported
(E. W. Zinser, M. L. Wolfe, S. J. Bowman, E. M. Thomas, V. E. Groppi,
J. P. Davis, D. P. Thompson, and T. G. Geary, manuscript submitted for
publication) that paraherquamide and 2-deoxy-paraherquamide act as
antagonists of nematode nAChRs and mammalian neuronal nAChRs.
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Fig. 1.
Structure of anthelmintics paraherquamide, bephenium,
levamisole and pyrantel. In paraherquamide, note the presence of the
groups connecting N* in position 19 and O* in position 2 with a
similarities to acetylcholine. The O* is missing in
2-deoxy-paraherquamide.
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Nematode nAChRs have some pharmacological similarities to vertebrate
neuronal receptors, but there are important differences. This is
fortunate because it allows drugs like levamisole and pyrantel to be
used for therapeutic purposes. Levamisole and pyrantel are potent
anthelmintics that act as selective agonists on nematode nAChRs
(Martin et al., 1996). Nematode nAChRs, like vertebrate neuronal
nAChRs, are taken to be composed of a pentamer of subunits, and a
number of different subtypes are present on muscle (Robertson et al.,
1999; Richmond and Jorgensen, 1999). The distinctive pharmacology of
some nematode nAChRs seems to relate to the distinctive molecular structure of the agonist binding site formed by part of the
-subunit (Fleming et al., 1997).
In this article, we report the quantitative investigation of the
cholinergic antagonistic effects of paraherquamide and
2-deoxy-paraherquamide, using the simple competitive antagonist
model on nematode nAChRs in Ascaris body wall muscle. We
report that paraherquamide is a potent antagonist in nematodes that
distinguishes levamisole- and nicotine-sensitive nAChRs of nematodes
and that 2-deoxy-paraherquamide distinguishes levamisole- and
bephenium-sensitive receptors. These findings are important because
they demonstrate multiple subtypes of acetylcholine receptor with
different anthelmintic sensitivities in a parasitic nematode and they
further define the mode of action for these anthelmintics.
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Materials and Methods |
Ascaris Preparation.
Adult Ascaris
suum were collected weekly from the IPB packing plant
(Storm Lake, IA) and returned to the laboratory in Locke's solution at around 35°C in a metal vacuum flask. The
Ascaris were used for the contraction studies within 72 h because the ability to contract vigorously to cholinergic agonists
declined after this period.
Two 2-cm body-flap preparations, one dorsal and one ventral, were made
from each Ascaris female from the region anterior to the
genital pore. The gut was teased away from the muscle strip and the
lateral lines were removed from the edge of the flaps to produce two
preparations (one dorsal and one ventral). Each flap, composed of the
muscle field and the cut nerve cord, was monitored isometrically by
attaching a force transducer in an experimental bath, maintained at
37°C, containing 10 ml of Ascaris Ringer (23 mM NaCl, 110 mM Na-acetate, 24 mM KCl, 6 mM CaCl2, 5 mM
MgCl2, 11 mM glucose, 5 mM HEPES, pH 7.6, with
NaOH), and bubbled with nitrogen. Eight baths were used simultaneously.
After dissection, the preparations were allowed to equilibrate for 15 min under an initial tension of 2.0 g. The antagonist was then added to the preparation 15 min before the application of the first
concentration of the agonist. We ran two controls (no antagonist) and
three replicate antagonist concentrations during each experiment (eight
preparations). The dorsal and ventral flaps were assigned randomly in
the experiments to reduce any potential error associated with
differences in receptor populations. The agonists were added cumulatively with 2- to 3-min intervals between applications, and the
responses were steady changes in tension. Figure
2B is a representative trace of a control
agonist concentration-response plot with bephenium. Results for
individual flaps were rejected if the maximum change in tension, at the
highest agonist concentration, did not exceed 0.5 g. The mean
maximum tension of the preparations, produced at each concentration of
antagonist, was not significantly different (p > 0.05, F-test). The responses for each concentration were expressed as the
percentage of the maximum tension produced by each individual flap
preparation. The addition of small volumes of agonist did not
significantly reduce the antagonist concentration in the tissue bath.
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Fig. 2.
A, A. suum in hand for scale. B,
representative Ascaris muscle contraction
cumulative-concentration-response trace. The trace shows the effect of
cumulative addition of bephenium to the muscle strip.
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Drugs.
Paraherquamide and 2-deoxy-paraherquamide were
obtained from Pharmacia Animal Health (Kalamazoo, MI). Nicotine
hemi-sulfate, levamisole hydrochloride, pyrantel citrate, bephenium
hydroxynaphthoate, and dihydro-
-erythroidine were purchased from
Sigma-Aldrich (St. Louis, MO). Pyrantel tartrate, bephenium
hydroxynaphthoate, paraherquamide, and 2-deoxy-paraherquamide were
dissolved in DMSO and added to the bath. Concentrations of DMSO did not
exceed 0.1% in the bath. Control experiments demonstrated that this
concentration of DMSO had no effect on concentration-response
relationships (data not shown).
Recording and Analysis.
Changes in isometric muscle tension
responses were monitored using a PowerLab System (ADInstruments Pty
Ltd., Castle Hill, Australia) that consists of the PowerLab hardware
unit and Chart for Windows software (Microsoft, Redmond, WA). The
system allows for recording, displaying, and analysis of experimental
data. Sigmoid concentration-response curves for each individual flap preparation at each concentration of antagonist were described by the
Hill equation.
![% <UP>Response</UP>=1/(1+[<UP>EC</UP><SUB>50</SUB>/X<SUB><UP>A</UP></SUB>]<SUP>n<SUB><UP>H</UP></SUB></SUP>)<UP>,</UP>](/content/vol302/issue3/fulltext/853/img001.gif)
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(1)
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where EC50 is the concentration of agonist
(XA) producing 50% of the maximum
response and nH is the Hill
coefficient (slope). On the limited occasions when desensitization was
evident (exclusively in control or low antagonist conditions), the
maximum response was taken as 100%, and responses at higher agonist
concentrations set at 100% to prevent the desensitization phenomenon
from biasing the concentration-response plots. Prism 2.01 (GraphPad
Software, San Diego, CA) was used to estimate the constants
EC50 and nH in
eq. 1 by nonlinear regression for each preparation. The effect of
antagonist concentration on nH values
was tested using analysis of variance. The pEC50
was calculated as the negative logarithm of EC50.
To illustrate the agonist concentration-response relationship at each
concentration of antagonist, responses are plotted using the mean ± S.E. percent responses (n = 6-10 flap
preparations), and the lines of fit are obtained for the figure
displays by nonlinear regression without constraining the lines to be
parallel. These lines of fit were not used for estimation for the
pKB values. If a compound behaves as a
simple competitive antagonist, estimation of
pKB, the negative logarithm of the
dissociation constant of the antagonist, is best made by nonlinear
regression.
![<UP>pEC</UP><SUB>50</SUB>=<UP>−log</UP> ([X<SUB><UP>B</UP></SUB>]+10<SUP>−<UP>p</UP>K<SUB><UP>B</UP></SUB></SUP>)−<UP>log</UP> C](/content/vol302/issue3/fulltext/853/img002.gif)
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(2)
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where pEC50 values are as before,
XB is the concentration of the
antagonist, and 
log C is a constant and is the difference between the antagonist pKB and the
agonist control curve pEC50. pKB and log C were
estimated by nonlinear regression as before. The advantage of
estimating pKB with this method is
that there is no over-reliance on the control concentration-response
relationship for estimating dose ratios for the Schild plot. Also, an
error estimate for pKB can be made
from the covariance matrix and is expected to be normally distributed
(Lew and Angus, 1995).
The slope factor, equivalent to the slope of the Schild plot, was
estimated using nonlinear regression and the following equation.
![<UP>pEC</UP><SUB>50</SUB>=−<UP>log</UP> ([X<SUB><UP>B</UP></SUB>]<SUP>N</SUP>+10<SUP>−<UP>p</UP>K<SUB><UP>B</UP></SUB></SUP>)−<UP>log C</UP>](/content/vol302/issue3/fulltext/853/img003.gif)
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(3)
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where N is equivalent to the slope of the Schild plot
and the other variables are the same as before.
To display the effect of the antagonists paraherquamide and
2-deoxy-paraherquamide on the agonist EC50
values, Clark plots of log EC50 versus log
([XB] + pKB) were used. This display allows the distribution of the data to be compared with the ideal for simple
competitive block.
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Results |
Dihydro--Erythroidine.
Richmond and Jorgensen (1999) have
described the presence of nicotine- and levamisole-sensitive
cholinergic receptors on muscle of Caenorhabditis elegans
and have used single concentrations of the antagonist
dihydro--erythroidine to block electrophysiological responses to
nicotine but not levamisole. We tested, in Ascaris, the
effects of different concentrations of
dihydro--erythroidine on the contraction-responses to
nicotine and levamisole.
Figure 3 shows the effect of different
concentrations (10-100 µM) of dihydro--erythroidine on the
concentration-response relationships of levamisole and nicotine.
Dihydro--erythroidine had no detectable effect on the levamisole
response (N = 7, 5, 5, and 5 for the 0, 10, 30, and 100 µM concentrations of dihydro--erythroidine) but produced a modest
parallel shift to the right, in the concentration-response curves for
nicotine (N = 8 for each concentration of
dihydro--erythroidine). The dose-ratio produced by 100 µM
dihydro--erythroidine was 2.16. If the antagonism is taken to be
competitive, we can make an estimate for the antagonist
pKB using eq. 4.
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(4)
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where dr is the dose-ratio and XB
is the concentration of dihydro--erythroidine. The
pKB for nicotine was 4.07. The
selective effect of dihydro--erythroidine suggests the presence of
nicotine- and levamisole-sensitive receptors in Ascaris and
in C. elegans. However, the modest
pKB of dihydro--erythroidine shows
that it is not potent in Ascaris.
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Fig. 3.
Dihydro--erythroidine (DHE) as an antagonist of
nicotine (A) and levamisole (B). Note that dihydro--erythroidine
produces some dose-dependent antagonism of the effects of nicotine but
has no effect on the levamisole response (n  5 for control and each antagonist concentration).
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Paraherquamide.
We found that paraherquamide was a more potent
antagonist than dihydro--erythroidine in Ascaris, and we
therefore examined the antagonism in greater detail. We selected
agonists that were cholinergic anthelmintics belonging to different
chemical classes for further examination. We chose (Fig. 1) the tobacco
alkaloid nicotine, the imidazothiazole levamisole, the
tetrahydropyrimidine pyrantel, and the quaternary nitrogen compound bephenium.
Figure 4 shows the antagonistic effects
of paraherquamide (0.03-30 µM) on concentration-response plots for
nicotine (N = 8, 5, 5, and 5 for the 0, 0.3, 3, and 30 µM paraherquamide), levamisole (N = 8, 5, 6, and 5 for 0, 0.3, 3, and 30 µM paraherquamide), pyrantel (N = 6, 5, 6, and 5 for 0, 0.3, 3, and 30 µM paraherquamide) and
bephenium (N = 7, 6, 8, 7, and 3 for 0, 0.3, 3, 10, and
30 µM paraherquamide). The antagonism produced clear shifts to the right in all the concentration-response curves. As a test for the
parallel nature of the shifts, we fitted all the concentration-response plots of single flap preparations (n = 5-8 at each
antagonist concentration) with eq. 1, to estimate
nH and EC50. We
then tested the effect of antagonist concentration on
nH and found no significant effect
(p > 0.05, F-tests) for nicotine, levamisole,
pyrantel, or bephenium and concluded that the shifts could be taken as
parallel.
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Fig. 4.
Concentration-response plots for the agonists
nicotine (A), levamisole (B), pyrantel (C), and bephenium (D) in the
presence of different concentrations of paraherquamide
(n 5 for control and each paraherquamide
concentration).
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Since the concentration-response curves were shifted to the right in a
parallel manner, we used the simple competitive antagonist model (eq.
2) to describe the data. We also estimated the parameters of eq. 3
using nonlinear regression to make an estimate of the Schild slope
factor, N.
Table 1 shows the values that were
estimated for nicotine, levamisole, and pyrantel. The Schild slopes for
these agonists were not significantly different from 1. The slope for
bephenium was significantly less than 1, suggesting deviation from the
simple competitive antagonist model. For reasons we discuss later, we described the antagonism of all the agonists with the simple
competitive model using eq. 2.
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TABLE 1
Paraherquamide parameter and error estimates for the Schild slope
factor N in eq. 3, the negative log of the antagonist
dissociation constant pKB, in eq. 2, with degrees of
freedom, and the goodness of fit R2
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The mean ± S.E. values for the parameters of eq. 2 are shown in
Table 1 along with their confidence limits. Interestingly, the goodness
of fit, R2, showed that the fit for
bephenium was comparable to nicotine and levamisole. As a further test
of the fit of the simple competitive antagonist model, we plotted log
EC50 versus log ([B] + KB) as the Clark plot.
EC50 values were obtained from eq. 1,
B is the concentration of paraherquamide, and
KB is the antagonist dissociation constant
(the antilog of pKB). Figure
5 shows these plots for nicotine,
levamisole, pyrantel, and bephenium. These plots show the relationship
between the experimental data and the line predicted by the simple
competitive antagonist model.
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Fig. 5.
Clark plots for the agonists nicotine (A), levamisole
(B), pyrantel (C), and bephenium (D). Ordinate: mean ± S.E. log
EC50. Abscissa: log (paraherquamide concentration + agonist
dissociation constant). Note the degree of fit between the observed
values and the line predicted by the simple competitive block model.
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The important point that we can make here is that the
pKB of paraherquamide for nicotine is
5.86 ± 0.14, and this is significantly less (p < 0.001, t test, degrees of freedom 43-52) than for
levamisole (6.61 ± 0.19), pyrantel (6.50 ± 0.11), or
bephenium (6.75 ± 0.15). The observations demonstrate that the
receptors activated by nicotine are not the same as those activated by
levamisole, pyrantel, and bephenium.
2-Deoxy-Paraherquamide.
We also tested the
paraherquamide analog 2-deoxy-paraherquamide (see Fig. 1). We found
that like paraherquamide it was an antagonist of nicotine, levamisole,
pyrantel, and bephenium. Figure 5 shows the effect of
2-deoxy-paraherquamide on the log-concentration response plots.
Concentration-response plots for levamisole (N = 6, 6, 6, and 6 for 0, 0.3, 3, and 30 µM antagonist), pyrantel (N = 6, 8, 7, and 8 for 0, 0.3, 3, and 30 µM
antagonist), and bephenium (N = 7, 6, 6, 6, and 6 for
0, 0.03, 0.3, 3, and 30 µM antagonist), but not nicotine
(N = 9, 10, 9, and 30 for 0, 0.3, 3, and 30 µM
antagonist) were shifted to the right in a parallel manner by
2-deoxy-paraherquamide. Again, as a test for the parallel nature of the
shift, we fitted all the concentration-response plots of single
preparations with eq. 2. We then tested the effect of
2-deoxy-paraherquamide concentrations on
nH; we found no significant effect
(p > 0.05, F-tests) for levamisole, pyrantel, or
bephenium. The effect of 2-deoxy-paraherquamide on
nH of the nicotine slopes was
significant (p < 0.0004, F = 10.2, degrees
of freedom 3,18), an observation inconsistent with a simple competitive
antagonism model.
We estimated N using eq. 3. Table
2 shows the mean values and 95%
confidence limits for N. The slope, N, for
nicotine was clearly less than 1, with a mean value of 0.39 (95%
confidence limits 0.10-0.69), again emphasizing that a simple
competitive action was not adhered to. As with paraherquamide, we again
fitted the simple competitive model of antagonism to describe the
action of the antagonist and to estimate the
pKB values with eq. 2. Table 2 shows
the mean values and error estimates for the
pKB values for levamisole, pyrantel,
and bephenium. We do not include the error estimates for nicotine
because the simple competitive antagonism model did not describe the
experimental data well, and R2 was 0.50. To illustrate the fit of the experimental data to the simple
competitive antagonist model and the estimated dissociation constant,
we again used the Clark plots (Fig.
6). The
plots suggest that the model is suitable and describes the data for
levamisole, pyrantel, and bephenium.
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TABLE 2
2-Deoxy-paraherquamide parameter and error estimates for the Schild
slope factor N in eq. 3, the negative log of the antagonist
dissociation constant pKB, eq. 2, with degrees of
freedom, and the goodness of fit R2
Errors for nicotine are omitted because of the poor fit of the
antagonism to the simple competitive model.
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Fig. 6.
Concentration-response plots for the agonists
nicotine (A), levamisole (B), pyrantel (C), and bephenium (D) in the
presence of different concentrations of 2-deoxy-paraherquamide [0
(control), 0.3, 3, and 30 µM] (n 6 for each
agonist/antagonist combination).
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Fig. 7.
Clark plots for the agonists levamisole (A), pyrantel
(B), and bephenium (C). Ordinate: mean ± S.E. log
EC50. Abscissa: log (2-deoxy-paraherquamide concentration + agonist dissociation constant). Note the relationship between the
observed values and the line predicted by the simple competitive model
of competitive antagonism.
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The pKB values estimated for
2-deoxy-paraherquamide are less than the
pKB values of paraherquamide for the
same agonist showing that paraherquamide is more potent than
2-deoxy-paraherquamide as an antagonist in A. suum. Also, the pKB of
2-deoxy-paraherquamide was significantly greater with bephenium as
agonist than with levamisole and pyrantel as agonist. This implies that
bephenium does not act on the same population of nAChRs as levamisole
and pyrantel.
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Discussion |
Use of the Simple Competitive Antagonism Model.
We used
classical pharmacological techniques to analyze the antagonist action
of paraherquamide and 2-deoxy-paraherquamide. The simple model of
competitive action is based on the law of mass action, using the
reaction scheme below.
where, R is the receptor not occupied, RA* is the receptor
occupied by one molecule of agonist, RB is the receptor occupied by one
molecule of antagonist. KB and KA are the dissociation constants of the
antagonist and agonist, and * indicates activation of the receptor. In
the presence of agonist and antagonist, equation 2 (see Materials
and Methods) should describe the degree of antagonism.
This simple model may not be universally valid for ion-channels that
have two or more binding sites for agonist and antagonist molecules.
Under these conditions, the slope of the Schild plot is predicted to be
reduced under the law of mass action and under limiting conditions is
described by the modified Schild equation (Williams et al., 1988),
which has a slope less than 1. For the paraherquamide antagonism of
bephenium and the 2-deoxy-paraherquamide antagonism of nicotine, we saw
that the Schild slope factor was less than 1. One explanation then for
the reduced slope factor may lie along the lines prescribed by Williams
et al. (1988).
When a Monod-Wyman-Changeux allosteric model for competitive antagonism
is used for the operation of the receptor ion-channel, the dose-ratio
turns out to be a linear function of the antagonist concentration, even
in the general case (Colquhoun, 1973). So the explanation for when the
slope factors are less than one may lie elsewhere and relate to the
presence of more than one nAChR receptor subtype. If there are multiple
subtypes of nAChR present in the preparation and the agonist and
antagonists are not sufficiently selective, it is possible, at low
agonist concentrations, that the KB value
of the antagonist for receptors with high agonist affinity dominates
the shift in the concentration-response curve. At high concentrations
of antagonist, the KB of the antagonist for low-agonist-affinity receptors may dominate the shift in the concentration-response curve. This could reduce the Schild slope factor
and/or give rise to nonparallel shifts in concentration-response plots,
as we saw with the antagonism of nicotine by 2-deoxy-paraherquamide.
By using eq. 2, we have imposed a slope factor of 1 for all the
agonists to estimate the pKB for
reasons of parsimony and because the approach also reduces over
estimates of pKB (Black and Shankley,
1985). Thus, despite some known uncertainties, we feel justified in
using the competitive model. Waud and Parker (1971) have commented that
"I do not blush when caught using a competitive kinetic analysis
heuristically when the underlying model may in fact be more along the
allosteric lines... if you tell me the
KB of a new antagonist, I know a lot
about the drug." The conclusions we draw in this article are based on
the assumption that agonists acting on identical receptors will be
affected to the same degree by the antagonist. Classically, agonists
that act on the same receptors are expected to produce the same
pKB (Arunlakshana and Schild, 1959).
Competitive Action of Paraherquamide and 2-Deoxy-Paraherquamide and
nAChR Subtypes.
In this study, we have been able to show that
paraherquamide and 2-deoxy-paraherquamide can behave as competitive
antagonists at nematode nAChRs. We know that paraherquamide is a potent
anthelmintic (Shoop et al., 1990). The potency of the competitive
antagonist action seen here, with dissociation constants in the
micromolar range, suggests that competitive nAChR antagonism
contributes to the anthelmintic properties of this compound.
2-Deoxy-paraherquamide lacks a carbonyl group at the 2-position
(Fig. 1) and was around 10× less potent as an antagonist in our
assays. In whole-worm preparations with a cuticle barrier to penetrate
this order of potency is reversed (Thompson et al., 1996), perhaps for
reasons of access. Our observations here suggest that the presence of a
carbonyl group, which presumably acts in hydrogen bonding to the
receptor, is important for binding. The classic model of the
cholinergic receptor consists of a site which binds the quaternary
nitrogen, N+, and an osteophilic site that binds
to =O of acetylcholine; the separation between the
N+ and =O is a chain length of four atoms. We can
see a similar structure in paraherquamide, starting with the carbonyl
group in position 2 and ending in the methylated nitrogen in position 19 (Fig. 1). This portion of paraherquamide might bind to the nAChR. We
point out that the amino acid structure of the ligand-binding has now
been resolved at the atomic level, and the quaternary nitrogen of
ligands is thought to stack onto tryptophan (position 143)
making cation-
interactions (Brejc et al., 2001).
Interestingly, our analysis revealed that paraherquamide and
2-deoxy-paraherquamide showed pKB
values that varied significantly when different agonists were used. We
saw that paraherquamide discriminated between receptors sensitive to
nicotine and receptors sensitive to levamisole, pyrantel, and
bephenium. This observation is consistent with the observations of
Richmond and Jorgensen (1999), who described the presence of nicotine-
and levamisole-sensitive receptors on C. elegans
muscle. We found that 2 deoxy-paraherquamide had a
pKB for bephenium that was
significantly different to that of levamisole and pyrantel. Again the
evidence suggests that the receptors acted on by levamisole and
pyrantel are not the same as those of bephenium. It is interesting to
point out that a comparison of levamisole-sensitive and
levamisole-resistant isolates of Haemonchus contortus showed
that muscle contraction became less sensitive to levamisole with
resistance but did not change its sensitivity to bephenium (Sangster et
al., 1991). Both these lines of evidence support the view that
bephenium-sensitive receptors are different to levamisole-sensitive receptors.
Multiple Subtypes of nAChRs in Mammals and Nematodes and
Selective Ligands.
Ionotropic acetylcholine receptors are taken to
be pentameric structures (Corringer et al., 2000; Sharples and
Wonnacott, 2001). In vertebrate muscle, the subunits , , 
, and
form the ion-channels with a stoichiometry of
2, , , at the endplate. In
vertebrate neuronal receptors, the channels are usually formed by a
heteropentameric combination of and subunits, 2 to 7, 9,
10, and 2 to 4 in mammals, with an additional subunit, 8, in
avian species. Homomeric combinations of some subunits may occur,
e.g., by 7, 8, or 9, but most channels are composed of and
subunits. The rules governing the ability to combine to form
functional ion-channels vary with the structure of the subunit.
The ligand binding sites (Corringer et al., 2000: Brejc et al.,
2001) of individual nAChR receptors are composed of six amino acid
loops, three (loops A, B, and C) from the subunit and three (D, E,
and F) from the adjacent subunit. Since nAChRs have two or more subunits, there are two or more ligand binding sites. Different nAChRs
formed by different and subunit combinations have different
agonist binding sites because the loops, ABC and DEF, will vary with
the particular and subunits that form the ion-channel receptor.
It is also a consequence that nAChRs composed of two identical subunits and three dissimilar subunits will have two nonequivalent
binding sites. Consequently, selective agonists and antagonists are
expected to distinguish between receptor subtypes and between
nonequivalent sites on an individual nAChR.
A distinct but related family of nAChR subunits has been found in
C. elegans in which there are 42 candidate nAChR
subunits (Fleming et al., 1996, 1997; Baylis et al., 1997; Bargmann,
1998). The subunits include: UNC-39, UNC-63, ACR-16 or Ce21, and
ACR-3. The subunits include: ACR-1, ACR-2, LEV-1, and UNC-29. An
nAChR, sensitive to levamisole, may be composed of UNC-38 as an
-subunit along with UNC-29 and LEV-1 on muscle. Expression of ACR-2 + UNC-38, UNC-38 + ACR-3, and UNC-38 + UNC-29 + LEV-1 in
Xenopus oocytes produces small currents in response to
levamisole. In contrast, expression of ACR-16, which is 47% identical
to chicken 8, in Xenopus oocytes, produces channels
sensitive to acetylcholine but insensitive to levamisole or pyrantel
(Ballivet et al., 1996). In the intact nematode, levamisole pyrantel,
morantel, and oxantel activate nAChRs on Ascaris muscle
(Martin et al., 1996), and single channel recordings of nAChRs in
Oesophogostomum dentatum reveal the
presence of four subtypes on somatic muscle (Robertson et al., 1999).
There is therefore evidence for the presence of multiple subtypes of
nAChR in nematodes with ligands that can be selective for different
subtypes of receptor.
Significance of nAChR Receptor Subtypes for Anthelmintic
Resistance.
There is strong evidence for nAChR receptor subtypes
in vertebrates and, now, nematodes and that the different subtypes have different sensitivities to ligands. Also, we have presented evidence that nicotine, levamisole, and bephenium are selective ligands for
different subtypes. Sangster et al. (1991) have described isolates of
levamisole resistant H. contortus that have a
reduced sensitivity to levamisole but not to bephenium. We suggest that one form of levamisole resistance is associated with a switch from
levamisole-sensitive nAChRs to other nAChR subtypes. This mode of
anthelmintic resistance would not be revealed as a change in the amino
acid sequence of particular nAChR subunits (Hoekstra et al., 1997) but
as a change in proportion of expressed nAChR subtypes.
The effect of paraherquamide and 2-deoxy-paraherquamide on whole
nematodes seems to be that of paralysis by competitive antagonist action at different nAChR subtypes. If the potency of these antagonists is sufficient against all of the nAChR subtypes present in nematode parasites, these compounds could be useful for the control of types of
levamisole resistant nematodes associated with changes in proportion of
nAChR subtypes.
We are pleased to acknowledge the support of National
Institutes of Health Grant RO1 A147194-02 awarded to R.J.M.
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Abstract
Introduction
molecular cloning and functional expression.
Recept Channels
5:
149-158[Medline].
