thunberginol-a and Hemolysis

The role of the signal transmission pathway of thunberginol A (TA) in mast cell degranulation was examined using rat peritoneal mast cells. First of all, we investigated the cellular distribution of TA using fluorescent microscopy. This indicated that TA is immediately incorporated into cells and distributes in cytosol around the nucleus. We then investigated the effect of TA on mast cell protein tyrosine phosphorylation, which is part of the signal transduction cascade for degranulation. TA non-specifically inhibited the tyrosine phosphorylation induced by compound 48/80 (Co. 48/80), at 10 to 100 microM, and orthovanadate/hydrogen peroxide at more than 50 microM in a dose-dependent manner. As far as the intracellular Ca2+ change in fluo-3-loaded cells was concerned, TA (10 microM) suppressed the rise in Ca2+ induced by antigens, ionomycin and thapsigargin, while TA did not suppress the rise induced by Co. 48/80. This evidence suggests that TA mainly inhibits extracellular Ca2+ influx, but TA does not act on the intracellular Ca2+ mobilization from the endoplasmic reticulum. We also investigated the influence of TA on the cytoskeleton and membrane changes using mast cells and erythrocytes. TA (10 microM) inhibited the cytoskeletal assembly formation in dicyanovinyl julolidin-loaded mast cells induced by Co. 48/80. Moreover, TA suppressed the hypotonic hemolysis of erythrocytes, from 3 to 1000 microM, in a dose-dependent manner. These results suggest that inhibition of protein tyrosine phosphorylation, extracellular Ca2+ influx and cytoskeletal assembly formation, and membrane stabilization are involved in the inhibitory effect of TA in mast cell degranulation.

Topics: Animals; Calcium; Cattle; Cell Degranulation; Cell Membrane; Coumarins; Cytoskeleton; Erythrocytes; Hemolysis; In Vitro Techniques; Isocoumarins; Magnoliopsida; Mast Cells; Phosphorylation; Plant Extracts; Rats; Signal Transduction; Tyrosine