Insulin induced the formation of ruffling membranes in cultured KB cells (a cell strain derived from human epidermoid carcinoma) within 1-2 min after its addition. The ruffled regions were stained strongly with antibody to actin but not that to tubulin . Pretreatment of KB cells with agents disrupting microfilaments (cytochalasins), but not with those disrupting microtubules (colcemid, nocodazole, and colchicine) completely inhibited the formation of ruffling membranes . Pretreatment of KB cells with dibutyryl cyclic AMP, but not with dibutyryl cyclic GMP, also inhibited the formation of ruffling membranes . Addition of insulin enhanced Na+-dependent uptake of a system A amino acid («-amino isobutyric acid ; AIB) by the cells within 5 min after the addition, and decreased the cyclic AMP content of the cells. Treatments that inhibited insulin-induced formation of ruffling membranes of KB cells also inhibited insulin-induced enhancement of their AIB uptake . From these observations, the mechanism of insulin-induced formation of ruffling membranes and its close correlation with AIB transport are discussed .Various kinds of nonmuscle cells, such as fibroblasts (1,21,24,58), macrophages (39, 50), osteoclasts (64), glial cells (5), aortic endothelial cells(2), megakaryocytes~(35), and epithelial cells (8,23), show ruffling movements (see also reviews in references 58, 62). Ruffles are rapid movements with irregular fluctuation of protrusion and withdrawal of the margins of the cell surface. The ruffled areas ofthese nonmuscle cells are known to contain actin, a-actinin, fimbrin, and filamin (see review in reference 62). In areas of ruffling, networks of microfilaments composed ofactin are prominent, but myosin, tropomyosin, and microtubules are depleted or absent (see review in reference 62). Membrane rufflings are believed to be closely concerned with locomotive movements (58), although the detailed relationship is still unknown (1, 8, 21). Membrane rufflings are also believed to be concerned with phagocytosis (12,39,40,50,64) and cytoplasmic spreading on the glass substratum (23, 57) of various kinds ofnonmuscle cells. But it is still obscure whether membrane rufflings perform other cellular functions and whether some hormonal factors affect their formation and activity .While culturing KB cells (a cell strain derived from human epidermoid carcinoma) as sparse monolayers in flasks, we unexpectedly found that almost all ICB cells formed ruffling THE JOURNAL OF CELL BIOLOGY~VOLUME 98 MARCH 1984 807-809 ® The Rockefeller University Press~0021-9525/84/98/0801/09 $1 .00 membranes immediately after the culture medium was replaced with fresh medium. Thus, we examined what components) of the fresh medium was necessary for the formation of ruffling membranes and found that insulin was especially effective. Next we examined what physiological functions) the insulin showed concomitant with the formation ofruffling membranes and found that increased uptake of an amino acid by the ICB cells induced by insulin was closely...

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Intravenous injection of the thermostable direct hemolysin (lethal toxin) produced by Vibrio parahaemolyticus caused rapid death of rats. Studies by electroencephalography and electrocardiography showed that after intravenous injection of the toxin the electroencephalogram remained normal for quite a long time after the heart of the animals had stopped beating. Depression of intraatrial and intraventricular conduction of electrical activation, including atrioventricular block, was observed in electrocardiograms of animals injected with the toxin. The toxin was also found to be toxic to cultured mouse heart cells. When it was added to the medium, the beating rhythm of the cultured heart cells increased temporarily and then soon stopped abruptly. The effect of the toxin on cultured mouse heart cells was blocked by preincubation of the toxin with a ganglioside mixture. From these results it is concluded that the thermostable direct hemolysin (lethal toxin) has cardiotoxic activity, and thus administration of the toxin causes rapid death of animals.

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