Myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] acts as a full agonist for Ca2+ release in saponin-permeabilised SH-SY5Y neuroblastoma cells. Studies were conducted in the presence of myo-inositol hexakisphosphate (InsP6, 10 μM), to inhibit the Ins(1,3,4,5)P4-3-phosphatase catalysed back conversion of Ins(1,3,4,5)P4 to Ins(1,4,5)P3. HPLC analysis confirmed that Ins(1,3,4,5)P4 releases the entire content of Ins(1,4,5)P3-sensitive intracellular Ca2+ stores, independent of 3-phosphatase activity. Further we utilised racemic myo-inositol 1,4,5-trisphosphate-3-phosphorothioate [dl-Ins(1,3,4,5)P4-3S], a novel intrinsically Ins(1,3,4,5)P4-3-phosphatase resistant Ins(1,3,4,5)P4 analogue. DL-Ins(1,3,4,5)P4-3S specifically displaced [3H]Ins(1,4,5)P3 from bovine adrenal cortex Ins(1,4,5)P3 binding sites (IC50 = 889 nM, compared to Ins(1,4,5)P3,1C50 = 4.4 nM and Ins(1,3,4,5)P4, IC50 = 152 nM). dl-Ins(1,3,4,5)P4-3S was a full agonist for Ca2+ release (EC50 = 4.7 μM), being 90- and 2-fold less potent than Ins(1,4,5)P3 and Ins(1,3,4,5)P4 (with InsP6), respectively. dl-Ins(1,3,4,5)P4-3S will be an important tool for identification of potentially exclusive Ins(1,3,4,5)P4 second messenger functions, since its resistance to 3-phosphatase action precludes the inconvenient artefact of steady state Ins(1,4,5)P3 generation.
