Pittet D, Lew D P, Mayr G W, Monod A, Schlegel W
Infectious Diseases Division, University Hospital, Genève, Switzerland.
J Biol Chem. 1989 May 5;264(13):7251-61.
The mechanisms by which the chemotactic peptide formyl-methyl-leucyl-phenyl-alanine stimulates Ca2+ influx across the plasma membrane were investigated in the human promyelocytic cell line HL-60, induced to differentiate with dimethyl sulfoxide. Ca2+ influx was determined: (a) from the initial rate of Mn2+ influx, apparent from the quenching of intracellular quin2 or fura-2 fluorescence; (b) from the rate of the elevation of cytosolic free calcium, [Ca2+]i, upon readdition of Ca2+ to cells previously stimulated in the absence of extracellular Ca2+. [3H]Inositol tris-, tetrakis-, and pentakisphosphates were analyzed by a high performance liquid chromatography procedure which was optimized for the separation of inositol tetrakisphosphates, yielding three predominant isomers: inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), inositol 1,4,5,6-tetrakisphosphate, and inositol 1,3,4, 6-tetrakisphosphate. Both the kinetics and agonist dose dependence of Ca2+ influx stimulation correlated closely with the corresponding receptor-mediated variations of [Ca2+]i either in the presence or in the absence of extracellular Ca2+. Of the different inositol phosphates determined in parallel and under the same conditions, accumulation of [3H]Ins(1,3,4,5)P4 correlated best with Ca2+ influx both temporally and in its dose dependence in the presence or in the absence of extracellular Ca2+; inositol 1,3,4-trisphosphate was also correlated but to a lesser extent. Attenuations of [Ca2+]i elevations by decreasing extracellular Ca2+ or by increasing the cytosolic Ca2+ buffering capacity with quin2 led to parallel inhibition of Ca2+ influx and Ins(1,3,4,5)P4 production.
在经二甲基亚砜诱导分化的人早幼粒细胞系HL-60中，研究了趋化肽甲酰基-甲基-亮氨酰-苯丙氨酸刺激Ca2+跨质膜内流的机制。通过以下方法测定Ca2+内流：(a)根据Mn2+内流的初始速率，从细胞内喹啉2或氟罗-2荧光的淬灭情况判断；(b)根据先前在无细胞外Ca2+的情况下受到刺激的细胞重新加入Ca2+后，胞质游离钙[Ca2+]i升高的速率判断。通过一种高效液相色谱法分析[3H]肌醇三磷酸、四磷酸和五磷酸，该方法针对肌醇四磷酸的分离进行了优化，产生三种主要异构体：肌醇1,3,4,5-四磷酸(Ins(1,3,4,5)P4)、肌醇1,4,5,6-四磷酸和肌醇1,3,4,6-四磷酸。无论是在有细胞外Ca2+还是无细胞外Ca2+的情况下，Ca2+内流刺激的动力学和激动剂剂量依赖性都与相应的受体介导的[Ca2+]i变化密切相关。在相同条件下平行测定的不同肌醇磷酸中，[3H]Ins(1,3,4,5)P4的积累在时间上及其剂量依赖性方面，无论在有细胞外Ca2+还是无细胞外Ca2+的情况下，都与Ca2+内流相关性最好；肌醇1,3,4-三磷酸也有相关性，但程度较小。通过降低细胞外Ca2+或用喹啉2增加胞质Ca2+缓冲能力来减弱[Ca2+]i升高，会导致Ca2+内流和Ins(1,3,4,5)P4产生的平行抑制。
1)甲酰基-甲硫氨酰-亮氨酰-苯丙氨酸对Ca2+内流的激活取决于[Ca2+]i的升高，后者由细胞内储存的Ca2+动员引发；2)Ins(1,3,4,5)P4是维持分化的HL-60细胞中受体介导的Ca2+内流激活的有力候选者。
