Glycine blocks the increase in intracellular free
Ca2+ due to vasoactive mediators in hepatic parenchymal
cells
Wei
Qu1,2,
Kenichi
Ikejima1,
Zhi
Zhong1,
Michael
P.
Waalkes2, and
Ronald G.
Thurman1
1 Laboratory of Hepatobiology and Toxicology,
Department of Pharmacology, University of North Carolina at Chapel
Hill, Chapel Hill 27599 - 7365; and 2 Inorganic
Carcinogenesis Section, Laboratory of Comparative Carcinogenesis,
National Cancer Institute at the National Institute of Environmental
Health Sciences, Research Triangle Park, North Carolina 27709
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ABSTRACT |
Recently, glycine
has been shown to prevent liver injury
after endotoxin treatment in vivo. We demonstrated that ethanol and endotoxin stimulated Kupffer cells to release PGE2, which
elevated oxygen consumption in parenchymal cells. Because glycine has
been reported to protect renal tubular cells, isolated hepatocytes, and
perfused livers against hypoxic injury, the purpose of this study was
to determine whether glycine prevents increases in intracellular free
Ca2+ concentration ([Ca2+]i) in
hepatic parenchymal cells by agonists released during stress, such as
with PGE2 and adrenergic hormones. Liver parenchymal cells isolated from female Sprague-Dawley rats were cultured for 4 h in
DMEM/F12 medium, and [Ca2+]i in individual
cells was assessed fluorometrically using the fluorescent calcium
indicator fura 2. PGE2 caused a dose-dependent increase in
[Ca2+]i from basal values of 130 ± 10 to maximal levels of 434 ± 55 nM. EGTA partially prevented this
increase, indicating that either extracellular calcium or agonist
binding is Ca2+ dependent. 8-(Diethylamino)octyl
3,4,5-trimethoxybenzoate (TMB-8), an agent that prevents the release of
Ca2+ from intracellular stores, also partially blocked the
increase in [Ca2+]i caused by
PGE2, suggesting that intracellular Ca2+ pools
are involved. Together, these results are consistent with the
hypothesis that both the intracellular and extracellular
Ca2+ pools are involved in the increase in
[Ca2+]i caused by PGE2.
Interestingly, glycine, which activates anion (i.e., chloride)
channels, blocked the increase in [Ca2+]i due
to PGE2 in a dose-dependent manner. Low-dose strychnine, an
antagonist of glycine-gated chloride channel in the central nervous
system, partially reversed the inhibition by glycine. When
extracellular Cl
was omitted, glycine was much less
effective in preventing the increase in
[Ca2+]i due to PGE2.
Phenylephrine, an
1-type adrenergic receptor agonist,
also increased [Ca2+]i, as expected, from
159 ± 20 to 432 ± 43 nM. Glycine also blocked the increase
in [Ca2+]i due to phenylephrine, and the
effect was also reversed by low-dose strychnine. Together, these data
indicate that glycine rapidly blocks the increase in
[Ca2+]i in hepatic parenchymal cells due to
agonists released during stress, most likely by actions on a
glycine-sensitive anion channel and that this may be a major aspect of
glycine-induced hepatoprotection.
intracellular calcium; prostaglandin E2; strychnine
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INTRODUCTION |
GLYCINE PROTECTS HEPATOCYTES from injury caused by
cold ischemia and markedly reduces cell damage caused by
anoxia, potassium cyanide, and t-butyl hydroxide
(16). Glycine also protects pulmonary artery endothelial
cells from neutrophil-mediated cell death (25) and human
umbilical vein endothelial cells from calcium- and hydrogen peroxide-induced cell death (27). Similarly,
glycine protects suspensions of rabbit renal proximal tubules from
hypoxia and a variety of toxic agents such as cyanide, antimycin A,
rotenone, and ouabain (15, 26). In addition, glycine
diminished hypoxic and cold ischemic injury to perfused dogs
(1, 4). Recently, studies from this laboratory showed that
glycine minimized reperfusion injury in a low-flow, reflow liver
perfusion model and improved survival after endotoxin shock in rats
(8, 29). Glycine also minimized alcohol-induced liver
injury in an enteral alcohol delivery model (i.e., Tsukamoto-French
model) (7). Therefore, glycine can prevent cell death
induced by anoxia, oxidative stress, and various toxic agents at the
cell, organ, and whole body levels in a variety of species.
Although it is well known that calcium acts as an intracellular second
messenger in many important physiological processes, cell injury can
also be caused by elevation of intracellular free Ca2+
concentration ([Ca2+]i). For example,
oxidative stress is associated with mobilization of Ca2+
from intracellular stores; the resulting increase in Ca2+
alters hepatocyte morphology (e.g., formation of surface blebs), which
precedes cell death (17). Many studies have clearly shown that in rat liver parenchymal cells,
-adrenergic agonists,
vasopressin, and angiotensin II initially mobilize intracellular
Ca2+, elevating [Ca2+]i
(3). Recently, we demonstrated that ethanol and endotoxin stimulated Kupffer cells to release PGE2, which elevated
oxygen uptake in parenchymal cells (20). It is well known
that hypoxia and free radicals follow alcohol-induced stimulation of
hepatic oxygen metabolism. Because glycine has been shown to be
cytoprotective and inhibit nonlysosomal calcium-dependent proteolysis
during anoxic injury of rat hepatocytes (16), the purpose
of this study was to determine whether glycine affects the increase in
intracellular free Ca2+ in hepatic parenchymal cells caused
by agonists released during stress, such as PGE2 and
adrenergic hormones.
Glycine, a nonessential amino acid, is an inhibitory neurotransmitter
in the centrol nervous system. It is well known that activation of the
neuronal glycine-gated chloride channel causes an influx of chloride
and hyperpolarizes the nerve cell membrane, making opening of
voltage-gated Ca2+ channels on the cell surface more
difficult, thereby diminishing the response to a variety of agonists
that depolarize the cell membrane (2, 11, 28). This study
led to the conclusion that activation of a chloride channel in hepatic
parenchymal cells by glycine, which hyperpolarizes the cell membrane
and diminishes increases in intracellular Ca2+
concentrations due to PGE2 or phenylephrine, is similar to
its action in the neuron.
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METHODS |
Experimental animals.
Female Sprague-Dawley rats (200-240 g) were allowed free access to
laboratory chow and tap water. All animals were given human care in
compliance with institutional guidelines.
Chemicals.
EGTA, 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8),
PGE2, phenylephrine, glycine, and strychnine (Str) were
purchased from Sigma (St. Louis, MO).
Isolation and culture of parenchymal cells.
Hepatic parenchymal cells were isolated from rat livers according to
the method of Pertoft and Smedsrod (18). Briefly, livers were isolated under pentobarbital anesthesia (60 mg/kg ip) and perfused
in a nonrecirculating system with calcium-free Krebs-Ringer-HEPES buffer that contained EGTA (0.5 mM) for 10 min (pH 7.4, 37°C). The
liver was then perfused with Krebs-Ringer-HEPES buffer containing 0.02% type IV collagenase (Sigma) for 6-8 min until the tissue surrounding each lode became detached from the parenchyma. The liver
was placed in cold buffer, and hepatic parenchymal cells were dispersed
by gentle shaking and separated from other cells and liver debris by
centrifugation at 50 g for 2 min. Cells were subsequently
washed with Krebs-Henseleit bicarbonate buffer and collected by
centrifugation at 50 g for 2 min (21).
Viability of hepatic parenchymal cells was assessed routinely by light
microscopy and uptake of trypan blue and routinely exceeded 90%.
Isolated hepatic parenchymal cells were resuspended in DMEM/F12 culture medium and seeded onto 25-mm glass coverslips in 60-mm culture dishes
for [Ca2+]i measurements and cultured at
37°C in a 5% CO2 atmosphere. Cultured parenchymal cells
were used for this study within 8 h of isolation.
Measurement of intracellular calcium.
[Ca2+]i in individual parenchymal cell was
assessed fluorometrically using the fluorescent calcium indicator fura
2 and a microspectrofluorometer (22). Parenchymal cells
were incubated in DMEM/F12 culture medium that contained 5 µM fura
2-AM (Molecular Probes, Eugene, OR) and 0.06% Pluronic F127 (BASF
Wyandotte, Wyandotte, MI) at 37°C for 30-40 min. Coverslips
plated with parenchymal cells were rinsed and placed in a chamber with
Krebs-Ringer-HEPES buffer containing 1 mM MgSO4 and 5 mM
glucose at 25°C. Changes in fluorescence intensity of fura 2 at
excitation wavelengths of 340 and 380 nm were monitored in individual
cells with a PTI fluorescence analytical system (Photon Technology
International, South Brunswick, NJ) interfaced with a NIKON Diaphot
inverted microscope (23). Each value was corrected by
subtracting the system dark-noise and autofluorescence, assessed by
quenching fura 2 fluorescence with Mn2+.
[Ca2+]i was calculated as described by
Grynkiewicz et al. (6) and Ratto et al. (22)
from the equation [Ca2+]i = Kd ([R
Rmin]/[Rmax
R])(Fo/Fs), where
Fo/Fs is the ratio of fluorescent intensities
evoked by 380 nm light from fura 2 pentapotassium salt in buffered salt
solutions containing nanomolar Ca2+ and millimolar
Ca2+; R is the ratio of fluorescent intensities at
excitation wavelengths of 340 and 380 nm; and Rmin and
Rmax are values of R at nanomolar Ca2+ and
millimolar Ca2+, respectively. The values of these
constants were determined at the end of each experiment, and we used a
Kd (dissociation constant) value of 135 nM
(6).
In all experiments, hepatic parenchymal cells were incubated in
Krebs-Ringer-HEPES buffer and basal [Ca2+]i
was determined in the presence of 5 mM K+. Four to eight
separate parenchymal cells isolated from four rats were observed per
experiment for all data. From each 25-mm glass coverslip, one
individual cell was examined per field.
Measurement of chloride uptake.
Assays for chloride uptake were conducted as described previously
(13, 28). Briefly, isolated hepatic parenchymal cells (1 × 106 cells/ml) were plated on glass coverslips
and allowed to adhere for 1 h at 37°C. Medium was replaced with
buffer (in mM: 20 HEPES, 118 NaCl, 4.7 MgSO4, 2.5 CaCl2, and 10 glucose) and allowed to equilibrate for 10 min at room temperature. Coverslips were gently blotted dry and
incubated in a Petri dish with 2 ml of buffer containing 2 µCi/ml
36Cl in the presence of glycine (10 mM) and/or Str (10 µM) for 5 s. The coverslips were washed with ice-cold buffer,
which terminated chloride uptake. Radioactivity was detected by
liquid-scintillation spectroscopy using a Backman LC6000SC
scintillation counter (Beckman Instruments, Fullerton, CA). Flux
measured in glycine-free buffer was substracted from all values to
account for basal chloride movement across the cell membrane as well as
trapped radioactive chloride.
Statistical analysis.
Student's t-test or ANOVA followed by Tukey's honestly
significant difference multiple-comparison test were used as
appropriate. Differences were considered significant when the
P value was <0.05.
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RESULTS |
PGE2 increases intracellular
Ca2+ in isolated parenchymal cells in a
dose-dependent manner.
The increase of [Ca2+]i due to
PGE2 in an individual representative parenchymal cell is
illustrated in Fig. 1. PGE2
(10 nM) increased intracellular free Ca2+ about fourfold in
freshly isolated rat parenchymal cells. PGE2 caused a
dose-dependent increase in intracellular Ca2+ (Fig.
2). The concentration that caused a
half-maximal increase was ~1 nM PGE2.

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Fig. 1.
Effect of PGE2 on intracellular
Ca2+ ([Ca2+]i) in an isolated
parenchymal cell. Parenchymal cells isolated from a normal rat were
cultured for 4 h in DMEM/F12 medium, and
[Ca2+]i was assessed fluorometrically using
the fluorescent calcium indicator fura 2 as described in
METHODS. PGE2 (10 nM) was added as indicated by
the horizontal bar and arrow. The data are representative of
experiments repeated 8 times.
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Fig. 2.
Dose-response relationship between PGE2 and
[Ca2+]i. Experimental conditions as described
in Fig. 1. PGE2 was added at concentrations indicated on
the abcissa, and [Ca2+]i in freshly isolated
rat parenchymal cells was measured. Data are expressed as the
means ± SE (n = 8).
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Effect of EGTA and TMB-8 on PGE2-induced increases in
intracellular Ca2+.
The effect of EGTA and TMB-8 on PGE2-induced increases in
[Ca2+]i in isolated parenchymal cells is
shown in Fig. 3. To test the dependence
of PGE2-induced intracellular Ca2+ increase on
extracellular Ca2+, the external solution was replaced with
Ca2+-free Krebs-Ringer-HEPES buffer containing 1 mM EGTA, a
membrane-permeant chelator that is useful for the determination of
calcium in the presence of magnesium. The Ca2+-free
external solution prevented the increase in
[Ca2+]i caused by PGE2 by nearly
40%, suggesting that either extracellular calcium is involved or
agonist-binding is Ca2+ dependent. When isolated
parenchymal cells were incubated with 200 µM TMB-8, an agent that
blocks release of Ca2+ from intracellular stores, for 10 min before addition of 10 nM PGE2, the increase in
[Ca2+]i caused by PGE2 was almost
totally prevented, supporting the hypothesis that intracellular calcium
release occurs in response to PGE2. Because the major
intracellular calcium release channel in hepatocytes is well known to
be the inositol 1,4,5-trisphosphate (IP3) receptor,
isolated hepatic parenchymal cells were pretreated with heparin (10 µg/ml), an inhibitor of IP3-induced Ca2+
release, for 10 min, then treated with PGE2. Heparin also
almost totally prevented the increase in
[Ca2+]i caused by PGE2,
suggesting that PGE2-induced increase in
[Ca2+]i is involved in
IP3-induced Ca2+ release in hepatic parenchymal
cells. Furthermore, a mixture of EGTA (1 mM) and TMB-8 (200 µM)
pretreated cells for 10 min, then treated with PGE2 (10 nM). As expected, a mixture of these inhibitors totally blocked
the [Ca2+]i increase due to PGE2.

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Fig. 3.
Effect of EGTA and 8-(diethylamino)octyl
3,4,5-trimethoxybenzoate (TMB-8) on PGE2-induced increases
in [Ca2+]i. The external buffer solution was
replaced with Ca2+-free Krebs-Ringer-HEPES buffer
containing 1 mM EGTA, and intracellular free Ca2+ was
measured following addition of PGE2 in freshly isolated rat
parenchymal cells. In addition, isolated parenchymal cells were
preincubated with 200 µM TMB-8 for 10 min before addition of 10 nM
PGE2 and [Ca2+]i was measured.
Data are expressed as the means ± SE (n = 5).
a P < 0.05 for comparison between basal
and peak values by ANOVA. b P < 0.05 for comparison between PGE2 treatment and PGE2
with Ca2+-free buffer or TMB-8 treatment by ANOVA.
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Effect of glycine on PGE2-induced increases in
intracellular Ca2+.
Parenchymal cells were preincubated with 1 mM glycine for 3-4 min
before addition of PGE2 (10 nM), and intracellular free Ca2+ concentrations were measured. Glycine (1 mM), which
activates an anion (chloride) channel, significantly diminished the
increase in [Ca2+]i due to PGE2
(Fig. 4). Peak values were only 30% as
high in the presence of glycine as in its absence. Furthermore, glycine diminished the increase in [Ca2+]i due to
PGE2 in a dose-dependent manner (Fig.
5). The concentration of glycine that
reduced PGE2-induced [Ca2+]i to a
half-maximal level was ~0.8 mM. High concentrations of glycine (10 mM) totally blocked the increase in [Ca2+]i
due to PGE2, yet glycine per se had no effect on
[Ca2+]i in isolated parenchymal cells
(130 ± 10 nM in basal vs. 136 ± 8 nM in glycine treatment).
To address the specific effect of glycine, parenchymal cells were also
preincubated with alanine (1 mM) or valine (1 mM) for 3-4 min
before addition of PGE2 (10 nM), and
[Ca2+]i was measured. Neither alanine nor
valine, when used at the same concentration as glyicne, had an effect
on PGE2-induced increases in
[Ca2+]i in isolated liver parenchymal cells
(Fig. 6). Furthermore, alanine or valine
per se had no effect on [Ca2+]i in isolated
liver parenchymal cells. Thus the effect of glycine appears to be
relatively specific.

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Fig. 4.
Glycine minimizes increases in intracellular free
Ca2+ due to PGE2. Experimental conditions as
described in Fig. 1. Parenchymal cells were preincubated with 1 mM
glycine for 3-4 min before addition of PGE2
concentration (10 nM), and [Ca2+]i was
measured. Data are expressed as means ± SE (n = 4).
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Fig. 5.
Effect of glycine concentration on
PGE2-induced increases in [Ca2+]i
by isolated parenchymal cells. Experimental conditions as described in
Fig. 1. Isolated parenchymal cells were preincubated with various
concentrations of glycine indicated on the abcissa for 3-4 min
before addition of PGE2 (10 nM), and intracellular free
Ca2+ was measured. Peak values of
[Ca2+]i were plotted. Data are expressed as
the means ± SE (n = 4). *P < 0.01 compared with control group by ANOVA; r, correlation
coefficient.
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Fig. 6.
Effect of various amino acids on PGE2-induced
increases in [Ca2+]i. Experimental conditions
as described in Fig. 1. Cultured parenchymal cells were incubated with
1 mM glycine, alanine, or valine for 3 min before addition of
PGE2, and peak values of [Ca2+]i
after stimulation by PGE2 (10 nM) were measured as
described in METHODS. Data are expressed as means ± SE (n = 4). a P < 0.01 for comparison between basal and peak values.
b P < 0.01 for comparison between
PGE2 treatment and PGE2 with glycine.
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Str and chloride-free media reverse the effect of glycine on
PGE2-induced increases in intracellular
Ca2+.
It is well known that at low concentrations, Str is a glycine
antagonist, whereas high concentrations of Str act as a glycine agonist
(9). Therefore, isolated liver parenchymal cells were incubated with Str (10 µM or 1 mM) for 3-4 min before addition of 10 mM glycine. Three minutes later, PGE2 (10 nM) was
added and [Ca2+]i was measured. Low
concentrations of Str (10 µM) partially reversed the inhibitory
effect of glycine on PGE2-induced increases in [Ca2+]i (Fig.
7). When extracellular Cl
was replaced with gluconate, the inhibitory effect of glycine on the
increase in [Ca2+]i due to PGE2
was reduced by about one-third. However, high concentrations of Str (1 mM) most likely act as an anion channel agonist similar to glycine
(Fig. 7). Str by itself had no effect on
[Ca2+]i in isolated parenchymal cells. For
instance, [Ca2+]i in isolated parenchymal
cells is 117 ± 8 nM with low concentration of Str treatment (10 µM) and 133 ± 9 nM with high concentration of Str treatment (1 mM). Radiolabeled chloride is used routinely in cells to provide hard
evidence for movement of ions from the extracellular to the
intracellular space (28). Accordingly, hepatic parenchymal
cells were incubated with 36Cl treated with glycine (10 mM). Indeed, glycine increased chloride uptake by hepatic parenchymal
cells about threefold. This increase of chloride influx by glycine was
reduced significantly by Str (10 µM) pretreatment. This result
provides direct evidence that hepatic parenchymal cells express
glycine-gated chloride channels.

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Fig. 7.
Strychnine (Str) and chloride-free buffer reverse the
effect of glycine on PGE2-induced increases in
[Ca2+]i. Experimental conditions as described
in Fig. 1. Isolated parenchymal cells were preincubated with Str (10 µM or 1 mM), a glycine-gated chloride channel antagonist in the
central nervous system, for 3-4 min before addition of 10 mM
glycine. PGE2 (10 nM) was added, and intracellular free
Ca2+ was measured. In some experiments, extracellular
Cl was replaced with gluconate. Data are expressed as the
means ± SE (n = 4-5, ANOVA test).
a P < 0.01 for comparison between basal
and peak values. b P < 0.01 for
comparison between PGE2 treatment and PGE2 with
glycine or high concentrations of Str (1 mM).
c P < 0.01 for comparison between
glycine treatment and low concentrations of Str (10 µM) with glycine
or chloride-free buffer with glycine.
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Glycine prevents the increase in intracellular free
Ca2+ due to phenylephrine.
It is well known that in rat liver parenchymal cells, the
-adrenergic agonists vasopressin and angiotensin II initially
stimulate Ca2+ mobilization from intracellular storage
depots, which elevates [Ca2+]i
(3). The second message for this mobilization in rat
parenchymla cells is IP3 (3). Studies were
performed to examine whether glycine could affect adrenergic
agonist-induced elevation in [Ca2+]i. Results
showed phenylephrine (10 µM), which acts via
1-adrenergic receptors, increases
[Ca2+]i from a basal concentration of ~130
to ~430 µM (Fig. 8). However, glycine
blocked the increase in [Ca2+]i due to
phenylephrine (Fig. 9). Low
concentrations of Str (10 µM) partially reversed the inhibitory
effect caused by glycine (Fig. 9). When extracellular Cl
was omitted, glycine was also much less effective. However, high concentrations of Str (1 mM) most likely act as an anion channel agonist similar to glycine (Fig. 9).

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Fig. 8.
Glycine prevents the increase in intracellular free
Ca2+ due to phenylephrine. Experimental conditions as in
Fig. 1. Parenchymal cells were preincubated with 1 mM glycine for
3-4 min before addition of phenylephrine (10 µM), and
[Ca2+]i was measured. Data are expressed as
the means ± SE (n = 4).
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Fig. 9.
Str and chloride-free buffer reverse the effect of
glycine on phenylephrine-induced increases in
[Ca2+]i. Experimental conditions as described
in Fig. 1. Isolated parenchymal cells were preincubated with Str (10 µM or 1 mM) for 3-4 min before addition of 10 mM glycine.
Phenylephrine (10 µM) was added, and intracellular free
Ca2+ was measured. In some experiments, extracellular
Cl was replaced with gluconate. Data are expressed as
means ± SE (n = 4-5, ANOVA test).
a P < 0.01 for comparison between basal
and peak values. b P < 0.01 for
comparison between phenylephrine treatment and phenylephrine with
glycine or high concentrations of Str (1 mM).
c P < 0.01 for comparison between
glycine treatment and low concentrations of Str (10 µM) with glycine
or chloride-free buffer with glycine.
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DISCUSSION |
PGs are locally acting hormones that have a remarkable variety of
physiological actions in nearly all mammalian tissues
(19). Four subtypes of PGE2 receptors
(EP1, EP2, EP3, and
EP4) have been characterized pharmacologically on the basis
of the relative agonist potencies of a number of different analogs of
PGE2 from at least one species (10, 19). The
specific receptor subtypes are coupled to different signal-transduction
pathways. For instance, EP1 receptors are coupled to
inositol phospholipid turnover, phosphatidylinositol-4,5-bisphosphate in the plasma membrane to produce two intracellular messengers: myoinositol IP3 and 1,2-diacylglycerol. IP3
causes release of intracellular Ca2+ from intracellular
stores, producing a rapid rise in cytosolic Ca2+.
EP2 and EP4 receptors act via Gs
proteins to mediate an increase in cAMP, whereas EP3
receptors are coupled to Gi and decrease cAMP (12,
19). Previously, Qu et al. (20) demonstrated that alcohol and endotoxin stimulated hepatic Kupffer cells to release PGE2, which, in turn, stimulated oxygen uptake in isolated
parenchymal cells. This study demonstrated that PGE2
increased intracellular calcium about fourfold in isolated rat liver
parenchymal cells. It is most likely that in hepatic parenchymal cells,
PGE2 stimulates increases in
[Ca2+]i via the EP1 subclass of
receptors. It has been reported that hepatic parenchymal cells maintain
intracellular total and cytosolic free Ca2+ levels by entry
of Ca2+ through channels, extrusion of Ca2+ by
an outwardly directed Ca2+ pump, and controlled
sequestration into intracellular pools (5). In this
regard, this study demonstrated that the PGE2-induced increase in [Ca2+]i is dependent on
extracellular calcium, because a Ca2+-free external
solution together with EGTA perturbed the increase in
[Ca2+]i due to PGE2. On the other
hand, TMB-8, an intracellular calcium antagonist, also prevented the
increase in [Ca2+]i caused by
PGE2, suggesting that intracellular calcium release is also
involved in the cellular response to PGE2. On the basis of
these results, it appears that PGE2 not only affects influx of calcium from the extracellular space but also affects calcium release from intracellular stores. However, because the exact mechanism
by which Ca2+ is released from intracellular organelles
remains unclear (5), additional research will be needed
for the elucidation of the exact mechanisms by which vasoactive
mediator-stimulated increases in Ca2+ entry into the cell
and regulation of Ca2+ from intracellular pools.
Glycine, a nonessential amino acid, has been reported to be protective
against hypoxia, ischemia, and various cytotoxic substances in
renal proximal tubules via glycine-gated chloride channels (22). It was reported that dietary glycine prevents liver
and lung injury due to lethal does of lipopolysaccharide (LPS) in the
rat (8). Previous studies (9, 28) from our
laboratory showed that both Kupffer cells and neutrophils contain a
glycine-gated chloride channel. Recently, studies (13)
from this laboratory have demonstrated that dietary glycine prevents
peptidoglycan polysaccharide-induced reactive arthritis in the rat by
reducing cytokine release from macrophages by increasing chloride
influx via a glycine-gated chloride channel. More recently, Turecek et al. (24a) reported that glycine induced large
Str-sensitive ionic currents whose reversal potential shifted with
changes in concentration of intracellular Cl
, indicating
that glycine activated the Cl
channel. The
present study showed that glycine significantly reduced the increase in
[Ca2+]i due to PGE2 or the
adrenergic compound phenylephrine in hepatic parenchymal cells. The
structurally related amino acids alanine and valine had no effect,
suggesting that the effect of glycine is specific and the inhibitory
effect of glycine on PGE2- or phenylephrine-induced increases in [Ca2+]i in liver parenchymal
cells occurs via glycine receptors, as in neurons. Glycine is well
known as an inhibitory neurotransmitter in the central nervous system,
and Str is an antagonist of a glycine-gated chloride channel
(11). Here, we demonstrated that low concentration of Str
(10 µM) largely reversed the inhibitory effect due to glycine, restoring the increase in [Ca2+]i due to
PGE2 or phenylephrine. These data provide clear evidence for the presence of glycine-gated chloride channels in rat hepatic parenchymal cells. Furthermore, to confirm parenchymal cells contain glycine-gated chloride channels similar to the central nervous system,
we used chloride-free buffer to prevent chloride influx. Indeed,
substitution of chloride with an impermeable anion, gluconate, largely
prevented the inhibitory effect of glycine on PGE2- or phenylephrine-induced increases in [Ca2+]i.
These results indicate that the effect of glycine is dependent on
extracellular chloride.
Because glycine can totally prevent increases in
[Ca2+]i due to PGE2 or
phenylephrine, it is suggested that glycine not only affects influx of
calcium from the extracellular space but also prevents release from
intracellular stores. The exact mechanism for this effect of glycine is
not well known. It has been reported that the IP3-gated
chloride channel on the endoplasmic reticulum may be inactivated when
the potential difference across the membrane is increased
(14). Therefore, one possible explanation is that influx of chloride across the cell membrane also increases the potential difference across the endoplasmic reticulum, making the
IP3 receptor-mediated calcium channel more difficult to
open (28).
It has been reported that high concentrations of Str are protective
against hypoxic injury in renal tubules (15) and in the
perfused liver (29) and reduce increases in
[Ca2+]i in Kupffer cells (9),
which is paradoxical to its effects on the glycine-gated chloride
channel (28). In this study, a high concentration of Str
(1 mM) also prevented increases in [Ca2+]i
due to PGE2 or phenylephrine, essentially like glycine. On the basis of these data, it is concluded that Str at high
concentrations is also an agonist for the glycine-gated chloride
channel in liver parenchymal cells.
In accordance with the results of the present study, we proposed a
possible mechanism of action of glycine in rat liver parenchymal cells
(Fig. 10). When hepatic parenchymal
cells are stimulated with vasoactive mediators such as PGE2
or phenylephrine, signal-transduction pathways are activated that
change the potential differences across both the cell and endoplasmic
reticulum membranes, resulting in the increase in intracellular
calcium. In the presence of glycine, a glycine-gated chloride channel
is activated causing an influx of chloride, leading to
hyperpolarization of the parenchymal cell membrane, which makes calcium
channels on the plasma membrane more difficult to open and inhibits the
influx of calcium. Moreover, influx of chloride could also inactivate
the IP3-gated calcium channels and block release of calcium
from intracellular stores. The increase in intracellular calcium caused
by vasoactive mediators, such as PGE2 or phenylephrine, is
indeed reduced by glycine. Furthermore, glycine has been reported to
have several benefitial effects, including protection against toxicity
induced by anoxia, oxidative stress, and various toxic agents at the
cell, organ, and whole body levels (15, 16, 25, 26). For
instance, it was demonstrated that a diet enriched with glycine
protects against endotoxin (LPS)-induced lethality, hypoxia-reperfusion
injury after liver transplantation, D-galactosamine-mediated liver injury, and experimental
arthritis (13, 24). This study demonstrated that glycine
perturbed the increase in [Ca2+]i in
hepatocytes caused by agonists released during stress, such as
PGE2 and adrenergic hormones. Thus inhibition of
Ca2+ signals through glycine-gated chloride channels using
glycine as a dietary supplemental nutrient could represent a new
strategy for prevention of liver injury due to production of
stress-related mediators.

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|
Fig. 10.
Scheme depicting the possible mechanisms by which
glycine blocks the increase in intracellular free Ca2+ due
to PGE2 and phenylephrine in hepatic parenchymal cells.
Glycine blocks the increase in intracellular free Ca2+ due
to PGE2 and phenylephrine most likely by mechanisms
involving glycine-sensitive Cl channel, inositol
1,4,5-trisphosphate (IP3)-activated Ca2+ efflux
and receptor-operated Ca2+ influx. -, -adrenergic
receptor; GLY, Glycine; G, G protein; PLC, Phospholipase C;
PIP2, phosphatidylinositol-4,5-bisphosphate.
|
|
 |
ACKNOWLEDGEMENTS |
We thank Drs. L. K. Keefer, J. Liu, and J. Pi for critical
review of this manuscript.
 |
FOOTNOTES |
This work was supported by National Institute on Alcohol Abuse and
Alcoholism Grants AA-09156 and AA-03624.
Address for reprint requests and other correspondence: W. Qu, Inorganic Carcinogenesis Section, NCI at NIEHS, P.O. Box 12233, Mail Drop F0-09, 111 Alexander Dr., Research Triangle Park, NC 27709 (E-mail: qu{at}niehs.nih.gov).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
August 14, 2002;10.1152/ajpgi.00197.2002
Received 24 May 2002; accepted in final form 12 August 2002.
 |
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