Insect Control: Biological and Synthetic Agents - Index of

226 6: The Spinosyns: Chemistry, Biochemistry, Mode of Action, and Resistance
Figure 12 Pharmacological separation of cockroach nicotinic acetylcholine receptor (nAChR) subtypes and the action of
spinosyn A on both subtypes. (a) Combined current in an isolated cockroach neuron and the pure nAChD current remaining after
specific block of nAChN receptors by 100 pM MLA. (b) In the presence of 100 nM imidacloprid (IMI), only the nAChN current remains in
a cell with both receptor subtypes. (c) When nAChN is blocked by the inclusion of 100 pM MLA in the bath, 10 mM spinosyn A slightly
blocks the nAChD current. (d) nAChN current isolated with 100 nM IMI, alone and in the presence of increasing concentrations
of spinosyn A (Salgado, unpublished data).
of ACh, and these effects increased dramatically
with increasing concentrations of spinosyn A.
Although the nAChN receptors do not desensitize,
the control current drooped approximately 15%
during the ACh pulse, possibly due to redistribution
of ions near the membrane. At 3 nM spinosyn A,
this droop was eliminated and at higher concentrations
the current actually increased up to 10%
during the ACh pulse, because of a synergistic interaction
between spinosyn A and ACh on the nAChN
receptor. This synergistic effect can also be seen from
the fact that the current after the ACh pulses in the
presence of spinosyn A decays much more slowly
than in the control, an effect also noted by Salgado
et al. (1997). The effect of spinosyn A on the nAChN
receptors in Figure 12d was quantified by measuring
the fraction of unmodified receptors remaining,
taken as the amplitude of the rapid increase
in current in the first second of ACh application as
a fraction of the peak current in the control. For
example, for the 10 nM spinosyn A treatment in
Figure 12d, the result is 59% unmodified and for
100 nM it is 10%. The EC50 (concentration for
50% activation) for activation of nAChN receptors
by spinosyn A, measured in the presence of 100 nM
IMI to block nAChD receptors (Figure 12d) was
10 1nM (n ¼ 3). Because of the synergistic interaction
of spinosyns with competitive nicotinic
agonists, the EC50 for activation of nAChN
receptors was also measured in the absence of IMI,
giving a value of 27 4nM (n ¼ 4) (Salgado and
Saar, 2004).
The synergistic interaction between spinosyn A
and classical, competitive agonists acting at the ACh
binding site shows that spinosyns are in fact allosteric
agonists of nAChN. This is further supported by
the fact that a-BGTX blocks the spinosyn-induced
current noncompetitively and that spinosyn A does
not displace binding of [ 3 H]a-BGTX (Salgado et al.,
1997). Under certain circumstances the synergism
between spinosad and classical nicotinic agonists,
such as neonicotinoids, is demonstrable in practice
(Andersch et al., 2003).
Note also in Figure 12d that the total current in
the presence of 100 nM spinosyn A and 100 mM IMI
is more than twice the total current evoked by ACh
alone; spinosyn A is a more efficacious agonist than
ACh and can therefore have very strong effects on
cells that have a significant number of nAChN
receptors. The sensitivity of any cell to the agonistic
effects of spinosyns on nAChN receptors will be
dependent on the number of those receptors and
the input resistance of the cell, which together
determine what proportion of the receptors need
to be activated to significantly depolarize the cell
and change its firing pattern. Symptoms arise in
cockroaches at an internal aqueous concentration
of 20 nM, at which 45% of nAChN receptors are