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(1,4,4)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)
Wojcikiewicz
R J
RJ
Department of Pharmacology and Therapeutics, University of Leicester, England.
Cooke
A M
AM
Potter
B V
BV
Nahorski
S R
SR
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D004558:Electric Stimulation; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007294:Inositol / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007295:Inositol Phosphates / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D009447:Neuroblastoma; D009946:Organothiophosphorus Compounds / Q000494:pharmacology*
D007295:Inositol Phosphates; D009946:Organothiophosphorus Compounds; C066055:inositol 1,4-bisphosphate 5-phosphorothioate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D007294:Inositol; D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol