Lärfars G, Gyllenhammar H
Department of Hematology, Karolinska Institute at Huddinge University Hospital, Sweden.
J Lab Clin Med. 1998 Jul;132(1):54-60. doi: 10.1016/s0022-2143(98)90025-7.
The production of nitric oxide (NO) may play an important role in functional responses of the human polymorphonuclear neutrophil granulocytes (PMNs). Others have described the presence of both an inducible, Ca2+-independent and a constitutionally expressed, Ca2+-dependent nitric oxide synthase (NOS) in human PMNs. However, the conditions for production and release of NO in human PMNs are still largely unknown. We assessed mechanisms for activation of NO release from human PMNs and particularly the dependence on extracellular and intracellular Ca2+. We addressed this question by applying a variety of agonists with known and differing mechanisms of activation in PMNs and measuring the released NO by two highly sensitive and specific real-time methods for detection of NO, the oxidation of oxyhemoglobin to methemoglobin and an electrochemical method. We found that human PMNs activated with the surface receptor-dependent agonist, N-formyl-methionyl-leucyl-phenylalanine (fMLP); the calcium ionophore, A23187; or the direct stimulator of protein kinase C, phorbol myristate acetate (PMA), produced NO which was inhibited by a specific NOS inhibitor, NG-monomethyl-L-arginine. The NO production induced by fMLP or A23187 was dependent on the presence of extracellular Ca2+, but this was not the case for PMA. The stimulatory effect of fMLP was almost completely inhibited by Bordetella pertussis toxin. These results indicate an NOS activity in purified human PMNs in vitro, and the transduction mechanisms for the agonists used show strong similarity with previously known pathways for other neutrophil functions.
一氧化氮（NO）的产生可能在人类多形核中性粒细胞（PMNs）的功能反应中起重要作用。其他人已经描述了人类PMNs中存在一种诱导型、不依赖Ca2+的一氧化氮合酶（NOS）和一种组成性表达、依赖Ca2+的一氧化氮合酶。然而，人类PMNs中NO产生和释放的条件仍 largely未知。我们评估了人类PMNs中NO释放的激活机制，特别是对细胞外和细胞内Ca2+的依赖性。我们通过应用多种具有已知且不同激活机制的激动剂来激活PMNs，并通过两种高度敏感和特异的实时检测NO的方法——将氧合血红蛋白氧化为高铁血红蛋白和一种电化学方法，来测量释放的NO，从而解决了这个问题。我们发现，用表面受体依赖性激动剂N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（fMLP）、钙离子载体A23187或蛋白激酶C的直接刺激剂佛波酯（PMA）激活的人类PMNs产生了NO，而这种NO被一种特异性NOS抑制剂NG-单甲基-L-精氨酸所抑制。fMLP或A23187诱导的NO产生依赖于细胞外Ca2+的存在，但PMA诱导的NO产生情况并非如此。fMLP的刺激作用几乎完全被百日咳毒素抑制。这些结果表明在体外纯化的人类PMNs中存在NOS活性，并且所使用的激动剂的转导机制与先前已知的其他中性粒细胞功能途径有很强的相似性。
