Insect Control: Biological and Synthetic Agents - Index of

86 3: Neonicotinoid Insecticides
recorded from isolated cockroach neurons, where
imidacloprid exhibited an EC50 of 0.36 mM (Orr
et al., 1997). Acetamiprid, nitenpyram, and the natural
toxin epibatidine exhibited an EC50 of between
1 and 2 mM (Table 5). Similar values were also
observed for the cockroach preparation, with an
EC50 between 0.5 and 0.7 mM for epibatidine and
acetamiprid (Orr et al., 1997). Nithiazine had the
lowest potency, with an EC50 of about 10 mM. The
neonicotinoids, clothianidin, and nitenpyram were
full agonists, whereas acetamiprid, epibatidine,
and imidacloprid were partial agonists. The maximal
response elicited by saturable concentrations of
imidacloprid (100 mM) was only 15% of the maximal
current obtained by 1000 mM ACh. In earlier
work on isolated cockroach neurons (Orr et al.,
1997) and isolated locust neurons (Nauen et al.,
1999b), it was also found that imidacloprid acted
as a partial agonist on insect nAChRs. The partial
agonistic action of imidacloprid was also observed
with chicken a4b2 nAChRs, and on a hybrid nAChR
formed by the coexpression of a Drosophila a subunit
(SAD) with the chicken b2 subunit in Xenopus
oocytes (Matsuda et al., 1998). Imidacloprid activates
very small inward currents in clonal rat phaeochromocytoma
(PC 12) cells, thus also indicating
partial agonistic actions (Nagata et al., 1996).
Furthermore, single-cell analysis revealed that
imidacloprid activates predominatly a subconductance
of approximately 10 pS, whereas acetylcholine
activated mostly the high conductance state
with 25 pS. Multiple conductance states were also
observed in an insect nAChR reconstituted into planar
lipid bilayers (Hancke and Breer, 1986) and on
locust neurons (van den Breukel et al., 1998).
3.6.3. Correlation between Electrophysiology and
Radioligand Binding Studies
There is a good correlation between electrophysiological
measurements, using isolated Heliothis neurons,
and radioligand binding studies on housefly
head membranes regarding the affinity of different
ligands to nAChRs (Figure 22). This good correlation
for the neonicotinoids may indicate that houseflies
(binding data) and tobacco budworms
(electrophysiology) have similar binding sites for
imidacloprid and related compounds. Biochemical
investigations using [ 3 H]imidacloprid as a radioligand
in a number of different insect membrane
preparations, e.g., from Periplaneta americana,
Lucilia sericata, D. melanogaster, Manduca sexta,
H. virescens, Ctenocephalides felis, M. persicae, and
N. cincticeps, indicate that many (if not all) insects
have high specific imidacloprid binding sites with
nearly identical Kd values of 1–10 nM (Lind et al.,
Figure 22 Comparison between electrophysiological and
binding potencies of different neonicotinoids and nicotinoids.
Electrophysiological data were obtained from neuron cell
bodies isolated from the CNS of Heliothis virescens. pEC 50 values
(¼ log M) correspond to the half maximal activation of nAChR by
each agonist. Binding data: pI50 values (¼ log M)correspondto
the concentration of cold ligand displacing 50% of bound [ 3 H]imidacloprid
from housefly head membranes. (Reproduced from
Nauen, R., Ebbinghaus-Kintscher, A., Elbert, A., Jeschke, P.,
Tietjen, K., 2001. Acetylcholine receptors as sites for developing
neonicotinoid insecticides. In: Iishaaya, I. (Ed.), Biochemical
Sites of Insecticide Action and Resistance. Springer, New York,
pp. 77–105.)
1998). However, it was also found that only the
homopteran species seems to have an additional
very high affinity binding site.
In general, the pI50 values obtained by the displacement
of specifically bound [ 3 H]imidacloprid
from housefly head membranes were two to four
orders of magnitude higher than the electrophysiologically
determined pEC 50 values obtained from
isolated Heliothis neurons. Similar differences in
biochemical binding and functional assay studies
were also observed for different vertebrate nAChRs
(review: Holladay et al., 1997). A possible explanation
for this phenomenon might be provided by
considering the following: it is generally accepted
that each nAChR can exist in multiple states, i.e., a
resting state, an active (open) state, and one or more
desensitized state(s), each of which has different
affinities for ligands. The active state has a low
affinity for ACh (Kd ranging from about 10 mM to
1000 mM), whereas the desensitized state(s) has a
higher affinity (Kd ranging from about 10 nM to
1 mM) for nicotinic ligands (Lena and Changeux,
1993).
The kinetics of the transitions between these
states have been resolved for Torpedo nAChR
in vitro. The rate of isomerization between the resting
and active state lies in the ms to ms timescale, and