Wojcikiewicz R J, Cooke A M, Potter B V, Nahorski S R
Department of Pharmacology and Therapeutics, University of Leicester, England.
Eur J Biochem. 1990 Sep 11;192(2):459-67. doi: 10.1111/j.1432-1033.1990.tb19248.x.
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)
采用电通透的[3H]肌醇标记的SH-SY5Y人神经母细胞瘤细胞，来研究两种合成的、对磷酸酶有抗性的1,4,5-三磷酸肌醇[Ins(1,4,5)P3]类似物对细胞膜来源的[3H]Ins(1,4,5)P3或外源性[5-32P]Ins(1,4,5)P3代谢的影响。将通透的SH-SY5Y细胞在37℃下与卡巴胆碱和鸟苷5'-[γ-硫代]三磷酸一起孵育5分钟，导致[3H]磷酸肌醇磷脂水平降低，[3H]肌醇磷酸积累增加，其中4-磷酸肌醇、1,4-二磷酸肌醇、Ins(1,4,5)P3和1,3,4,5-四磷酸肌醇分别约占增加量的79%、16%、3%和2%。刺激后1-磷酸肌醇水平未升高，4-磷酸肌醇也未迅速转化为肌醇。在平行孵育中，类似物DL-1,4,5-三磷酸硫代肌醇（DL-InsP3S3）和DL-1,4-二磷酸肌醇5-磷酸硫代物（DL-InsP3S）以及合成的外消旋Ins(1,4,5)P3（DL-InsP3）改变了回收的[3H]肌醇磷酸的分布，在毫摩尔浓度下导致[3H]Ins(1,4,5)P3增加10-15倍。然而，在合成的D-Ins(1,4,5)P3（大于或等于5 mM）存在时，[3H]Ins(1,4,5)P3代谢的抑制程度最大，此时[3H]Ins(1,4,5)P3约占总[3H]肌醇磷酸增加量的50%。因此，在这些条件下，至少50%的[3H]肌醇磷酸来自[3H]磷脂酰肌醇4,5-二磷酸。外源性[5-32P]Ins(1,4,5)P3的[32P]Pi释放也受到DL-InsP3S3、DL-InsP3S和DL-InsP3的抑制，半最大抑制浓度分别约为50 microM、160 microM和240 microM。这些作用比这些化合物对[3H]肌醇磷酸积累的影响强约十倍，表明外源性和细胞膜来源的Ins(1,4,5)P3没有均匀混合。这些发现表明DL-InsP3S3和DL-InsP3S抑制Ins(1,4,5)P3 5-磷酸酶。相比之下，合成的DL-InsP3和D-Ins(1,4,5)P3的作用是由于同位素稀释。虽然DL-InsP3S3是外源性或细胞膜来源的Ins(1,4,5)P3去磷酸化的最有效抑制剂，但它是3-激酶催化的Ins(1,4,5)P3磷酸化的最弱抑制剂。同样，虽然效力比DL-InsP3S3低约50倍，但2,3-二磷酸甘油酸抑制Ins(1,4,5)P3 5-磷酸酶活性，对Ins(1,4,5)P3 3-激酶活性显然没有影响。（摘要截断于400字）
