Glycine-gated chloride channels in neutrophils attenuate calcium influx and superoxide production.
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+)](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 [Ca(2+)](i). Therefore, the purpose of this study was to determine if glycine affected agonist-induced increases in [Ca(2+)](i) in rat neutrophils. The effect of glycine on increases in [Ca(2+)](i) 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+)](i), 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+)](i) 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+)](i) 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+)](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 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+)](i) and diminish oxidant production by this important leukocyte.
Wheeler
M
M
Department of Pharmacology, Center for Alcohol Studies, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, USA.
Stachlewitz
R F
RF
Yamashina
S
S
Ikejima
K
K
Morrow
A L
AL
Thurman
R G
RG
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion; D065101:Chloride Channel Agonists; D018118:Chloride Channels / Q000097:blood*; D002712:Chlorides / Q000097:blood / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D005260:Female; D005998:Glycine / Q000494:pharmacology*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013331:Strychnine / Q000494:pharmacology; D013481:Superoxides / Q000097:blood*
D065101:Chloride Channel Agonists; D018118:Chloride Channels; D002712:Chlorides; D008070:Lipopolysaccharides; D013481:Superoxides; D004533:Egtazic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D013331:Strychnine; D002118:Calcium; D005998:Glycine