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


    ABSTRACT
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ABSTRACT
INTRODUCTION
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DISCUSSION
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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 alpha 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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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, alpha -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.


    METHODS
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INTRODUCTION
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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.


    RESULTS
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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).

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.

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.

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.

Glycine prevents the increase in intracellular free Ca2+ due to phenylephrine. It is well known that in rat liver parenchymal cells, the alpha -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 alpha 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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

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. alpha -, alpha -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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 283(6):G1249-G1256