Intracellular singlet oxygen generation by phagocytosing neutrophils in response to particles coated with a chemical trap.
Intracellular singlet oxygen generation by phagocytosing neutrophils in response to particles coated with a chemical trap.
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DOI：
10.1016/s0021-9258(18)42228-4
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发表时间：
1992-07
期刊：
影响因子：
--
通讯作者：
M. J. Steinbeck;Ahsan U. Khan;M. J. Karnovsky
中科院分区：
文献类型：
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作者：
M. J. Steinbeck;Ahsan U. Khan;M. J. Karnovsky
为了确定中性粒细胞（PMN）在吞噬过程中是否产生单线态氧（O2（1 δ g）），用O2（1 δ g）的特异性化学捕集剂9，10-二苯基蒽（DPA）包被玻璃珠。单线态氧而不是其它活性氧物质以kr = 1.3 × 10（6）M-1 s-1的速率与DPA快速反应，形成稳定的产物DPA-内过氧化物（Corey，E. J.，和泰勒，W. C.等（1964）J. Am. 86，3881-3882; Wasserman，H. H、Scheffer，J.R.，和库珀，J.L.等（1972）J. Am. Soc.94，4991-4996; Turro，N. J.，周，M.- F.、和Rigaudy，J.（1981）J. Am. 103，7218-7224）。DPA-内过氧化物的产生通过紫外光谱法测定为在355 nm处DPA吸光度的降低。将DPA的吸光度归一化为苝的吸光度，苝作为非反应性内标物包含在珠上的涂层中。在本研究中，DPA和二萘嵌苯包被的珠最初允许粘附到纤连蛋白包被的盖玻片上。然后将PMN加入到珠包被的盖玻片中，并使其在37 ℃下粘附并吞噬珠1小时。在一些实验中，加入4 B-佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）（1 ng/2.5 x 10（7）个细胞/ml）（一种已知的PMN NADPH氧化酶激活剂）作为共刺激剂。经计算，吞噬刺激的PMN产生的O2（1 Δ g）量为11.3 +/- 4.9 nmol O2（1 Δ g）/1.25 × 10（6）个细胞。低剂量PMA共刺激使O2（1 Δ g）的产生增加至14.1 +/- 4.1 nmol/1.25 x 10（6）个细胞。这些量平均在一起代表响应于DPA-和二萘嵌苯-涂覆的珠粒的PMN消耗的总氧的约19 +/-5.0%。DPA与O2（1 δ g）反应的特异性通过升温至120 ℃来证实，这从DPA-内过氧化物释放O2（1 δ g），再生母体DPA化合物（Wasserman等人，1972; Turro等人，1981）和355 nm处的吸光度。此外，β-胡萝卜素，一种O2（1 Δ g）的亲淬灭剂，被包括在一些珠制剂的涂层中;使用这些珠的测定显示在355 nm处的吸光度没有变化。单线态氧的产生髓过氧化物酶也测量使用包被珠测定和结果表明，这是一个主要的途径，单线态氧产生吞噬刺激的中性粒细胞。（400字处截断摘要）
To determine if singlet oxygen (O2(1 delta g)) is produced by neutrophils (PMNs) during the process of phagocytosis, glass beads were coated with a specific chemical trap for O2(1 delta g), 9,10-diphenylanthracene (DPA). Singlet oxygen, but not other reactive oxygen species, reacts rapidly with DPA at a rate of kr = 1.3 x 10(6) M-1 s-1 to form a stable product, DPA-endoperoxide (Corey, E. J., and Taylor, W. C. (1964) J. Am. Chem. Soc. 86, 3881-3882; Wasserman, H. H., Scheffer, J. R., and Cooper, J. L. (1972) J. Am. Chem. Soc. 94, 4991-4996; Turro, N. J., Chow, M.-F., and Rigaudy, J. (1981) J. Am. Chem. Soc. 103, 7218-7224). The production of DPA-endoperoxide was determined by ultraviolet spectroscopy as a decrease in DPA absorbance at 355 nm. The absorbance of DPA was normalized to the absorbance of perylene, which was included in the coating on the beads as a nonreactive, internal standard. In the present study, DPA- and perylene-coated beads were initially allowed to adhere to fibronectin-coated coverslips. PMNs were then added to the bead-coated coverslips and allowed to adhere and phagocytose the beads for 1 h at 37 degrees C. In some experiments, 4B-phorbol-12-myristate-13-acetate (PMA) (1 ng/2.5 x 10(7) cells/ml), a known activator of the PMN NADPH-oxidase, was added as a co-stimulant. The amount of O2(1 delta g) produced by phagocytically stimulated PMNs was calculated to be 11.3 +/- 4.9 nmol of O2(1 delta g)/1.25 x 10(6) cells. Low dose PMA co-stimulation increased the production of O2(1 delta g) to 14.1 +/- 4.1 nmol/1.25 x 10(6) cells. Averaged together these amounts represent approximately 19 +/- 5.0% of the total oxygen consumed by PMNs in response to DPA- and perylene-coated beads. The specificity of the DPA reaction with O2(1 delta g) was confirmed by warming to 120 degrees C, which releases O2(1 delta g) from the DPA-endoperoxide, regenerating the parent DPA compound (Wasserman et al., 1972; Turro et al., 1981) and the absorbance at 355 nm. In addition, beta-carotene, an avid quencher of O2(1 delta g), was included in the coating of some bead preparations; assays in which these beads were used showed no change in the absorbance at 355 nm. Singlet oxygen production by myeloperoxidase was also measured using the coated bead assay and the results suggest that this is a major pathway by which singlet oxygen is generated in phagocytically stimulated PMNs.(ABSTRACT TRUNCATED AT 400 WORDS)