In cell injury due to hypoxia (anoxia) or chemical energy depletion (in the following collectively named as hypoxic cell injury), it is generally accepted that protection by glycine requires the presence of the amino acid but does not rely on its metabolism, on protein synthesis, on changes in cytosolic calcium or on the maintenance of the intracellular pH and GSH pool, and that glycine does neither support energy (ATP) generation nor help to diminish energy consumption (Baines et al., 1990; Weinberg et al., 1990a; 1991b; 1994; Dickson et al., 1992; Garza-Quintero et al., 1993; Brecht and de Groot, 1994; Churchill et al., 1995; Sakaida et al., 1996; Nagatomi et al., 1997). On the other hand, glycine (and also alanine) decreases proteolysis in the hypoxic cells, especially the one catalysed by Ca2+-dependent, non-lysosomal proteases (including calpains) (Dickson et al., 1992; Nichols et al., 1994; Tijsen et al., 1997). Inconsistent results have been reported for the effects of glycine on the accelerated phospholipid degradation (Venkatachalam et al., 1995; Sakaida et al., 1996) and on plasma membrane blebbing under conditions of hypoxia and reoxygenation (Dickson et al., 1992; Garza-Quintero et al., 1993; Brecht and de Groot, 1994; Venkatachalam et al., 1996).
In energy-depleted isolated renal proximal tubules, strychnine and other glycine receptor antagonists – at millimolar levels, well above micromolar and submicromolar concentrations that antagonize the neuronal glycine receptor – were as protective as glycine (Aleo and Schnellmann, 1992; Miller and Schnellmann, 1993; Moran and Schnellmann, 1997). Both, strychnine and glycine prevented chloride uptake, and chloride channel inhibitors provided protection as well. Based on these results, involvement of the glycine receptor in cytoprotection was proposed assuming that at high (cytoprotective) concentrations, glycine inhibits opening of the chloride channel. Protection by strychnine from hypoxic injury has subsequently been shown for several cell types, including hepatocytes, and for the isolated perfused liver (Currin et al., 1996; Zhong et al., 1996; Carini et al., 1997; Zhang et al., 2003). Moreover, in kidney epithelial (MDCK) cells, chloride channel blockers proved to be protective as well (Venkatachalam et al., 1996), and in energy-depleted hepatocytes, chloride-free medium was described to be protective (Carini et al., 1997). In further support of an essential role of the glycine receptor in the protective function of glycine, in HEK-293 cells lacking the glycine receptor, protection by glycine was restored by transfection with the α1-subunit of the glycine receptor (Pan et al., 2005). Furthermore, in the transfected HEK-293 cells and in MDCK cells (possessing the receptor), protection by glycine could be suppressed by RNA interference with the α1-subunit of the glycine receptor.
In obvious contradiction to the above proposal that glycine protects by inhibiting opening of a chloride channel of a putative glycine receptor, in hypoxic isolated (cultured) rat hepatocytes and energy-depleted MDCK cells and neuronal (PC-12) cells, removal of extracellular chloride did not protect while glycine was clearly protective (Venkatachalam et al., 1996; Frank et al., 2000; Zhang et al., 2003). Likewise, reperfusion of cold-stored rat livers with warm chloride-free buffer did not reduce reperfusion-induced injury of non-parenchymal cells and was without effect on the protection provided by glycine under these conditions (Currin et al., 1996). Furthermore, in hepatocytes and endothelial cells, even stimulation of chloride influx by glycine has been reported (at high, protective concentrations and inhibitable by strychnine at micromolar concentrations) (Yamashina et al., 2001; Qu et al., 2002; Yamashina et al., 2007). On the other hand, it is still unclear whether functional glycine receptors do exist in hepatocytes, endothelial and renal cells (Froh et al., 2002; van den Eynden et al., 2009).
Based on the results of their experiments with MDCK cells (protection by glycine, strychnine, and a variety of chloride channel blockers but lack of protection by chloride-free medium), Venkatachalam and coworkers proposed that glycine-gated chloride channel receptors are central components of multimeric proteins forming plasma membrane pores under injurious conditions (pathological pores), but that these pores are unrelated to the chloride channel activity of the glycine receptor (Venkatachalam et al., 1996). Glycine was suggested to prevent the formation of these pores. Using the same experimental model and studying the permeability characteristics of fluoresceinated dextrans of graded molecular size, they subsequently showed that the membrane defects evolve from small pores permeable only to propidium iodide (668 Da) and the smallest dextrane (4000 Da), before enlarging with time to become permeable to dextrans up to 145 000 Da (Dong et al., 1998). In these experiments, pore formation could not only be prevented by glycine but also by a membrane-impermeant homobifunctional ‘nearest-neighbour’ cross-linking agent and, in later experiments, by an impermeant strychnine derivative (Dong et al., 2001). Evidence for the formation of pathological pores, unrelated to the chloride channel activity of the glycine receptor, under conditions of energy deficiency and its prevention by glycine has also been presented in experiments with other cell types. In isolated cultured rat hepatocytes, glycine prevented a hypoxia-induced influx of the cations sodium, cobalt and nickel and an efflux of the anion Newport Green (Frank et al., 2000). In cultured hepatic sinusoidal endothelial cells, upon induction of chemical hypoxia, there was a delayed increase in the permeability of the plasma membrane to the anionic fluorophores calcein and lucifer yellow followed, with a time lag, by increased permeabilities to the cation propidium and to high molecular weight dextrans (40–2000 kDa) (Nishimura and Lemasters, 2001). These alterations were largely decreased or prevented by glycine. Entry of anions through the pathological pores (paralleled by sodium entry due to inhibition of Na,K-ATPase and opening of monovalent cation channels) was suggested to lead to cell swelling and bleb formation by colloid osmotic forces, ultimately resulting in plasma membrane rupture and thus full permeability to both low and high molecular weight solutes.
Overall, there is compelling evidence that cytoprotection mediated by glycine in hypoxic cell injury results from prevention of an increased permeability of the plasma membrane. The underlying structural alterations and the mechanism of their prevention by glycine, however, are largely unknown. Although most likely being formed, a pathological pore has never been identified, presumably because such a pore is not a fixed (permanent) entity, but several of such pores of different composition and characteristics develop in the course of injury (Figure 5). Since, however, agonists of the glycine receptor other than glycine but also antagonists of the glycine receptor as well as inhibitors of chloride channels proved to be protective as well, a relationship between the glycine receptor or components of this receptor and the formation of a pathological pore appears to be likely. However, all these protective compounds had to be applied at high concentrations, and cytoprotection was also achieved by compounds, such as alanine and serine (Figure 1), which are closely related to glycine but only poorly interact with the glycine receptor (Baines et al., 1990; Garza-Quintero et al., 1990; 1993; Mandel et al., 1990; Weinberg et al., 1990b; 1992; Silva et al., 1991; Dickson et al., 1992; Heyman et al., 1992a; Paller and Patten, 1992; Marsh et al., 1993; Brecht and de Groot, 1994; Nichols et al., 1994; Frank et al., 2000; Zhang et al., 2003). According to these results, and still in line with the other results, at least under certain conditions proteins only structurally related to components of the glycine receptor (but not components of the glycine receptor themselves) appear to be involved in the formation of the pathological pores.
Figure 5. Formation of pathological plasma membrane pores during hypoxic injury and its prevention by glycine. During hypoxic cell injury, pathological pores are formed in the plasma membrane with increasing size. These pores are composed of different proteins, with components of GlyR presumably playing a central role. Glycine prevents the formation of these pores by binding to the GlyR components but possibly also to other structurally related proteins.
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The formation of pathological plasma membrane pores and its prevention by glycine accounts for several of the effects of glycine on injurious alterations in hypoxic cells such as inhibition of the influx of sodium or calcium and, partly that way, of the activation of proteases and phospholipases. The formation of the plasma membrane pores (in their final forms), however, is a very late event in hypoxic cell injury (Dong et al., 1998; Frank et al., 2000; Nishimura and Lemasters, 2001) as also indicated by the observation that addition of glycine 1 h after energy depletion still provided effective protection (Dickson et al., 1992). This implies, on the other hand, that other injurious cellular alterations triggered by ATP depletion but not affected by glycine occur prior (upstream) to the formation of the pathological plasma membrane pores and/or independent of plasma membrane pore formation. An important example for the latter possibility appears to be the opening of the mitochondrial permeability transition pore and/or the development of other mitochondrial defects such as damage to complex I (Qian et al., 1997; Weinberg et al., 1997; 2000; Park et al., 2011). Due to these lesions, mitochondrial energy production may already be severely (irreversibly) impaired at a time point when the plasma membrane still maintains its selective permeability due to the protection provided by glycine, thus paradoxically indicating a still viable cell. Accordingly, withdrawal of glycine without recovery of the mitochondrial function led to a rapid loss of plasma membrane protection and thus cell death (Weinberg et al., 1997; 2000; Park et al., 2011).
There are, however, some observations, which, at least at first sight, are not compatible with the assumption that glycine protects from hypoxic cell injury by preventing the formation of plasma membrane pores. Examples are an involvement of ERK1/2 and Akt signalling pathways in glycine cytoprotection (Jiang et al., 2011) or direct inhibition of cytosolic proteases already at 2 mM glycine (Ferguson et al., 1993). The significance of these observations, however, remains to be established.
The results of the few studies on the protection by glycine against cell injury due to ROS or upon reoxygenation (reperfusion) provide only very limited and partly inconclusive information on the underlying protective mechanism. Thus, there is some evidence that glycine cannot protect from membrane injury due to lipid peroxidation (Sogabe et al., 1996). On the other hand, an increase in cellular GSH achieved by pre-treatment with glycine may contribute to its protective function against oxidative challenge due to tert-butylhydroperoxide treatment (Howard et al., 2010). In isolated hepatocytes where cell injury was induced by combining hypoxia–reoxygenation with restoration of the pH from 6.2 to 7.4, protection by glycine, added at reoxygenation/pH restoration, was independent of the presence of chloride in the medium, and no evidence for an effect of glycine on mitochondrial permeability transition pore opening was found (Qian et al., 1997). In contrast, under comparable conditions, opening of the permeability transition pore appears to be prevented by glycine in cardiomyocytes (Ruiz-Meana et al., 2004). In these experiments, glycine even blocked opening of the transition pore in the isolated mitochondria.
Inhibition of the inflammatory response
Thurman and coworkers were the first to suggest that glycine protects from ischaemia–reperfusion injury by inhibiting activation of macrophages and other cells of the immune system and thus the inflammatory response (Figure 4). In experiments with rat Kupffer cells, the resident macrophages in the liver, they demonstrated that their activation by lipopolysaccharides (LPS) was largely diminished by glycine with a half-maximal effect at c. 200 µM glycine (Ikejima et al., 1997). Since glycine blunted the LPS-induced increase in the cytosolic calcium concentration, and since the glycine effects were prevented by 1 µM strychnine or chloride-free buffer, they proposed that glycine activates a glycine-gated chloride channel, which hyperpolarizes the plasma membrane and thus inhibits calcium influx and activation of the Kupffer cells, similar to its action in neuronal cells (Figure 3); in these experiments, a high concentration of strychnine (1 mM) mimicked the glycine effects, while 1 mM alanine was without any effect.
Meanwhile, the existence of glycine-gated chloride channels on cells of the immune system, especially on macrophages and neutrophils, is generally accepted (Froh et al., 2002; van den Eynden et al., 2009). Inhibition of activation by glycine has been shown for neutrophils (half-maximal effect around 0.3 mM) (Wheeler et al., 2000), splenic macrophages (half-maximal inhibition at 0.55 mM) (Li et al., 2001), alveolar macrophages (half-maximal effect already at around 10 µM) (Wheeler and Thurman, 1999) and cultured mononuclear cells (CD4+ T lymphocytes) (half-maximal effect at c. 1 mM) (Bruck et al., 2003). On the other hand, for Kupffer cells, a lack of blockade of LPS-induced TNF-α formation by glycine has been reported (Currin et al., 1996), and in peritoneal macrophages, glycine pre-treatment for hours to days even improved TNF-α and NO formation following activation by LPS, an effect that was suggested to be mediated by neutral amino acid transporters (Carmans et al., 2010). In accordance with its capability of inhibiting cells of the immune system, glycine has been shown to protect from a variety of injurious processes where inflammation decisively triggers or amplifies the injurious process such as in endotoxin shock (Wheeler et al., 1999; Zhong et al., 2003).
Blood plasma glycine concentration has been reported to vary between 170 and 330 µM both in humans and in experimental animals (Evins et al., 2000; Iresjöet al., 2006; Petrat et al., 2011). After a meal, plasma glycine concentration may increase from 250 to 330 µM (Iresjöet al., 2006). The structurally related amino acids alanine, serine and taurine are present in blood plasma at concentrations around 220 to 620, 70 to 180 and 40 to 100 µM respectively (Iresjöet al., 2006). Due to active uptake, the intracellular glycine concentrations are significantly higher than the extracellular levels (Weinberg et al., 1991c; Weinberg, 1992). Exceptionally high glycine concentrations exist in the kidney with more than 20 mM glycine in the tubular cells of the rabbit renal cortex. The glycine concentrations in blood plasma and the extracellular space equilibrate within minutes (Hahn et al., 1999; Hahn, 2006a). The half-life of glycine in the blood depends on the dose administered and may vary between half an hour and several hours. The majority of glycine administered is taken up by cells and metabolized, primarily in the liver. Only a minor amount is excreted in the urine.
Remarkably, the physiological presence of glycine under in vivo conditions has not been taken into consideration in the discussion of its protective mechanism(s) so far. The only exception is the kidney, where the very high glycine content of the tubule cells has been suggested to be responsible for the missing protective effect of glycine treatment under in vivo conditions (Weinberg, 1992). Upon energy depletion, glycine of the tubule cells is assumed to leak into the extracellular space reaching concentrations high enough to fully protect (still viable) cells from hypoxic injury. Upon reperfusion, however, this protection should be lost due to washout of glycine.
Studies on the protective properties of glycine and related compounds in primary cells, in cell lines and in isolated perfused or stored organs have been, as far as we can see, exclusively performed in the absence of blood, plasma or serum, using buffered salt solutions as incubation, perfusion or storage medium. Under these conditions, not only in cells but also in perfused or stored organs, direct cytoprotection should be the preferred mode of protection by glycine (Figure 4). In primary cells and cell lines, glycine necessarily prevents injury this way. In perfused or stored organs, protection is not only achieved by glycine but also by glycine receptor antagonists and glycine-related compounds, which only poorly activate the glycine receptor (Baines et al., 1990; Silva et al., 1991; Heyman et al., 1992a). In addition, in the absence of blood, the immune response is impaired and thus should contribute less to the injurious process. In marked contrast to the in vitro systems, clear evidence for the involvement of direct cytoprotection in the protection provided by glycine against ischaemia–reperfusion injury under in vivo conditions is missing.
In contrast to direct cytoprotection, protection from ischaemia–reperfusion injury provided by inhibition of the inflammatory response should mainly play a role in the presence of blood and thus under in vivo conditions, and here due to its requirement of oxygen especially in the reperfusion phase (Figure 4). In line with this mechanism of protection, in those in vivo experiments where glycine protected from ischaemia–reperfusion injury (and where parameters of the immune system were determined), a concomitant decrease in inflammation has been reported (Zhong et al., 1999; Mauriz et al., 2001; Wang et al., 2004; Rentsch et al., 2005; Duenschede et al., 2006; Liu et al., 2006; Yamanouchi et al., 2007; Schaefer et al., 2008; Bruns et al., 2011; Sheth et al., 2011). In all cases, however, the cause–effect relationship remained unclear. A decrease in the inflammatory response may merely result from a decrease in upstream injurious events such as a decrease in cell injury due to direct cytoprotection by glycine. On the other hand, protection by glycine receptor agonists other than glycine (Schemmer et al., 2005; Kincius et al., 2007; Bruns et al., 2011) and the lack of protection by alanine (Zhong et al., 1999) support the notion that inhibition of the inflammatory response is the decisive protective mechanism of glycine against ischaemia–reperfusion injury in vivo.
Both, direct cytoprotection and inhibition of the inflammatory response (Figure 4), occurred at half-maximal glycine concentrations of around 0.4 mM (see above), that is somewhat above the physiological range of the plasma (interstitial) glycine concentration. Accordingly, both protective mechanisms should be already operative under normal (physiological) in vivo conditions. Thus, to provide significant additional protection, glycine doses high enough to increase the plasma glycine concentration close to 1 mM should be required. In accordance with this postulation, plasma glycine concentrations of 1 mM and above were indeed achieved in the vast majority of the in vivo studies where protection by glycine was reported (see above). On the other hand, there is a significant number of studies where protection against ischaemia–reperfusion injury was already attained at low glycine or taurine doses (den Butter et al., 1993a; Zhong et al., 1999; Wang et al., 2004; Kincius et al., 2007; Petrat et al., 2011; Sheth et al., 2011). At these low-dose treatment regimens, the plasma glycine concentration does not increase to a level to elicit (additional) direct cytoprotection or inhibition of the inflammatory response. Thus, at least under these conditions, the mechanism of protection by glycine against ischaemia–reperfusion injury remains elusive, and alternative mechanisms of protection need to be considered such as stimulation of intracellular protective signalling pathways like those mediated by Akt (PKB) and glycogen synthase kinase 3β (GSK3β) (Heusch et al., 2008). This possibility was studied in intestinal ischaemia–reperfusion injury in rats, however, with a negative finding (unpubl. results). Ischaemia and reperfusion (10 min and 5 min) significantly increased phosphorylation of Akt without altering phosphorylation of GSK3β. Pre-treatment with 20 mg glycine per kilogram (i.v. infusion for 30 min before ischaemia) was without any effect on the phosphorylation of both kinases.