Kay Overgaard-Hansen

Hypoxanthine as inhibitor of uric acid transport in human erythrocytes

Abstract Hypoxanthine has a powerful inhibitory effect on uric acid transport through the human erythrocyte membrane. This inhibition is, in the case of influx of labelled uric acid, shown to be competitive in nature. The mechanism underlying the efflux of uric acid from erythrocytes seems to have two components, one sensitive to hypoxanthine and one which is independent of hypoxanthine concentrations above a certain level. Such a system is kinetically different from the commonly accepted carrier transport of monosaccharides. An effect of estrogens on the uric acid transport in erythrocytes has recently been reported. These results are discussed in relation to the above findings.

Purine derivatives as inhibitors of uric acid transport into human erythrocytes

Abstract The effect of several derivatives of purine and 8-aza-purine on uric acid transport into human erythrocytes has been investigated. The 6-substituted derivatives of purine which were investigated have approximately the same inhibitory effect as unsubstituted purine. This was also noted with 2,6-substituted derivatives if one or both positions were occupied by an amino group. With hydroxy groups in the 2- and 6-positions, the compound (xanthine) was only half as effective as an inhibitor of uric acid transport. The 2,6-substituted derivatives of 8-aza-purine had the same inhibitory effect as the corresponding derivatives of purine the 6-substituted derivatives of 8-aza-purine inhibited uric acid transport considerably less than the corresponding purine ones. From the finding of mutual inhibition of the transports of hypoxanthine and adenine and from earlier studies it is concluded that the purine and 8-aza-purine derivatives inhibit uric acid transport by competition for a common transport system.

Net uptake of orthophosphate in Ehrlich ascites tumor cells in the presence of purine riboside may be rate limiting for the expansion of the pool of ribonucleotides

Incubation with adenosine or with structural analogs thereof may in several cell types under some conditions result in the cellular accumulation of abundant amounts of the corresponding triphosphates. In the present work we have found that incubation of cells at high concentrations of orthophosphate (Pi) results in increased intracellular levels thereof, although they become not as high as the extracellular concentration. In the presence of purine riboside (nebularine, Pr) and high concentration of Pi the intracellular Pi is, however, kept at a low steady-state level, probably because it immediately upon uptake is being trapped primarily as the triphosphate of purine riboside. The latter compound accumulates at a constant rate for at least 1 h. The rate of accumulation of the sum of phosphate residues present in Pi, adenosine phosphates and purine riboside phosphates appears to be proportional to the extracellular concentration of Pi and to be highly dependent on pH (6.5 and 7.0 being optimal and 7.9 nonpermissible) but it is unaffected by substitution of Na+ by choline.

Synergistic effect of 3'-deoxyadenosine N1-oxide and adenosine deaminase inhibitors on growth of Ehrlich ascites tumor cells in vivo.

SummaryThe simultaneous administration of 3′-deoxyadenosine Nl-oxide (3′-dANO) and the adenosine deaminase inhibitors erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) or 2′-deoxycoformycin (2′-dCF) to mice bearing Ehrlich ascites tumor cells resistant to 3′-dANO resulted in 80%–90% inhibition of tumor growth in vivo. 3′-dANO and 2′-dCF increased the survival time of tumor-bearing mice by a factor of 2. In vitro studies showed that the 3′-dANO resistant Ehrlich cells initiate the metabolism of 3′-dANO by a reduction to 3′-deoxyadenosine, which is converted primarily to 3′-deoxyinosine by adenosine deaminase and, to a small extent, phosphorylated to the cell toxic agent 3′-dATP. By the addition of EHNA or 2′-dCF it was possible to block the formation of 3′-deoxyinosine, resulting in a profound stimulation in the accumulation of 3′-dAtP. The development of resistance to 3′-dANO was studied in cell cultures and found to be accompanied by changes in the enzyme activities of the reductase, the adenosine kinase, and the adenosine deaminase.

Studies on the mechanism of cytotoxicity of 3′-deoxyadenosine N1-oxide in different strains of Ehrlich ascites tumor cells

SummaryThe correlation between the metabolic processing of 3′-deoxyadenosine N1-oxide (3′-dANO) in vitro and its effect on tumor growth in vivo has been investigated in seven different strains of Ehrlich ascites tumor cells.The metabolism of 3′-dANO is initiated by reduction to 3′-deoxyadenosine (3′-dA). This process is the rate-limiting process. The 3′-dA does not accumulate, but is converted to 3′-deoxyadenosine triphosphate (3′-dATP) or 3′-deoxyinosine (3′-dI). The ratio between 3′-dATP and 3′-dI inosine corresponds to the ratio between the activities of adenosine kinase and adenosine deaminase in the cell.Two of the cell lines were markedly inhibited by 3′-dANO in vivo. In these cells the accumulation of 3′-dATP was 1.4–2.2 nmol/h per mg cells, which accounts for the major part of the metabolized 3′-dANO. Five of the cell lines were not inhibited by 3′-dANO and the formation of 3′-dATP was 5–10 times less in these than in the sensitive strains. The low level of 3′-dATP is caused primarily by a low ratio between the activities of adenosine kinase and adenosine deaminase, which is 15 time less than in the sensitive cell lines. The rate of reduction of 3′-dANO seems to be of minor importance.These results indicate a correlation between the inhibition of tumor growth by 3′-dATP and the ability of the cell to accumulate 3′-dATP from 3′-dANO and show that this conversion is determined solely by the rate of reduction of 3′-dANO (3′-dANO reductase activity) and the ratio between the activities of adenosine kinase and adenosine deaminase in the cell. Consequently, the estimation of these enzyme activities in cell lysate of a given tumor can be used to predict whether the tumor is susceptible to inhibition by 3′-dANO.

Differential effect of N-carboxymethylisatoylation on the DNA polymerase activity, the 5′ → 3′-exonuclease activity and the 3′ → 5′-exonuclease activity of DNA polymerase I of Escherichia coli

Abstract Treatment of native DNA polymerase I of Escherichia coli with the acylating agent N- carboxymethylisatoic acid anhydride (NCMIA) results under specific conditions in a rapid loss of polymerase activity, an increase in 5′ → 3′-exonuclease activity and in unchanged 3′ → 5′-exonuclease activity. When a nucleoside triphosphate and Mg2+ was present the polymerase activity was completely protected against the effect of NCMIA. Treatment with higher concentration of the acylating agent under these conditions led to a loss of 3′ → 5′-exonuclease activity without any appreciable loss of polymerase activity. Treatment with NCMIA of the two catalytically active fragments of the enzyme led to very similar results. In this case both the polymerase activity and the 3′ → 5′-exonuclease activity deteriorated more rapidly on treatment with the acylating reagent. The increase in 5′ → 3′-exonuclease activity as a result of modification of the native enzyme appeared to be due to a change in the optimum conditions with regard to concentration of the assay buffer used. These changes are very similar to those seen when the polymerase is cleaved by limited proteolysis. From the results obtained it is concluded that NCMIA reacts primarily with a site at or near the triphosphate-Mg2+ complex binding site, leading to an almost complete loss of polymerase activity. The acylating reagent reacts also with another group on the native enzyme resulting in a modification of the 5′ → 3′-exonuclease activity, and at high concentrations with a group leading to a slow loss of 3′ → 5′-exonuclease activity.

Distribution of 3H within purine nucleotides of Ehrlich mouse ascites tumour cells after intraabdominal injection of myo-[2-3H]inositol

In Ehrlich mouse ascites tumour cells, exposed intra-abdominally to [2-3H]inositol, ATP and GTP presented enough aberrant 3H-label to cause potential interference in the chromatographic analysis of inositol phosphates involved in signal transduction. After acid extraction and charcoal adsorption/desorption the nucleotides were dephosphorylated, enriched with [U-14C]adenosine, and exposed to purine-nucleoside specific enzymes. Reverse phase HPLC and radioactivity measurement demonstrated that for adenosine about 82% of total stable 3H label was in ribose and thus about 18% in adenine. For guanosine about 89% was in ribose and 11% in guanine. This aberrant 3H labelling could be avoided using [1-3H]inositol. Copyright

Expansion of the cellular content of ribonucleoside triphosphates induces cell shrinkage and KCl loss in Ehrlich ascites tumor cells and in Chinese hamster ovary cells

The conversion to corresponding triphosphate derivatives of various ribonucleosides has been studied in Ehrlich ascites tumor cells and in Chinese hamster ovary cells under conditions that are optimal for cellular uptake of orthophosphate. The initial cellular uptake of orthophosphate is followed by a cellular loss of Cl- which might be consistent with a H2PO4-/Cl- exchange mechanism. Subsequent addition of ribonucleosides to the medium leads to cellular accumulation of the corresponding triphosphate and to a concomitant loss of KCl and to sustained cell volume reduction. The latter two events are quite unspecific with regard to the nucleobase moiety of the ribonucleoside triphosphate accumulated (adenine, guanine and purine being almost equally effective) and they depend in a rather simple way on the increase of the cellular content of these compounds. The KCl loss seems to depend on opening of the separate K+ and Cl- channels. The pharmacological profile of the putative ion channels could not be identified in spite of experiments with conventional blockers. In the case of purine riboside the accumulation of the corresponding triphosphate and concomitant loss of KCl and cell water may be followed by a regain of cell volume due to a continued purine riboside triphosphate accumulation, which apparently depends on the uptake of orthophosphate by cotransport with Na+ and which for osmotic reasons is accompanied by the uptake of water and hence volume increase. The possibility that the nucleoside triphosphate induced opening of a putative Cl- channel may be due to a direct effect of triphosphate on a channel protein is discussed.

Toxicity and metabolism of 3′-deoxyadenosine N1-oxide in mice and Ehrlich ascites tumor cells

SummaryThe toxic effect of 3′-deoxyadenosine N1-oxide (3′-dANO) on mice, on their different organs, and on Ehrlich ascites tumor cells was studied. In both healthy and tumour-bearing animals, the lethal dose for 10% of the mice receiving i. p. injections (LD10) of 3′-dANO was estimated to be about 300 mg/kg×4 days in one mouse strain (Theiller). In another mouse strain (NMRI), we obtained a markedly higher LD10 value (675 mg/kg×5 days). At nonlethal doses (250 mg/kg×4 days), we observed reversible neurological symptoms on days 4–12 after treatment, but no macroscopical or microscopical changes was detected in the brain, heart, thymus, lung, lymph node, spleen, liver, kidney, bone marrow, or gastrointestinal tract. At doses of 450 mg/kg×4 days, severe neurological symptoms were observed, and atony of the gastrointestinal canal and damage to the kidney and liver were registered. Even at doses that were lethal to the mice, no histopathological change was observed in the bone marrow or in the gastrointestinal canal. Pharmacokinetics studies showed that after the i.p. injection of 3′-dANO, the maximal plasma concentration was reached after 10 min, after which it declined showing a half-life of about 40 min. A transient accumulation of 3′-deoxyadenosine triphosphate (3′-dATP) was observed within 24 h in the liver and kidney, with the maximal concentration being reached after about 2–3 h. 3′-dANO was excreted partly as the unchanged substance and partly as the metabolite 3′-deoxyinosine within 24 h. Flow-cytometric DNA analysis of Ehrlich tumor cells treated either in vitro or in vivo with 3′-dANO revealed no therapy-induced change in the cell-cycle perturbations, which indicates that cells were randomly killed during all phases of the cycle.