Traynor-Kaplan A E, Thompson B L, Harris A L, Taylor P, Omann G M, Sklar L A
Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037.
J Biol Chem. 1989 Sep 15;264(26):15668-73.
We recently showed that phosphatidylinositol trisphosphate (PIP3) was present in a unique lipid fraction generated in neutrophils during activation. Here, we demonstrate that the band containing this fraction isolated from thin layer chromatography consists primarily of PIP3 and that only small amounts of radiolabeled PIP3 exist prior to activation. In addition, high performance liquid chromatography of deacylated phospholipids from stimulated cells reveals an increase in a fraction eluting ahead of glycerophosphoinositol 4,5-P2. After removal of the glycerol we found that it coeluted with inositol 1,3,4-P3 when resubjected to high performance liquid chromatography. Thus, we have detected a second, novel form of phosphatidylinositol bisphosphate in activated neutrophils, PI-(3,4)P2. The elevation of PIP3 through the formyl peptide receptor is blocked by pretreatment with pertussis toxin, implicating mediation of the increase in PIP3 by a guanosine triphosphate-binding (G) protein. The rise in PIP3 is not secondary to calcium elevation. Buffering the rise in intracellular calcium did not diminish the increase in PIP3. The elevation of PIP3 appears to occur during activation with physiological agonists, its level varying with the degree of activation. Leukotriene B4, which elicits many of the same responses as stimulation of the formyl peptide receptor but with minimal oxidant production, stimulates a much attenuated rise in PIP3. Isoproterenol, which inhibits oxidant production also reduces the rise in PIP3. Hence formation of PI(3,4)P2 and PIP3 (presumed to be PI(3,4,5)P3) correlates closely with the early events of neutrophil activation.
我们最近发现，磷脂酰肌醇三磷酸（PIP3）存在于中性粒细胞激活过程中产生的一种独特脂质组分中。在此，我们证明，从薄层色谱法分离出的含有该组分的条带主要由PIP3组成，且在激活前仅存在少量放射性标记的PIP3。此外，对受刺激细胞的脱酰基磷脂进行高效液相色谱分析发现，在甘油磷酸肌醇4,5-P2之前洗脱的一个组分有所增加。去除甘油后，我们发现当再次进行高效液相色谱分析时，它与肌醇1,3,4-P3共洗脱。因此，我们在激活的中性粒细胞中检测到了第二种新型磷脂酰肌醇二磷酸，即PI-(3,4)P2。通过甲酰肽受体导致的PIP3升高被百日咳毒素预处理所阻断，这表明PIP3的增加是由鸟苷三磷酸结合（G）蛋白介导的。PIP3的升高并非继发于钙升高。缓冲细胞内钙的升高并未减少PIP3的增加。PIP3的升高似乎发生在生理性激动剂激活过程中，其水平随激活程度而变化。白三烯B4引发的许多反应与甲酰肽受体刺激相同，但氧化剂产生极少，它刺激的PIP3升高幅度要小得多。抑制氧化剂产生的异丙肾上腺素也会降低PIP3的升高。因此，PI(3,4)P2和PIP3（推测为PI(3,4,5)P3）的形成与中性粒细胞激活的早期事件密切相关。
