VanUffelen B E, de Koster B M, Van den Broek P J, VanSteveninck J, Elferink J G
Department of Medical Biochemistry, Leiden University, The Netherlands.
J Leukoc Biol. 1996 Jul;60(1):94-100. doi: 10.1002/jlb.60.1.94.
We studied the effect of exogenous nitric oxide (NO) on migration of rabbit peritoneal neutrophils. Exogenous NO enhanced random migration of neutrophils in a concentration-dependent way. An optimally stimulatory effect was observed with 0.5 microM NO, whereas at higher NO concentrations the enhancing effect decreased again. NO caused a rapid and transient increase in intracellular guanosine-3',5'-cyclic monophosphate (cGMP) levels. The enhancing effect of NO on random migration was largely reversed by the inhibitors of cGMP accumulation, LY-83583 and methylene blue, and by the antagonists of cGMP-dependent protein kinase, 8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-cGMPS) and 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS). These observations strongly suggest that the enhancement of random migration by NO is mediated by cGMP and cGMP-dependent protein kinase. The effect of NO on migration did not occur in the absence of extracellular calcium. Although NO did not induce a measurable elevation of intracellular free calcium, pre-incubation with the intracellular calcium chelator Fura-2/AM abolished the enhancing effect of NO. It appears therefore that a small change in the level of cytoplasmic free calcium does play a role in the enhancement of random migration by NO. High concentrations of NO were found to inhibit chemotaxis induced by an optimal concentration of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). This inhibitory effect was also dependent on the presence of extracellular calcium. A role for cGMP in the inhibition of fMLP-induced chemotaxis by NO is not supported by our measurements of intracellular cGMP levels. In contrast to the effects on fMLP, NO did not affect chemotaxis induced by the phorbol ester PMA. In conclusion, we show that NO, not derived from NO donors but applied directly, may stimulate or inhibit neutrophil migration, dependent on the concentration. The enhancing effect of NO on random migration is mediated by cGMP, which emphasizes the importance of this second messenger as a modulator of neutrophil functional.
我们研究了外源性一氧化氮（NO）对兔腹膜中性粒细胞迁移的影响。外源性NO以浓度依赖的方式增强中性粒细胞的随机迁移。在0.5微摩尔/升的NO浓度下观察到最佳刺激效果，而在更高的NO浓度下，增强作用再次减弱。NO导致细胞内鸟苷-3',5'-环磷酸（cGMP）水平迅速短暂升高。cGMP积累抑制剂LY-83583和亚甲蓝，以及cGMP依赖性蛋白激酶拮抗剂8-溴鸟苷-3',5'-环磷硫酰酯、Rp-异构体（Rp-8-Br-cGMPS）和8-（4-氯苯硫基）-鸟苷-3',5'-环磷硫酰酯（Rp-8-pCPT-cGMPS）在很大程度上逆转了NO对随机迁移的增强作用。这些观察结果强烈表明，NO对随机迁移的增强作用是由cGMP和cGMP依赖性蛋白激酶介导的。在没有细胞外钙的情况下，NO对迁移没有影响。虽然NO没有引起细胞内游离钙的可测量升高，但用细胞内钙螯合剂Fura-2/AM预孵育消除了NO的增强作用。因此，细胞质游离钙水平的微小变化似乎在NO增强随机迁移中起作用。发现高浓度的NO抑制由最佳浓度的趋化肽N-甲酰甲硫氨酰亮氨酰苯丙氨酸（fMLP）诱导的趋化作用。这种抑制作用也依赖于细胞外钙的存在。我们对细胞内cGMP水平的测量不支持cGMP在NO抑制fMLP诱导的趋化作用中起作用。与对fMLP的影响相反，NO不影响佛波酯PMA诱导的趋化作用。总之，我们表明，直接应用而非来自NO供体的NO可根据浓度刺激或抑制中性粒细胞迁移。NO对随机迁移的增强作用由cGMP介导这一点，强调了这种第二信使作为中性粒细胞功能调节剂的重要性。
