Genistein inhibits lysosomal enzyme release by suppressing Ca2+influx in HL-60 granulocytes

Abstract

The tyrosine kinase inhibitor genistein (5–200 μM) suppressed Ca2+-dependent fMLP (1 μM) and ATP (100 μM)-induced release of the lysosomal enzyme, β–glucuronidase from neutrophil-like HL–60 granulocytes. Agonist-induced Ca2+mobilization resulted from the release of intracellular Ca2+stores and the influx of extracellular Ca2+. Genistein (200 μM) suppressed fMLP (1 μM) and ATP (100 μM)-induced Ca2+mobilization, by 30–40%. Ca2+release from intracellular stores was unaffected by genistein, however, genistein abolished agonist-induced Ca2+(Mn2+) influx. Consistent with these findings, genistein (200 μM) or removal of extracellular Ca2+(EGTA 1 mM), inhibited Ca2+-dependent agonist-induced β–glucuronidase release by similar extents (about 50%). In the absence of extracellular Ca2+, genistein had a small additional inhibitory effect on fMLP and ATP-induced β–glucuronidase release, suggesting an additional inhibitory site of action. Genistein also abolished store-operated (thapsigargin-induced) Ca2+(Mn2+) influx. Neither fMLP nor ATP increased the rate of Mn2+influx induced by thapsigargin (0.5 μM). These data indicate that agonist-induced Ca2+influx and store-operated Ca2+influx occur via the same genistein-sensitive pathway. Activation of this pathway supports approximately 50% of lysosomal enzyme release induced by either fMLP or ATP from HL–60 granulocytes.