Jerzy Barankiewicz

Menadione-Induced Oxidative Stress in Bovine Heart Microvascular Endothelial Cellsa

Objective: Oxidative stress from increased production of reactive oxygen species or decreased efficiency of inhibitory and scavenger systems may contribute to vascular injury. In this study, we developed an in vitro model of vascular injury by menadione‐induced oxidative stress in bovine heart microvascular endothelial cells.

Alteration of purine metabolism by AICA-riboside in human B lymphoblasts.

The effect of 5-amino-4-imidazole-carboximide (AI-CA)-riboside on different pathways of purine metabolism (biosynthesis de novo, salvage pathways, adenosine metabolism, ATP catabolism) was studied in human B lymphoblasts (WI-L2). AICA-Riboside markedly decreased intracellular levels of 5-phosphoribosyl-1-pyrophosphate and in consequence affected purine biosynthesis de novo and purine salvage pathways. AICA-riboside inhibited incorporation of glycine into purine nucleotides, but when formate was used as the precursor of purine biosynthesis de novo, a biphasic effect was observed. The incorporation of formate into purine nucleotides was increased by AICA-riboside at concentrations up to 2 mM but decreased at higher concentrations. Salvage of the purine bases adenine, hypoxanthine, and guanine was markedly inhibited and utilization of extracellular adenosine in B lymphoblasts was reduced by AICA-riboside. AICA-riboside increased ribose 1-phosphate concentrations and increased degradation of prelabeled ATP. No effect on the intracellular levels of orthophosphate was found. Proliferation of WI-L2 lymphoblasts was only slightly affected at concentrations of AICA-riboside below 500 microM but markedly inhibited by higher concentrations.

Impairment of nucleotide metabolism by iron-chelating deferoxamine☆

The effect of deferoxamine on nucleotide metabolism in HL-60 leukemic cells was studied to explore the mechanism of its antiproliferation activity. It was found that in intact cells deferoxamine markedly inhibited the ribonucleotide reduction and incorporation of bases (adenine, hypoxanthine), ribonucleosides (inosine, guanosine) and deoxyribonucleosides (thymidine, deoxyadenosine, deoxyguanosine) into nucleic acids. Although deferoxamine did not inhibit thymidine and uridine incorporation into free nucleotides, inhibition of hypoxanthine and adenine incorporation into nucleotides as well as inhibition of nucleotide biosynthesis de novo was found. Nucleotide catabolism, protein synthesis, and intracellular levels of ribonucleotides were not affected significantly by deferoxamine. These results showed that deferoxamine selectively affects several specific reactions of nucleotide metabolism. Inhibition of ribonucleotide reduction, inhibition of ribonucleotide and deoxyribonucleotide incorporation into nucleic acids, as well as inhibition of purine biosynthesis, may alter significantly cellular physiology and, therefore, contribute significantly to the antiproliferative activity of deferoxamine.

Selective protection of tubercidin toxicity by nitrobenzyl thioinosine in normal tissues but not in human neuroblastoma cells

SummaryTubercidin, an adenosine analogue, is toxic to human neuroblastoma cell lines, to peripheral blood mononuclear cells (PBMCs), and to myeloid colony-forming cells (CFU-C) as tested by a short-term labeled precursor uptake and by a clonogenic assay. When it was co-administered with a potent purine transport inhibitor, nitrobenzyl thioinosine (NBTI), the cytotoxic effect of tubercidin was abolished in PBMCs but not in neuroblastoma cells. Studies of nucleoside transport in neuroblastoma cells demonstrate that although [3H]NBTI binds to the plasma membrane of these cells, the transport of thymidine into the cells is only partially inhibited in the presence of excess NBTI. These data imply that neuroblastoma cells contain a nucleoside transport mechanism which is insensitive to NBTI. “Host protection” with a nucleoside transport inhibitor such as NBTI, may allow effective therapy with otherwise toxic dosages of tubercidin and other cytotoxic nucleosides in patients with neuroblastoma.

Purine Metabolism in Rat Macrophages

The association of deficiencies of purine metabolic enzymes with immunodeficiency diseases has stimulated research in purine metabolism of lymphoid cells. Up to now our knowledge about purine metabolism of other cells of the immune system, e.g., macrophages, is relatively limited. During phagocytosis, macrophages excrete large amounts of uric acid1. The excretion of uric acid suggests that nucleotide catabolism may be predominant in phagocytosing macrophages. On the other hand, purine bases and nucleosides formed during digestion of nucleic acids may be transported across the phagolysosomal membrane and reutilized into intracellular nucleotide pools. Indeed activities of several purine salvage enzymes along with enzymes of purine catabolism have been found in macrophage extracts1,2.

Endogenous Adenosine Formation can Regulate Human Neutrophil Function

Neutrophils, in addition to their role in host defense, can cause injury to normal tissues during inflammatory processes. Oxygen radicals and secreted proteases, in particular, are responsible for some aspects of neutrophil-mediated injury to endothelial cells and cardiomyocytes. A variety of neutrophil functions, including adhesion and reactive oxygen species production, are inhibited by adenosine (Ado) (Cronstein, 1991 and Cronstein, et al., 1992). Furthermore, inhibition of neutrophil adhesion by adenosine regulating agents like acadesine and adenosine kinase (AK) inhibitors (Firestein, et al., 1994) appears to be mediated by Ado, since it is reversed by the addition of adenosine deaminase (ADA) or Ado receptor antagonists. Although Ado and adenine nucleotides can be released at inflammatory sites during platelet aggregation or from endothelial cells during ischemic stress conditions, little is known about Ado formation by human neutrophils. To determine if neutrophils can serve as an endogenous Ado source and thereby provide an autocrine stimulus, we evaluated purine metabolism and Ado formation in human neutrophils.

Inhibition of nucleoside transport by reactive oxygen species in bovine heart microvascular endothelial cells.

Cellular homeostasis depends on the structural and functional integrity of the membrane bilayer. Damage to the membrane can interfere with vital cellular processes, including signal transduction, molecular recognition, maintenance of the membrane potential, cellular metabolism and transport of molecules. Modification of the membrane bilayer by reactive oxidative species (ROS) is a major contributor to membrane damage and has been implicated in many pathological processes. In a number of diseases, injury to endothelial cells is mediated by oxidative species generated during ischemia/reperfusion or by activated neutrophils. During ischemia and oxidant injury, several metabolic events occur, including depletion of intracellular ATP and formation of a number of purine catabolites including inosine (Ino), hypoxanthine (Hyp) and adenosine (Ado) (Halliwell and Gutteridge, 1990). Accumulated Hyp can be oxidized by xanthine oxidase when the tissue is oxygenated, causing rapid generation of Superoxide and hydrogen peroxide. On the other hand, formation of Ado is especially important because it has vasodilatory and antiinflammatory activity. Alterations in nucleoside transport (NT) by ROS might have an effect on extracellular Ado concentration in vivo. Reduced Ado transport could elevate Ado concentrations, especially when Ado is formed extracellularly. Inhibition of NT could also diminish extracellular nucleoside uptake required for the reconstitution of intracellular nucleotide pools after cellular stress. Decreased nucleoside efflux might protect cells by allowing more efficient intracellular nucleoside salvage.

Regulation of Adenosine Concentrations by Acadesine (Aica-Riboside) in Human B-Lymphoblasts

A large number of physiological functions of adenosine including regulation of coronary vasodilation, (1) neurotransmission (2) and immune response (3) have been described and discussed (4–8). However, the mechanism(s) involved in adenosine formation remain controversial. Adenosine found in the extracellular environment can be formed by: a) the intracellular process of AMP dephosphorylation and SAH hydrolysis resulting in adenosine release across the cell membrane; b) extracellular degradation of adenine nucleotides released from the tissues; c) both intracellular and extracellular mechanisms occurring simultaneously.

Can Adenosine Deaminase Inhibitors be Cytoprotective Agents

Adenosine (Ado) is a well recognized nucleoside that possesses a number of beneficial properties including cardioprotective, cerebroprotective, anti-inflammatory and analgesic activities.1

Z-Nucleotides Formation in Human and Rat Cells

Little is known about the formation and role of Z-nucleotides (ZMP,ZDP,ZTP) in rat and human cells. In cells having an active purine de novo biosynthesis pathway, ZMP (AICA-riboside monophosphoribonucleoside) is formed naturally as one of the final intermediates in IMP biosynthesis. ZMP, when synthesized by this pathway, is then efficiently formylated by 5-amino-4-imidazole-carboxamide 5′ribonucleotide transformylase (EC 2.1.2.3) and therefore the intracellular level of ZMP is usually very low and is not detectable by HPLC (1–3). ZMP can also be formed when cells with an active or absent de novo purine biosynthesis pathway are exposed to acadesine (AICA-riboside) or RICA-base. ZMP is formed from acadesine after phosphorylation, whereas formation from AICA-base requires the transfer of the phosphoribosyl group from PRPP. It has already been established that adenosine kinase (AK) is responsible for the phosphorylation of acadesine to ZMP, and AK inhibited or AK deficient cells are unable to metabolize acadesine (4–7). ZMP, when formed from acadesine, can be further metabolized by alternative pathways depending on its intracellular concentrations (2). At low ZMP concentrations, metabolism of ZMP follows predominantly the steps of de novo synthesis of IMP and other purines. At intermediate concentrations of ZMP, net retrograde flux through adenylosuccinate lyase results in sZMP (5-amino-4imidazole-N-succinocarboxamide ribonucleotide) formation, while at high ZMP concentrations, accumulation of ZMP, ZDP and ZTP becomes significant.