Cookies on this website
We use cookies to ensure that we give you the best experience on our website. If you click 'Continue' we'll assume that you are happy to receive all cookies and you won't see this message again. Click 'Find out more' for information on how to change your cookie settings.

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(1,4,4)P3. Incubation of permeabilised SH-SY5Y cells for 5 min at 37 degrees C with carbachol and guanosine 5'-[gamma-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 (greater than or equal to 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]Pi 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 microM, 160 microM and 240 microM 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 cell-membrane-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.(ABSTRACT TRUNCATED AT 400 WORDS)

Type

Journal article

Journal

Eur J Biochem

Publication Date

11/09/1990

Volume

192

Pages

459 - 467

Keywords

Animals, Carbachol, Cell Line, Cell Membrane, Cell Membrane Permeability, Electric Stimulation, Guanosine 5'-O-(3-Thiotriphosphate), Humans, Inositol, Inositol 1,4,5-Trisphosphate, Inositol Phosphates, Neuroblastoma, Organothiophosphorus Compounds