Nitrobenzylthioinosine: an in vivo inhibitor of pig erythrocyte energy metabolism.

The potential role of plasma nucleosides as metabolic energy substrates for pig erythrocytes, which are impermeable to glucose, was investigated in vivo by infusion of anesthetized pigs with nitrobenzylthioinosine phosphate (NBMPR-P), a soluble prodrug form of the specific nucleoside transport inhibitor, nitrobenzylthioinosine. NBMPR-P administration (1 or 10 mg X kg-1 X h-1) led to complete in vivo blockade of erythrocyte nucleoside transport activity and was associated with a dramatic decrease in the erythrocyte [ATP]-to-[ADP] ratio from 11.4 at time 0 to 2.9 after 4 h (mean results from 3 animals). Plasma inosine concentrations increased progressively from 2-4 microM at time 0 to 20-70 microM after 4 h of drug administration. In contrast, plasma adenosine concentrations remained less than 0.4 microM in all samples. These data suggest that pig erythrocytes utilize plasma inosine as their physiological energy substrate.

Effects of 6-aminonicotinamide on cell growth, poly(ADP-ribose) synthesis and nucleotide metabolism.

The purpose of this study was to determine if the cytotoxic effects of 6-aminonicotinamide are solely the result of an inhibition of poly(ADP-ribose) synthesis. The effects of 6-aminonicotinamide on cell growth, poly(ADP-ribose) synthesis and nucleotide concentrations were compared with the effect of 3-aminobenzamide, a more potent inhibitor of poly(ADP-ribose) synthesis. The growth of L1210 cells was not inhibited by 1 mM 3-aminobenzamide and was only slightly inhibited by 5 mM 3-aminobenzamide even though poly(ADP-ribose) synthesis, as measured by the N-methyl N-nitrosourea induced depletion of NAD+, was inhibited substantially. In contrast, 6-aminonicotinamide was found to be a potent inhibitor of L1210 and CHO cell growth. A 5 mM concentration of 3-aminobenzamide had no effect on purine and pyrimidine ribonucleotide concentrations or on the ATP to ADP ratio, but it did cause a slight elevation of NAD+. 6-Aminonicotinamide at 0.01 mM caused a depletion of purine and pyrimidine nucleotides and NAD+ as well as a reduction in the ATP to ADP ratio. 6-Aminonicotinamide at 1 mM caused a substantial inhibition of purine nucleotide synthesis from [14C] glycine but did not stimulate ATP breakdown. We conclude that inhibition of poly(ADP-ribose) synthesis caused little growth inhibition in itself and that the effects of 6-amininicotinamide on nucleotide metabolism were sufficient to produce an inhibition of both cell growth and DNA repair.

Nucleoside transport and metabolism in erythrocytes from the Yucatan miniature pig. Evidence that inosine functions as an in vivo energy substrate.

Erythrocytes from the Yucatan miniature pig, like those from the normal domestic pig, lack functional glucose transporters and were unable to utilize plasma glucose as an energy source. In contrast, inosine and adenosine entered the cells rapidly. The nucleoside transporter responsible for this uptake was identified as a band 4.5 polypeptide (5000 copies per cell; apparent Mr 45 000-66 000). Inosine concentrations in the physiological plasma range (1.6-2.5 microM) were found to maintain normal erythrocyte ATP levels and ATP/ADP ratios during prolonged in vitro incubation of cells at 37 degrees C, an effect that was blocked by the specific nucleoside transport inhibitor, nitrobenzylthioguanosine. In the absence of extracellular nucleoside, cells 'protected' themselves against some of the consequences of deprivation of energy substrate by glycolyzing the ribose moiety of inosine produced during ATP catabolism. Although erythrocytes from the miniature pig were capable of utilizing extracellular adenosine as an energy substrate, plasma samples from these animals contained less than 0.4 microM adenosine. It is concluded that inosine is a major physiological energy source of pig erythrocytes.

Regulation of purine metabolism in lymphocytes.

Three general questions regarding nucleosides and lymphocytes are discussed: (a) Why are so many measurements being made of adenosine deaminase activity, what do the results mean, and why is there still disagreement about some of the conclusions; (b) what do we understand about nucleosides and lymphocyte death; and (c) to what extent do we really understand nucleoside and nucleotide metabolism in lymphocytes? Experimental studies show that treatment of mice with deoxycoformycin, to produce accumulation of deoxyadenosine, leads to rapid thymus involution, elevated dATP concentrations in thymus and liver, and inhibition of adenosylhomocysteine hydrolase in these tissues. Deoxyguanosine inhibits the growth of mouse lymphoma L5178Y cells, and this toxicity is prevented by deoxycytidine plus adenine. In cells treated with deoxyguanosine, concentrations of both GTP and dGTP are elevated, and this is not affected by deoxycytidine. Adenine, however, reduces GTP concentrations to normal, and prevents most of the elevation in dGTP concentrations. Contrary to previous belief, it has been demonstrated that lymphocytes and nucleated bone marrow cells will synthesize purine nucleotides de novo if incubated in an appropriate medium; carbon dioxide is particularly important for this process.

Specificity of inhibitors of poly(ADP-ribose) synthesis. Effects on nucleotide metabolism in cultured cells.

The effects of inhibitors of poly(ADP-ribose) synthesis on cell growth and several parameters of nucleotide metabolism have been determined. At concentrations which produced similar inhibitions of poly(ADP-ribose) synthesis, 3-acetylaminobenzamide (1 mM) had no effect on L1210 cell growth, 3-aminobenzamide (5mM) was slightly inhibitory and 3-methoxybenzamide (5 mM) was a potent inhibitor of growth. During a 2-h incubation, none of the inhibitors affected ribo- or deoxyribonucleotide concentrations in cells treated with or without N-methyl-N-nitrosourea; however, N-methyl-N-nitrosourea treatment reduced dCTP concentrations by 50%. During a 24-hr incubation, 3-aminobenzamide and 3-acetylaminobenzamide did not lower ribonucleotide concentrations in cells grown with either undialyzed or dialyzed serum. In contrast, 3-methoxybenzamide caused a depletion of UTP in cells grown with undialyzed serum and caused a depletion of all purine and pyrimidine ribonucleotides in cells grown with dialyzed serum. 3-Aminobenzamide and 3-acetylaminobenzamide had no effect on the conversion of hypoxanthine to ATP and GTP but did slightly inhibit incorporation of formate into ATP and GTP. 3-Methoxybenzamide inhibited incorporation of both hypoxanthine and formate into purine ribonucleotides. 3-Aminobenzamide, 3-acetylaminobenzamide, and 3-methoxybenzamide all inhibited glycine incorporation into ATP and GTP and reduced both the incorporation of thymidine into DNA and the apparent specific activity of the dTTP pool. We conclude that inhibition of poly(ADP-ribose) synthesis causes little or no growth inhibition and has no effect on purine or pyrimidine nucleotide synthesis de novo. The effect of all the inhibitors on glycine and formate metabolism may be related to an inhibition of ADP-ribose synthesis or may be a secondary effect of the inhibitors. The growth inhibition and the reduction in nucleotide concentration caused by 3-methoxybenzamide are apparently secondary effects of this drug and may result from an inhibition of phosphoribosyl pyrophosphate synthesis.

Compartmentation of intracellular nucleotides in mammalian cells.

The important role of nucleotides in cellular metabolism requires that serious consideration be given to the question of the homogeneity or inhomogeneity of nucleotide pools in cells. The purpose of this review is to summarize the existing evidence for compartmentation of nucleotide pools, discuss the limitations of this evidence, and to discuss the implications of compartmentation for the interpretation of nucleotide concentration measurements. Evidence for nucleotide compartmentation comes from the following types of evidence: compartmentation of RNA precursors; compartmentation of deoxynucleoside triphosphates; mitochondrial compartmentation; the existence of tightly bound nucleotides; pools derived from alternative synthetic routes; compartmentation in cyclic nucleotide metabolism; channeling in the synthesis of pyrimidine nucleotides; and others. The types of evidence adduced for compartmentation will be considered critically and in detail, and alternative explanations considered, as well. Implications of the data and hypotheses on nucleotide compartmentation for the interpretation of nucleotide pool measurements in various types of experiments will be discussed.

Treatment of multiple myeloma with deoxycoformycin.

A comparison of adenosine deaminase activity in intact human plasma cells and lymphocytes in vitro showed that plasma cells had at least as much activity of this enzyme as did T or non-T lymphocytes. This observation led us to examine the effectiveness of deoxycoformycin in the treatment of multiple myeloma. Thirteen patients with advanced refractory myeloma were treated with deoxycoformycin at 5 mg/m2 daily for 3 days every 2 weeks until response or progression. Of the seven evaluable patients who received more than one cycle of therapy, two had a greater than 50% reduction in the level of myeloma protein and two had a demonstrable reduction in soft tissue disease. Toxicity consisted of marked nausea, anorexia lasting several days, and mild transient confusion in some patients. Plasma levels of deoxyadenosine and adenosine peaked on day 4 or 5 with average values of 1.9 and 0.6 microM, respectively. Red cell levels of dATP reached approximately 40% of ATP levels. The viability of plasma cells was shown to be greatly reduced in in vitro incubations with deoxycoformycin and low levels of deoxyadenosine (ID50 of 6 microM).

Salvage of uridine in the mouse. Effect of uridine phosphorylase pretreatment.

Salvage of circulating nucleosides provides an alternative to de novo synthesis of nucleotides and may modify response to antimetabolites. We have investigated treatment with uridine phosphorylase as a means of inhibiting salvage of uridine in vivo. Examination of the metabolism of intravenous [3H] uridine in mice revealed that 30-40% was salvaged by conversion to uracil nucleotides and the remainder was catabolized. In contrast, less than 0.3% of intravenous [3H]uracil was salvaged. Addition of partially purified bacterial uridine phosphorylase to plasma produced a rapid phosphorolysis of uridine. In vivo, 1.5 hr after intravenous injection of 9 units of uridine phosphorylase, plasma activity (1.3 units/ml) was 65-fold greater than that of control mice. Pretreatment with uridine phosphorylase prior to administration of [3H]uridine produced a marked (65-92%) but incomplete inhibition of salvage of uridine in all tissues examined. The dose required to produce 50% inhibition of uridine salvage at 1 hr was 2 to 2.5 units/mouse. The inhibition of nucleoside salvage by this approach may permit an evaluation of the role of nucleoside salvage in the supply of cellular nucleotides and the effects of concurrent inhibition of de novo and salvage pathways for nucleotide synthesis.

Ultrasensitive assay for ribonucleoside triphosphates in 50-1000 cells. Application to studies with pyrazofurin and mycophenolic acid.

New methods have been developed for the measurement of ATP, GTP, and UTP pools of microscopic cell colonies of 50-1000 cells. These methods are based on stoichiometric generation of ATP from ADP with GTP and UTP serving as phosphate donors, followed by measurement of ATP with firefly luciferase. This technique has been used to generate dose-response curves describing the effects of mycophenolic acid and pyrazofurin on individual colonies of Chinese hamster ovary cells.

Salvage of circulating hypoxanthine by tissues of the mouse.

Hypoxanthine is an inefficient precursor of purine nucleotides in mouse tissues. In vitro, mouse erythrocytes salvage less than 10% of hypoxanthine (10 microM) added to whole blood in 30 min of incubation at 37 degrees C. In vivo, circulating hypoxanthine is rapidly degraded (greater than 90% in 10 min) to allantoin and uric acid. All tissues examined (other than erythrocytes) converted small amounts of hypoxanthine to nucleotides, with kidney and lung being the most active tissues examined. It is estimated that less than 2% of circulating hypoxanthine is salvaged in the mouse; the remainder is catabolized.

Nucleoside triphosphate specificity of firefly luciferase.

Twelve naturally occurring nucleoside triphosphates have been examined as substrates and inhibitors of the light-producing reaction of firefly luciferase. Deoxyadenosine 5'-triphosphate was 1.7% as effective relative to ATP as a substrate, whereas all others tested were less than 0.1% as effective as ATP. At concentrations normally present in mammalian cell extracts no interference with ATP measurements results from these nucleotides.

Ultrasensitive assay of RNA: application to 100-500 cells.

A new method has been developed to measure RNA over the range 0.5 to 25 ng. This procedure involves quantitative hydrolysis of RNA to 5'-monophosphates, conversion of the 5'-AMP to 5'-ATP, and measurement of the ATP by the firefly luciferase assay. This method can be applied to acid precipitated macromolecules without interference from DNA. The measurement of the RNA content of CHO cell colonies of less than 100 cells is described.

Variation in erythrocyte purine metabolism among mouse strains.

Erythrocytes of five strains of mice had ATP concentrations of ca 2.7 mumol/ml packed cells, while those of CBA mice were 23% lower, and those of BALB/C mice were 40% lower. The ratio of the concentrations of ATP and GTP were ca 3.3 in four strains but greater than 27 in three other strains. When erythrocytes from different mouse strains were incubated with radioactive precursors, appreciable strain differences were found in the apparent activities of adenine and hypoxanthine-guanine phosphoribosyltransferase, adenosine kinase, adenosine deaminase, guanine deaminase and xanthine oxidase. The activities of adenosine deaminase and guanine deaminase in sera of mice of different strains also varied.

Comparison of purine and pyrimidine metabolism in G1 and S phases of HeLa and Chinese hamster ovary cells.

Detailed quantitative studies of purine and pyrimidine metabolism in G1 and S phases of synchronized HeLa and Chinese hamster ovary cells have been carried out. Concentrations of ribonucleoside triphosphates increased approximately in proportion to the increase in cell size as cells moved from G1 to S phase. Deoxyribonucleoside triphosphate concentrations were low in G1 phase and increased 2.5- to 10-fold in S phase. Pathways and rates of metabolism of radioactive adenine, guanosine, deoxyadenosine, deoxyguanosine, uridine, cytidine, deoxyuridine, deoxycytidine, and thymidine were determined by measuring the incorporation of each precursor into individual acid-soluble nucleotides and RNA and DNA bases. Cell-cycle or size-dependent differences were detected in many of the parameters studied.

Deoxyadenosine triphosphate accumulation in erythrocytes of deoxycoformycin-treated mice.

The accumulation of deoxyadenosine triphosphate (dATP) in erythrocytes of mice treated with the adenosine deaminase inhibitor deoxycoformycin was studied in an attempt to establish and evaluate a model system for the study of at least some biochemical aspects of hereditary adenosine deaminase deficiency. Mouse erythrocytes in vitro readily phosphorylated deoxyadenosine to dATP, and this nucleotide was relatively stable once formed. dATP accumulated in vivo in mice treated with deoxycoformycin both as a function of dose from 0.25 to 10 mg/kg, and with time after administration. Major sources of the deoxyadenosine used for dATP formation in vivo appear to be normoblast nuclei produced during erythropoiesis, and dying cells; minor sources would appear to include dietary DNA, overproduction of deoxyribonucleotides, and DNA repair.

Relationship between ribo- and deoxyribonucleotide concentrations and biological parameters in cultured Chinese hamster ovary cells.

The relationship between ribo- and deoxyribonucleotide concentrations, growth rate and cell visibility has been studied. Nucleotide pools were manipulated using mycophenolic acid, pyrazofurin, phosphonoacetyl-L-aspartate (PALA) or thymidine. Growth rate, cell viability, nigrosin exclusion, progression through the cell cycle, and the rates of nucleic acid synthesis were measured. The observed qualitative relationships between nucleotide concentrations and growth rate can be rationalized in terms of the effects of changes in substrate availability on nucleic acid synthesis. However, there is no discernible relationship between changes in nucleotide concentrations and the loss of cell viability. More detailed studies will be required to elucidate each step in the complex process leading from the initial changes in nucleotide concentrations to cell death.