BIPHASIC FORMATION OF INOSITOL PHOSPHATES IN OPSONIZED ZYMOSAN-STIMULATED HUMAN NEUTROPHILS

Stimulation by serum-opsonized zymosan (SOZ) typically causes a biphas ic 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 C a2+. It was the aim of this study to elucidate the underlying mechanis m of this atypical Ca2+ response. For this reason we analysed the prod uction of inositol phosphates (InsPs) in myo-[H-3]inositol labelled ce lls. Stimulation by SOZ within 10 a transiently elevated inositol tris phosphate (InsP(3)) by 1.50-fold. This response was followed by a seco nd, more sustained 1.55-foId rise in InsP(3) by 90 s. A similar, bipha sic pattern of inositol tetrakisphosphate (InsP(4)) formation with 1.1 5- and 1.35-fold increases, respectively, was observed. The SOZ-induce d formation of InsP(3) was unaffected by the removal of extracellular Ca2+ by 1.4 mM EGTA. In contrast, the early accumulation of InsP(4) wa s stronger and more prolonged and no second rise over the baseline lev el was seen in the absence of extracellular Ca2+. Under these conditio ns, the sudden exposure of Fura-2 AM loaded, SOZ-stimulated neutrophil s to extracellular Ca2+ at a time point where InsP(4) was the predomin ant InsP resulted in a marked increase in [Ca2+](i). Recalcification a t a time point when InsP(3) was the major InsP had no effect on [Ca2+] (i). These findings suggest that in SOZ-stimulated neutrophils (1) the transient, first accumulation of InsP(3) mediates the slow Ca2+ relea se from internal pools, and (2) the second, more pronounced formation of InsP(4) triggers the Ca2+ influx. It is hypothesised that the unusu al second messenger response to SOZ stimulation may reflect the activa tion of several receptor types coupled to parallel signalling pathways .