avermectin-b(1)a and 1-(4-ethynylphenyl)-4-propyl-2-6-7-trioxabicyclo(2.2.2)octane

[(3)H]Ethynylbicycloorthobenzoate ([(3)H]EBOB), a high affinity radioligand for the noncompetitive blocker site of the GABA(A) receptor, is used here for quantitative autoradiography to determine regional binding in mouse brain and the effects on this binding of administering toxic doses of endosulfan, fipronil, and avermectin B(1a) (AVM). Animals were euthanized 4-8 min after 1 LD50 or 2 LD50 doses of the two channel blockers and 32 min after 1 LD50 or 4 LD50 doses of the channel activator AVM. Specific binding of [(3)H]EBOB was determined for 20-microm brain sections as the difference in labeling on incubation with 2 nM [(3)H]EBOB either alone (total binding) or with 5 microM alpha-endosulfan (nonspecific binding). The highest specific labeling was observed for layers I and IV of the cerebral cortex, the globus pallidus, and the medial septal nucleus/nucleus of the vertical limb of the diagonal band. Dose-dependent inhibition by endosulfan was highest in the nucleus accumbens and least in the cerebellum and periaqueductal gray matter. Fipronil had much less effect on binding even at severely toxic doses. AVM increased [(3)H]EBOB binding in most regions and was the only one of the three agents inhibiting in vitro [(3)H]strychnine binding to the glycine receptor. In summary, the noncompetitive blocker site was strongly inhibited with dose dependence and regional selectively by alpha-endosulfan but was generally poorly inhibited or activated by fipronil and was activated by avermectin.

Topics: Animals; Brain; Bridged Bicyclo Compounds, Heterocyclic; Endosulfan; Ivermectin; Mice; Molecular Structure; Pyrazoles; Receptors, GABA-A; Strychnine; Tritium

Avermectin B1a (AVM B1a), a widely used insecticide and acaricide, is reported to both activate and inhibit gamma-aminobutyric acidA (GABA(A)) receptor function in mammalian brain. This study attempts to resolve these seemingly contradictory results by examining the binding properties of AVM B1a and its effects on the GABA-gated chloride channel with primary cultures of rat cerebellar granule neurons as a model system. Specific binding of [3H]AVM B1a in intact neuron cultures is time- and concentration-dependent and is displaceable by AVM analogs. Scatchard analysis of [3H]AVM B1a binding reveals high- and low-affinity sites with K(D) values of 5 and 815 nM, respectively. AVM B1a alters the binding of [3H]ethynylbicycloorthobenzoate at the noncompetitive blocker site in a biphasic manner; activation is evident with 10 to 300 nM AVM B1a after 5 to 10 min incubation and inhibition with an IC50 of 866 nM after 60 min incubation. Consistent with this observation, 36Cl- influx is stimulated by AVM B1a at 3 to 100 nM and inhibited at 1 to 3 microM. GABA-stimulated 36Cl- influx is completely blocked by both [3H]ethynylbicycloorthobenzoate and 12-ketoendrin (two GABA-gated chloride channel blockers) and AVM B1a at 1 to 1.5 microM. Also, 36Cl- influx induced by AVM B1a at 10 nM is suppressed by the two channel blockers. Thus, AVM B1a binds to two different sites in the GABA-gated chloride channel with dual effects, i.e., activating the channel on binding to the high-affinity site and blocking it on further binding to the low-affinity site.

Topics: Animals; Anthelmintics; Binding Sites; Bridged Bicyclo Compounds, Heterocyclic; Cells, Cultured; Cerebellum; Chloride Channels; Ion Channel Gating; Ivermectin; Rats; Receptors, GABA-A