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Studies on the mechanism of action of tityustoxin
4th International Symposium on Animal, Plant and Microbial Toxins
93
Fitm x-MmA, L., CuNaA-Maio, J. R., GomEz, M. V., TAiu, W. L., MBA, T. A. and FuTvxo-Nm, H. A., Department of Physiology and Biophysics, Institute of Biological Sciences, Box 2486, Belo Horizonte, Brazil . STUDIES ON THE MECHANISM OF ACTION OF TITYUSTOXIN The contraction of the isolated rat ileum produced by tityustoxin (Ts= was explained by the release of acetylcholine (ACh) and probably substance P (CUNHA-mELo et al., Toxicon 11, 81, 1973), whereas the relaxation of the atropinized duodenum was due, at least in part,to the release of catecholamines (CALmrm and FREmE-MATA, Ciénc. Cult . S Paulo 24, suppl., 287,1972 ; CuNHA-MEro et al. 1973). To study further the mechanism of action of TsTX, a new series of experiments were conducted. Tetrodotoxin ('ITX) prevents the contraction of the isolated guinea-pig and rat ilea produced by TsTX (HENmQuEs et ai., CMnc. Cult. S Paulo 25, supl ., 150, 1973 ; Fam$-MAiA et al., Clênc. Cult., S Paulo 25, supl., 279, 1973). In the present experiments using the rat ileum, TsTX (5 jig per ml) was without effect when added to the organ bath containing Tyrode's solution and TTX (2 x 10-' M). However, after the Tyrode's solution (plus TTX) was washed out and replaced by a solution without TTX, a strong contraction of the preparation was recorded. Addition of TTX to the bath brought the muscle back to its original tone . These experiments seem to indicate that TTX links weakly to its receptor in the nervemembrane and can be easily removed by washing, whereas the binding of TsTX is stronger and resists washing. This hypothesis was tested by experiments in which slices of rat and guinea-pig ilea were incubated during 30 min with TsTX . The amount of ACh (nmoles per g wet tissue) released spontaneously (RS) or by the action of TsIX (without or with addition of TT)Q was bio-assayed (for the technique see Gorrnz et al., J. Neurochern. 20, 1051, 1973). The results (mean f S.D.) were thefollowing: in the rat, (1) RS : 5-9 f 1-3 ; (2) TTX: 5-2 f 2-1 ; (3) TsTX : 10-1 :L 1-9 ; (4) TsTX -h TTX: 5-5 f 2-2. In the guinea pig, (1) RS : 0-8 f 0-1 ; (2) TTX : 0-8 f 0-1 ; (3) TsTX : 9-8 ± 2-0; (4) TsTX -h TTX: 1-0 f 0-4. After the first incubation the slices were washed 3 times with the incubation medium and re-incubated (30 min) without further addition of TsTX or TTX. The results were : in the rat, (1) RS : 5-7 f 0-9; (2) slices previously incubated with TsTX and TTX : 9-4 ± 1-2, and in the guinea-pig, (1) RS : 3-2 ± 0-7 ; (2) slices previously incubated with TsTX and TTX: 13-3 f 0-9. Based on these results, we postulate that TsTX acts in at least two points of the nerve membrane, one of which is presumably the sodium channel. A comparative study of the effects of acetylcholine (ACH, 1 x 10 -7 M) and TsTX (3 hg per ml) on the rat and guinea-pig ileum was also conducted. Rat ileum was 7-8 times less sensitive to ACh than the guinea-pig ileum; however, the rat ileum was 2-1 times more sensitive to TsTX than the guinea-pig ileum. Since the above incubation experiments showed that TsTX releases about 2 times more ACh from the guinea-pig than from the rat ileum it is probable that TsTX releases other substances, besides ACh, in the rat. The addition of TsTX to the rat duodenum, immersed in a Tyrode's solution containing atropine (1-5 x 10-7 M) and guanethidine (2 x 10'' M) produced a small relaxation followed by contraction. Substance P (1-2 units per ml) and ATP (2 x 10-4 M) produced similar effects. It is therefore suggested that TsTX produces part of its effectsin the gut by release of substance P and/or ATP. To study the possible action of TsTX on the small and large granular vesicles of the Auerbach plexus, two segments of the rat ileum were immersed in an isolated bath containing Jalon's solution and toxin (15 pg perml) was added to one of these baths (9 experiments) . The granular and clear vesicles were counted in 1300 transversely cut axons and the number of vesicles per 100 ltms of axonal area was estimated. In the control group, 1571 granular or dense vesicles (DV) and 1471 clearvesicles (CV) were found per loo pm' of axonal area, whereas in the treated group the results were : 1228 DV and 1874 CV . The difference between the distributions of the vesicles was statistically significant (X' test), indicating that the toxin reduced the number of the granular vesicles and increased the number of the clearvesicles. Therefore, it seems likely that the action of TsTX on the axon resulted in the release of catecholamines and probably substance P from the granular vesicles. Finally, TsTX (1-3 hg perml) andepinephrine (30-50 pg per ml) added to a bath containing rat or cat spleen strips produced contraction of the preparation. The second or third dose of TsTX failed to produce an effect (tachyphylaxis), whereas the effect of epinephrine was potentiated . The mechanism of epinephrine potentiation is under investigation. FuNATsu, G., YosxiTrAxe, S., MIYAUCIU, S . IsmGuxo, M. and FuNATsu, M., Laboratory of Biochem., Faculty of Agriculture, Kyushu University, Fukuoka, Japan. STRUCTURE AND FUNCTION OF RICIN D Ricin D, a major toxicprotein in castor bean (F. communis), consists of two differentpolypeptide chainsIle-chain and Ala-chain-linked with a single disulfide bond . These two polypeptide chains have been separated by DEAF-cellulose column chromatography after reduction followed by carboxymethylation and the toxicitites of ROCM-Ile and ROCM-Ala chains have been found to be about 1/30 and 1/300 of that of ricin D, respectively. TOXICON 1975 Vol. 13
93
Fitm x-MmA, L., CuNaA-Maio, J. R., GomEz, M. V., TAiu, W. L., MBA, T. A. and FuTvxo-Nm, H. A., Department of Physiology and Biophysics, Institute of Biological Sciences, Box 2486, Belo Horizonte, Brazil . STUDIES ON THE MECHANISM OF ACTION OF TITYUSTOXIN The contraction of the isolated rat ileum produced by tityustoxin (Ts= was explained by the release of acetylcholine (ACh) and probably substance P (CUNHA-mELo et al., Toxicon 11, 81, 1973), whereas the relaxation of the atropinized duodenum was due, at least in part,to the release of catecholamines (CALmrm and FREmE-MATA, Ciénc. Cult . S Paulo 24, suppl., 287,1972 ; CuNHA-MEro et al. 1973). To study further the mechanism of action of TsTX, a new series of experiments were conducted. Tetrodotoxin ('ITX) prevents the contraction of the isolated guinea-pig and rat ilea produced by TsTX (HENmQuEs et ai., CMnc. Cult. S Paulo 25, supl ., 150, 1973 ; Fam$-MAiA et al., Clênc. Cult., S Paulo 25, supl., 279, 1973). In the present experiments using the rat ileum, TsTX (5 jig per ml) was without effect when added to the organ bath containing Tyrode's solution and TTX (2 x 10-' M). However, after the Tyrode's solution (plus TTX) was washed out and replaced by a solution without TTX, a strong contraction of the preparation was recorded. Addition of TTX to the bath brought the muscle back to its original tone . These experiments seem to indicate that TTX links weakly to its receptor in the nervemembrane and can be easily removed by washing, whereas the binding of TsTX is stronger and resists washing. This hypothesis was tested by experiments in which slices of rat and guinea-pig ilea were incubated during 30 min with TsTX . The amount of ACh (nmoles per g wet tissue) released spontaneously (RS) or by the action of TsIX (without or with addition of TT)Q was bio-assayed (for the technique see Gorrnz et al., J. Neurochern. 20, 1051, 1973). The results (mean f S.D.) were thefollowing: in the rat, (1) RS : 5-9 f 1-3 ; (2) TTX: 5-2 f 2-1 ; (3) TsTX : 10-1 :L 1-9 ; (4) TsTX -h TTX: 5-5 f 2-2. In the guinea pig, (1) RS : 0-8 f 0-1 ; (2) TTX : 0-8 f 0-1 ; (3) TsTX : 9-8 ± 2-0; (4) TsTX -h TTX: 1-0 f 0-4. After the first incubation the slices were washed 3 times with the incubation medium and re-incubated (30 min) without further addition of TsTX or TTX. The results were : in the rat, (1) RS : 5-7 f 0-9; (2) slices previously incubated with TsTX and TTX : 9-4 ± 1-2, and in the guinea-pig, (1) RS : 3-2 ± 0-7 ; (2) slices previously incubated with TsTX and TTX: 13-3 f 0-9. Based on these results, we postulate that TsTX acts in at least two points of the nerve membrane, one of which is presumably the sodium channel. A comparative study of the effects of acetylcholine (ACH, 1 x 10 -7 M) and TsTX (3 hg per ml) on the rat and guinea-pig ileum was also conducted. Rat ileum was 7-8 times less sensitive to ACh than the guinea-pig ileum; however, the rat ileum was 2-1 times more sensitive to TsTX than the guinea-pig ileum. Since the above incubation experiments showed that TsTX releases about 2 times more ACh from the guinea-pig than from the rat ileum it is probable that TsTX releases other substances, besides ACh, in the rat. The addition of TsTX to the rat duodenum, immersed in a Tyrode's solution containing atropine (1-5 x 10-7 M) and guanethidine (2 x 10'' M) produced a small relaxation followed by contraction. Substance P (1-2 units per ml) and ATP (2 x 10-4 M) produced similar effects. It is therefore suggested that TsTX produces part of its effectsin the gut by release of substance P and/or ATP. To study the possible action of TsTX on the small and large granular vesicles of the Auerbach plexus, two segments of the rat ileum were immersed in an isolated bath containing Jalon's solution and toxin (15 pg perml) was added to one of these baths (9 experiments) . The granular and clear vesicles were counted in 1300 transversely cut axons and the number of vesicles per 100 ltms of axonal area was estimated. In the control group, 1571 granular or dense vesicles (DV) and 1471 clearvesicles (CV) were found per loo pm' of axonal area, whereas in the treated group the results were : 1228 DV and 1874 CV . The difference between the distributions of the vesicles was statistically significant (X' test), indicating that the toxin reduced the number of the granular vesicles and increased the number of the clearvesicles. Therefore, it seems likely that the action of TsTX on the axon resulted in the release of catecholamines and probably substance P from the granular vesicles. Finally, TsTX (1-3 hg perml) andepinephrine (30-50 pg per ml) added to a bath containing rat or cat spleen strips produced contraction of the preparation. The second or third dose of TsTX failed to produce an effect (tachyphylaxis), whereas the effect of epinephrine was potentiated . The mechanism of epinephrine potentiation is under investigation. FuNATsu, G., YosxiTrAxe, S., MIYAUCIU, S . IsmGuxo, M. and FuNATsu, M., Laboratory of Biochem., Faculty of Agriculture, Kyushu University, Fukuoka, Japan. STRUCTURE AND FUNCTION OF RICIN D Ricin D, a major toxicprotein in castor bean (F. communis), consists of two differentpolypeptide chainsIle-chain and Ala-chain-linked with a single disulfide bond . These two polypeptide chains have been separated by DEAF-cellulose column chromatography after reduction followed by carboxymethylation and the toxicitites of ROCM-Ile and ROCM-Ala chains have been found to be about 1/30 and 1/300 of that of ricin D, respectively. TOXICON 1975 Vol. 13