Effect of lead ions on rat erythrocyte purine content.

The influence of short-term exposure to lead on the energetic status of erythrocytes in rats is reported in this study. The male Wistar rats selected for this study drank water containing 1% lead(II) acetate and/or intraperitoneal injections of 1 or 2 mg/kg body wt every 4 d starting on the eighth of the experiment, over a period of 1 mo. The whole-blood lead concentration measured after 4 wk was 1.51-35.31 microg/dL. The concentrations of adenosine, adenosine triphosphates, diphosphates, and monophosphates (ATP, ADP, and AMP), guanine triphosphates, diphosphates and monophosphates (GTP, GDP, and GMP), guanosine (Guo), inosine (Ino), inosine monophosphate (IMP), hypoxantine (Hyp), and nicotinamide dinucleotide and its phosphate (NAD(+) and NADP(+)) were determined by high-performance liquid chromatography (HPLC). The mean concentrations of ATP, GTP, NAD(+), and NADP(+) and those of adenylate (AEC) and guanylate (GEC) were significantly reduced in erythrocytes from the animals exposed to lead when compared to untreated controls. These results suggest that a lead ion disrupts the erythrocyte energy pathways. The decreases of NAD(+) and ATP could be used as an indicator of the extent of exposure to low levels of lead.

Clastogenic inosine nucleotide as components of the chromosome breakage factor in scleroderma patients.

In the present study, we attempted to identify the chemical nature of the clastogenic factor (CF) from patients with progressive systemic sclerosis (scleroderma). Computerized mass spectrometry of clastogenic fractions obtained by HPLC of plasma ultrafiltrates detected molecular peaks compatible with inosine triphosphate and inosine diphosphate (ITP and IDP). The concomitant detection of IDP, together with ITP, and the absence of these peaks in nonclastogenic fractions and corresponding control fractions are arguments in favor of a biological relevance of these observations. The most important confirmation came from the clastogenic effect of commercial ITP and IDP added to the culture medium of the test cultures. The induction of chromatid type damage by these substances in lymphocytes exposed in the G0 phase of their cell cycle and the prevention of this damage by superoxide dismutase are analogous to the observations with CF.

Relationship between adenosine deaminase activity (ADA) and inosinemonophosphate (IMP) accumulation in fresh erythrocytes incubated with adenosine and dipyridamole.

Fresh human erythrocytes were incubated in two media: a) adenosine (10 mM), pyruvate (10 mM), phosphate (50 mM) (APP medium); b) APP medium enriched with 100 mumol/l dipyridamole (APPD) medium. The amount of IMP in fresh erythrocytes was 0.18 +/- 0.09 mumol/g Hb, after incubation in APP medium it was 1.52 +/- 0.78 mumol/g Hb, and after incubation in APP medium it was 1.52 +/- 0.78 mumol/g Hb, and after incubation in APPD the amount was 5.28 +/- 0.94 mumol/g Hb. ADA activity was measured simultaneously. The mean activity (+/- SD) of ADA fresh red cells was 1.29 +/- 0.36 U/g Hb, after 2 h incubation in APP medium it was 1.71 +/- 0.38 U/g Hb, and after 2 h incubation in APPD medium an activity of 2.68 +/- 0.95 U/g Hb was found. A highly significant correlation between the accumulation of IMP and the activity of ADA in fresh erythrocytes (r = 0.93; p = less than 0.001) and in erythrocytes incubated in APPD medium (r = 0.97; p = less than 0.001) was found.

Calcium-induced conversion of adenine nucleotides to inosine monophosphate in human red cells.

1. When inosine-fed human red cells are permeabilized to calcium by exposure to the ionophore A23187, progressively larger proportions of the cell population become irreversibly depleted of ATP as calcium influx is increased (Brown & Lew, 1983; García-Sancho & Lew, 1988b). When calcium influx is over 30 mmol/(l cells.h), all cells become ATP depleted and calcium equilibrated (E cells) (García-Sancho & Lew, 1988b). When calcium influx is lower, E cells co-exist with cells able to maintain normal ATP and low calcium contents in vigorous pump-leak balance (B cells). The experiments reported here investigate why calcium-induced ATP depletion of E cells is irreversible. 2. The inosine monophosphate (IMP) content of cells after 30 min of calcium permeabilization increased with the magnitude of the calcium load, roughly in inverse proportion to the fall in ATP. The calcium-induced increase in IMP was confined to the fraction of cells which became osmotically resistant after SCN- treatment (H cells), and which contained the E cells. 3. Cell nucleotides were measured after calcium permeabilization [( A23187]c = 100 mumol/l cells) in substrate-free media with different [Ca2+]o (0-0.5 mM). Calcium entry caused rapid ATP fall, AMP and IMP accumulation, and delayed ADP fall at all [Ca2+]o concentrations. Initial IMP formation increased with [Ca2+]o along a sigmoid saturation-like curve whereas AMP accumulation and ATP fall were maximal at [Ca2+]o = 20 microM and declined at the higher [Ca2+]o. The rate of IMP formation correlated positively with cell ATP and negatively with cell AMP at all [Ca2+]o values. 4. The AMP deaminase activity of red cell lysates was reversibly increased over tenfold by calcium. Half-maximal stimulation was observed at a Ca2+ concentration of about 50 microM. 5. These results suggest that the irreversibility of calcium-induced ATP depletion results from irreversible trapping of the adenine nucleotide as IMP, and help explain the mechanism of E cell formation.

Assay of 6-thioinosinic acid and 6-thioguanine nucleotides, active metabolites of 6-mercaptopurine, in human red blood cells.

A highly sensitive reversed-phase high-performance liquid chromatographic assay, with ultraviolet detection, for 6-thioinosinic acid and the 6-thioguanine nucleotides (6TGNs) was developed. The 6TGNs are major red blood cell metabolites of the immunosuppressive agent azathioprine and the cytotoxic drugs 6-thioguanine and 6-mercaptopurine. The assay is based on the specific extraction, via phenyl mercury adduct formation, of the thiopurine released on acid hydrolysis of the thionucleotide metabolite. Red blood cell 6TGN concentrations in eighteen leukaemic children receiving chronic 6-mercaptopurine chemotherapy were measured and compared to a previously published spectrophotofluorometric assay. Linear regression analysis gave r = 0.991; P less than 0.001; y = 40 + 0.94x.

Studies on IMP degradation and ribose 1-phosphate utilization in human erythrocytes.

1. Intact human red cells do not attack exogenous IMP. The nucleotide is readily broken down by the soluble erythrocyte fraction to inosine, hypoxanthine and ribose 1-phosphate, with a pH optimum of approx. 6.2. 2. Ribose 1-phosphate can be actively reutilized, in the presence of ATP and hypoxanthine, to give IMP, at pH 7.4. The velocity of the IMP salvage synthesis dramatically increases at more alkaline pH values. 3. The two curves relating the velocities of IMP breakdown and of IMP synthesis as a function of hydrogen ion concentration intersect at pH 7.4. 4. The observations might be relevant in the process of purine transport by red cells.

Accumulation of inosine 5'-monophosphate in human erythrocytes incubated with inosine.

The changes in inosine 5'-monophosphate (IMP) and adenine nucleotides were studied in human erythrocytes incubated with glucose alone, or with glucose plus inosine in the presence or absence of monoiodoacetate, a strong inhibitor of the glycolytic pathways of the cells. HPLC was useful in analyzing the changes in these nucleotides. When the cells were incubated with glucose and inosine for several hours at pH 7.0, 37 degrees C, they produced much IMP, while the cells incubated with glucose and monoiodoacetate accumulated AMP rapidly within 2 hours, accompanied with the decomposition of ADP and ATP; but IMP was not accumulated. When the cells were incubated with glucose and inosine in the presence of monoiodoacetate, AMP increased more rapidly to about 1400 mu mols/ml of cells within 1 hour and then gradually decreased. ATP was consumed completely and ADP decreased correspondingly. However, IMP was produced in this case, though its accumulation was not so high as in the case with glucose plus inosine without monoiodoacetate. The production of IMP depended on the pH of the cell suspension, i.e., more IMP was produced at higher pHs.

Purine metabolism in normal and high-ITP human erythrocytes. Attempts to evaluate the ability to store the cells.

Storage of erythrocyte units from donors with ITP pyrophosphohydrolase deficiency have been studied and compared with units from normal donors. Verifying other investigations the incidence of this genetic disorder was found to be as high as about 3%. Hemolysis in the units was higher than in other units and there was a tendency to low total adenylate concentration. It is suggested that blood centers should organize a quality assurance program where one of the aims should be to detect genetic disorders that make the erythrocytes from the donors less suitable for long term liquid storage.

Incorporation of adenosine and adenine into hypoxanthine nucleotides of fresh red blood cells.

Incorporation of adenosine and adenine into hypoxanthine nucleotides of fresh red blood cells was monitored using 8-14C-adenosine and 8-14C-adenine added to the incubation medium containing adenosine, pyruvate and inorganic phosphate (APP medium). Using 8-14C-adenosine it was shown that 21.7% of the isotope contained in the incubation medium penetrated red blood cells. Of that quantity about 50% becomes incorporated into nucleotides. Of the isotope 5.3% was found in hypoxanthine nucleotides (1.3% in ITP and 4.0% in IMP). During incubation of red blood cells in APP medium fortified with the 8-14C-adenine about 95% of isotope penetrated into cells and 60% of that quantity became incorporated into nucleotides. In hypoxanthine nucleotides only trace amounts of isotope were found (0.12% in IMP and 0.13% in ITP).

Effect of dipyridamole on adenine incorporation into hypoxanthine nucleotides of fresh red blood cells.

An investigation was carried out on the penetration of [8-14C]adenine into fresh human red blood cells and of adenine incorporation into hypoxanthine nucleotides of red blood cells incubated in: 1) a medium containing adenosine, pyruvate, inorganic phosphate and NaCl, and 2) APP medium containing 1 X 10(-4) M dipyridamole (APPD medium). It was found that dipyridamole inhibits by about 45% the penetration of adenine into the red blood cells, and by 18% the incorporation of the isotope into the nucleotides of the cells under study. The inhibition of nucleotide synthesis and incorporation of the isotope into them did not apply to IMP, whose content--following erythrocyte incubation in APPD medium--increased 3.5 times, i.e. from 1.52 to 5.30 mumole/g Hb. At the same time there was an increase of the isotope count from 0.12% in IMP isolated from APP incubated erythrocytes to 0.34% in IMP synthesized in APPD incubated erythrocytes. Erythrocyte incubation in APPD medium reduced ITP synthesis by about 53% relative to its synthesis observed after erythrocyte incubation in APP medium equal to 1.40 mumole per g Hb.

Effect of dipyridamole on adenosine incorporation into hypoxanthine nucleotides of fresh human red cells.

Fresh human red cells were incubated for 2 hours in a medium containing adenosine, pyruvate and inorganic phosphate (APP medium), or in APP medium supplemented with 10(-4) M dipyridamole (APPD medium). No measureable amount of ITP was found in fresh red cells, and the average IMP content in these cells was 0.18 +/- 0.09 mumol/g Hb. After 2 hours incubation in APP medium, the IMP content increased almost 8.5-fold to 1.52 +/- 0.78 mumol/g Hb. Under these conditions the ITP level also increased to 1.40 +/- 0.84 mumol/g Hb. After 2 hours incubation of red cells in APPD medium, the average IMP content increased to 5.30 +/- 2.33 mumol/g Hb, about 3.5 times that found in APP medium. At the same time ITP content was about 53.6% lower, that is 0.65 mumol/g Hb. In red cells incubated in APPD medium, penetration of 8-14C-adenosine decreased by 50%, and incorporation of this nucleotide into the pool of all free nucleotides also decreased by 18.2% as compared to red cells incubated in APP medium. It is concluded that IMP is probably formed directly from AMP gained by the phosphorylation of adenosine during its penetration.

Pyrroline-5-carboxylate stimulates the conversion of purine antimetabolites to their nucleotide forms by a redox-dependent mechanism.

The activation of purine antimetabolites to their respective nucleotides is a step critical to their effectiveness as chemotherapeutic agents. Erythrocytes, with their relatively simple purine metabolism, are useful as a model for identifying mechanisms which enhance this 5-phosphoribosyl 1-pyrophosphate (P-Rib-PP)-dependent activation. We previously showed that pyrroline-5-carboxylate, a physiologic intermediate in the interconversions of proline, ornithine, and glutamate, markedly stimulated the pentose phosphate pathway, increased the formation of P-Rib-PP, and increased purine incorporation into nucleotides. We now report that the events initiated by pyrroline-5-carboxylate markedly increased the activation of 6-thiohypoxanthine, 6-thioguanine, and azathioprine to their respective nucleotides in intact human erythrocytes. The mechanism of this effect was directly demonstrated in studies using the conversion of hypoxanthine to inosine monophosphate as a model for pyrroline-5-carboxylate-mediated stimulation of P-Rib-PP-dependent nucleotide formation. Since the P-Rib-PP-dependent activation of these chemotherapeutic agents may be important to their clinical effectiveness, the events initiated by pyrroline-5-carboxylate may provide new insight into the nature of tumor sensitivity and resistance to these agents.

Adenine-induced hypoxanthine release from IMP-enriched human erythrocytes.

Adenine uptake and hypoxanthine release by IMP-enriched human erythrocytes has been studied. The presence of IMP within the erythrocytes leads to an increase in the rate of adenine incorporation. Adenine is taken up by IMP-enriched erythrocytes as AMP, even when intracellular 5-phosphoribosyl-1-pyrophosphate concentration is undetectable and too low to allow IMP synthesis from hypoxanthine. During adenine uptake and AMP synthesis, hypoxanthine is released by the cells. The possibility that 5-phosphoribosyl-1-pyrophosphate, necessary for AMP synthesis, is formed through the hypoxanthine guanine phosphoribosyltransferase-catalyzed IMP pyrophosphorolysis is considered.

Hypoxanthine nucleotides synthesis in fresh and stored human erythrocytes.

Human erythrocytes incubated in inosine-pyruvate-phosphate (IPP) medium are able to accumulate great amounts of inosine monophosphate (IMP) and inosine triphosphate (ITP). Accumulation of ITP is low in freshly drawn cells. It increases during blood storage in acid-citrate-dextrose solution. Addition of labelled inosine or hypoxanthine to the IPP medium allowed to show the synthesis of small amounts of inosine diphosphate and the appearance of radioactivity in the ATP fraction. Conditions of incubation and the pathways of hypoxanthine nucleotides synthesis are discussed.

Preservation of red blood cells with purines and nucleosides. III. Synthesis of adenine, guanine, and hypoxanthine nucleotides.

Purine nucleotides of fresh human red cells and of red cells during storage at 4 degrees and 25 degrees C with additions of adenine, guanine, guanosine and inosine were estimated by HPLC. Six nucleotides were found in red cells: ATP, ADP, AMP, GTP, GDP, and IMP. The adenine nucleotides represented 92 per cent of the total purine nucleotides, guanine nucleotides 7 per cent and IMP less than 1 per cent. In red cells stored with adenine the total concentration of purine nucleotides increased to 125 per cent of the normal value. An adenine-free but guanine and guanine + inosine containing medium caused a decrease of the concentration of purine nucleotides by 10 to 20 per cent. When red cells were stored without adding guanine or guanosine the content of the guanine nucleotides decreased from 0.32 to 0.17 mumol/g Hb due to the decrease in the GTP content, but the GDP concentration increased slightly. In CPD-AG blood, however, the concentration of guanine nucleotides increased considerably up to 0.6 mumol/g Hb. IMP was estimated in all investigated stored red cells. In CPD-A and in CPD-AG blood 0.4 mumol/g Hb were produced during 3 weeks of storage, but twice of that in CPD-AI blood. The principles of the synthesis and the degradation of purine nucleotides in stored red cells are discussed in detail.

High levels of inosine monophosphate in the erythrocytes of elasmobranchs.

The acid soluble organic phosphates of the erythrocytes of three species of elasmobranchs were assayed by chromatography on Dowex 1 anion exchange columns. Organic phosphates in the peaks eluted from these columns were identified by their ultraviolet absorption spectra and by further chromatography on paper. All three species are unusual amongst the vertebrates in that their erythrocytes contain high levels of inosine monophosphate (IMP). IMP has little effect on the oxygen affinity of the hemoglobins of the two species tested.

Synthesis of ITP and ATP and regeneration of 2,3-DPG in human erythrocytes incubated with adenosine, pyruvate and inorganic phosphate.

In the stored, 2,3-DPG depleted human erythrocytes incubated for four hours in a medium containing adenosine (10 mM), pyruvate (10 mM), and inorganic phosphate (50 mM) the regeneration of 2,3-DPG reached the value of five times higher than the physiological concentration and the ATP synthesis exceeded four times the physiological level. It has been also found that these erythrocytes are able to synthesize hypoxanthine nucleotides, namely IMP to 58 and ITP to 61 mumoles per 100 ml of erythrocytes.

Factors affecting inosinate synthesis and inosine triphosphate accumulation in human erythrocytes.

Measurements of rates of inosinate synthesis from radioactive hypoxanthine by human erythrocytes show a large degree of individual variation. Rates of inosinate synthesis also vary with the pH and phosphate concentration of the incubation medium. This may be due to changes in the rate of phosphoribosyl pyrophosphate synthesis, and the stimulatory effect of phosphate on this process seems to be more important than the inhibitory effect of 2,3-diphodphoglycerate. The rate of inosinate synthesis, and especially the extent of accumulation of inosine triphosphate, increase disproportionately with time of incubation up to at least 24 h. Storage of erythrocytes also tends to increase inosinate synthesis and inosine triphosphate accumulation.

Hydrogen peroxide lowers ATP levels in platelets without altering adenyalte energy charge and platelet function.

H2O2 irreversibly reduced metabolic platelet ATP levels with a corresponding accumulation of hypoxanthine. This process was enhanced by sodium azide or potassium cyanide and by increasing H2O2 concentrations. The adenylate energy charge was unaltered when less than two thirds of the metabolic ATP had disappeared but decreased markedly when more ATP disappeared. Platelet shape change, primary aggregation, dense granule and alpha-granule secretion were unaffected by H2O2-induced lowering of ATP provided that the adenylate energy charge did not fall by more than 5%; at greater adenylate energy charge reduction, platelet functions were inhibited. These results indicate that cell functions depend more on adenyalte energy charge than on the ATP level and expands the applicability of this view from bacterial systems to a mammalian cell, the human platelet.