Cyclic GMP is rapidly formed a few seconds after binding of chemotactic signailing molecules to specific receptors on the cell surface of Dictyostelium amoebae. This phenomenon could be mimicked by addition of a pulse of Ca2+ to ~~eabili~d amoebae. The con~ntration of Ca 2+ for haIf-maximal response was 60 FM. Other ions (K+, Na+, Mg*+ or Mn2+) had no effect. A pulse of 5 PM IP, produced a cyclic GMP response of similar magnitude but IP, elicited no response. The data provide strong support for the hypothesis that cell surface receptor binding induces cyclic GMP formation by liberating Caz+ from internal stores.(Dictyostelium) cyclic GMP Chemotaxis Ca*+ INTRODUCTlONAmoebae of the cellular slime mould, Dictyostelium discoideum, respond chemotactically to signalling molecules such as folate during the foodseeking phase [l], and CAMP during the subsequent starvation phase [2] when they aggregate to form motile multicellular organisms [3]. These chemoattractants bind to separate cell surface receptors, yet appear to induce a group of internal responses common to both types of stimulus 141.Following a pulse of exogenous CAMP or folate, two of the initial responses identified are the polymerisation of actin associated with the cytoskeleton, peaking 3-5 s after stimulation (5lo], and the transient formation of cyclic GMP, which peaks 9-12 s after CAMP or folate binding , and is rapidly degraded by a specific cGMP phosphodiesterase [ 11,121. This cGMP formation has been implicated in chemotactic movement by studies which indicate its rapid accumulation following pulsing of several species of cellular slime mould (including D. ~is~~ide~rn, D. ~~~~e~rn~ and Po~ys~~o~dyfium violaceum) with their specific chemoattractants Abbreviations: IP,, inositol 1,4,5trisphosphate; IP2, inositol 1,4-bisphosphate [ 13,141. It is aIso strongly implicated in chemotaxis by the finding that streamer F mutants of D. discoideum (which lack the specific cCiMP phosphodiesterase and hence produce a large peak of cCMP in response to CAMP-receptor binding that persists for at least 1 min at elevated levels) show a chemotactic movement period that is greatly lengthened compared to the parental wild type f10, 15,16].Recently, studies in this laboratory have produced indirect evidence for the involvement of Ca'+ in chemotaxis in D. discoideum [17-191 and have revealed that IP3, when added to permeabilised amoebae, can mimic the action of chemoattractants on normal intact amoebae in inducing cGMP formation [ZO]. As IP3 in mammalian systems acts via release of intracellular Ca'+ 1211, it seemed worthwhile to study the direct effect of pulses of Ca2+ on the formation of cGMP in permeabilised amoebae. MATERIALS AND METHODS MaterialsIP3 (potassium salt) and IPz (potassium salt) were obtained from Amersham. Saponin and EGTA were obtained from Sigma.