Effects of P-Bungarotoxin and Tityustoxin on Uptake and Release of Neurotransmitters

Information available on the molecular mechanism of neurotransmitter release is minimal. However, it has been demonstrated that purified preparations of brain synaptosomes are extremely useful for biochemical studies on the uptake and release of neurotransmitters (de Belleroche & Bradford, 1972; Wonnacott & Marchbanks, 1976). Moreover, electrophysiological and pharmacological studies have shown that 8-bungarotoxin and tityustoxin, polypeptides from snake and scorpion venoms respectively, block neurotransmission by acting primarily on the presynaptic nerve membrane and specifically affecting transmitter release (Chang et al., 1973; Warnick er al., 1976). 8-Bungarotoxin causes an initial increase in transmitter release followed by complete blockade, which is probably due to an inability to release the contents of synaptic vesicles (Chang et al., 1973); at higher concentrations it causes swelling of mitochondria in the axon terminals (Chen & Lee, 1970; Sen et al., 1976). In contrast, tityustoxin increases release due to depolarization of the nerve terminal, by opening or prolonging the closing of the sodium channels (its action is inhibited by tetrodotoxin); this is followed by complete inhibition of release, probably due to ultrastructural changes in the nerve (Warnick e f al., 1976; Diniz et al., 1974). In the present communication we describe the effects of highly purified preparations of 8-bungarotoxin and tityustoxin on the uptake and release of two putatative neurotransmitters, glutamate and y-aminobutyrate by using purified synaptosomes from brain cortex. 8-Bungarotoxin was purified from the venom of Bungarus multicinctus by chromatography on CM-Sephadex ((2-50) with a convex gradient (0.05-0.9~) of ammonium acetate; complete purification was achieved by preparative isoelectric focusing on Sephadex G-75. Its molecular weight, as determined by gel filtration, was 23000; n o detectable phospholipase activity was present (Lawrence et a/., 1974). After polyacrylamide-gel electrophoresis or isoelectric focusing of the purified toxin, under native conditions, a single protein species was obtained. On dodecyl sulphate/polyacrylamidegelelectrophoresis the toxin gave two polypeptidechains withmol.wts. 12000and 14000. Its toxicity was demonstrated by intraperitoneal injection into mice (LDSo 0.01 ,ug/g body wt .) and also by measuring its ability t o block neuromuscular transmission in frog nerve-sartorius muscle preparation (OS,~g/ml produced complete blockade in 6.5 h a t 20°C). Tityustoxin was purified from the venom of the Brazilian yellow scorpion (Tityus serrulatus) and characterized as previously described (Diniz et al., 1974). Its LD,, measured in mice was 0.1 pg/g body weight. Synaptosomes from rat brain cortex were purified by sucrose-density-gradient centrifugation and their respiration rates measured by the methods described by Bradford (1969). Suspensions of synaptosomes (2-4mg of protein/ml) were made in Krebs phosphate medium(or the latter solution without calcium and with0.2m~-EGTA)containing ['4C]glutamate ( 0 . 2 2 , ~ ~ ; sp. radioactivity 276Ci/mol) or y-amin~['~C]butyrate ( 0 . 1 3 , ~ ~ ; sp. radioactivity 224Ci/mol) and incubated with and without added toxins a t 37°C for periods up t o 75min. Uptake of radioactivity was measured by scintillation counting of the supernatants prepared from samples removed at appropriate intervals. (Figs. l a and 16); in addition, a filtration assay method (Wernicke et al., 1974) was used (Fig. l c ) . For studying release, a bulk synaptosome suspension was preloaded by incubation with ['4C]glutamate (2,YM; sp. radioactivity 125Ci/mol) or y-aminobutyrate ( 0 . 6 5 ~ ~ ; sp. radioactivity 224Ci/mol) a t 37°C for 20min followed by washing, resuspension and separation into samples. After incubation at 37°C for 15min, non-stimulated and stimulated (in the presence of 55 mM-K+) release was monitored by measuring the radioactivity in the supernatants as described above; toxin was added