Wheeler M, Stachlewitz R F, Yamashina S, Ikejima K, Morrow A L, Thurman R G
Department of Pharmacology, Center for Alcohol Studies, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, USA.
FASEB J. 2000 Mar;14(3):476-84. doi: 10.1096/fasebj.14.3.476.
Recently, it was demonstrated that liver injury and TNF-alpha production as a result of endotoxin (lipopolysaccharide, LPS) were attenuated by feeding animals a diet enriched with glycine. This phenomenon was shown to be a result of, at least in part, activation of a chloride channel in Kupffer cells by glycine, which hyperpolarizes the cell membrane and blunts increases in intracellular calcium concentrations (Ca(2+)) similar to its action in the neuron. It is well known that hepatotoxicity due to LPS has a neutrophil-mediated component and that activation of neutrophils is dependent on increases in Ca(2+). Therefore, the purpose of this study was to determine if glycine affected agonist-induced increases in Ca(2+) in rat neutrophils. The effect of glycine on increases in Ca(2+) elicited either by the bacterial-derived peptide formyl-methionine-leucine-phenylalanine (FMLP) or LPS was studied in individual neutrophils using Fura-2 and fluorescence microscopy. Both FMLP and LPS caused dose-dependent increases in Ca(2+), which were maximal at 1 microM FMLP and 100 microgram/ml LPS, respectively. LPS increased intracellular calcium in the presence and absence of extracellular calcium. Glycine blunted increases in Ca(2+) in a dose-dependent manner with an IC(50) of approximately 0.3 mM, values only slightly higher than plasma levels. Glycine was unable to prevent agonist-induced increases in Ca(2+) in chloride-free buffer. Moreover, strychnine (1 microM), an antagonist of the glycine-gated chloride channel in the central nervous system, reversed the effects of glycine (1 mM) on FMLP- or LPS-stimulated increases in Ca(2+). To provide hard evidence for a glycine-gated chloride channel in the neutrophil, the effect of glycine on radioactive chloride uptake was determined. Glycine caused a dose-dependent increase in chloride uptake into neutrophils with an ED(50) of approximately 0.4 mM, an effect also prevented by 1 microM strychnine. Glycine also significantly reduced the production of superoxide anion from FMLP-stimulated neutrophils. Taken together, these data provide clear evidence that neutrophils contain a glycine-gated chloride channel that can attenuate increases in Ca(2+) and diminish oxidant production by this important leukocyte.
最近有研究表明，给动物喂食富含甘氨酸的饮食可减轻内毒素（脂多糖，LPS）所致的肝损伤和TNF-α生成。该现象至少部分是由于甘氨酸激活了库普弗细胞中的氯离子通道，这使其细胞膜超极化，并抑制细胞内钙浓度（Ca(2+)）的升高，类似于其在神经元中的作用。众所周知，LPS所致的肝毒性有中性粒细胞介导的成分，且中性粒细胞的激活依赖于Ca(2+)的升高。因此，本研究的目的是确定甘氨酸是否会影响大鼠中性粒细胞中激动剂诱导的Ca(2+)升高。使用Fura-2和荧光显微镜在单个中性粒细胞中研究了甘氨酸对细菌衍生肽甲酰甲硫氨酸-亮氨酸-苯丙氨酸（FMLP）或LPS诱导的Ca(2+)升高的影响。FMLP和LPS均引起Ca(2+)的剂量依赖性升高，分别在1 microM FMLP和100 microgram/ml LPS时达到最大值。LPS在有和没有细胞外钙的情况下均增加细胞内钙。甘氨酸以剂量依赖性方式抑制Ca(2+)的升高，IC(50)约为0.3 mM，该值仅略高于血浆水平。在无氯缓冲液中，甘氨酸无法阻止激动剂诱导的Ca(2+)升高。此外，士的宁（1 microM），一种中枢神经系统中甘氨酸门控氯离子通道的拮抗剂，可逆转甘氨酸（1 mM）对FMLP或LPS刺激的Ca(2+)升高的影响。为了为中性粒细胞中的甘氨酸门控氯离子通道提供确凿证据，测定了甘氨酸对放射性氯摄取的影响。甘氨酸导致中性粒细胞对氯的摄取呈剂量依赖性增加，ED(50)约为0.4 mM，1 microM士的宁也可阻止该效应。甘氨酸还显著降低了FMLP刺激的中性粒细胞中超氧阴离子的产生。综上所述，这些数据提供了明确的证据，表明中性粒细胞含有甘氨酸门控氯离子通道，该通道可减弱Ca(2+)的升高并减少这种重要白细胞的氧化剂产生。
