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Glycine‐gated chloride channels in neutrophils attenuate calcium influx and superoxide production

Recently, it was demonstrated that liver injury and TNF‐a 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 ([Ca2+]i) 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 [Ca2+]i. Therefore, the purpose of this study was to determine if glycine affected agonist‐induced increases in [Ca2+]i in rat neutrophils. The effect of glycine on increases in [Ca2+]i elicited either by the bacterial‐derived peptide formyl‐methi‐onine‐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 [Ca2+]i, which were maximal at 1 μM FMLP and 100 μg/ml LPS, respectively. LPS increased intracellular calcium in the presence and absence of extracellular calcium. Glycine blunted increases in [Ca2+]i in a dose‐dependent manner with an IC50 of ~0.3 mM, values only slightly higher than plasma levels. Glycine was unable to prevent agonist‐induced increases in [Ca2+]i in chloride‐free buffer. Moreover, strychnine (1 μM), 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 [Ca2+]i. 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 neutro‐phils with an ED50 of ~0.4 mM, an effect also prevented by 1 μM 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 [Ca2+]i and diminish oxidant production by this important leukocyte.—Wheeler, M., Stachlewitz, R. F., Yamashina, S., Ikejima, K., Morrow, A. L., and Thurman, R. G. Glycine‐gated chloride channels in neutrophils attenuate calcium influx and superoxide production. FASEB J. 14, 476–484 (2000)