Purine Nucleoside Phosphorylase Release Research Articles

Purine Nucleoside Phosphorylase Inhibition Attenuates the Progression of Anemia and Organ Damage in Sickle Cell Mice

Purine nucleoside phosphorylase (PNP) converts guanosine to guanine, inosine to hypoxanthine, and 2'-deoxyguanosine to guanine, and leads to the downstream formation of the pro-oxidant and vasculotoxic hypoxanthine and xanthine. Red blood cells are rich in PNP, yet PNP's role in the pathogenesis of sickle cell disease (SCD) is not known. Our group showed that patients with SCD have increased PNP release and activity, leading to accelerated guanosine/inosine metabolism and increased production of vasculotoxic hypoxanthine/xanthine (Bilan VP et al. 2018). Thus, we proposed that PNP may have a major pathogenic role in red blood cell sickling, hemolytic angioproliferation, cell aggregation and end organ damage, and that PNP inhibition may be beneficial in the treatment of SCD. The highly potent PNP inhibitor 8-aminoguanine is the product of the conversion of the endogenous guanosine metabolite and pro-drug 8-aminoguanosine (8-AG). We investigated the safety and efficacy of short-term (8 weeks) and chronic (20 weeks) treatment of Townes sickle mice (SS) and their non-sickling controls (AA) with 8-AG (60mg/kg/day administered ad libitum in the drinking water). We collected blood and urine samples at multiple intervals using metabolic cages. To determine 8-AG body disposition and PNP inhibition, urine samples were analyzed for purines by liquid chromatography - tandem mass spectrometry (LC-MS/MS). Both AA and SS mice tolerated 8-AG well with no evidence of toxicity. In both AA (n= 15) and SS (n=40) mice there was very efficient conversion of 8-AG that resulted in a 40-50-fold increase in urinary 8-aminoguanine concentrations (p<0.001). In 8-AG treated SS mice we observed reduced hypoxanthine levels, with an increased inosine/hypoxanthine ratio. Additionally, guanosine was increased, guanine was reduced, and an increased guanosine/guanine ratio was observed (p<0.05), suggesting potent PNP inhibition. In SS mice, the 8-week treatment with 8-AG significantly reduced hemoglobinuria (p<0.05) and albuminuria (p<0.05), attenuated left ventricular (LV, p<0.01) and right ventricular (RV, p<0.05) hypertrophy, limited hepatomegaly (p<0.001), and improved RV contractility (p<0.05), with a trend towards reduced lung weights and splenomegaly. In SS mice, 20-week treatment with 8-AG significantly slowed the progression of anemia as evidenced by attenuated age-related drops in hemoglobin and hematocrit levels, and it attenuated hemoglobinuria and albuminuria while significantly reducing splenomegaly (p=<0.05) and hepatomegaly (p<0.05, Figure). Semi-quantitative histopathological analysis suggested reduced iron deposition (Prussian blue stain, Figure), and reduced ischemic (H&E stain) and fibrotic (trichrome stain, Figure) injury in the spleen and liver from SS mice treated with 8-AG. Our data suggest that the PNP inhibitor 8-AG may have a pharmacological benefit in SCD and may warrant further investigation as a multi-pronged treatment approach for SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Release of soluble and vesicular purine nucleoside phosphorylase from rat astrocytes and microglia induced by pro-inflammatory stimulation with extracellular ATP via P2X7 receptors

Purine nucleoside phosphorylase (PNP), a crucial enzyme in purine metabolism which converts ribonucleosides into purine bases, has mainly been found inside glial cells. Since we recently demonstrated that PNP is released from rat C6 glioma cells, we then wondered whether this occurs in normal brain cells. Using rat primary cultures of microglia, astrocytes and cerebellar granule neurons, we found that in basal condition all these cells constitutively released a metabolically active PNP with Km values very similar to those measured in C6 glioma cells. However, the enzyme expression/release was greater in microglia or astrocytes that in neurons. Moreover, we exposed primary brain cell cultures to pro-inflammatory agents such as lipopolysaccharide (LPS) or ATP alone or in combination. LPS alone caused an increased interleukin-1β (IL-1β) secretion mainly from microglia and no modification in the PNP release, even from neurons in which it enhanced cell death. In contrast, ATP administered alone to glial cells at high micromolar concentrations significantly stimulated the release of PNP within 1 h, an effect not modified by LPS presence, whereas IL-1β secretion was stimulated by ATP only in cells primed for 2 h with LPS. In both cases ATP effect was mediated by P2X7 receptor (P2X7R), since it was mimicked by cell exposure to Bz-ATP, an agonist of P2X7R, and blocked by cell pre-treatment with the P2X7R antagonist A438079. Interestingly, ATP-induced PNP release from glial cells partly occurred through the secretion of lysosomal vesicles in the extracellular medium. Thus, during inflammatory cerebral events PNP secretion promoted by extracellular ATP accumulation might concur to control extracellular purine signals. Further studies could elucidate whether, in these conditions, a consensual activity of enzymes downstream of PNP in the purine metabolic cascade avoids accumulation of extracellular purine bases that might concur to brain injury by unusual formation of reactive oxygen species.

Evidence for purine nucleoside phosphorylase (PNP) release from rat C6 glioma cells.

Intracellular purine turnover is mainly oriented to preserving the level of triphosphate nucleotides, fundamental molecules in vital cell functions that, when released outside cells, act as receptor signals. Conversely, high levels of purine bases and uric acid are found in the extracellular milieu, even in resting conditions. These compounds could derive from nucleosides/bases that, having escaped to cell reuptake, are metabolized by extracellular enzymes similar to the cytosolic ones. Focusing on purine nucleoside phosphorylase (PNP) that catalyzes the reversible phosphorolysis of purine (deoxy)-nucleosides/bases, we found that it is constitutively released from cultured rat C6 glioma cells into the medium, and has a molecular weight and enzyme activity similar to the cytosolic enzyme. Cell exposure to 10μM ATP or guanosine triphosphate (GTP) increased the extracellular amount of all corresponding purines without modifying the levels/activity of released PNP, whereas selective activation of ATP P2Y1 or adenosine A2A metabotropic receptors increased PNP release and purine base formation. The reduction to 1% in oxygen supply (2h) to cells decreased the levels of released PNP, leading to an increased presence of extracellular nucleosides and to a reduced formation of xanthine and uric acid. Conversely, 2h cell re-oxygenation enhanced the extracellular amounts of both PNP and purine bases. Thus, hypoxia and re-oxygenation modulated in opposite manner the PNP release/activity and, thereby, the extracellular formation of purine metabolism end-products. In conclusion, extracellular PNP and likely other enzymes deputed to purine base metabolism are released from cells, contributing to the purinergic system homeostasis and exhibiting an important pathophysiological role.

Effects of hypothermic machine perfusion on rat liver function depending on the route of perfusion.

The aim of this study was to evaluate the efficacy of hypothermic machine perfusion (HMP) to preserve rat livers according to the route of perfusion, i.e., via portal vein, hepatic veins (retrograde), or hepatic artery. Livers were preserved for 24 or 48 hr by simple cold storage (SCS) or by HMP. Preservation solution was supplemented with (HMP) or without (SCS) hydroxyethyl starch. After preservation, grafts were reperfused for 2 hr with an oxygenated Krebs-Henseleit bicarbonate buffer. After 24 hr of preservation, total glutathione concentrations in HMP livers were similar (1287+/-37, 1418+/-118, and 1471+/-62 nmol/g in hepatic artery, portal vein, and hepatic vein HMP livers, respectively) and higher than in the SCS (833+/-118 nmol/g, P<0.05) group. These higher total glutathione values were due to higher reduced glutathione concentrations. ATP concentrations in the liver tissue were similar in HMP groups (0.75+/-0.4, 0.64+/-0.1, and 0.77+/-0.1 micromol/g in hepatic artery, portal vein, and hepatic vein HMP livers, respectively) and higher than in SCS (0.32+/-0.06 micromol/g, P<0.05). After 2 hr of normothermic reperfusion, bile production in the HMP portal and HMP retrograde groups were similar (391+/-29 ml and 372+/-25 ml) and higher than in the HMP artery or SCS groups (275+/-25 ml and 277+/-32 ml, respectively; P<0.05). Aspartate transaminase, alanine transaminase, lactate dehydrogenase, and purine nucleoside phosphorylase release into the perfusate of HMP portal and HMP retrograde perfused livers was similar and significantly lower compared to the HMP artery and SCS groups. At the end of reperfusion, no statistical differences were found for glutathione concentration and energetic reserves in the livers of each group. After 48 hr of preservation, livers from the HMP portal and HMP retrograde groups did significantly better than livers from the HMP artery or SCS groups. This study confirms the superiority of HMP over SCS to preserve the liver graft. It shows that retrograde perfusion is similar to PV perfusion and that perfusion by HA is less beneficial.

Antioxidants and gadolinium chloride attenuate hepatic parenchymal and endothelial cell injury induced by low flow ischemia and reperfusion in perfused rat livers

The objective of this study was to determine whether Kupffer cells contribute to parenchymal and endothelial cell damage induced by ischemia-reperfusion in perfused rat livers. Parenchymal and endothelial cell injury were determined by measuring activities of lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP), respectively, in the effluent perfusate of livers subjected to 60 min of low flow ischemia followed by 30 min of reperfusion. Upon reperfusion, LDH and PNP activities increased significantly within the first 10 min of reperfusion and remained elevated over control values throughout the duration of reperfusion. Pretreatment with gadolinium chloride, an inhibitor of Kupffer cell function, significantly decreased LDH and PNP efflux during reperfusion by approximately 60% and 50%, respectively. When Kupffer cells were stimulated by vitamin A pretreatment, PNP efflux was doubled during reperfusion. Vitamin E pretreatment attenuated LDH and PNP release by approximately 70% during reperfusion compared to enzyme release in untreated livers. Moreover, the water-soluble antioxidants superoxide dismutase and desferrioxamine reduced reperfusion injury, whereas catalase had no effect on enzyme release. These results demonstrate that superoxide anions released from Kupffer cells are involved in oxidative damage to endothelial cells as well as hepatocytes during the early stages of hepatic reperfusion.

Treatment of experimental ischemia/reperfusion injury with S-adenyosylmethionine: evidence that donor pretreatment complements other regimens.

Triple therapy with S-adenosylmethionine (SAM) (given to the donor animal, included in University of Wisconsin solution [UW], and added to the reperfusing medium) has been shown to reduce the sequential cold and warm ischemia/reperfusion injuries characteristic of the liver transplantation procedure. To clarify the actions of SAM during different stages of ischemia/ reperfusion, we have compared its benefit in five dosage regimens, using perfused rat livers after sequential periods of 24 hr cold and 20 min rewarming ischemia. When added only to UW, the presence of SAM throughout ischemia improved hepatic blood flow by 26% after 15 min of reperfusion versus no treatment (2.32+/-0.18 vs. 1.84+/-0.11 ml/min/g liver, P<0.05). SAM also improved blood flow by 23% during the 3-hr perfusion overall (P<0.05). Oxygen consumption and the release of purine nucleoside phosphorylase (PNP) were decreased (both P<0.05). When added to both UW and the perfusate, SAM additionally increased bile production at 15 min (7.14+/-1.21 vs. 2.31+/-0.74 mg/h/g liver, P<0.01). By pretreating the liver donor with SAM in vivo, and including it in the preservation and reperfusing media, it was possible to prolong and amplify the benefits on blood flow (P<0.001) and bile production (P<0.05) and to sustain glucose uptake (P<0.01). An acute exposure to SAM, when used in saline to flush UW from the graft before reperfusion, increased blood flow at 15 min (by 68%) and over a 3-hr period (both P<0.001), but no indices of metabolic activity were improved. Oxygen consumption and PNP release were both decreased (P<0.05). When added to the perfusate (present throughout reperfusion), SAM increased blood flow at 15 min (58%) and over a 3-hr period (P<0.01 in both cases). Net glucose uptake was increased (P<0.05), whereas oxygen consumption (P<0.001) and PNP release fell (P<0.05). Actions of SAM achieved acutely and over the intermediate- and long-term all seem to underlie its benefits in reducing ischemia/reperfusion injuries.

No attenuation of ischemic and reperfusion injury in Kupffer cell-depleted, cold-preserved rat livers.

Activated Kupffer cells (KC) have been implicated in the damage sustained by preserved liver grafts during ischemia and reperfusion. The aim of this study was to compare ischemia/reperfusion injury in preserved, KC-depleted rat livers and preserved control livers, with special regard to sinusoidal endothelial cell (SEC) injury. Wistar rats were injected with liposome-encapsulated dichloromethylene diphosphonate, 48 hr before hepatectomy, to eliminate KC, or were withheld this pretreatment (controls). Livers were flushed with cold University of Wisconsin solution and after 0, 8, 16, or 24 hr of storage at 4 degrees C, were reperfused in a recirculation system with 200 ml of oxygenated Krebs-Henseleit solution at 37 degrees C for 90 min. Damage to SEC was measured by the uptake of hyaluronic acid (HA) from the perfusate and release of purine nucleoside phosphorylase (PNP). Perfusate samples were, furthermore, analyzed for aspartate aminotransferase (AST) and tumor necrosis factor-alpha. Carbon particles were infused in the perfusate to determine the phagocytotic capacity of KC. Biopsies were taken for histological examination and sections were stained with ED2 monoclonal antibodies to confirm the absence of KC. After 90 min of reperfusion, immediately after cold flush (t0), the uptake of HA was 72.2+/-2.3% and 69.3+/-1.3% in KC-depleted livers and in control livers, respectively (n.s.). After 8 hr of storage, HA uptake was 21.6+/-4.5% and 34.6+/-8.0%, respectively (n.s.). After 16 and 24 hr of storage and reperfusion, no uptake of HA was found in either KC-depleted or control livers, indicating abolished SEC function. PNP activities in the perfusate were higher in control livers (after 8 and 24 hr of storage), presumably due to release from damaged KC. No difference was found in AST and no tumor necrosis factor-alpha was measured in the perfusates of normal and KC-depleted livers. Electron microscopic studies showed that after 8 and 24 hr of storage and reperfusion, KC were activated and were able to phagocytose colloidal carbon. Our conclusion was that the elimination of Kupffer cells did not result in reduction of ischemic and reperfusion damage in livers preserved up to 24 hr, as assessed in vitro by SEC uptake of HA, PNP release, and AST release.

Evaluation of purine nucleoside phosphorylase release as a measure of hepatic endothelial cell injury

With emerging data that endothelial cell (EC) injury is the limiting factor in liver preservation and hepatic function, a simple and reliable biochemical technique for monitoring EC injury is needed. Measurement of purine nucleoside phosphorylase (PNP) release into the circulation from perfused liver has been proposed as such a method. However, our experiments with perfused rat liver did not display a clear or direct relationship between PNP release and endothelial cell injury. Therefore, we re-examined the suitability of using PNP as a measure of nonparenchymal injury by measuring its distribution in purified populations of hepatocytes, ECs, and Kupffer cells (KCs) and correlating cell injury and enzyme release in short-term cultures at 37°C of each cell type. Purified cells were incubated (4 × 10 6 cells/mL) in oxygen or nitrogen saturated Wisconsin solution or Krebs buffer for 6 hours, with cell viability and PNP release assayed every 2 hours. ECs had the lowest specific activity (27 ± 9 U/ mg protein; mean ± standard error of the mean [Sem]) compared with both hepatocytes (115 ± 15) and KCs (66 ± 18). Despite a decrement in EC and KC viability over time in each incubation solution, there was poor correlation between time of incubation and PNP release ( r = .01 to .22), and between cell viability and PNP release ( r = .01 to .16). In contrast, PNP release from incubated hepatocytes correlated with the length of incubation ( r = .57 to .78) as well as cell injury ( r = .63 to .77) in all four test solutions. This data suggest that PNP release is unlikely to specifically reflect EC injury in the intact liver.

Evaluation of purine nucleoside phosphorylase release as a measure of hepatic endothelial cell injury

With emerging data that endothelial cell (EC) injury is the limiting factor in liver preservation and hepatic function, a simple and reliable biochemical technique for monitoring EC injury is needed. Measurement of purine nucleoside phosphorylase (PNP) release into the circulation from perfused liver has been proposed as such a method. However, our experiments with perfused rat liver did not display a clear or direct relationship between PNP release and endothelial cell injury. Therefore, we re-examined the suitability of using PNP as a measure of nonparenchymal injury by measuring its distribution in purified populations of hepatocytes, ECs, and Kupffer cells (KCs) and correlating cell injury and enzyme release in short-term cultures at 37 degrees C of each cell type. Purified cells were incubated (4 x 10(6) cells/mL) in oxygen or nitrogen saturated. Wisconsin solution or Krebs buffer for 6 hours, with cell viability and PNP release assayed every 2 hours. ECs had the lowest specific activity (27 +/- 9 U/mg protein; mean +/- standard error of the mean [SEM]) compared with both hepatocytes (115 +/- 15) and KCs (66 +/- 18). Despite a decrement in EC and KC viability over time in each incubation solution, there was poor correlation between time of incubation and PNP release (r = .01 to .22), and between cell viability and PNP release (r = .01 to .16). In contrast, PNP release from incubated hepatocytes correlated with the length of incubation (r = .57 to .78) as well as cell injury (r = .63 to .77) in all four test solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Resistance of endothelial cells to anoxia-reoxygenation in isolated guinea pig hearts

The release of cytosolic enzymes from myocardial and endothelial cells in the anoxic-reoxygenated guinea pig heart was investigated. Isolated hearts were perfused with Tyrode solution in the Langendorff mode. Sixty-minute anoxic perfusion with or without glucose (5 mM) was followed by 15-min normoxic perfusion with glucose. The losses of purine-nucleoside phosphorylase (PNP) from endothelial cells and of lactate dehydrogenase (LDH) and creatine kinase (CK) from the mass of myocardial cells were determined. After 30-min anoxia, the release of LDH and CK but not of PNP increased. Reoxygenation after 60-min anoxia with glucose caused a partial recovery of tissue ATP but also an increase in leakage of LDH (11% of total in 15 min) and CK (10%) and a sudden rise in coronary resistance, indicating contracture development ("oxygen paradox"). PNP release remained low (0.5%). In hearts subjected to glucose-free anoxia, ATP levels did not rise during 15-min reoxygenation, contracture development was delayed, and the release of LDH and CK was diminished (3.1 and 2.7%, respectively). Leakage of PNP was again low (0.5%). The results indicate that cardiomyocytes are more severely injured by anoxia-reoxygenation than the coronary endothelium. The rapidly developing reoxygenation-induced injury of cardiomyocytes seems to be an energy-dependent phenomenon, since it was attenuated in hearts deprived of substrate in anoxia.

167 D-XYLULOSE-INDUCED DEPLETION OF ATP AND Pi AND INCREASE IN PRPP IN ISOLATED RAT HEPATOCYTES

Xylitol provokes hepatic ATP catabolism by trapping Pi as glycerol 3-P (Woods & Krebs, Biochem. J. 134, 437-43, 1973). The dependence of the latter upon an increase in the NADH/NAD+ ratio, due to oxidation of xylitol to D-xylulose (D-Xyl), raised the question whether D-Xyl depletes ATP. Incubation of hepatocytes with 5 mM D-Xyl decreased ATP by 80 % within 5 min and by 30 % with 5 mM xylitol. Intracellular Pi decreased by 66 % within 5 min with both compounds, but reincreased 3-fold faster and reached supraphysiological levels with D-Xyl. D-Xyl was used at the rate of 5 Mmol/min/g of cells, i.e. 3-fold faster than xylitol. With D-Xyl, glycerol 3-P was barely modified but Xyl 5-P increased from 0.02 to 1.4 μ mol/g of cells. The main cause of the ATP− and Pi-depleting effect of D-Xyl was an accumulation of sedoheptulose 7-P which increased from 0.3 to 8 μ mol/g after 10 min. Ribose 5-P increased from 0.03 to 0.50 μmol/g with D-Xyl, and to 0.16 μmol/g with xylitol. Ribose 1-P also accumulated, albeit extracellulary, because of release of purine nucleoside phosphorylase from damaged cells, acting on inosine which diffused out. D-Xyl increased PRPP from a basal value of 6 ± 1 nmol/g to 509 ±72, as compared to 42 ± 11 with xylitol. It is concluded that the markedly higher PRPP level reached with D-Xyl results from a higher activity of PRPP synthetase, owing to a more marked increase of intracellular Pi and of ribose 5-P, combined with other, still unknown factors.