The dependence on Ca2+ of phosphatidylinositol breakdown and enzyme secretion in rabbit neutrophils stimulated by formylmethionyl-leucylphenylalanine or ionomycin.
作者信息
Cockcroft S, Bennett J P, Gomperts B D
出版信息
Biochem J. 1981 Dec 15;200(3):501-8. doi: 10.1042/bj2000501.
We have measured the breakdown of [3H]phosphatidylinositol in rabbit neutrophils prelabelled with [3H]glycerol by a pulse-chase procedure. With a view to defining a possible causal relationship between phosphatidylinositol breakdown and enzyme secretion in these cells, we have compared the characteristics of both these processes induced by either the receptor-directed agonist formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) or the Ca2+-ionophore ionomycin. 2. The dependence on fMet-Leu-Phe concentration of phosphatidylinositol breakdown and secretion is identical (half-maximal at 0.3 nM). This is 30-fold less than that required for half-maximal occupation of receptors. 3. Both secretion and breakdown of phosphatidylinositol due to fMet-Leu-Phe are modulated by extracellular Ca2+. The sensitivity to Ca2+ of both processes is enhanced by pretreatment to deplete cell Ca2+. The concentration of Ca2+ required to cause half-maximal effects of both processes in Ca2+-depleted cells on stimulation with 1nM-fMet-Leu-Phe is 100 microM. Ionomycin-stimulated secretion and breakdown of phosphatidylinositol are completely dependent on extracellular Ca2+ over similar concentration ranges. 4. Both secretion and phosphatidylinositol breakdown due to fMet-Leu-Phe approach completion by 10s. With ionomycin these processes are slower, terminating by 2 min. 5. In the presence of [32P]Pi, labelling of [32P]phosphatidic acid reaches a maximum 15 min after stimulation with either fMet-Leu-Phe or ionomycin. This precedes the labelling of [32P]phosphatidylinositol and shows the expected precursor-product relationship. 6. We conclude from these results that in rabbit neutrophils a rise in cytosol [Ca2+] is both sufficient and necessary to cause secretion and phosphatidylinositol breakdown. In cells depleted of Ca2+, the occupation of receptors by fMet-Leu-Phe is without effect on these two processes.
摘要
我们通过脉冲追踪法测量了用[3H]甘油预标记的兔中性粒细胞中[3H]磷脂酰肌醇的分解情况。为了确定这些细胞中磷脂酰肌醇分解与酶分泌之间可能的因果关系，我们比较了由受体导向激动剂甲酰甲硫氨酰 - 亮氨酰 - 苯丙氨酸（fMet - Leu - Phe）或钙离子载体离子霉素诱导的这两个过程的特征。2. 磷脂酰肌醇分解和分泌对fMet - Leu - Phe浓度的依赖性是相同的（半最大效应浓度为0.3 nM）。这比受体半最大占有率所需浓度低30倍。3. 由于fMet - Leu - Phe导致的磷脂酰肌醇的分泌和分解均受细胞外Ca2 +调节。通过预处理耗尽细胞内Ca2 +可增强这两个过程对Ca2 +的敏感性。在用1 nM - fMet - Leu - Phe刺激时，使Ca2 +耗尽的细胞中这两个过程产生半最大效应所需的Ca2 +浓度为100 microM。离子霉素刺激的磷脂酰肌醇分泌和分解在相似浓度范围内完全依赖于细胞外Ca2 +。4. 由于fMet - Leu - Phe导致的分泌和磷脂酰肌醇分解在10秒时接近完成。对于离子霉素，这些过程较慢，在2分钟时终止。5. 在存在[32P]Pi的情况下，在用fMet - Leu - Phe或离子霉素刺激后15分钟，[32P]磷脂酸的标记达到最大值。这先于[32P]磷脂酰肌醇的标记，并显示出预期的前体 - 产物关系。6. 我们从这些结果得出结论，在兔中性粒细胞中，胞质溶胶[Ca2 +]的升高对于引起分泌和磷脂酰肌醇分解既是充分的也是必要的。在Ca2 +耗尽的细胞中，fMet - Leu - Phe对受体的占据对这两个过程没有影响。
