Previous studies indicated that in Dictyostelium amoebae signal transmission from cell surface cyclic AMP receptors to intracellular events concerned with chemotaxis involves inositol 1,4,5-trisphosphate (1,4,5-IP3): micromolar amounts of 1,4,5-IP3 or Ca2+ were found to mimic the effects of chemoattractants and 1,4,5-IP3 triggered release of Ca2+ from non-mitochondrial stores. Here we report a more direct test of the involvement of inositol phosphates. Intact amoebae were labelled with high specific activity [3H]inositol, then stimulated with the chemoattractant cyclic AMP at 22 degrees C and rapidly assayed for phosphorylated inositol products formed. Labelled IP3 was found to accumulate transiently after a pulse of 50 nM-cyclic AMP, with a peak at 15 s after stimulation and some (inconclusive) evidence for a more rapidly formed peak at 5 s or less. Inositol bisphosphate (IP2) showed a transient shallow peak at about 20 s. When the events of signal transmission were slowed down by incubation at 4 degrees C, the rapidly formed IP3 peak could be consistently seen at 5 s after stimulation and the second peak at 25–30 s. Further resolution of the IP3 peaks indicated the presence of IP4, which represented a major fraction of the peak accumulated at 5 s (4 degrees C). The results provide an important link in the chain of evidence connecting the cell surface cyclic AMP receptors, via IP3, with the Ca2+-activated events of chemotaxis.

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