THE USE OF FURA-2 TO DETERMINE THE RELATIONSHIP BETWEEN CYTOPLASMIC FREE CA-2+ AND OXIDASE ACTIVATION IN RAT NEUTROPHILS
THE USE OF FURA-2 TO DETERMINE THE RELATIONSHIP BETWEEN CYTOPLASMIC FREE CA-2+ AND OXIDASE ACTIVATION IN RAT NEUTROPHILS
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DOI：
10.1016/0143-4160(88)90034-6
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发表时间：
1988-02-01
期刊：
影响因子：
4
通讯作者：
HALLETT, MB
中科院分区：
文献类型：
--
作者：
ALMOHANNA, FA;HALLETT, MB
大鼠中性粒细胞与Fura-2-乙酰氧基甲酯(Fura-2/AM)孵育后，Fura-2几乎完全进入细胞质。尽管颗粒中还存在Fura-2/AM酯酶活性，但只有1.5%的细胞相关Fura-2位于这些细胞器中。Fura-2从中性粒细胞中以可接受的低速率泄漏0.16+-。在37度时为0.05%min-1。当细胞内Fura-2的浓度达到500µm时，虽然细胞内的ATP含量下降到大约50%，但对氧化酶的激活没有影响。该多肽f-Met-Leu-Phe(Fmlp)在340 nm和380 nm处产生了激发的荧光强度变化，这与胞内钙离子从静息水平94+-升高相一致。13海里到768海里+-。173 nm(n=6)。细胞内的Fura-2浓度需要大于1 mM才能有效地缓冲这种上升，据估计，在高信号：自体荧光比(100µm)所需的细胞内Fura-2浓度下，细胞的细胞质钙缓冲能力仅增加10%。多肽诱导的胞质内游离钙离子的升高先于酶的激活几秒钟，反应的大小取决于钙离子升高的程度，大约在一半时达到最大激活。600毫米。因此，这些数据与胞浆钙离子在触发中性粒细胞氧化酶激活中的第二信使作用是一致的。
Incubation of rat neutrophils with fura-2-acetoxy-methyl ester (fura-2/AM) resulted in the loading of fura-2 almost exclusively into the cytoplasm. Despite the additional presence of fura-2/AM esterase activity in the granules, only 1.5% of cell-associated fura-2 was located within these organelles. Fura-2 leaked from neutrophils at an acceptably low rate 0.16 .+-. 0.05% min-1 at 37.degree. C. At intracellular concentrations of fura-2 up to 500 .mu.M, there was no effect on oxidase activation; although the cellular ATP content was reduced to approximately 50%. The peptide, f-met-leu-phe (fmlp), 1 .mu.M, produced intensity changes of fluorescence excited at 340 nm and 380 nm which were consistent with a cytoplasmic Ca2+ rise from the resting level of 94 .+-. 13 nM to 768 .+-. 173 nM (n = 6). Intracellular concentrations of fura-2 greater than 1 mM were required to buffer effectively this rise, and it was estimated that at an intracellular fura-2 concentration required for a high signal:autofluorescence ratio (100 .mu.M) the cytoplasmic Ca2+ buffering capacity of the cells was increased by only 10%. The rise in cytoplasmic free Ca2+ induced by the peptide preceded activation of the oxidase by several seconds, and the magnitude of the response was dependent on the extent of the Ca2+ rise, half-maximal activation being achieved at approx. 600 mM. These data were therefore consistent with a secondary messenger role for cytoplasmic Ca2+ in triggering neutrophil oxidase activation.