Demkow U, Winklewski P, Potapinska O, Popko K, Lipinska A, Wasik M
Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Warsaw Medical University, Warsaw, Poland.
J Physiol Pharmacol. 2008 Dec;59 Suppl 6:219-29.
The goal of the study was to evaluate the process of neutrophil activation via Ca(2+)-mediated transduction signal and its modification by insulin. The study was performed with the use of isolated peripheral blood neutrophils obtained from 20 healthy volunteers. Isolated granulocytes were stimulated by fMLP or insulin alone, or by both substances added to the medium in combinations: fMLP + insulin (after 20 min) or insulin + fMLP (after 20 min). To explore the mechanism of intracellular Ca(2+) changes, receptor signal transduction pathway was blocked by tyrosine kinase inhibitors: tyrphostin 25 and genistein. fMLP evoked fast intracellular increase of free Ca(2+) concentration in neutrophils, compared with the resting state (P< 0.001). Insulin did not cause any changes in intracellular Ca(2+) when was added to the previously fMLP stimulated cells. Prestimulation with insulin significantly decreased fMLP-induced intracellular free Ca(2+) concentration compared with fMLP alone (P<0.01). A strong correlation was observed between initial intracellular Ca(2+) concentration after incubation with insulin and the response to fMLP (P<0.0001). The tyrphostin 25 did not influence the Ca(2+) concentration in control granulocytes, but inhibited the fMLP-induced intracellular Ca(2+) increase when added before fMLP (P<0.05). In a Ca(2+)-free medium, a strong relationship between intracellular Ca(2+) and the response to fMLP after incubation with tyrphostin was found (P<0.001) The genistein did not influence the intracellular Ca(2+) in non-stimulated cells. However, it inhibited the fMLP-induced Ca(2+) increase when added before fMLP (P<0.05). The genistein added to the suspension of cells after fMLP stimulation did not influence intracellular Ca(2+) level. A positive correlation was found between the initial intracellular Ca(2+) and the response to fMLP of genistein preincubated cells. This effect was seen in both Ca(2+)-rich, and Ca(2+)-free medium We conclude that insulin is a potent immunomodulator and its signaling pathways are mediated by Ca(2+) concentration changes. The process of intracellular Ca(2+) changes following insulin signaling is, at least partly, tyrosine kinase-related. Derangements in the concentration of intracellular Ca(2+) may represent a link between the mechanisms of insulin resistance in diabetes.
本研究的目的是评估中性粒细胞通过Ca(2+)介导的转导信号激活的过程及其被胰岛素修饰的情况。该研究使用了从20名健康志愿者获取的分离外周血中性粒细胞来进行。分离的粒细胞分别用fMLP或胰岛素单独刺激，或两种物质以组合形式添加到培养基中刺激：fMLP +胰岛素（20分钟后）或胰岛素 + fMLP（20分钟后）。为了探究细胞内Ca(2+)变化的机制，受体信号转导途径被酪氨酸激酶抑制剂阻断： tyrphostin 25和金雀异黄素。与静息状态相比，fMLP引起中性粒细胞内游离Ca(2+)浓度快速升高（P<0.001）。当将胰岛素添加到先前用fMLP刺激的细胞中时，未引起细胞内Ca(2+)的任何变化。与单独使用fMLP相比，用胰岛素预刺激显著降低了fMLP诱导的细胞内游离Ca(2+)浓度（P<0.01）。在用胰岛素孵育后的初始细胞内Ca(2+)浓度与对fMLP的反应之间观察到强相关性（P<0.0001）。tyrphostin 25对对照粒细胞中的Ca(2+)浓度没有影响，但在fMLP之前添加时可抑制fMLP诱导的细胞内Ca(2+)升高（P<0.05）。在无Ca(2+)培养基中，在用tyrphostin孵育后发现细胞内Ca(2+)与对fMLP的反应之间存在强相关性（P<0.001）。金雀异黄素对未刺激细胞中的细胞内Ca(2+)没有影响。然而，在fMLP之前添加时可抑制fMLP诱导的Ca(2+)升高（P<0.05）。在fMLP刺激后添加到细胞悬液中的金雀异黄素不影响细胞内Ca(2+)水平。在用金雀异黄素预孵育的细胞中，初始细胞内Ca(2+)与对fMLP的反应之间发现正相关。在富含Ca(2+)和无Ca(2+)的培养基中均可见此效应。我们得出结论，胰岛素是一种有效的免疫调节剂，其信号通路由Ca(2+)浓度变化介导。胰岛素信号后细胞内Ca(2+)变化的过程至少部分与酪氨酸激酶相关。细胞内Ca(2+)浓度的紊乱可能代表糖尿病中胰岛素抵抗机制之间的联系。
