Hemolysis and hyperpotassemia in rat induced by the thermostable direct hemolysin of Vibrio parahaemolyticus.

Thermostable direct hemolysin purified from cultured filtrates of a pathogenic strain of Vibrio parahaemolyticus (1) has been reported to have potent cardiotoxic and hemolytic activities (2, 3). Intravenous injection of small doses (10 to 20 ug/kg) of hemolysin killed the rats within several hr. Electrocardiograms of rats after administration (i.v.) of hemolysin showed significant changes due to depression of intra-atrial and intra-ventricular conduction of electrical activation (4). Moreover, hemolysin was toxic to cultured myocardial cells of rats and mice, and the beating of cultured cells was stopped by the addition of low concen trations of hemolysin to the medium (5). Hemolytic activity of hemolysin was found in the erythrocytes of rat, dog, mouse, monkey, and man (6) as listed in decreasing order of activity. It is conceivable that both toxicity to the heart and to eryrhrocytes originates from the binding of hemolysin to a receptor which has been considered to be the gangliosides (7, 8) on the membrane of these cells and that cardiac arrest was caused by the depolarization through direct action of hemolysin on myocardial cells (4). It has been known that hemolysin caused potassium leakage from erythrocytes of mice prior to hemolysis (9). We thus investigated the relation between hemolysis and potassium leakage in the erythrocytes of rats by electron microscopy. Purified hemolysin was kindly supplied by K. Fujiwara (Tsukuba University), T. Miwa tani and Y. Takeda (Osaka University). Wistar male rats weighing 300-400 g were anesthetized with urethane (1 .2 g/kg i.p.). The femoral vein was cannulated with a polyethylene tube for intravenous application of hemolysin and blood sampling. Assay of hemolysis was performed by determining the absorbance of plasma at 540 nm with a spectrophotometer (Hitachi Perkin-Elmer 139). The relative ratio of the absorbance of plasma to that of totally hemolyzed solution was expressed as the percentage of hemolysis. Assay of potassium levels in the same plasma was performed by atomic absorption spectrometry (Perkin-Elmer 403). In the in vitro experiment, fresh rat blood was washed three times with saline and then suspended in saline. Hemolysin at a final concentration of 0.2 /cg/ml was added and the suspension incubated with gentle shaking at 37°C. Assays of hemolysis and potassium levels were performed by the same methods as in the in vivo experiment. Morphological changes of erythrocytes were observed by scanning electron microscopy and the carbon replica technique. Samples of erythrocytes from the suspension were fixed