Studies on the Interaction of Surangin B with Insect Mitochondria, Insect Synaptosomes, and Rat Cortical Neurones in Primary Culture

The effects of the insecticidal coumarin surangin B on mitochondrial function, transmitter release from synaptosomes, and whole-body ATP levels have been examined in insects. The interaction of this compound with rat cortical neurones in primary culture was also investigated using the whole-cell patch-clamp technique. Surangin B (1 μM) blocked state 4 (basal) respiration in cricket thoracic muscle mitochondria respiring on succinate. No inhibition of basal oxygen consumption was detected when glutamate or proline was used as substrate; however, surangin B increased state 4 respiration in the presence of a pyruvate:malate substrate combination. State 3 (ADP-driven) respiration using glutamate, proline, or the pyruvate:malate combination was sensitive to inhibition by surangin B (1 μM). In blowfly flight muscle mitochondria, concentration-response experiments with proline as the substrate indicated a state 3 respiration IC50of 40 nM for surangin B. Unlike rotenone, surangin B was a very effective blocker of ADP-driven respiration and uncoupler [carbonyl cyanide chlorophenyl-hydrazone (CCCP)]-stimulated state 3 respiration in blowfly mitochondria respiring on succinate. In contrast to oligomycin, surangin B blocked state 4 respiration and its inhibitory effect on state 3 respiration was not reversed by CCCP. Our experiments on isolated components of the electron transport chain revealed minimal effects of surangin B on sites I and III and extensive inhibition of coupling site II. Whole-insect ATP levels were reduced in crickets dosed topically with 6 μg of surangin B. Cricket synaptosomes showed a marked stimulation of neurotransmitter release when exposed to surangin B (EC50= 3 μM). This effect was not blocked by TTX but was enhanced in Ca2+-free saline. Miniature EPSC frequency was dramatically increased in rat cortical neurones within 2–4 min of application of surangin B (1 μM). Responses were more pronounced in calcium-free saline with 500 μM Cd2+. Surangin B-induced increases in miniature EPSC frequency were associated with an irreversible block of spontaneous inhibitory and excitatory synaptic currents. This investigation suggests that blockade of a complex III component, depletion of ATP stores, and massive presynaptic transmitter release resulting in irreversible loss of postsynaptic sensitivity are likely to be important mechanisms underlying the development of paralysis in insects treated with surangin B.