STUDIES ON THE INTERACTION OF SURANGIN-B WITH INSECT MITOCHONDRIA, INSECT SYNAPTOSOMES, AND RAT CORTICAL-NEURONS IN PRIMARY CULTURE

The effects of the insecticidal coumarin surangin B on mitochondrial f unction, transmitter release from synaptosomes, and whole-body ATP lev els have been examined in insects. The interaction of this compound wi th rat cortical neurones in primary culture was also investigated usin g the whole-cell patch-clamp technique. Surangin B (1 mu M) blocked st ate 4 (basal) respiration in cricket thoracic muscle mitochondria resp iring on succinate. No inhibition of basal oxygen consumption was dete cted when glutamate or proline was used as substrate; however, surangi n B increased state 4 respiration in the presence of a pyruvate:malate substrate combination. State 3 (ADP-driven) respiration using glutama te, proline, or the pyruvate:malate combination was sensitive to inhib ition by surangin B (1 mu M). In blowfly flight muscle mitochondria, c oncentration-response experiments with proline as the substrate indica ted a state 3 respiration IC50 of 40 nM for surangin B. Unlike rotenon e, surangin B was a very effective blocker of ADP-driven respiration a nd uncoupler [carbonyl cyanide chlorophenyl-hydrazone (CCCP)]-stimulat ed state 3 respiration in blowfly mitochondria respiring on succinate. In contrast to oligomycin, surangin B blocked state 4 respiration and its inhibitory effect on stale 3 respiration was not reversed by CCCP . Our experiments on isolated components of the electron transport cha in revealed minimal effects of surangin B on sites I and III and exten sive inhibition of coupling site II. Whole-insect ATP levels were redu ced in crickets dosed topically with 6 mu g of surangin B. Cricket syn aptosomes showed a marked stimulation of neurotransmitter release when exposed to surangin B (EC50 = 3 mu M). This effect was not blocked by TTX but was enhanced in Ca2+-free saline. Miniature EPSC frequency wa s dramatically increased in rat cortical neurones within 2-4 min of ap plication of surangin B (1 mu M). Responses were more pronounced in ca lcium-free saline with 500 mu M Cd2+. Surangin B-induced increases in miniature EPSC frequency were associated with an irreversible block of spontaneous inhibitory and excitatory synaptic currents. This investi gation 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 importa nt mechanisms underlying the development of paralysis in insects treat ed with surangin B. (C) 1998 Academic Press.