Purine metabolism in pigs and humans and its implications for xenotransplantation

We compared concentrations of nucleotide substrates and activities of enzymes of nucleotide metabolism in pig and human blood, heart, and kidney. The most important difference was lower ecto-5-nucleotidase (ESN) activity in both pig hearts and kidney. Furthermore, higher hypoxanthine, inosine, adenine, and uracil, but lower uridine and uric acid concentrations were observed in pig blood as compared to human. A twofold increase in UTP concentration has been observed in pig hearts following 4 h perfusion with human blood. Purine metabolism is an important target for genetic and pharmacological manipulation during xenotransplantations.     

Mammalian mismatches in nucleotide metabolism: implications for xenotransplantation

Acute humoral rejection (AHR) limits the clinical application of animal organs for xenotransplantation. Mammalian disparities in nucleotide metabolism may contribute significantly to the microvascular component in AHR; these, however remain ill-defined. We evaluated the extent of species-specific differences in nucleotide metabolism. HPLC analysis was performed on venous blood samples (nucleotide metabolites) and heart biopsies (purine enzymes) from wild type mice, rats, pigs, baboons, and human donors. Ecto-5′-nucleotidase (E5′N) activities were 4-fold lower in pigs and baboon hearts compared to human and mice hearts while rat activity was highest. Similar differences between pigs and humans were also observed with kidneys and endothelial cells. More than 10-fold differences were observed with other purine enzymes. AMP deaminase (AMPD) activity was exceptionally high in mice but very low in pig and baboon hearts. Adenosine deaminase (ADA) activity was highest in baboons. Adenosine kinase (AK) activity was more consistent across different species. Pig blood had the highest levels of hypoxanthine, inosine and adenine. Human blood uric acid concentration was almost 100 times higher than in other species studied. We conclude that species-specific differences in nucleotide metabolism may affect compatibility of pig organs within a human metabolic environment. Furthermore, nucleotide metabolic mismatches may affect clinical relevance of animal organ transplant models. Supplementation of deficient precursors or application of inhibitors of nucleotide metabolism (e.g., allopurinol) or transgenic upregulation of E5'N may overcome some of these differences.     

Differences in Activities of the Enzymes Of Nucleotide Metabolism and its Implications for Cardiac Xenotransplantation

Xenotransplantation is one be possible solution for a severe shortage of human organs available for transplantation. However, only a few studies addressed metabolic compatibility of transplanted animal organs. Our aim was to compare activities of adenosine metabolizing enzymes in the heart of different species that are relevant to clinical or experimental xenotransplantation. We noted fundamental differences: ecto-5′nucleotidease (E5′N) activity was 4-fold lower in pig and baboon hearts compared to the human hearts while mouse activity was compatible with human and rat activity was three times higher than human. There also were significant differences in AMP-deaminase (AMPD), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities. We conclude that differences in nucleotide metabolism may contribute to organ dysfunction after xenotransplantation.     

Differences in activities of the enzymes of nucleotide metabolism and its implications for cardiac xenotransplantation

Xenotransplantation is one be possible solution for a severe shortage of human organs available for transplantation. However, only a few studies addressed metabolic compatibility of transplanted animal organs. Our aim was to compare activities of adenosine metabolizing enzymes in the heart of different species that are relevant to clinical or experimental xenotransplantation. We noted fundamental differences: ecto-5' nucleotidease (E5' N) activity was 4-fold lower in pig and baboon hearts compared to the human hearts while mouse activity was compatible with human and rat activity was three times higher than human. There also were significant differences in AMP-deaminase (AMPD), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities. We conclude that differences in nucleotide metabolism may contribute to organ dysfunction after xenotransplantation.     

Extracellular Adenine Nucleotide Catabolism in Heart Valves

Enzymes of extracellular nucleotide catabolism, such as ecto-nucleoside triphosphate diphosphohydrolase (eNTPD), ecto-5′-nucleotidase (e5NT) and ecto-adenosine deaminase (eADA), control extracellular concentrations of adenine nucleotides and adenosine, furthermore in that way regulate inflammation, immune response, and platelets aggregation. In valves, disturbances of these processes may lead to their dysfunction and calcification. The aim of this study was to analyze the distribution of enzymes, which are engaged in extracellular nucleotide metabolism on the surface of pig aortic and pulmonary valves in relation to activities in the vessel wall. Activity of e5NT was two times higher on the surface of the aortic valve in comparison to the aorta. The same relation between activity of this enzyme in the pulmonary valve and pulmonary artery can be observed. In contrast, eADA activity on the valve surface is much lower than in the vessel wall. No significant differences were observed between the activity of eNTPD on the valve and the vessel surface. This highlights that pattern of enzymes activities favors the production and retention of adenosine on the valve surface and that its alterations could play a role in valve pathology.     

Activities of purine converting enzymes in heart,liver and kidney mice LDLR−/− and Apo E−/−

Nucleotide metabolism plays a major role in a number of vital cellular processes such as energetics. This, in turn, is important in pathologies such as atherosclerosis.

Three month old atherosclerotic mice with knock outs for LDLR and apolipoprotein E (ApoE) were used for the experiments. Activities of AMP-deaminase (AMPD), ecto5′-nucleotidase (e5NT), adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) were measured in heart, liver and kidney cortex and medulla by analysing conversion of substrates into products using HPLC.

The activity of ecto5′-nucleotidase differ in hearts of LDLR^?/? and ApoE^?/? mice with no differences in ADA and AMPD activity. We noticed highest activity of e5NT in kidney medulla of the models.

This model of atherosclerosis characterize with an inhibition of enzyme responsible for production of protective adenosine in heart but not in other organs and different metabolism of nucleotides in kidney medulla.     

Exposure to human blood inactivates swine endothelial ecto-5'-nucleotidase

Ecto-5'-nucleotidase (E5'N) is an extracellular enzyme forming anti-inflammatory and immunosuppressive adenosine. We evaluated whether confrontation of pig heart and endothelial cells with human blood changes the activity of E5'N. Pig hearts were perfused ex vivo with fresh human blood for 4 h. Pig aortic endothelial cells (PAEC) were incubated in vitro with human plasma for 3 h. Ex vivo perfusion of pig heart with fresh human blood resulted in a decrease in E5'N activity to 62% and 61% of initial in wild-type and transgenic pig hearts, respectively. PAEC activity of E5'N decreased to 71% and 50% of initial after 3 h exposure to heat-inactivated and active complement human plasma, respectively, while it remained constant in controls. Pig heart activity of E5'N decreased following exposure to human blood, which may affect adenosine production and exacerbate hyperacute and vascular rejection.     

Metabolism of arachidonic acid in isolated glomeruli from pig kidney

Arachidonic acid metabolism in isolated glomeruli from pig kidney was investigated. Arachidonic acid metabolism via cyclooxygenase was studied by three different methodological approaches: radioimmunoassay (RIA), high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). By all these techniques, the major prostaglandins (PG) formed by pig glomeruli appeared to be 6-keto-PGF1 alpha and PGF2 alpha, the former being the most abundant. RIA and GC-MS also detected lower amounts of thromboxane B2 (TxB2) and PGE2. This emphasises the similarity with human glomeruli, in which the main cyclooxygenase product has indeed been reported to be 6-keto-PGF1 alpha. The lipoxygenase activity in isolated pig glomeruli, as studied by HPLC, generated 15-HETE, 12-HETE and 5-HETE. These data demonstrate that isolated glomeruli from pig kidney possess cyclooxygenase as well as lipoxygenase activity. Since a marked functional similarity exists between human and pig kidney, the pig can be regarded as a good model for studying the influence of arachidonic acid metabolites on glomerular pathophysiology.     

Loss of ecto-5'nucleotidase from porcine endothelial cells after exposure to human blood: Implications for xenotransplantation

The endothelial cell surface expression of ecto-5'-nucleotidase (E5'N, CD73) is thought to be essential for the extracellular formation of cytoprotective, anti-thrombotic and immunosuppressive adenosine. Decreased E5'N activity may play a role in xenograft acute vascular rejection, preventing accommodation and tolerance mechanisms. We investigated the extent of changes in E5'N activity and other enzymes of purine metabolism in porcine hearts or endothelial cells when exposed to human blood or plasma and studied the role of humoral immunity in this context. Pig hearts, wild type (WT, n = 6) and transgenic (T, n = 5) for human decay accelerating factor (hDAF), were perfused ex vivo with fresh human blood for 4 h. Pig aortic endothelial cells (PAEC) were exposed for 3 h to autologous porcine plasma (PP), normal (NHP) or heat inactivated human plasma (HHP), with and without C1-inhibitor. Enzyme activities were measured in heart or endothelial cell homogenates with an HPLC based procedure. The baseline activity of E5'N in WT and T porcine hearts were 6.60 +/- 0.33 nmol/min/mg protein and 8.54 +/- 2.10 nmol/min/mg protein respectively (P < 0.01). Ex vivo perfusion of pig hearts with fresh human blood for 4 h resulted in a decrease in E5'N activity to 4.01 +/- 0.32 and 4.52 +/- 0.52 nmol/min/mg protein (P < 0.001) in WT and T hearts respectively, despite attenuation of hyperacute rejection in transgenic pigs. The initial PAEC activity of E5'N was 9.10 +/- 1.40 nmol/min/mg protein. Activity decreased to 6.76 +/- 0.57 and 4.58 +/- 0.47 nmol/min/mg protein (P < 0.01) after 3 h exposure of HHP and NHP respectively (P < 0.05), whereas it remained unchanged at 9.62 +/- 0.88 nmol/min/mg protein when incubated with PP controls. C1-inhibitor partially preserved E5'N activity, similar to the effect of HHP. Adenosine deaminase, adenosine kinase and AMP deaminase (other enzymes of purine metabolism) showed a downward trend in activity, but none were statistically significant. We demonstrate a specific decrease in E5'N activity in pig hearts following exposure to human blood which impairs adenosine production resulting in a loss of a cytoprotective phenotype, contributing to xenograft rejection. This effect is triggered by human humoral immune responses, and complement contributes but does not fully mediate E5'N depletion.     

Abnormalities in uridine homeostatic regulation and pyrimidine nucleotide metabolism as a consequence of the deletion of the uridine phosphorylase gene

We report in the present study the critical role of uridine phosphorylase (UPase) in uridine homeostatic regulation and pyrimidine nucleotide metabolism, employing newly developed UPase-/- mice. Our data demonstrate that the abrogation of UPase activity led to greater than a 6-fold increase in uridine concentrations in plasma, a 5-6-fold increase in lung and gut, and a 2-3-fold increase in liver and kidney, as compared with wild type mice. Urine uridine levels increased 24-fold normal in UPase-/- mice. Uridine half-life and the plasma retention of pharmacological doses of uridine were significantly prolonged. Further, in these UPase-/- mice, abnormal uridine metabolism led to disorders of various nucleotide metabolisms. In the liver, gut, kidney, and lung of UPase-/- mice, total uridine ribonucleotide concentrations increased 2-3 times as compared with control mice. Cytidine ribonucleotides and adenosine and guanosine ribonucleotides also increased, although to a lesser extent, in these organs. Most significant deoxyribonucleotide changes were present in the gut and lung of UPase-/- mice. In these tissues, dTTP concentration increased more than 4-fold normal, and dCTP, dGTP, and dATP concentrations rose 1-2 times normal. In kidney, dTTP concentration increased 2-fold normal, and dCTP and dGTP concentrations rose less than 1-fold normal. In addition, the accumulated uridine in plasma and tissues efficiently reduced 5-fluorouracil host toxicity and altered the anesthetic effect of pentobarbital. These data indicate that UPase is a critical enzyme in the regulation of uridine homeostasis and pyrimidine nucleotide metabolism, and 5-fluorouracil activity.     

Hepatocyte nuclear factors as possible C-reactive protein transcriptional inducer in the liver and white adipose tissue of rats with experimental chronic renal failure

Little is known about the effects of coffee that are not related to the presence of caffeine. The aim of the study was to analyse changes in kidney function and nucleotide metabolism related to high intake of decaffeinated coffee. Mice consumed decaffeinated coffee extract for two weeks. Activities of AMP deaminase, ecto5′-nucleotidase, adenosine deaminase, purine nucleoside phosphorylase were measured in kidney cortex and medulla by analysis of conversion of substrates into products using HPLC. Concentration of nucleotides in kidney cortex, kidney medulla and serum were estimated by HPLC. Activity of ecto5′-nucleotidase increased from 0.032 ± 0.006 to 0.049 ± 0.014 nmol/mg tissue/min in kidney cortex of mice administered high-dose decaffeinated coffee (HDC) together with increase in cortex adenosine concentration and decrease in plasma creatinine concentration. HDC leads to increased activity of ecto5′-nucleotidase in kidney cortex that translates to increase in concentration of adenosine. Surprisingly this caused improved kidney excretion function.     

Gene expression of D-amino acid oxidase in rabbit kidney

Although D-amino acid oxidase (DAO) [EC 1.4.3.3] activity in rabbit kidney extract was undetectable, protein immunoreactive toward rabbit anti-pig kidney DAO antiserum and RNAs that hybridized with fragments of human and pig DAO cDNAs were detected distinctly in the rabbit kidney. A cDNA clone, RD22, was isolated from the rabbit kidney cDNA library by hybridization with a fragment of human DAO cDNA. Analysis of the nucleotide sequence revealed a 2,018 nucleotide sequence encoding a protein consisted of 347 amino acids. The number of amino acid residues was identical with those of human and pig DAOs, and the amino acid sequence showed 80 and 83% identity with pig and human DAOs, respectively. RNAs that hybridized with RD22 DNA fragment also existed in rabbit kidney, and their sizes were the same as those of the RNAs detected with the human and pig DAO cDNA fragments. RD22-derived protein was hardly synthesized by an in vitro expression system. However, a cDNA fragment lacking most of the 5'-untranslated region and its mutants containing base changes around the initiation codon did direct protein synthesis. Moreover, the protein derived from the partial cDNA fragment containing a large part of the coding region sequence showed immunoreactivity toward anti-pig DAO antiserum. The results suggest that one of the causes of the very poor synthesis of DAO protein in rabbit kidney is translational suppression in the synthetic process.     

Effect of AMP-Deaminase 3 Knock-Out in Mice on Enzyme Activity in Heart and Other Organs

Recent findings suggest that inhibition of AMP-deaminase (AMPD) could be effective therapeutic strategy in heart disease associated with cardiac ischemia. To establish experimental model to study protective mechanisms of AMPD inhibition we developed conditional, cardiac specific knock-outs in Cre recombinase system. AMPD3 floxed mice were crossed with Mer-Cre-Mer mice. Tamoxifen was injected to induce Cre recombinase. After two weeks, hearts, skeletal muscle, liver, kidney, and blood were collected and activities of AMPD and related enzymes were analyzed using HPLC-based procedure. We demonstrate loss of more than 90% of cardiac AMPD activity in the heart of AMPD3 -/- mice while other enzymes of nucleotide metabolism such as adenosine deaminase, purine nucleoside phosphorylase were not affected. Surprisingly, activity of AMPD was also reduced in the erythrocytes and in the kidney by 20%–30%. No change of AMPD activity was observed in the skeletal muscle and the liver.     

Altered NADH and improved function by anesthetic and ischemic preconditioning in guinea pig intact hearts

NADH increases during ischemia because O(2) shortage limits NADH oxidation at the electron transport chain. Ischemic (IPC) and anesthetic preconditioning (APC) attenuate cardiac reperfusion injury. We examined whether IPC and APC similarly alter NADH, i.e., mitochondrial metabolism. NADH fluorescence was measured at the left ventricular wall of 40 Langendorff-prepared guinea pig hearts. IPC was achieved by two 5-min periods of ischemia and APC by exposure to 0.5 or 1.3 mM sevoflurane for 15 min, each ending 30 min before 30 min of global ischemia. During ischemia, NADH initially increased in nonpreconditioned control hearts and then gradually declined below baseline levels. This increase in NADH was lower after APC but not after IPC. The subsequent decline was slower after IPC and APC. On reperfusion, NADH was less decreased after IPC or APC, mechanical and metabolic functions were improved, and infarct size was lower compared with controls. Our results indicate that IPC and APC cause distinctive changes in mitochondrial metabolism during ischemia and thus lead to improved function and tissue viability on reperfusion.     

Exercise improves plasma lipid profiles and modifies lipoprotein composition in guinea pigs

These studies were conducted to determine the effects of exercise training on plasma lipoprotein levels and metabolism in the guinea pig to evaluate potential utilization of this model for studies of exercise-mediated effects on the regulation of sterol and lipoprotein metabolism and atherosclerosis regression. Male guinea pigs (n = 5 per group) were randomly assigned to either a control or an exercise group. The exercise protocol consisted of a 7-week training program, 5 days/wk on a rodent treadmill. Final speed and duration were 33 meters/min for 30–40 min per session. Guinea pigs in the exercise group had 33% lower plasma triacylglycerol concentrations (P < 0.01), 66% higher HDL cholesterol levels (P < 0.05) and 31% lower plasma free fatty acids (P < 0.05) than guinea pigs from the non-exercised group. In addition, lipoprotein lipase activity in the heart was 50% higher (P < 0.025) in guinea pigs allocated to the exercise protocol. Exercise training resulted in modifications in composition and size of lipoproteins. The concentrations of free cholesterol in LDL and HDL were higher in the exercised guinea pigs. The LDL peak density values were lower in guinea pigs from the exercise group compared to controls suggesting that exercise training resulted in larger LDL particles. In contrast, no significant effects due to exercise were observed in hepatic cholesterol concentrations, hepatic HMG-CoA reductase activity or LDL binding to guinea pig hepatic membranes. These data indicate that exercise had a more pronounced effect on the intravascular processing of lipoproteins than on hepatic cholesterol metabolism. In addition, the pattern of changes in guinea pig lipoprotein metabolism, in response to exercise training, was similar to reported effects in humans.     

Effect of nitrobenzylthioinosine accumulated by the heart in rats and guinea pigs on the release of adenine nucleotide breakdown products during ischemia and reperfusion

The uptake kinetics of nitrobenzyl thioinosine (NBTI), a nucleoside transport inhibitor, was studied in the isolated Langendorf-perfused guinea pig and rat hearts. In rats the rate constant of NBTI uptake was higher and the extent of NBTI accumulation was less than in guinea pig hearts. Heart-accumulated NBTI inhibited the total release of adenine nucleotide degradation products (ANDP) during reperfusion 25 min after global ischemia. The effect was more pronounced in guinea-pig hearts-in accordance with observed higher myocardial concentration of NBTI. Unlike other ANDP, the release adenosine by guinea-pig hearts was unchanged and that by rat hearts increased. In spite of significant NBTI-induced decrease of ANDP losses recovery of contractile function during reperfusion was not observed to improve.     

Anti-proliferative activity of L-651,582 correlates with calcium-mediated regulation of nucleotide metabolism at phosphoribosyl pyrophosphate synthetase

L-651,582, 5-amino-[4-(4-chlorobenzoyl)-3,5-dichlorobenzyl]-1, 2,3-triazole-4-carboxamide, is an antiproliferative and antiparasitic agent which inhibits nucleotide metabolism in mammalian cells. The drug equivalently inhibited 3H-hypoxanthine, 14C-adenine, and 14C-formate incorporation into nucleotide pools in Madin-Darby bovine kidney (MDBK) cells, suggesting depletion of the supply of phosphoribosyl pyrophosphate, (PRPP), required for each of these independent pathways. Inhibition of nucleotide metabolism correlated with inhibition of proliferation for three cell types with differing sensitivities toward the drug. L-651,582 inhibited incorporation of 3H-hypoxanthine into nucleotide pools with either glucose, uridine, or ribose as carbon source suggesting a block at PRPP synthetase, rather than a block in a pathway supplying ribose-5-phosphate. PRPP synthetase was not inhibited directly by the compound, indicating regulation of the enzyme in intact cells. Drug treatment did not kill cells but reduced the fraction of cells in S and G2/M while increasing the population in G1. Inhibition of uptake of 45Ca was demonstrated at concentrations identical to those required for inhibition of nucleotide metabolism or proliferation. Inhibition of cellular PRPP biosynthesis rates were also observed using EGTA to lower calcium levels. These data suggest a previously unrecognized link between calcium entry, the regulation of nucleotide biosynthesis at PRPP synthetase, and the rate of proliferation of mammalian cells.     

Iron and copper metabolism in analbuminaemic rats fed a high-iron diet

The metabolism of iron and copper in male Nagase analbuminaemic (NA) and Sprague Dawley (SD) rats was compared. Relative liver weight was higher and spleen weight significantly lower in NA than SD rats. In NA rats, red blood cell count, haemoglobin and haematocrit were lower, whereas plasma transferrin, total iron-binding capacity and mean corpuscular haemoglobin were higher when compared with SD rats. Iron concentrations in plasma, liver, kidneys and heart were higher, and those in the spleen and tibia were lower, in NA rats. The iron concentrations in liver and spleen were positively correlated with the amount of brown pigment as observed histopathologically. Bile flow as well as biliary iron and copper excretion were higher in NA than SD rats. Copper concentrations in liver, kidneys and plasma were higher in NA rats. Plasma levels of ceruloplasmin were about two-fold higher in NA rats. The feeding of a high-iron diet reduced kidney copper concentrations in both strains of rats, which was associated with a decrease in the absorption and biliary excretion of copper.     

Urinary excretion patterns of 5-hydroxyindole-3-acetic acid and 5-hydroxytryptophol in various animal species: implications for studies on serotonin metabolism and turnover rate

The concentrations of the serotonin metabolites 5-hydroxyindole-3-acetic acid (5HIAA) and 5-hydroxytryptophol (5HTOL) were determined in spot urine samples of 12 mammalian and one fish species (cat, cow, dog, ferret, golden hamster, guinea pig, horse, monkey, mouse, rabbit, rainbow trout, rat, sheep) and compared with human data. The highest urinary concentrations of 5HTOL were found in the Sprague-Dawley rat (mean 9.5 micromol/L) and NMRI mouse (8.2 micromol/L), and the lowest in rainbow trout, cynomolgus macaque, and human urine (approximately 0.1 micromol/L). The highest 5HIAA concentrations were found in hamster (89.3 micromol/L) and mouse (85.2 micromol/L), and the lowest in rainbow trout, horse and sheep (range 2.0-3.7 micromol/L). Several species showed 5HIAA concentrations similar to that normally observed in human urine (approximately 5-40 micromol/L). This study demonstrated wide inter- and intra-species variations in the urinary concentrations of 5HIAA and 5HTOL, both separately and in the sum of concentrations. The 5HTOL/5HIAA ratio, which is used as an easily accessible index of the relative importance of the reductive and oxidative pathways for serotonin metabolism, also varied considerably between different species. This observation confirms that the much higher urinary 5HTOL/5HIAA ratio in rats (mean 0.35) compared with humans (< 0.01) is due to a higher baseline formation of 5HTOL in the rat. The monkey, ferret, hamster, and rabbit most closely resembled humans in this respect, and at least the two latter species appear to be more suitable than rats as animal models for studying serotonin metabolism and turnover rate, and the metabolic interaction with ethanol.     

Metabolic pathway of 4-pyridone-3-carboxamide-1β-d-ribonucleoside and its effects on cellular energetics

4-pirydone-3-carboxamide-1β-d-ribonucleoside (4PYR) is an endogenous nucleoside that could be converted to triphosphates, diphosphates, monophosphates and an analogue of NAD − 4PYRAD. Elevated level of these compounds have been reported in chronic renal failure, cancer and active HIV infection. However, little is known about the effect on cell functionality and the metabolic pathways. This study tested effects of 4PYR in different cell types on nucleotide, energy metabolism and clarified enzymes that are involved in conversions of 4PYR.We have found that human neuroblastoma cells, human malignant melanoma cells, human adipose-derived stem cells, human bone marrow-derived stem cells, human dermal microvascular endothelial cells and human embryonic kidney cells, were capable to convert 4PYR into its derivatives. This was associated with deterioration of cellular energetics. Incubation with 4PYR did not affect mitochondrial function, but decreased glycolytic rate (as measured by extracellular acidification) in endothelial cells. Silencing of adenosine kinase, cytosolic 5′-nucleotidase II and nicotinamide nucleotide adenylyltransferase 3, blocked metabolism of 4PYR. Incubation of endothelial cells with 4PYR decreased AMP deaminase activity by 40%.The main finding of this paper is that human cells (including cancer type) are capable of metabolizing 4PYR that lead to deterioration of energy metabolism, possibly as the consequence of inhibition of glycolysis. This study, it was also found that several enzymes of nucleotide metabolism could also contribute to the 4PYRconversions