Evidence for reactive nitrogen species formation in the gingivomucosal tissue.

An increase in nitric oxide production has been demonstrated in periodontitis. Here we investigated the potential role of nitric-oxide-derived nitrating species (such as peroxynitrite) in a rat model of ligature-induced periodontitis. Formation of 3-nitrotyrosine, the stable product formed from tyrosine reacting with nitric-oxide-derived nitrating species, was detected in the gingivomucosal tissue. 3-Nitrotyrosine immunohistochemical analysis revealed a significant elevation in the number of immunopositive leukocytes, and higher immunoreactivity of the gingival ligaments and epithelium in the ligated than in the contralateral (control) side. On both sides, several 3-nitrotyrosine-positive bands and, on the ligated side, a unique 52-kDa 3-nitrotyrosine-positive band were detected by Western blot. However, in the sterile gingivomucosal tissue of rat pups, no 3-nitrotyrosine or inducible nitric oxide synthase immunoreactivity was found. Analysis of these data suggests that resident bacteria of the gingivomucosal tissue induce an increase in reactive nitrogen species, which is greatly enhanced by plaque formation in periodontitis.

Evidence for the expression of cyclooxygenase-2 enzyme in periodontitis.

We investigated the role of the inducible isoform of cyclooxygenase (COX-2) in a rat model of periodontitis using a selective COX-2 inhibitor NS-398. Periodontitis was produced by a silk ligature placed around the lower left 1st molar. Animals were treated with NS-398 (3 mg kg(-1) i.p., 2 times per day for 7 days) or vehicle. At Day 8, the gingivomucosal tissues encircling the mandibular 1st molars were removed on both sides for COX-2 immunohistochemistry, measurement of plasma extravasation by the Evans blue technique, and alveolar bone loss by videomicroscopy. Immunohistochemical analysis revealed numerous strongly COX-2-positive cells in the subepithelial tissues in the ligated side and only a few COX-2-reactive cells in the contralateral (control) side. Ligation significantly increased Evans blue extravasation in the gingivomucosal tissue and alveolar bone destruction compared to the control side. NS-398 treatment significantly reduced the plasma extravasation and alveolar bone resorption of the ligated side compared to vehicle administration. The present results suggest that COX-2 is induced by periodontitis, and plays an important role in gingival inflammation and alveolar bone destruction. In a previous study (Br J Pharmacol 1998;123:353-60) we found the expression of the inducible isoform of nitric oxide synthase in this model. Therefore, based on our own data and the literature, we propose that selective inhibition of these inducible enzymes might be a basis for adjunctive therapy, or new therapeutic approaches in periodontitis.

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.

Inosine inhibits inflammatory cytokine production by a posttranscriptional mechanism and protects against endotoxin-induced shock.

Extracellular purines, including adenosine and ATP, are potent endogenous immunomodulatory molecules. Inosine, a degradation product of these purines, can reach high concentrations in the extracellular space under conditions associated with cellular metabolic stress such as inflammation or ischemia. In the present study, we investigated whether extracellular inosine can affect inflammatory/immune processes. In immunostimulated macrophages and spleen cells, inosine potently inhibited the production of the proinflammatory cytokines TNF-alpha, IL-1, IL-12, macrophage-inflammatory protein-1alpha, and IFN-gamma, but failed to alter the production of the anti-inflammatory cytokine IL-10. The effect of inosine did not require cellular uptake by nucleoside transporters and was partially reversed by blockade of adenosine A1 and A2 receptors. Inosine inhibited cytokine production by a posttranscriptional mechanism. The activity of inosine was independent of activation of the p38 and p42/p44 mitogen-activated protein kinases, the phosphorylation of the c-Jun terminal kinase, the degradation of inhibitory factor kappaB, and elevation of intracellular cAMP. Inosine suppressed proinflammatory cytokine production and mortality in a mouse endotoxemic model. Taken together, inosine has multiple anti-inflammatory effects. These findings, coupled with the fact that inosine has very low toxicity, suggest that this agent may be useful in the treatment of inflammatory/ischemic diseases.

Traumatic brain injury in mice deficient in poly-ADP(ribose) polymerase: a preliminary report.

Poly (ADP-ribose) polymerase (PARP) is a ubiquitous nuclear enzyme that, when activated by free-radical induced DNA damage, contributes to energy failure and cell death in models of central nervous system ischemia and reperfusion. PARP contributes to neuronal cell death in vivo after cerebral ischemia/reperfusion, however, the role of PARP in the pathogenesis of traumatic brain injury (TBI) is unknown. We hypothesized that, compared to wild type mice (+/+), mice deficient in PARP (-/-) would have reduced motor and cognitive deficits after TBI. Mice underwent controlled cortical impact (CCI) (6 m/s, 1.2 mm depth) and were tested for motor (d 1-5) and cognitive (d 14-18) function after CCI. PARP -/- mice demonstrated improved motor performance and improved cognitive function after CCI (both p < 0.05 compared to +/+). This is the first study to evaluate a role for PARP in functional outcome after TBI. The results suggest a detrimental role for PARP in the pathogenesis of TBI.

Glycine inhibits growth of T lymphocytes by an IL-2-independent mechanism.

Previously, it was shown that glycine prevented increases in intracellular calcium ([Ca2+]i) in Kupffer cells. Since Kupffer cells and T lymphocytes are derived from the same pluripotent stem cell, it was hypothesized that glycine would prevent increases in [Ca2+]i in lymphocytes and inhibit cell proliferation. Lymphocyte proliferation was measured in one-way MLC with spleen cells from DA and Lewis rats and in enriched T lymphocyte preparations stimulated by immobilized anti-CD3 Ab. Glycine caused a dose-dependent decrease in cell proliferation to about 40% of control. Con A caused a dose-dependent increase in [Ca2+]i in Jurkat cells which was blunted maximally with 0.6 mM glycine. The effect of glycine was dependent on extracellular chloride and reversed by strychnine, an antagonist of the glycine-gated chloride channel. Similar results were obtained with rat T lymphocytes stimulated by anti-CD3 Ab. Surprisingly, glycine had no effect on IL-2 production in the mixed lymphocyte culture; therefore, the effect of glycine on IL-2-dependent proliferation was tested. Glycine and rapamycin caused dose-dependent decreases in IL-2-stimulated growth of Ctll-2 cells to about 60% and 40%, respectively, of control. Moreover, glycine also inhibited the IL-2-stimulated growth of rat splenic lymphocytes. It is concluded that glycine blunts proliferation in an IL-2-independent manner. This is consistent with the hypothesis that glycine activates a glycine-gated chloride channel and hyperpolarizes the cell membrane-blunting increases in [Ca2+]i that are required for transcription of factors necessary for cell proliferation.

Reduction of cognitive and motor deficits after traumatic brain injury in mice deficient in poly(ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase (PARP), or poly-(ADP-ribose) synthetase, is a nuclear enzyme that consumes NAD when activated by DNA damage. The role of PARP in the pathogenesis of traumatic brain injury (TBI) is unknown. Using a controlled cortical impact (CCI) model of TBI and mice deficient in PARP, the authors studied the effect of PARP on functional and histologic outcome after CCI using two protocols. In protocol 1, naive mice (n = 7 +/+, n = 6 -/-) were evaluated for motor and memory acquisition before CCI. Mice were then subjected to severe CCI and killed at 24 hours for immunohistochemical detection of nitrated tyrosine, an indicator of peroxynitrite formation. Motor and memory performance did not differ between naive PARP +/+ and -/- mice. Both groups showed nitrotyrosine staining in the contusion, suggest ing that peroxynitrite is produced in contused brain. In protoco 2, mice (PARP +/+, n = 8; PARP -/-, n = 10) subjected to CCI were tested for motor and memory function, and contusion volume was determined by image analysis. PARP -/- mice demonstrated improved motor and memory function after CC versus PARP +/+ mice (P < 0.05). However, contusion volume was not different between groups. The results suggest a detri mental effect of PARP on functional outcome after TBI.

Amino acids in rinse effluents as a predictor of graft function after transplantation of fatty livers in rats.

There are too few reliable markers by which one can predict future function of a liver before implantation. Consequently, the purpose of this study was to test the hypothesis that amino acids in rinse-effluents could predict transplant outcome in marginal fatty livers from rats. Amino acids were measured in the rinse effluent from the livers immediately after harvest and graft preparation or cold storage. Amino acids in the effluent were twice as high in ethanol-treated animals compared to those in nonfatty controls. Ethanol-treated fatty livers survived for no longer than 7 days after transplantation while 83% of nonfatty controls survived (P < 0.05). In subsequent studies, the cold-storage time was decreased to 6 h to determine whether failing fatty livers released more amino acid than grafts that would function normally. There was a significant increase in amino acids in the effluent of fatty grafts compared to controls. Moreover, the sum of the four selected amino acids (alanine, valine, histidine, leucine) was lower than 23 nmol/g liver in functional livers, whereas failing grafts had totals significantly higher than 25 nmol/g liver. The sum of the four amino acids correlated well with 24 h post-transplant serum AST levels (r = 0.78, P < 0.0001). So we can conclude that amino acid release can serve as a useful marker of graft viability and reliably predicts survival.

Activation of nuclear factor-kappaB during orthotopic liver transplantation in rats is protective and does not require Kupffer cells.

Reperfusion after liver transplantation results in the induction of tumor necrosis factor-alpha (TNFalpha) as well as activation of the stress-associated signaling proteins, c-Jun N-terminal kinase (JNK), activating protein-1 (AP-1), and nuclear factor-kappaB (NF-kappaB). To test the hypothesis that Kupffer cells are involved in the activation of signal transduction cascades during rat liver transplantation, Kupffer cells were depleted from donor liver using gadolinium chloride (GdCl3), and then the activation of JNK, AP-1, and NF-kappaB were assessed after transplantation. The results showed that GdCl3 treatment did not inhibit the activation of these stress signals, although transplanted livers were depleted of Kupffer cells and partially protected from reperfusion injury. Interleukin-6 (IL-6) and IL-10 messenger RNAs (mRNAs) were induced by transplantation, and the induction was suppressed by Kupffer cell depletion. The induction of TNFalpha mRNA and serum protein during liver transplantation was unaffected by GdCl3. These results show that Kupffer cells are not a major source of TNFalpha production after liver transplantation and that stress-signaling protein activation occurs independently of Kupffer cells. Transplantation strongly activates the transcription factor NF-kappaB, which blocks TNFalpha-mediated apoptosis in hepatocytes in vitro. To assess the role of NF-kappaB activation during liver transplantation, the IkappaBalpha superrepressor was expressed in donor livers using adenoviral-mediated gene transfer. Inhibition of NF-kappaB resulted in increased serum alanine aminotransferase levels after 3 hours of transplantation. In addition, the blockade of NF-kappaB resulted in increased histological tissue injury and increased hepatic terminal deoxyribonucleotide transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining, indicating apoptosis. These results show that NF-kappaB activation has a protective role in the transplanted liver.

Glycine and uridine prevent D-galactosamine hepatotoxicity in the rat: role of Kupffer cells.

Extrahepatic factors, such as increased gut permeability and bacteria from the gut, have been shown to play a role in D-galactosamine toxicity in rats. Because bacterial endotoxin activates Kupffer cells, the purpose of this study was to clarify the role of Kupffer cells in the mechanism of D-galactosamine hepatotoxicity in rats and determine whether uridine, a compound that rescues animals from D-galactosamine toxicity, affects Kupffer cells. Rats were fed control or glycine (5%) containing diets to prevent Kupffer cell activation or treated with gadolinium chloride (GdCl3, 20 mg/kg) to destroy Kupffer cells selectively before injection of D-galactosamine (500 mg/kg, intraperitoneally). D-galactosamine caused panlobular focal hepatocellular necrosis, polymorphonuclear cell infiltration, and increased serum transaminases significantly at 24 hours. Dietary glycine or pretreatment with GdCl3 prevented these effects. D-galactosamine caused a transient increase in circulating endotoxin that was maximal at 1 hour and was blunted significantly by dietary glycine. Additionally, antisera to tumor necrosis factor-alpha (TNF-alpha) prevented hepatotoxicity caused by D-galactosamine. Moreover, apoptosis in hepatocytes caused by D-galactosamine occurred before necrosis (6 hours) and was prevented by glycine, GdCl3, TNF-alpha antiserum, and uridine. Thus, it was hypothesized that TNF-alpha from Kupffer cells causes apoptosis after D-galactosamine administration in the rat. Indeed, increases in TNF-alpha messenger RNA (mRNA) were detected as early as 2.5 hours after D-galactosamine treatment. Previous work proposed that uridine blocks D-galactosamine toxicity by preventing inhibition of mRNA synthesis. In view of these results, the possibility that uridine might affect Kupffer cells was investigated. Uridine significantly blunted the increase in [Ca2+]i and release of TNF-alpha caused by endotoxin in isolated Kupffer cells and prevented apoptosis caused by D-galactosamine treatment in vivo. These data support the hypothesis that uridine prevents D-galactosamine hepatotoxicity not only by rescuing the hepatocyte in the late phases of the injury but also preventing TNF-alpha release from Kupffer cells thereby blocking apoptosis that occurs early after D-galactosamine treatment. Taken together, these data strongly support the role of Kupffer cell activation by endotoxin early after D-galactosamine treatment as an important event in the mechanism of hepatotoxicity in the rat.

Taurine blunts LPS-induced increases in intracellular calcium and TNF-alpha production by Kupffer cells.

Activation of Kupffer cells by lipopolysaccharide (LPS) plays a pivotal role in the onset of pathophysiological events that occur during endotoxemia and intracellular calcium ([Ca2+]i) is involved in LPS-stimulated cytokine production. Recently, it was shown that Kupffer cells contain a glycine-gated chloride channel. Because taurine, a ubiquitous sulfur-containing beta-amino acid, acts similarly to glycine in neurons by causing hyperpolarization, it was hypothesized that taurine would act via a similar mechanism, blunting the LPS-induced increase in [Ca2+]i in Kupffer cells. To test this hypothesis, Kupffer cells were isolated from female Sprague-Dawley rats and cultured for 24 h. LPS-induced changes in [Ca2+]i were monitored fluorometrically in single cells, whereas levels of tumor necrosis factor alpha (TNF-alpha) released by Kupffer cells after exposure to LPS were measured by enzyme-linked immunosorbent assay. Taurine significantly blunted the LPS-induced increase in [Ca2+]i in a dose-dependent manner (IC50, 0.1 mM). This effect was reversed by strychnine (1 microM) and was prevented when chloride was removed from the extracellular media. Moreover, taurine increased 36Cl- uptake by Kupffer cells in a dose-dependent manner (EC50, 0.2 mM). Furthermore, strychnine (1 microM) reversed the effect of taurine on 36Cl- uptake. These results indicate that taurine activates a glycine-gated chloride channel in Kupffer cells causing chloride influx. In addition, LPS-induced TNF-alpha production was reduced by more than 40% by taurine, an effect that was also reversed by strychnine. In conclusion, taurine blocks the increase in [Ca2+]i due to LPS and significantly reduces TNF-alpha production by mechanisms involving chloride influx into the Kupffer cell.

Generation of lipid free radicals by adherent leukocytes from transplanted rat liver.

The production of free radicals in blood correlates with primary nonfunction of transplanted livers, but the source of the free radicals is unknown. The purpose of this study was to determine if adherent leukocytes in the transplanted liver are responsible for the radicals detected in blood. First, a new method to harvest adherent leukocytes from the liver without enzymatic digestion was developed and characterized by transplanting livers from ethanol-treated rats, which increases primary nonfunction, and from saline-treated controls. Free radicals were then detected in isolated leukocytes using the spin-trapping technique and electron spin resonance (ESR) spin spectroscopy. Livers were perfused with a balanced salt solution (200 ml), followed by a Ca(2+)-free solution containing EGTA and heparin (400 ml). Perfusion with Ca(2+)-free buffer removed greater than 90% of all adherent leukocytes from saline-treated livers and nearly 80% of all leukocytes from fatty livers without removing Kupffer cells. Transplanted fatty livers from rats given ethanol contained significantly more adherent leukocytes (5.0 x 10(7) cells/liver) than grafts from control donors (3.2 x 10(7) cells/liver) and almost double the number of adherent neutrophils and monocytes. Moreover, adherent white blood cells from transplanted livers produced the same three free radical species that have been detected previously in blood; however, cells from ethanol-treated livers produced about five times more radical adducts. These data show that adherent white blood cells produce free radicals that are important in the mechanism of primary graft nonfunction.

Cyclosporin A increases hypoxia and free radical production in rat kidneys: prevention by dietary glycine.

The major side effect of cyclosporin A is severe nephrotoxicity. It is likely that cyclosporin A causes vasoconstriction leading to hypoxia-reperfusion injury; therefore, these experiments were designed to attempt to obtain physical evidence for hypoxia and free radical production in kidney following cyclosporin A. Rats were treated daily with cyclosporin A (25 mg/kg ig) for 5 days, and pimonidazole, a hypoxia marker, was injected 2 h after the last dose of cyclosporin A. A dose of alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) was injected 3 h after cyclosporin A to trap free radicals. Cyclosporin A doubled serum creatinine and decreased glomerular filtration rates by 65% as expected. Pimonidazole adduct binding in the kidney was increased nearly threefold by cyclosporin A, providing physical evidence for tissue hypoxia. Moreover, cyclosporin A increased 4-POBN/radical adducts nearly sixfold in the urine but did not alter levels in the serum. Glycine, which causes vasodilatation and prevents cyclosporin A toxicity, minimized hypoxia and blocked free radical production; however, it did not alter cyclosporin A blood levels. These results demonstrate for the first time that cyclosporin A causes hypoxia and increases production of a new free radical species exclusively in the kidney. Therefore, it is concluded that cyclosporin A causes renal injury by mechanisms involving hypoxia-reoxygenation, effects which can be prevented effectively by dietary glycine.

Role of free radicals in primary nonfunction of marginal fatty grafts from rats treated acutely with ethanol.

Acute treatment with one large dose of ethanol, which mimics binge drinking, causes marginal fatty liver and decreases survival significantly after liver transplantation in rats, yet mechanisms remain unclear. Therefore, we evaluated the possible role of free radicals in primary nonfunction caused by acute ethanol. Female donor rats were administered ethanol (5 g/kg orally) 20 hr before explantation, and grafts were stored in UW cold storage solution for 24-42 hr before implantation. Free radicals were trapped with alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone after transplantation, and adducts were detected using electron spin resonance spectrometry. Ethanol increased a carbon-centered radical adduct in bile approximately 2-fold and elevated serum lipid hydroperoxides approximately 4-fold. Ethanol also increased transaminase release 3.7-fold and decreased bile production by 55%. Catechin, a free radical scavenger, minimized the increase in free radicals, blunted transaminase release, and elevated bile production significantly, indicating that free radical production plays an important role in ethanol-induced fatty graft injury. GdCl3 (20 mg/kg intravenously), a selective Kupffer cell toxicant, largely blocked the increases in free radical and lipid hydroperoxide production caused by ethanol. In addition, ethanol nearly doubled white blood cell adhesion after transplantation, leading to increased superoxide production in fatty grafts. GdCl3 largely blocked leukocyte adhesion as well as superoxide production. Allopurinol, an inhibitor of xanthine oxidase, also diminished free radical production, blunted transaminase release, and improved bile production in fatty grafts significantly. Taken together, we conclude that free radical formation increases in ethanol-induced fatty grafts due mainly to activation of Kupffer cells and increased adhesion of white blood cells. Antioxidants can effectively block free radical formation and minimize injury to marginal fatty grafts caused by binge drinking.

Development and characterization of a new model of tacrine-induced hepatotoxicity: role of the sympathetic nervous system and hypoxia-reoxygenation.

Tacrine is an acetylcholinesterase inhibitor approved for the treatment of Alzheimer's disease. Unfortunately, reversible hepatotoxicity in about 30% of patients at therapeutic doses limits clinical use. The purpose of this study was to develop and characterize a model of tacrine hepatotoxicity to begin to understand the mechanisms of injury. Rats were given tacrine (10-50 mg/kg, intragatrically) and killed 24 hr later. An increase in serum aspartate aminotransferase was observed up to 35 mg/kg and histology revealed pericentral necrosis and fatty changes. Aspartate aminotransferase was increased from 12 to 24 hr and returned to control values by 32 hr. Livers were perfused in a nonrecirculating system to measure oxygen uptake and trypan blue was infused at the end of each experiment to evaluate tissue perfusion. Time for trypan blue to distribute evenly throughout the liver 3 hr after tacrine treatment was significantly increased (6.9 +/- 1.3 min) compared to controls (1.0 +/- 0.3 min) reflecting decreased tissue perfusion. Tacrine also significantly increased the binding of a hypoxia marker, pimonidazole, in pericentral regions almost 3-fold, and increased portal pressure in vivo significantly. It is hypothesized that tacrine, by inhibiting acetylcholine breakdown in the celiac ganglion, increases sympathetic activity in the liver leading to vascular constriction, hypoxia and liver injury. To test this hypothesis, the hepatic nerve was severed and animals were allowed to recover before tacrine treatment. This procedure significantly reduced serum aspartate aminotransferase, time of dye distribution, pimonidazole binding and portal pressure. Furthermore, a free radical adduct was detected with spin trapping and electron spin resonance spectroscopy 8 hr after tacrine treatment, providing evidence for reoxygenation. When catechin (100 mg/kg, i.p.), a free radical scavenger, was given before tacrine, injury was decreased by about 45%. Furthermore, feeding 5% arginine in the diet significantly reduced portal pressure and time of dye distribution. These data are consistent with the hypothesis that tacrine hepatotoxicity is a hypoxia-reoxygenation injury mediated through the sympathetic nervous system.

Release of amino acids from fatty livers during organ harvest for transplantation.

Shortage of organ donors presents a perplexing problem in liver transplantation, and improved methods for evaluating the viability of organs prior to implantation are urgently needed. In the present study, the hypothesis was evaluated that grafts from fatty livers release more amino acids than non-fatty controls during organ harvest. Amino acids in graft rinse effluents at the time of harvest and after cold storage were measured by reverse-phase high performance liquid chromatography and compared with plasma aspartate aminotransferase (AST) levels and recipient survival. Twenty-four hours after transplantation of fatty livers, AST levels in recipient rats were increased more than two-fold compared to non-fatty controls (p < 0.01). Survival in the control group was 83 percent, whereas animals receiving fatty livers from ethanol-treated rats survived no longer than 7 days after transplantation (p < 0.05). The rate of release of amino acids from the liver explant was two-fold higher during the harvest procedure (0.5 h) than during the subsequent 23.5 hour cold storage period (435 +/- 70 vs. 186 +/- 14 nmol/ml/hr/g liver, p < 0.001). Further, in the early rinse effluent, amino acids were released about two-fold faster from fatty livers than from controls (p < 0.05). This study demonstrates that the release of amino acids from liver explants increases during the harvesting procedure and is about two-fold higher in fatty livers which fail after transplantation than in surviving controls. It is proposed that amino acid release from explants after organ harvest might serve as a useful marker to evaluate graft function prior to transplantation.

Kupffer cells contain a glycine-gated chloride channel.

Here the effect of glycine on intracellular Ca2+ concentration ([Ca2+]i) in cultured Kupffer cells stimulated with lipopolysaccharide (LPS) was investigated to assess the possibility that they contain a glycine-gated chloride channel. LPS (10 micrograms/ml) increased [Ca2+]i rapidly, with peak values reaching 307 +/- 29 nM. Glycine (1 mM) prevented this increase nearly completely. Low concentrations of strychnine (1 microM), a glycine receptor antagonist, reversed the inhibitory effect of glycine completely; however, high concentrations of strychnine (1 mM) mimicked glycine. The effects of glycine and high-dose strychnine were prevented when cells were incubated in chloride-free buffer. Furthermore, potassium (25 mM) and LPS depolarized the Kupffer cell plasma membrane, whereas glycine caused hyperpolarization and prevented depolarization due to potassium and LPS. Moreover, tumor necrosis factor-alpha (TNF-alpha) production in cultured Kupffer cells due to LPS was decreased significantly by glycine. Therefore, it is concluded that Kupffer cells contain a glycine-gated chloride channel similar to that described previously in the central nervous system. Prevention of increases in [Ca2+]i due to LPS by activation of chloride influx reduced synthesis and release of toxic mediators by Kupffer cells.

Prevention of cyclosporine-induced nephrotoxicity with dietary glycine.

BACKGROUND: The nonessential amino acid glycine has been used previously to prevent hypoxic and ischemic injury to kidney tissue in vitro. Furthermore, it was recently shown that glycine prevents activation of macrophages and neutrophils in vitro. Because there is some evidence that the immunosuppressant cyclosporine causes nephrotoxicity through a hypoxia-reoxygenation mechanism that could involve infiltration and activation of macrophages and neutrophils, we hypothesized that dietary glycine could prevent this injury. METHODS: Rats were fed a diet containing glycine (5%) or a control diet for 3 days before cyclosporine treatment. To produce nephrotoxicity, cyclosporine (25 mg/kg daily by gavage) was administered for 28 days while animals were maintained on glycine or control diets. Serum creatinine, urea, glomerular filtration rates, and kidney histology were evaluated in different treatment groups. RESULTS: All rats gained weight; however, overall weight gain in the cyclosporine, glycine, and cyclosporine+glycine groups was significantly less by about 40% compared with the control group. Diet consumption was not statistically different between the groups. As expected, cyclosporine caused kidney damage in the rats fed control diet, reflected in significantly elevated serum urea and creatinine. In addition, cyclosporine treatment decreased glomerular filtration rate by nearly 70%, caused proximal tubular dilation and necrosis as well as increased macrophage and neutrophil infiltration into the kidney. Dietary glycine prevented or minimized kidney damage due to cyclosporine in all parameters studied nearly completely. Furthermore, feeding glycine for up to 1 month had no detrimental effect on kidney function. CONCLUSIONS: Dietary glycine is a safe and effective treatment to reduce the nephrotoxicity of cyclosporine.

Reperfusion after liver transplantation in rats differentially activates the mitogen-activated protein kinases.

The injury resulting from cold ischemia and warm reperfusion during liver transplantation is a major clinical problem that limits graft success. Kupffer cell activation plays a pivotal role in reperfusion injury, and Kupffer cell products, including free radicals and tumor necrosis factor alpha (TNF-alpha), are implicated as damaging agents. However, the second messengers and signaling pathways that are activated by the stress of hepatic ischemia/reperfusion remain unknown. The purpose of this study is to assess the activation of the three known vertebrate mitogen activated protein kinase (MAPKs) and the activating protein 1 (AP-1) transcription factor in response to ischemia and reperfusion in the transplanted rat liver. There was a potent, sustained induction of c-jun N-terminal kinase (JNK), but not of the related MAPKs extracellular signal-regulated kinases (ERK) or p38, upon reperfusion after transplantation. TNF-alpha messenger RNA (mRNA) levels and transcription factors AP-1 and nuclear factor-kappaB (NF-kappaB) were induced in the liver after 60 minutes of reperfusion. Finally, there was an elevation of ceramide, but not diacylglycerol or sphingosine, in the transplanted liver. Ceramide is a second messenger generated by TNF-alpha treatment and is an activator of JNK. Because JNK activation preceded the elevations in ceramide and TNF-alpha mRNA, these results suggest that increased hepatic TNF-alpha and ceramide may perpetuate JNK induction, but that they are not the initiating signals of JNK activation during reperfusion injury in the transplanted liver.

Destruction of Kupffer cells increases survival and reduces graft injury after transplantation of fatty livers from ethanol-treated rats.

This study investigated the role of Kupffer cells on survival and graft injury in transplanted fatty livers from rats treated acutely with ethanol. Donor rats were given ethanol (5 g/kg, by mouth) 20 hours before explantation, and liver grafts were preserved in University of Wisconsin cold storage solution for 24 to 42 hours prior to implantation. Blood samples were taken from the inferior vena cava for 3 hours after implantation. During this time, serum aspartate transaminase levels increased gradually from 122 U/L to 597 U/L in control rats, while ethanol treatment elevated values to 2,278 U/L. Gadolinium chloride (20 mg/kg, given intravenously to recipients 24 hours before explantation), a selective inactivator of Kupffer cells, minimized the increase in aspartate transaminase levels significantly. After implantation of grafts cold-stored for 42 hours, survival rates were 88% in control rats but only 33% in ethanol-treated rats. Gadolinium chloride improved survival nearly to control values. Ethanol nearly doubled white blood cell adhesion, an effect also largely blocked by gadolinium chloride. Further, alpha-(4-pyridyl 1-oxid)-N-tert-butylnitrone radical adducts detected in the bile were increased twofold by ethanol treatment. This effect was also reversed by gadolinium chloride. Taken together, these data indicate that survival is poorer and graft injury is greater in fatty livers from ethanol-treated rats. Inactivation of Kupffer cells minimized graft damage, most likely by improving hepatic microcirculation and diminishing lipid peroxidation.