Flubendiamide sulfoxide

II Flubendiamide sulfoxide [3-iodo-N-(2-methanesulfinyl-l,l-dimethyl-ethyl)-N -[2-methyl-4-(l,2,2,2-tetrafluoro-l-trifluoromethyl-ethyl)-phenyl -phthalamide ... 

This hypothesis was further supported using molecular biology and cellular tools where the insect ryanodine receptor gene was heterologously expressed in appropriate cells. In untransfected CHO cells, application of flubendiamide sulfoxide (a better soluble analogue) did not cause an [Ca ] increase (Figure 8). In CHO cells transfected with the RyR from Drosophila (CHO-RyR), flubendiamide sulfoxide induced Ca responses with similar kinetic responses to those found in Heliothis neurons (Figure 8). 

Figure 9. Effect of caffeine and phthalic diamides on mouse muscle cell line C2C12. representative [Ca ] traces of a Fura2-AM-loaded C2C12 cell during application of caffeine, flubendiamide sulfoxide, and again caffeine.

Figure I. Structures of the Phthalic Acid Diamides Employed in the Current Study (I, flubendiamide II, flubendiamide-sulfoxide)...

A Superimposed, representative responses induced by 30 pM of flubendiamide-sulfoxide, and by 10 pMACh, B The response to 1000 pM acetylcholine was completely prevented when Cc was removed from the application solution. In contrast, the [Ca Jc transients induced by flubendiamide-sulfoxide were nearly the same as under control conditions. 

Application of 30 pM flubendiamide-sulfoxide to isolated Heliothis neurons caused an increase in the cytosolic calcium concentration, [Ca ]c (Figure 2A), which was independent of the calcium concentration in the extracellular medium. 

Figure 3. Concentration-dependent intracellular calcium release elicited by flubendiamide sulfoxide (II). Amplitudes of the calcium transients were normalized to the maximum response evoked by 10 mM caffeine (n = 4).

The normalized amplitude heights of the calcium response induced by flubendiamide-sulfoxide were concentration-dependent with an apparent EC o of 0.7 pM (Figure 3). It is noteworthy that calcium transients could be specifically blocked with 50 pM ryanodine, but not with xestospongine, a blocker of IP3-gated Ce release channels (16). 

Figure 4. Effect of flubendiamide-sulfoxide on equilibrium saturation binding of fHJryanodine at two different calcium concentrations. Saturation binding was performed essentially as described earlier with 100 pM (A) or 800 pM (B) calcium, respectively. Control asst s (9) and assess including 1.0 pM of flubendiamide-sulfoxide (A.) were performed in quadruplicate and the data were fitted to the binding equation (Hill).

Remarkably, in the presence of 1.0 pM flubendiamide-sulfoxide, the resulting binding isotherm had a simple hyperbolic character (Hill coefficient, n = 1.0), which indicated a homogeneous population of independent sites. Ryanodine affmity was increased with an apparent dissociation constant Kd = 5.2 +/- 0.8 nM (4 experiments). However, Ae number of binding sites, Bn, . remained essentially imchanged (1072 fmol/mg) as compared to the control. 

When increasing the calcium concentration to 800 pM, specific binding of ryanodine could be fitted to the simple hyperbolic equation with n = 1.0 (Figure 4B). The Kd of ryanodine was calculated as 13.6 +/- 1.4 nM (4 experiments) d the receptor density was determined as 1086 finol/mg. Flubendiamide-sulfoxide (1.0 pM) decreased the apparent Kq slightly, but still significantly, to 7.9 +/- 0.9 nM (t-test p < 0.03). 

The phthalic acid diamides flubendiamide and the corresponding sulfoxide were synthesized by Nihon Nohyaku Co., Ltd., and Bayer CropScience AG. Ryanodine, Fura-2 acetoxymethyl ester (Fura-2 AM) and Fura-2 were obtained from Sigma and stored frozen as aliquots (stock solution ImM in DMSO). 

The current study shows that phthalic acid diamide insecticides, represented by flubendiamide and its sulfoxide analogue, activated ryanodine receptors present in isolated Heliothis neurons, as concluded from the following results. Firstly, calcium transients evoked by phthalic acid diamides were independent of the extracellular [Ca ], in contrast to the signals elicited by acetylcholine. This was interpreted as calcium release from intracellular stores of the endo(sarco)plasmic reticulum, which could in principle be mediated by two different release channels, namely the ryanodine receptor and the IP3 receptor. 

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