Chemoattractant and guanosine 5'-[gamma-thio]triphosphate induce the accumulation of inositol 1,4,5-trisphosphate in Dictyostelium cells that are labelled with [3H]inositol by electroporation.
The analysis of the inositol cycle in Dictyostelium discoideum cells is complicated by the limited uptake of [3H]inositol (0.2% of the applied radioactivity in 6 h), and by the conversion of [3H]inositol into water-soluble inositol metabolites that are eluted near the position of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] on anion-exchange h.p.l.c. columns. The uptake was improved to 2.5% by electroporation of cells in the presence of [3H]inositol; electroporation was optimal at two 210 microseconds pulses of 7 kV. Cells remained viable and responsive to chemotactic signals after electroporation. The intracellular [3H]inositol was rapidly metabolized to phosphatidylinositol and more slowly to phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. More than 85% of the radioactivity in the water-soluble extract that was eluted on Dowex columns as Ins(1,4,5)P3 did not co-elute with authentic [32P]Ins(1,4,5)P3 on h.p.l.c. columns. Chromatography of the extract by ion-pair reversed-phase h.p.l.c. provided a good separation of the polar inositol polyphosphates. Cellular [3H]Ins(1,4,5)P3 was identified by (a) co-elution with authentic [32P]Ins(1,4,5)P3 and (b) degradation by a partially purified Ins(1,4,5)P3 5-phosphatase from rat brain. The chemoattractant cyclic AMP and the non-hydrolysable analogue guanosine 5'-[gamma-thio]triphosphate induced a transient accumulation of radioactivity in Ins(1,4,5)P3; we did not detect radioactivity in inositol 1,3,4-trisphosphate or inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In vitro, Ins(1,4,5)P3 was metabolized to inositol 1,4- and 4,5-bisphosphate, but not to Ins(1,3,4,5)P4 or another tetrakisphosphate isomer. We conclude that Dictyostelium has a receptor- and G-protein-stimulated inositol cycle which is basically identical with that in mammalian cells, but the metabolism of Ins(1,4,5)P3 is probably different.
Van Haastert
P J
PJ
Cell Biology and Genetics Unit, University of Leiden, The Netherlands.
De Vries
M J
MJ
Penning
L C
LC
Roovers
E
E
Van der Kaay
J
J
Erneux
C
C
Van Lookeren Campagne
M M
MM
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D000242:Cyclic AMP / Q000494:pharmacology*; D004023:Dictyostelium / Q000187:drug effects / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D007294:Inositol / Q000378:metabolism*; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000032:analysis / Q000378:metabolism*; D007700:Kinetics; D010743:Phospholipids / Q000378:metabolism; D051381:Rats; D013268:Stimulation, Chemical; D013403:Sugar Phosphates / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology*
D007295:Inositol Phosphates; D010743:Phospholipids; D013403:Sugar Phosphates; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D007294:Inositol; D015544:Inositol 1,4,5-Trisphosphate; D006160:Guanosine Triphosphate; D000242:Cyclic AMP