Presynaptic Inhibition of [3H]Nitrendipine Binding and Modification of Membrane Properties by Insecticidal Dihydropyrazoles in Mammalian Brain

Abstract

RH-3421 and RH-5529 displace specifically bound [3H]nitrendipine in mouse brain synaptosomes, suggesting that dihydropyrazoles act at presynaptic L-type calcium channels in mammalian brain. Scatchard analysis revealed that both compounds interfere with binding of [3H]nitrendipine through a mechanism which reduces the number of available radioligand binding sites (Bmax) without altering the affinity (Kd) for remaining sites. In addition, there was no discernible change in either the rate of association of [3H]nitrendipine or in the rate of radioligand dissociation when the binding preparation was exposed to dihydropyrazoles. Such a binding profile indicates that dihydropyrazoles operate as noncompetitive blockers of [3H]nitrendipine binding without allosteric involvement. The effects of RH-3421 and RH-5529 on the physical properties of synaptic plasma membranes prepared from mouse brain were also evaluated using the fluorescence probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethyl-ammonium phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), which monitor the physical state of lipids in the membrane core and membrane surface, respectively. Exposure of synaptic membranes to RH-3421 and RH-5529 resulted in a reduction in fluorescence polarization of both probes, confirming that these lipid domains are accessible to dihydropyrazoles. Estimates of static and dynamic parameters show that lipid order and fluidity are modified within surface and core regions by dihydropyrazoles; however, the changes to regions probed by TMA-DPH appear more closely correlated with binding data. Overall, our results are consistent with the proposal that dihydropyrazoles reduce the availability of [3H]nitrendipine binding sites, possibly by modifying the physicostructural properties of the surface membrane. However, we do not exclude the possibility that dihydropyrazoles are irreversible inhibitors of [3H]nitrendipine binding or cause more subtle interference with regulatory biomolecules in the channel environment.