Biphasic formation of inositol phosphates in opsonized zymosan-stimulated human neutrophils

Stimulation by serum-opsonized zymosan (SOZ) typically causes a biphasic rise in the cytosolic free Ca2+ concentration ([Ca2+]i) of human neutrophils. It consists of an initial slow Ca2+ release from internal pools lasting for 60 s, followed by a rapid but sustained influx of Ca2+. It was the aim of this study to elucidate the underlying mechanism of this atypical Ca2+ response. For this reason we analysed the production of inositol phosphates (InsPs) in myo-[3H]inositol labelled cells. Stimulation by SOZ within 10 s transiently elevated inositol trisphosphate (InsP3) by 1.50-fold. This response was followed by a second, more sustained 1.55-fold rise in InsP3 by 90 s. A similar, biphasic pattern of inositol tetrakisphosphate (InsP4) formation with 1.15- and 1.35-fold increases, respectively, was observed. The SOZ-induced formation of InsP3 was unaffected by the removal of extracellular Ca2+ by 1.4 mM EGTA. In contrast, the early accumulation of InsP4 was stronger and more prolonged and no second rise over the baseline level was seen in the absence of extracellular Ca2+. Under these conditions, the sudden exposure of Fura-2 AM loaded, SOZ-stimulated neutrophils to extracellular Ca2+ at a time point where InsP4 was the predominant InsP resulted in a marked increase in [Ca2+]i. Recalcification at a time point when InsP3 was the major InsP had no effect on [Ca2+]i. These findings suggest that in SOZ-stimulated neutrophils (1) the transient, first accumulation of InsP3 mediates the slow Ca2+ release from internal pools, and (2) the second, more pronounced formation of InsP4 triggers the Ca2+ influx.(ABSTRACT TRUNCATED AT 400 WORDS)