PHARMACOLOGICAL IDENTIFICATION OF SODIUM CHANNEL TOXINS IN CULTURED PURPLE CLAM, HIATULA DIPHOS

PHARMACOLOGICAL IDENTIFICATION OF SODIUM CHANNEL TOXINS IN CULTURED PURPLE CLAM, HIATULA DIPHOS

An outbreak of poisoning from ingestion of purple clam (Hiatula diphos, Linnaeus 1771) occurred in Southern Taiwan in early January, 1986. Nearly half a hundred persons suffered from numbness of mouth and muscle paralysis about one hour after ingestion of the purple clam meat obtained from a brackish fish-culture pond. Three died before effective treatment could be offered. Although the symptoms indicated the possibility of saxitoxin-like chemicals in the purple clam as the cause of intoxication, chemical identification of the toxin was delayed because of the lack of authentic saxitoxin. No definite presence of dinoflagellates (Gonyaulax) could be proved either. Pharmacological identification of saxitoxin-like toxins was therefore devised by using the characteristic specific activity of this type of toxins on the Na-channel (Kao, 1966, 1972; Narahashi, 1972) and the specific interaction with another class of Na-channel toxins, crotamine and veratridine (Chang and Tseng, 1978; Chang et al., 1983; Hong and Chang, 1983). Tetrodotoxin was used as a reference toxin because it acts just like saxitoxin with almost the same potency except in some particular species (puffer fish and newt) and is commercially available. Isolated mouse phrenic nerve-diaphragm preparations were prepared for recording of twitch responses to both indirect and direct stimulations and for intracellular recording of transmembrane potentials as previously described (Chang et al. 1983; Hong and Chang, 1983). The organ bath contained 20 ml oxygenated Tryode solution at 37°C. Ten grams of the shell-removed whole purple clam, which was highly toxic to mice, were extracted by homogenization with 25 ml 0.1% acetic acid and heated on boiling water for 10 min to denature protein. The filtrate was made to 50 ml by adding 0.1% acteic acid. The final concentration of the clam extract added to the preparations thereafter refers to the fresh weight of purple clam. The twitch responses to indirect and to direct stimulations were completely blocked by clam extract in less than 4 min when the concentration was 0.5 mg/ml. At 0.1 mg/ml of purple clam, the indirect twitch response was depressed by more than 90% in 5 min whereas the direct response was reduced by about 40%, indicating the preferential susceptibility of the nerve to the toxin in the extract.