Cyclic AMP stimulates accumulation of inositol trisphosphate in Dictyostelium

Abstract

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.