EFFECT OF SUB-SKINNING CONCENTRATIONS OF SAPONIN ON INTRACELLULAR CA2+ AND PLASMA-MEMBRANE FLUIDITY IN CULTURED CARDIAC-CELLS

To determine the underlying mechanisms of the positive inotropic effect of sub-skinning concentrations of saponin, we studied changes in the intracellular Ca2+ ([Ca2+]i) and plasma membrane fluidity after exposure to digitonin (a representative saponin) in cultured cardiac cells. [Ca+]i was measured by use of the fluorescent calcium indicator Calcium Green-1. The membrane fluidity was evaluated by measuring the diffusion coefficient using the method of fluorescence recovery after photobleaching. 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate was used as the fluorescent probe. Digitonin at a sub-skinning concentration (0.1 to 1 muM) produced an increase in cell motion and an augmentation of [Ca2+]i. Membrane fluidity which is evaluated by the diffusion coefficient (from 0.34 . 10(-8) to 0.28 . 10(-8) cm2/s; P < 0.05), decreased in the presence of 0.2 muM digitonin while the cell maintained an augmented motion and an increased [Ca2+]i. The skinning concentration of digitonin (5 muM) produced a rapid contracture with a marked increase in [Ca2+]i. The membrane fluidity was further reduced (diffusion coefficient: 0.24 . 10(-8) cm2/s; P < 0.001). These results suggest that saponin at the sub-skinning concentration also causes holes in the plasma membrane by interaction with cholesterol, as was shown with the skinning concentration, and it increases [Ca2+]i, which thereby induces a positive inotropic effect.