M. Wheeler et al., Glycine-gated chloride channels in neutrophils attenuate calcium influx and superoxide production, FASEB J, 14(3), 2000, pp. 476-484
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 re
sult of, at least in part, activation of a chloride channel in Kupffer cell
s by glycine, which hyperpolarizes the cell membrane and blunts increases i
n intracellular calcium concentrations ([Ca2+](i)) similar to its action in
the neuron. It is well known that hepatotoxicity due to LPS has a neutroph
il-mediated component and that activation of neutrophils is dependent on in
creases in [Ca2+](i). Therefore, the purpose of this study was to determine
if glycine affected agonist-induced increases in [Ca2+](i) in rat neutroph
ils. The effect of glycine on increases in [Ca2+](i) elicited either by the
bacterial-derived peptide formyl-methionine-leucine-phenylalanine (FMLP) o
r LPS was studied in individual neutrophils using Fura-2 and fluorescence m
icroscopy, Both FMLP and LPS caused dose-dependent increases in [Ca2+](i),
which were maximal at 1 mu M FMLP and 100 mu g/ml LPS, respectively. LPS in
creased 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 similar to 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 mu M), 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 determ
ined. Glycine caused a dose-dependent increase in chloride uptake into neut
rophils with an ED50 of similar to 0.4 mM, an effect also prevented by 1 mu
M strychnine. Glycine also significantly reduced the production of superox
ide anion from FMLP-stimulated neutrophils. Taken together, these data prov
ide clear evidence that neutrophils contain a glycine-gated chloride channe
l that can attenuate increases in [Ca2+](i) and diminish oxidant production
by this important leukocyte.