Stimulatory Effects of Atorvastatin on Extracellular Nucleotide Degradation in Human Endothelial Cells

Endothelial degradation of extracellular nucleotides is known to be an important mechanism in regulation of thrombosis, inflammation and immune response. It is possible that this pathway is a target for pleiotropic drugs such as atorvastatin. We studied therefore the effect of atorvastatin on extracellular nucleotide degradation in human endothelial cells. Atorvastatin treatment of human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-1) resulted in significant increase in ATP breakdown and adenosine formation both if analysed in intact cell studies and as enzyme activity in cell lysates. We conclude that one of the beneficial effects of atorvastatin may include acceleration of extracellular nucleotide breakdown. This will attenuate nucleotide mediated pro-inflammatory effect and stimulate protective mechanisms of adenosine.     

Atorvastatin accelerates extracellular nucleotide degradation in human endothelial cells

HMG-CoA reductase inhibitors (statins) exert pleiotropic effects in the cardiovascular system beyond its cholesterol-lowering action. We aimed to investigate how atorvastatin affects extracellular nucleotide degradation in human endothelial cells, as increased activity of this pathway would facilitate conversion of pro-inflammatory nucleotides into anti-inflammatory adenosine. Primary cultures of human endothelial cells were treated with 1 μM, 10 μM and 100 μM atorvastatin for 24 h. Enzyme assays were performed as well as intact cell studies, to evaluate capacity of cells to degrade ATP to adenosine. Atorvastatin significantly increased ATP breakdown and adenosine formation in the medium of intact cells in a dose-dependent manner. The activities of ATPase, ADPase and ecto-5′-nucleotidase (eN) in cell homogenates following Atorvastatin treatment were also increased while no change was observed in the lactate dehydrogenase activity. We suggest a new mechanism of protective effect of atorvastatin by activation of endothelial enzymes involved in extracellular nucleotide degradation in human endothelial cells.     

Degradation of extracellular nucleotides and their analogs in HeLa and HUVEC cell cultures

The use of nucleotides and their analogs in the pharmacological studies of nucleotide receptors (P2 class) should be preceded by detailed studies on their degradation connected with ecto-enzymes of a given cell type. In the present studies we have analyzed stability of some phosphorothioate and phosphonate analogs of ATP and ADP in the HeLa epitheloid carcinoma and endothelial HUVEC cells cultures. Our studies have revealed that ecto-nucleotide pyrophosphatase (E-NPP) is one of the main enzymes involved in the extracellular degradation of ATP and other nucleotides in the HeLa cells. On the other hand, the ecto-ATPDase is responsible for the hydrolysis of extracellular nucleotides in human endothelial cell cultures, while the E-NPP-like enzymes of the HUVEC cells are not essential to this degradation. The concerted action of the aforementioned ecto-enzymes and nucleotide pyrophosphatase, 5'-nucleotidase and adenosine deaminase present in fetal bovine serum (FBS) supplied to the culture medium, results in partial or complete degradation of the phosphorothioate (ATPgammaS) and phosphonate analogs of adenosine nucleotides (alpha,beta-methylene-ATP and beta,gamma-methylene-ATP) in the cell cultures. Only ADPbetaS appears to be resistant to these enzymes. The influence of some nucleotides and their analogs on the proliferation of the HeLa cells in presence or absence of FBS is also discussed.     

Extracellular metabolism of nucleotides in neuroblastoma x glioma NG108-15 cells determined by capillary electrophoresis

1. The metabolism of extracellular nucleotides in NG108-15 cells, a neuroblastoma × glioma hybrid cell line, was studied by means of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC).2. In NG108-15 cells ATP, ADP, AMP, UTP, UDP, and UMP were hydrolyzed to the nucleosides adenosine and uridine indicating the presence of ecto-nucleotidases and ecto-phosphatases. The hydrolysis of the purine nucleotides ATP and ADP was significantly faster than the hydrolysis of the pyrimidine nucleotides UTP and UDP.3. ATP and UTP breakdown appeared to be mainly due to an ecto-nucleotide- diphosphohydrolase. ADP, but not UDP, was initially also phosphorylated to some extent to the corresponding triphosphate, indicating the presence of an adenylate kinase on NG108-15 cells. The alkaline phosphatase (ALP) inhibitor levamisole did not only inhibit the hydrolysis of AMP to adenosine and of UMP to uridine, but also the degradation of ADP and to a larger extent that of UDP. ATP and UTP degradation was only slightly inhibited by levamisole.4. These results underscore the important role of ecto-alkaline phosphatase in the metabolism of adenine as well as uracil nucleotides in NG108-15 cells. Dipyridamole, a potent inhibitor of nucleotide breakdown in superior cervical ganglion cells, had no effect on nucleotide degradation in NG108-15 cells.5. Dipyridamole, which is a therapeutically used nucleoside reuptake inhibitor in humans, reduced the extracellular adenosine accumulation possibly by allosteric enhancement of adenosine reuptake into the cells.     

The effects of pro- and anti-atherosclerotic factors on intracellular nucleotide concentration in murine endothelial cells

Abstract

Endothelial cell activation and dysfunction could lead to endothelial injury that is an important factor in the development of vascular diseases. Vascular injury is strongly associated with disturbed endothelial cell energetics and pyridine nucleotide pool. This study aimed to evaluate the effects of inflammatory stimuli (IL-6, LPS), uric acid, hyperglycemia, fatty acids, flavonoids, statins and nonsteroidal anti-inflammatory drugs on cellular concentration of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and nicotinamide adenine dinucleotide (NAD⁺) in cultured endothelial cells. Murine-immortalized heart endothelial cells (H5V cells) were treated with different concentrations of pro- and anti-atherosclerotic factors and intracellular concentration of nucleotides were measured using high performance liquid chromatography. Intracellular ATP concentration in H5V cells was not changed by inflammatory stimuli (IL-6 and LPS), uric acid, glucose, atorvastatin, acetylsalicylic acid, monounsaturated and polyunsaturated fatty acids. Only high concentration of palmitic acid (1?mM) and kaempferol (>0.1?mM) decreased intracellular ATP concentration. The concentration of intracellular ADP has not been altered by any of tested compounds. In turn, intracellular NAD⁺ pool was modified only by polyunsaturated fatty acids and atorvastatin. Linoleic acid, docosahexaenoic acid and atorvastatin increased cellular NAD⁺ concentration. Tested compounds have a small influence on murine endothelial cell energetics, but polyunsaturated fatty acids and atorvastatin increased intracellular NAD⁺ concentration that could be an important protective mechanism against endothelial cell injury.     

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     

Human brain endothelial cells are responsive to adenosine receptor activation

The blood–brain barrier (BBB) of the central nervous system (CNS) consists of a unique subset of endothelial cells that possess tight junctions which form a relatively impervious physical barrier to a large variety of blood components. Until recently, there have been no good in vitro models for studying the human BBB without the co-culture of feeder cells. The hCMEC/D3 cell line is the first stable, well-differentiated human brain endothelial cell line that grows independently in culture with characteristics that closely resemble those of resident human brain endothelial cells. As our previously published findings demonstrated the importance of adenosine receptor (AR) signaling for lymphocyte entry into the CNS, we wanted to determine if human brain endothelial cells possess the capacity to generate and respond to extracellular adenosine. Utilizing the hCMEC/D3 cell line, we determined that these cells express CD73, the cell surface enzyme that converts extracellular AMP to adenosine. When grown under normal conditions, these cells also express the A₁, A_2A, and A_2B AR subtypes. Additionally, hCMEC/D3 cells are responsive to extracellular AR signaling, as cAMP levels increase following the addition of the broad spectrum AR agonist 5′-N-ethylcarboxamidoadenosine (NECA). Overall, these results indicate that human brain endothelial cells, and most likely the human BBB, have the capacity to synthesize and respond to extracellular adenosine.     

Extracellular nucleotide catabolism in human B and T lymphocytes. The source of adenosine production

Extracellular nucleotide degradation was studied in intact human B and T lymphocyte subpopulations and in lymphoblastoid cell lines. Cells of B lymphocyte lineage showed high nucleotide degrading activity, whereas T lymphocytes were unable to degrade extracellular nucleotides. The external surface of B cells contained active sites of ecto-triphosphonucleotidase (ecto-ATPase), ecto-diphosphonucleotidase (ecto-ADPase), and ecto-monophosphonucleotidase (ecto-AMPase). The expression of all three ectoenzyme activities seemed closely associated with B cell development. ATPase and ADPase activities increase continuously during B cell maturation, ecto-AMPase activity, on the other hand, reaches maximal activity in late pre-B cells. These results combined with our previous studies of intracellular ATP catabolism (Barankiewicz, J., and Cohen, A. (1984) J. Biol. Chem. 259, 15178-15181) provide evidence that extracellular ATP catabolism may represent exclusive source for adenosine in lymphocytes. It is suggested that adenosine may serve as a means of communication between B and T cells in lymphoid organs, B lymphocytes being the sole producers of adenosine and T lymphocytes being the recipients of this signal.     

Biphasic cytotoxic mechanism of extracellular ATP on U-937 human histiocytic leukemia cells: involvement of adenosine generation

Since extracellular ATP can exhibit cytotoxic activity in vivo and in vitro, its application has been proposed as an alternative anticancer therapy. In this study we investigated the mechanisms of ATP-induced cytotoxicity in a human leukemic cell line (U-937). ATP added as a single dose exceeding 50 microM was cytostatic or even cytotoxic for U-937 cells. Interestingly, growth inhibition by ATP (50-3500 microM) showed a biphasic dose response. Up to 800 microM, ATP was cytotoxic in a dose-dependent manner (EC(50) 90 microM). In a range between 800 and 2500 microM, cell count was markedly higher despite the higher ATP concentrations. The cytotoxic effect of ATP could be antagonized by addition of uridine as a pyrimidine source and, alternatively, by addition of the nucleoside transmembrane inhibitor dipyridamole. The apoptosis-inducing adenosine A(3) receptor was not involved in measurable quantities, since (1) adenosine did not lead to an elevation of intracellular calcium levels, and (2) an unselective A(1-3) antagonist (ULS-II-80) could not abrogate the cytotoxic effect. Experiments monitoring extracellular nucleotide metabolism confirmed the assumption that the long-term production and continuous uptake of adenosine, which is extracellularly generated by degradation of ATP, led to an intracellular nucleotide imbalance with pyrimidine starvation. The biphasic dose response to higher ATP concentrations could be explained by the rapid degradation of lower ATP concentrations (300 microM) to adenosine by serum-derived enzymes, whereas higher concentrations (900 microM) only produced small amounts of adenosine due to forward inhibition of AMP hydrolysis by prolonged high ADP levels. FACS analysis revealed that at lower adenosine concentrations (300 microM) a reversible G(1) phase arrest of the cell cycle was induced, whereas higher concentrations (1000 microM) triggered apoptosis. Considering ATP as a potential cytostatic drug, our data have important implications concerning metabolic interactions of administered nucleotides.     

Adenine nucleotide degradation during energy depletion in human lymphoblasts. Adenosine accumulation and adenylate energy charge correlation.

Adenine nucleotide breakdown to nucleosides and purine bases was measured in cultures of human lymphoblastoid cells following: 1) the inhibition of oxidative phosphorylation in the absence of glucose or 2) the addition of 2-deoxyglucose. A mutant cell line, deficient in adenosine kinase, in the presence of an adenosine deaminase inhibitor was used to measure utilization of the two pathways of AMP catabolism involving initial action of either purine 5'-nucleotidase or AMP deaminase. In such a system the appearance of adenosine induced by the oxidative phosphorylation inhibitor, rotenone, implies that approximately 70% of AMP breakdown occurs via dephosphorylation. By the same method, deamination accounts for 82% of AMP breakdown when 2-deoxyglucose is added. The occurrence of AMP dephosphorylation is not correlated with elevated concentrations of substrate or with decreased concentrations of the inhibitors of 5'-nucleotidase, ATP and ADP. Dephosphorylation occurs if, and only if, the adenylate energy charge decreases to about 0.6 in these experiments. In cultures deprived of glucose and oxygen, adenine nucleotide degradation via dephosphorylation results in recovery of normal energy charge values.     

Adenosine uptake is the major effector of extracellular ATP toxicity in human cervical cancer cells

In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P2×7 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P2×7 levels, probably through its activation. Corroborating these data, blockage or knockdown of P2×7 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signaling—p53 increase, AMPK activation, and PARP cleavage—as well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells.     

Atorvastatin prevents endothelial dysfunction in high glucose condition through Skp2-mediated degradation of FOXO1 and ICAM-1

Objective

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor atorvastatin has been reported to exert vasculo-protective action in diabetes. We investigated the vasculo-protective mechanism of atorvastatin by evaluating its effect on two major pathogenic molecules, FOXO1 and ICAM1, mediated by S-phase kinase-associated protein 2 (Skp2) in diabetic endothelial dysfunction.

Extracellular adenine compounds, red blood cells and haemostasis: facts and hypotheses

Previously, the role of adenine nucleotides was thought to be confined to the intracellular space of the cell. Research of the last decades has revealed that nucleotides also occur in the extracellular milieu. This survey deals with extracellular adenine compounds in the blood, focussing on their role as chemical mediators in the haemostatic effect of red cells. Erythrocytes may act as pro-aggregatory cells by at least two chemical mechanisms. Firstly, they can enhance platelet aggregation by releasing adenosine diphosphate (ADP), a well known platelet stimulatory substance. ADP is set free when red cells are stressed mechanically, for instance by shear forces generated in the blood stream; ample experimental evidence supporting this view is summarized. Secondly, erythrocytes efficiently take up extracellular adenosine via their nucleoside transporters, thereby removing a potent inhibitor of platelet function. Extracellular adenosine occurs in the blood stream, either directly released from various tissues or as the end product of extracellular adenine nucleotide metabolism, e.g. after degradation of red cell-born ADP or ATP. Finally, a novel mechanism of action of the antithrombotic drug dipyridamole, which has very recently been put forward, is demonstrated. Dipyridamole inhibits platelet function indirectly by blocking the uptake of extracellular adenosine via the nucleoside transporter of red cells; increased adenosine levels in turn are responsible for the antiaggregatory effect of dipyridamole.     

Adherent leukocytes prevent adenosine formation and impair endothelial barrier function by Ecto-5'-nucleotidase/CD73-dependent mechanism

Extracellular purines are important signaling molecules that mediate both inflammatory (ATP, ADP) and anti-inflammatory (adenosine) effects in the vasculature. The duration and magnitude of purinergic signaling is governed by a network of purine-converting ectoenzymes, and endothelial and lymphoid cells are generally characterized by counteracting ATP-inactivating and ATP-regenerating/adenosine-eliminating, phenotypes, respectively. By using cultured human umbilical vein endothelial cells and normal or leukemic lymphocytes as an in vitro model of leukocyte-endothelial interactions, we have identified a link between the adhesion cascade and extracellular purine turnover. Upon adhesion, lymphocytes suppress endothelial purine metabolism via (i) inhibition of ecto-5'-nucleotidase/CD73-mediated AMP hydrolysis, (ii) rapid deamination of the remaining adenosine, and (iii) maintenance of the sustained pericellular ATP level through continuous nucleotide release and phosphotransfer reactions. Compensation of the loss of adenosine promotes vascular barrier function (measured as a paracellular flux of 70 kDa fluorescein isothiocyanate-dextran) and decreases transendothelial leukocyte migration. Together, these data show that adherent lymphocytes attempt to prevent adenosine formation in the endothelial environment that, as a consequence, may impair the vascular barrier function and facilitate the subsequent step of leukocyte transmigration into the tissue. These leukocyte adhesion-mediated shifts in the local nucleotide and nucleoside concentrations represent a previously unrecognized paracrine mechanism affecting the functional state of the targeted vascular endothelium and coordinately regulating lymphocyte trafficking between the blood and tissues.     

Influence of 4-pyridone-3-carboxamide-1Β-D-ribonucleoside (4PYR) on activities of extracellular enzymes in endothelial human cells

ABSTRACT

Previous studies demonstrated that human endothelial cells were capable to phosphorylate 4-pyridone-3-carboxamide-1β-D-ribonucleoside (4PYR) to monophosphate (4PYMP) and formed another metabolite—an analog of NAD (4PYRAD). Elevated levels of 4PYMP and 4PYRAD had an adverse effect on energy balance—depressed adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD) concentration in human endothelial cells. Ecto-enzymes such as ecto-nucleoside triphosphate diphosphohydrolase (eNTPD); ecto-5′-nucleotidase (e5’NT); and ecto-adenosine deaminase (eADA) are involved in controlling of inflammation and platelet aggregation. This study aimed to evaluate influence of 4PYR and its metabolites on activities of extracellular enzymes in human endothelial cells. Endothelial cells (endothelial cell line HMEC-1) were treated with 100 uM 4PYR for 0, 24, 48, or 72 hours. After incubation, intact HMEC-1 cells were incubated with suitable substrate. Simultaneously, in another path of experiments intracellular concentration of 4PYMP and 4PYRAD had been analyzed. Conversion of extracellular nucleotides into their products and intracellular concentration of 4PYMP and 4PYRAD were measured by high performance liquid chromatography (HPLC). We demonstrated that eNTPD and e5’NT activities increase after 72 hours of cell treatment with 4PYR as compared to control (0.40 ± 0.02 versus 0.29 ± 0.02 nmol/min/mg protein; 13.3 ± 0.6 versus 8.30 ± 0.34 nmol/min/mg protein, respectively, mean ± SEM). eADA activity decreases after 24 hours of cells treatment with 4PYR as compared to control (1.55 ± 0.06 versus 1.92 ± 0.13 nmol/min/mg protein, respectively, mean ± SEM). 4PYR and its derivatives have positive effect on ecto-enzymes related with ATP degradation pathway. We conclude that these increases in extracellular enzyme activities are an adaptive response to decreased intracellular ATP and NAD arising from 4PYR uptake. These changes may protect the cells from the inflammatory result of external ATP degradation.     

Epac1 regulates integrity of endothelial cell junctions through VE-cadherin

We have previously shown that Rap1 as well as its guanine nucleotide exchange factor Epac1 increases cell-cell junction formation. Here, we show that activation of Epac1 with the exchange protein directly activated by cAMP (Epac)-specific cAMP analog 8CPT-2'O-Me-cAMP (007) resulted in a tightening of the junctions and a decrease in the permeability of the endothelial cell monolayer. In addition, 007 treatment resulted in the breakdown of actin stress fibers and the formation of cortical actin. These effects were completely inhibited by siRNA against Epac1. In VE-cadherin knock-out cells Epac1 did not affect cell permeability, whereas in cells re-expressing VE-cadherin this effect was restored. Finally, the effect of Epac activation on the actin cytoskeleton was independent of junction formation. From these results we conclude that in human umbilical vein endothelial cells Epac1 controls VE-cadherin-mediated cell junction formation and induces reorganization of the actin cytoskeleton.     

Accelerated degradation of adenine nucleotide in erythrocytes of patients with chronic renal failure

Recently, we have shown that erythrocytes obtained from patients with chronic renal failure (CRF) exhibited an increased rate of ATP formation from adenine as a substrate. Thus, we concluded that this process was in part responsible for the increase of adenine nucleotide concentration in uremic erythrocytes. There cannot be excluded however, that a decreased rate of adenylate degradation is an additional mechanism responsible for the elevated ATP concentration. To test this hypothesis, in this paper we compared the rate of adenine nucleotide breakdown in the erythrocytes obtained from patients with CRF and from healthy subjects.Using HPLC technique, we evaluated: (1) hypoxanthine production by uremic RBC incubated in incubation medium: (a) pH 7.4 containing 1.2 mM phosphate (which mimics physiological conditions) and (b) pH 7.1 containing 2.4 mM phosphate (which mimics uremic conditions); (2) adenine nucleotide degradation (IMP, inosine, adenosine, hypoxanthine production) by uremic RBC incubated in the presence of iodoacetate (glycolysis inhibitor) and EHNA (adenosine deaminase inhibitor). The erythrocytes of healthy volunteers served as control.The obtained results indicate that adenine nucleotide catabolism measured as a hypoxanthine formation was much faster in erythrocytes of patients with CRF than in the cells of healthy subjects. This phenomenon was observed both in the erythrocytes incubated at pH 7.4 in the medium containing 1.2 mM inorganic phosphate and in the medium which mimics hyperphosphatemia (2.4 mM) and metabolic acidosis (pH 7.1). The experiments with EHNA indicated that adenine nucleotide degradation proceeded via AMP-IMP-Inosine-Hypoxanthine pathway in erythrocytes of both patients with CRF and healthy subjects. Iodoacetate caused a several fold stimulation of adenylate breakdown. Under these conditions: (a) the rate of AMP catabolites (IMP + inosine + adenosine + hypoxanthine) formation was substantially higher in the erythrocytes from patients with CRF; (b) in erythrocytes of healthy subjects degradation of AMP proceeded via IMP and via adenosine essentially at the same rate; (c) in erythrocytes of patients with CRF the rate of AMP degradation via IMP was about 2 fold greater than via adenosine.The results presented in this paper suggest that adenine nucleotide degradation is markedly accelerated in erythrocytes of patients with CRF.     

Induction of phosphodiesterase by cyclic adenosine 3':5'-monophosphate in differentiating Dictyostelium discoideum amoebae.

Cyclic adenosine 3':5'-monophosphate added to the starvation media of Dictyostelium discoideum amoebae induces both intracellular and extracellular phosphodiesterase activities of these cells. The induced enzyme activity appears several hours earlier than that in starved cells which have not been induced with cyclic nucleotide. In both cases, the appearance of enzyme is inhibited by cycloheximide, and actinomycin D, and daunomycin. The KmS for the extracellular enzyme(s) of nucleotide-induced and uninduced control cells are identical. The induction of enzyme activity seems specific for cyclic adenosine 3':5'-monophosphate since cyclic guanosine 3':5'-monophosphate, as well as other nucleotides, have no effect. No differences in the activity or excretion of either N-acetylglucosaminidase or the inhibitory of the extracellular phosphodiesterase are observed between cyclic adenosine 3':5'-monophosphate-induced and control cells. A direct activation of phosphodiesterase by cyclic adenosine 3':5'-monophosphate can be excluded, since the addition of this nucleotide to cell lysates has no effect on the enzyme activity.     

Nucleotide- and nucleoside-converting ectoenzymes: Important modulators of purinergic signalling cascade

The involvement of extracellular nucleotides and adenosine in an array of cell-specific responses has long been known and appreciated, but the integrative view of purinergic signalling as a multistep coordinated cascade has emerged recently. Current models of nucleotide turnover include: (i) transient release of nanomolar concentrations of ATP and ADP; (ii) triggering of signalling events via a series of ligand-gated (P2X) and metabotropic (P2Y) receptors; (iii) nucleotide breakdown by membrane-bound and soluble nucleotidases, including the enzymes of ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family, ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) family, ecto-5'-nucleotidase/CD73, and alkaline phosphatases; (iv) interaction of the resulting adenosine with own nucleoside-selective receptors; and finally, (v) extracellular adenosine inactivation via adenosine deaminase and purine nucleoside phosphorylase reactions and/or nucleoside uptake by the cells. In contrast to traditional paradigms that focus on purine-inactivating mechanisms, it has now become clear that "classical" intracellular ATP-regenerating enzymes, adenylate kinase, nucleoside diphosphate (NDP) kinase and ATP synthase can also be co-expressed on the cell surface. Furthermore, data on the ability of various cells to retain micromolar ATP levels in their pericellular space, as well as to release other related compounds (adenosine, UTP, dinucleotide polyphosphates and nucleotide sugars) gain another important insight into our understanding of mechanisms regulating a signalling cascade. This review summarizes recent advances in this rapidly evolving field, with particular emphasis on the nucleotide-releasing and purine-converting pathways in the vasculature.     

Expression of Human Ecto 5′ Nucleotidase in Pig Endothelial Cells and Its Implication for Adenosine Production and Xenotransplantation

Human endothelial activity of ecto-5′-nucleotidase (E5′N) is several times higher than in pig endothelial cells. This may have implication for xenotransplantation due to the role this enzyme plays in conversion of pro-inflammatory and pro-aggreggatory nucleotides into anti-inflammatory and anti-aggregatory adenosine. We have shown in this study that human E5′N can be functionally expressed in pig endothelial cells leading to increased adenosine production from both extracellular AMP and ATP. We suggest that E5′N expression in transgenic pigs for xenotransplantation may help to prolong graft survival.