Inhibition of inositol 1,4,5‐trisphosphate metabolism in permeabilised SH‐SY5Y human neuroblastoma cells by a phosphorothioate‐containing analogue of inositol 1,4,5‐trisphosphate

Electrically permeabilised [³H]inositol‐labelled SH‐SY5Y human neuroblastoma cells were employed to examine the effects of two synthetic, phosphatase‐resistant analogues of inositol 1,4,5‐trisphosphate [Ins(1,4,5)P₃] on the metabolism of cell membrane‐derived [³H]Ins(1,4,5)P₃ or exogenous [5‐³²P]Ins(1,4,5)P₃. Incubation of permeabilised SH‐SY5Y cells for 5 min at 37°C with carbachol and guanosine 5′‐[γ‐thio]triphosphate caused a decrease in [³H]phosphoinositol phospholipid levels and an increase in [³H]inositol phosphate accumulation with inositol 4‐phosphate, inositol 1,4‐bisphosphate, Ins(1,4,5)P₃ and inositol 1,3,4,5‐tetrakisphosphate comprising approximately 79%, 16%, 3% and 2%, respectively, of the increase. Inositol 1‐phosphate levels did not increase upon stimulation, nor was inositol 4‐phosphate converted rapidly to inositol. In parallel incubations, the analogues, DL‐inositol 1,4,5‐trisphosphorothioate (DL‐InsP₃S₃) and DL‐inositol 1,4‐bisphosphate 5‐phosphorothioate (DL‐InsP₃S), and synthetic racemic Ins(1,4,5)P₃ (DL‐InsP₃), altered the profile of the [³H]inositol phosphates recovered and led, at millimolar concentrations, to a 10–15‐fold increase in [³H]Ins(1,4,5)P₃. The extent of inhibition of [³H]Ins(1,4,5)P₃ metabolism was, however, greatest in the presence of synthetic D‐Ins(1,4,5)P₃ (5 mM), when [³H]Ins(1,4,5)P₃ comprised approximately 50% of the increase in total [³H]inositol phosphates. Thus, under these conditions, at least 50% of [³H]inositol phosphates were derived from [³H]phosphatidylinositol 4,5‐bisphosphate. [³²P]P_i release from exogenous [5‐³²P]Ins(1,4,5)P₃ was also inhibited by DL‐InsP₃S₃, DL‐InsP₃S and DL‐InsP₃, with half‐maximal inhibition at approximately 50 μM, 160 μM and 240 μM respectively. These actions were approximately ten times more potent than the effects of these compounds on [³H]inositol phosphate accumulation, indicating that homogenous mixing of exogenous and cellmembrane‐derived Ins(1,4,5)P₃ does not occur. These findings indicate that DL‐InsP₃S₃ and DL‐InsP₃S inhibit Ins(1,4,5)P₃ 5‐phosphatase. In contrast. the effects of synthetic DL‐InsP₃ and d‐Ins(1,4,5)P₃ are due to isotopic dilution. Whilst DL‐InsP₃S₃ was the most potent inhibitor of dephosphorylation of exogenous or cell‐membrane‐derived Ins(1,4,5)P₃, it was the weakest inhibitor of 3‐kinase‐catalysed Ins(1,4,5)P₃ phosphorylation. Similarly, although approximately 50 times less potent than DL‐InsP₃S₃, 2,3‐diphosphoglycerate inhibited Ins(1,4,5)P₃ 5‐phosphatase activity and was apparently without effect of Ins(1,4,5)P₃ 3‐kinase activity. These data show that both DL‐InsP₃S₃ and DL‐InsP₃S are potent inhibitors of Ins(1,4,5)P₃ 5‐phosphatase. In contrast, phosphorylation of Ins(1,4,5)P₃ was inhibited potently only by DL‐InsP₃S.