Alteration of lithium pharmacology through manipulation of phosphoadenosine phosphate metabolism.

Bisphosphate 3'-nucleotidase (BPNT1 in mammals and Met22/Hal2 in yeast) is one of five members of a family of signaling phosphatases united through a common tertiary structure and inhibition by subtherapeutic doses of the antibipolar drug lithium. Here we report a role for 3'-nucleotidase and its substrate, 3'-phosphoadenosine 5'-phosphate (PAP), in mediating the cellular effects of lithium. Lithium-induced inhibition of growth in yeast cells may be overcome by dose-dependent heterologous expression of human BPNT1. Disruption of the yeast 3'-nucleotidase gene or treatment of cells with lithium results in a >80-fold accumulation of PAP and leads to potent growth inhibition. These data indicate that the accumulation of a 3'-nucleotidase substrate, such as PAP, mediates the toxicity of lithium. To further probe this model we examined the growth inhibitory effects of lithium under conditions in which PAP biosynthetic machinery was concomitantly down-regulated. Disruption of met3 or met14 genes (ATP sulfurylase or phosphosulfate kinase), transcriptional down-regulation of MET3 through methionine addition, or administration of chlorate, a widely used cell-permeable sulfurylase inhibitor, function to reduce lithium-induced intracellular PAP accumulation and lithium toxicity; all of these effects were reversed by heterologous expression of human sulfurylase and kinase. Collectively, our data support a role for 3'-nucleotidase activity and PAP metabolism in aspects of lithium's mechanism of action and provide a platform for development of novel pharmacological modulators aimed at improving therapies for the treatment of bipolar disorder.

Regional localization of human ecto-5' nucleotidase to chromosome 6q14-q21.

Human and mouse hybrids that contain fragments of human chromosome 6 as translocations were analysed for expression of ecto-5' nucleotidase enzymic activity measured by the conversion of AMP to adenosine and for antigenicity recognized by a monoclonal antibody specific for the human isozyme. Both methods allow a regional assignment of ecto-5'nucleotidase to 6q14-q21.

Pyrimidine nucleotides impair phosphoribosylpyrophosphate (PRPP) synthetase subunit aggregation by sequestering magnesium. A mechanism for the decreased PRPP synthetase activity in hereditary erythrocyte pyrimidine 5'-nucleotidase deficiency.

The activity of phosphoribosylpyrophosphate (PRPP) synthetase (ATP: D-ribose-5-phosphate pyrophosphotransferase, EC 2.7.6.1) is decreased in the erythrocyte in hereditary pyrimidine 5'-nucleotidase (P5N) deficiency. Given the increased pyrimidine nucleotide content of the P5N-deficient erythrocyte, we evaluated the effects of prototypic pyrimidine nucleotides on the activity of PRPP synthetase. In normal hemolysate a 1.0 mM combination of cytidine tri-, di- and monophosphate (CTP/CDP/CMP) inhibited PRPP synthetase activity and changed the ribose 5-phosphate (R5P) saturation curve from a hyperbola to a biphasic shape. Untreated crude hemolysate from P5N-deficient erythrocytes showed a biphasic R5P kinetic curve. Since the activity of PRPP synthetase is dependent on its state of subunit aggregation, we examined PRPP synthetase subunit aggregation using gel permeation chromatography. P5N-deficient erythrocytes had a decreased absolute amount of aggregated PRPP synthetase and almost a total loss of disaggregated PRPP synthetase. Using normal hemolysate, 1 mM CTP/CDP/CMP interfered with the ability of 1.0 mM ATP and 2.0 mM MgCl2 to promote PRPP synthetase subunit aggregation. Increasing the MgCl2 to 6.0 mM overcame the inhibitory effect of CTP/CDP/CMP. Thus, the decreased PRPP synthetase activity of the P5N-deficient erythrocyte is due, at least in part, to the ability of the accumulated pyrimidine nucleotides to sequester magnesium and to interfere with the subunit aggregation of PRPP synthetase.

Aplastic crisis due to human B19 parvovirus infection in red cell pyrimidine-5'-nucleotidase deficiency.

Two siblings with chronic hemolytic anemia due to red cell pyrimidine-5'-nucleotidase (P-5'-N) deficiency, presented within a few days of each other with a febrile illness and pancytopenia. The cause of the aplastic crisis was an acute infection with human B19 parvovirus (B19 HPV) as proven by immunoelectron microscopy and DNA hybridization. This is the first report on the association of B19-HPV-related aplastic crisis with P-5'-N deficiency.

Erythrocyte pyrimidine 5'-nucleotidase and deoxynucleotidase isozymes: metallosensitivity and kinetics.

Erythrocytes from a subject with classical pyrimidine 5'-nucleotidase (P5N) deficiency (PND) possessed typically low nucleotidase activity with uridine 5'-monophosphate (UMP) and cytidine 5'-monophosphate (CMP) as the substrates with subnormal dephosphorylation of 2'-deoxy thymidine 5'-monophosphate (dTMP) and 2'-deoxyuridine 5'-monophosphate (dUMP) and intermediate activity with 2'-deoxycytidine 5'-monophosphate (dCMP). Contrary to previously reported data, there was enzyme activity with all five substrates. The metallosensitivities of the P5N isozymes using UMP, dUMP, dCMP and dTMP as the substrates were defined. Nucleotidase (P5N) is slightly more sensitive than 2'-deoxy-pyrimidine 5'-nucleotidase (dP5N) to inhibition by lead, copper and mercury. Chromium stimulated more enzyme activity with dCMP than other substrates. Apparent Michaelis constants (Km) were calculated for UMP, CMP, dUMP, dCMP, and dTMP for PND and control subjects. Optimal activity was at pH 7.0-7.8 when using UMP and CMP as the substrates but at pH 5.5-6.5 with dUMP, dCMP and dTMP as the substrates. The Km, pH optima and metallosensitivities were, however, consistent with electrophoretic evidence for two and probably three isozymes: P5N with maximal affinity for UMP and CMP; dP5N with maximal affinity for dUMP and dTMP; and a closely related dCMPase. The relative activity with representative substrates can be used to distinguish homozygotes and heterozygotes for P5N deficiency and subjects with heavy metal exposure.

Emergence of activated lymphocytes in CD4 and CD8 subpopulations of multiple myeloma: correlation with the expansion of suppressor T-cells (CD8+ OKM1+) and ecto-5'nucleotidase deficiency.

This study of CD4 and CD8 lymphocyte subpopulations in MM revealed increased proportions of cells expressing OKM1, Leu7, and HLA-DR antigens. In CD8 lymphocytes, there was a direct correlation between OKM1 and HLA-DR positivity. Two-colour analysis of purified CD8 subpopulations showed that HLA-DR was preferentially expressed by OKM1+ lymphocytes (suppressor cells), but a significant proportion of OKM1-lymphocytes (cytotoxic precursor cells) were also HLA-DR+. A significant proliferative activity found in CD2 lymphocytes was directly correlated with the proportion of HLA-DR+ cells in CD8 subpopulations only. CD8 lymphocytes displayed significantly lower 5'NT activity than those of normal subjects. This enzyme deficiency was correlated with the expansion of CD8 OKM1+ and HLA-DR+ cells; the increase in suppressor cells, as well as the emergence of activated cells, are associated with CD8 5'NT deficiency. In conclusion, the immunological pattern of CD4 and CD8 subpopulations in MM is significantly different from that in normal subjects. The implications of these data for the immune dysregulation occurring in MM are discussed.

Red cell pyrimidine 5' nucleotidase deficiency: determination of nucleotidase activity and nucleotide content using HPLC.

The hereditary deficiency of erythrocyte pyrimidine 5' nucleotidase has been investigated using an HPLC anion-exchange procedure designed to measure both red cell nucleotide content and enzymic activity. Red cell nucleotide profiles were determined using a low pH phosphate buffer salt-gradient system in 20 min, whereas a low pH buffer alone permitted the determination of enzymic activity with each of six different nucleotide substrates in less than 4 min. Both the red cell nucleotide profiles and the enzymic activity of haemolysates from two affected brothers and their children agreed well with previously published values. This unified approach should prove useful for detailed studies of deficiencies involving isoenzymes of pyrimidine nucleotidase.

Substrate specificity and pH sensitivity of deoxyribonucleotidase and pyrimidine nucleotidase activities in human hemolysates.

Residual 5'-nucleotidase activities in hemolysates from nine subjects with severe hereditary deficiency of pyrimidine nucleotidase (PyrNase) were compared to those in normal and reticulocyte-rich controls. Dephosphorylation rates of 12 potential ribo- and deoxyribomononucleotide substrates were measured as a function of pH. Data confirmed the existence of at least two isozymes of 5'-nucleotidase, PyrNase, and 2'-deoxy-5'-ribonucleotide phosphohydrolase (dNase) distinguishable by differences in maximal velocities, substrate preferences and restrictions, and pH optima. PyrNase was confirmed to be active principally with pyrimidine substrates (UMP = dCMP greater than CMP much greater than dTMP greater than dUMP) at a pH optimum of 7.5 +/- 0.1. dNase activity occurred with both purine and pyrimidine substrates and was maximal with deoxy analogs (dIMP much greater than dUMP greater than dGMP greater than dTMP = dAMP much greater than dCMP) at a pH optimum of 6.2, but slight cross-reactivity occurred with some nondeoxy substrates (IMP greater than GMP greater than UMP = XMP greater than CMP). PyrNase and dNase may be complementary systems that serve physiologically to clear the cytosol of RNA and DNA degradation products during maturation of erythroid elements by conversion of nucleotide monophosphates to diffusible nucleosides.

Erythrocyte-oxidized glutathione transport in pyrimidine 5'-nucleotidase deficiency.

The oxidized form of glutathione transport was studied in human erythrocytes in pyrimidine 5'-nucleotidase (P5N) deficiency, a disorder in which the amounts of CTP and UTP in the erythrocytes are elevated. The inhibition of ATP-requiring oxidized glutathione (GSSG) transport by CTP and UTP is believed to play a role in elevating the levels of the reduced form of glutathione (GSH) in the erythrocytes of patients with P5N deficiency. The current investigation was undertaken to determine if GSSG transport actually decreases in the erythrocytes of such patients. Erythrocytes from a 17-year-old patient and a 13-year-old patient with P5N deficiency hemolytic anemia and from ten normal subjects were used as materials for the experiment. Erythrocytes, which had been previously incubated with [3H]glycine, were incubated at 37 degrees C, and the rate of [3H]GSSG transported by the cells was estimated. The velocity of GSSG transport out of the erythrocytes was quite low in the patients, 3.17-3.65 nmol GSSG/ml erythrocytes/hr at 37 degrees C in one case, and 3.30 nmol GSSG/ml erythrocytes/hr in the other case, vs that in the normal controls (6.00 +/- 0.80 nmol GSSG/ml erythrocytes/hr; mean +/- SD). The activity of gamma-glutamylcysteine synthetase and glutathione synthetase did not decrease in the patients. Decreased transport activity of GSSG in addition to a normal synthesis rate for GSH may explain the increased concentration of erythrocyte GSH in P5N deficiency.

Multiple myeloma: ecto-5' nucleotidase deficiency of suppressor/cytotoxic (CD8) lymphocytes is a marker for the expansion of suppressor T cells.

In this study, 5'NT activity was investigated by radiochemical and cytochemical assays in T cell subpopulations of patients with multiple myeloma (MM). Cytotoxic/suppressor (CD8) and helper/inducer (CD4) lymphocytes were separated from peripheral blood by the panning technique, or sorted with a fluorescein activated cell sorter. 5'NT activity was significantly decreased in MM CD8 lymphocytes compared with the controls. By contrast, it was comparably low in CD4 subpopulations of normal controls and MM patients. The cytochemical assay indicated that a decreased number of 5'NT+ cells rather than a decreased activity per cell was responsible for 5'NT deficiency in MM CD8 lymphocytes. CD8 lymphocytes with the suppressor phenotype (OKMI+, granular cells) were significantly increased in MM. These data provide a phenotypic explanation for the enhanced suppressor activity displayed by T lymphocytes in MM. A significant correlation was found between the increase of suppressor cells and the decrease of 5'NT+ cells. 5'NT deficiency of CD8 lymphocytes can be a biochemical marker for the expansion of suppressor T cells.

Pyrimidine nucleoside monophosphate kinase hyperactivity in hereditary erythrocyte pyrimidine 5'-nucleotidase deficiency.

The pyrimidine nucleoside triphosphates (CTP, UTP) increase in the pyrimidine 5'-nucleotidase (P5N) deficient red blood cell (RBC) to a greater degree than do the pyrimidine nucleoside monophosphates (CMP, UMP). Pyrimidine nucleoside monophosphate (PNMP) kinase phosphorylates CMP and UMP to their respective phosphodiesters. We tested the hypothesis that increased PNMP kinase activity contributes to the disproportionate increase in CTP and UTP in the P5N deficient RBC. CMP and UMP kinase activities were increased in high reticulocyte (4.4 +/- 2.1 and 8.5 +/- 3.3 mumol/ml RBC per minute) compared to normal RBC (2.8 +/- 1.0 and 6.0 +/- 2.5 mumol/ml RBC per minute). P5N deficient RBC (n = 2) had significantly increased CMP and UMP kinase activities (14.0 and 26.5 mumol/ml RBC per minute). UMP and CDP-ethanolamine were able to increase the activity of CMP kinase in crude haemolysate and the activity of partially purified enzyme. Since the Km for CMP of CMP kinase was 33 mumol/l in P5N deficient RBC and since the CMP concentration is 25-90 mumol/l in the P5N deficient RBC, the enzyme should be nearly saturated with CMP in the P5N deficient RBC. Thus, PNMP kinase hyperactivity appears to contribute to the disproportionate increase in CTP and UTP in the P5N deficient RBC.

Decreased 5'-nucleotidase activity in suppressor (OKT8) T lymphocytes from homosexuals with AIDS-related complex: nonassociation with enhanced deoxynucleoside toxicity.

The purine metabolic enzymes adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and 5'nucleotidase (5NT) have been shown to be important for normal lymphocyte maturation. Abnormalities of these enzymes have been associated with hereditary as well as acquired immunodeficiency states. Enzyme activity was measured in helper (OKT4) and suppressor (OKT8) lymphocyte subsets from 10 homosexuals with AIDS-related complex (ARC) and in 10 healthy controls. There were no significant differences in either mean ADA activity or mean PNP activity between ARC OKT4 cells and control OKT4 cells and between ARC OKT8 cells and control OKT8 cells. By contrast, mean 5NT activity was slightly decreased in OKT4 cells from ARC patients compared with that of controls and more significantly diminished in ARC OKT8 cells compared with that of controls. Both deoxyadenosine and deoxyguanosine, when incubated separately with OKT4 and OKT8 cells in the presence of EHNA, an ADA inhibitor, did not significantly inhibit lymphocyte blastogenesis to a greater extent in ARC patients than in controls. Hence, the decreases in 5NT activity most likely reflect lymphocyte immaturity and are not associated with biochemical abnormalities leading to increased deoxynucleoside toxicity.

Chromatographic analysis of human erythrocyte pyrimidine 5'-nucleotidase from five patients with pyrimidine 5'-nucleotidase deficiency.

Human erythrocyte pyrimidine 5'-nucleotidase (P5N) was separated into two subclasses. P5N-I and P5N-II, by DEAE Bio-Gel A column chromatography. Their enzymological properties were studied using five normal subjects and five patients with different P5N deficiencies. Study of the normal subjects showed that P5N-I and P5N-II have distinctive properties, and P5N-II is similar to the 5'-nucleotidase in rat liver cytosol. The P5N-II from the five subjects with this deficiency had normal activity and other normal enzymological properties. However, the P5N-I from these patients had abnormal properties, including reduced activity. These variant enzymes had a high Michaelis constant for substrate cytidine 5'-monophosphate and were heat stable. The optimum pH was shifted towards the acidic side in two patients, towards the basic side in one, and was unchanged in the other two. These results strongly suggest that the main cause of P5N deficiency is an abnormality of P5N-I, probably arising from a structural gene mutation.

Red cell metabolism in hereditary pyrimidine 5'-nucleotidase deficiency: effect of magnesium.

Since pyrimidine nucleotides avidly bind magnesium, we tested the hypothesis that the haemolytic anaemia in hereditary pyrimidine 5'-nucleotidase (P5N) deficiency is due to a state of functional magnesium depletion in the red cell (RBC). In haemolysates from normal subjects, cytidine triphosphate (CTP) inhibited the activity of pyruvate kinase in a competitive manner for magnesium. The CTP Ki was 0.4 mmol/l. CTP inhibited the activity of hexokinase in a competitive manner for ATP (Mg-ATP2-) with a Ki of 4 mmol/l. The inhibitory effect of CTP on both enzymes was overcome by increasing the magnesium content of the test system. Since CTP appeared to inhibit enzymes which required magnesium as a cofactor or Mg-ATP2- as a substrate, we tested the effect of exogenous magnesium on the metabolism of P5N deficient RBC. The autohaemolysis test, the incubated Heinz body assay and the rate of glucose oxidation by the pentose phosphate shunt were abnormal in the intact RBC from a patient with hereditary P5N deficiency. The addition of MgCl2 (6-10 mmol/l) did not improve these abnormal in vitro measures of metabolism in the P5N deficient RBC. This lack of effect of exogenous magnesium may be due to the slow uptake of magnesium by the human RBC.

Mechanisms of adenosine 5'-monophosphate catabolism in human erythrocytes.

Uncertainties regarding the role of pyrimidine nucleotidase (PyrNase) in AMP catabolism were resolved by studies of erythrocytes from normal controls, controls with young mean cell ages, and patients with hereditary hemolytic anemia due to severe deficiency of PyrNase. Hemolysates from the latter exhibited undiminished capacity to dephosphorylate AMP over a broad range of pH, indicating that PyrNase was not directly involved. In each subject group, the rates of AMP dephosphorylation between pH 5.1 and 8.3 were indistinguishable from those of IMP, suggesting a potential role for AMP-deaminase, an erythrocyte enzyme that was stimulated by coformycin at pH 7.2. Quantitative analysis of catabolites in incubated hemolysates confirmed that AMP degradation preferentially occurred via deamination to IMP with subsequent dephosphorylation by another erythrocyte nucleotidase isozyme, deoxyribonucleotidase. Both AMP-deaminase and deoxyribonucleotidase have acidic pH optima with minimal activities at physiologic pH, suggesting that this pathway of AMP catabolism could accelerate depletion of the adenine nucleotide pool and thereby mediate the demise of senescent erythrocytes sequestered in the spleen.

Detection of pyrimidine 5'-nucleotidase deficiency using 1H- or 31P-nuclear magnetic resonance.

We describe here a further Japanese family with pyrimidine 5'-nucleotidase (P5'N) deficiency diagnosed using a nuclear magnetic resonance (NMR) spectrum, in Kumamoto prefecture where two families having the disease have been reported before. The specific spectra in 1H-NMR of P5'N deficient erythrocytes were due to three methyl protons of CDP-choline at 3.22 ppm and to H-2, H-8 and ribose-1' of pyrimidine nucleotide phosphate(s) in the lower fields (at 5.82 and 8.00 ppm). The other specificities in 31P-NMR spectra were due to CDP-choline, CDP-ethanolamine and UDP-glucose. Those spectra were not detected in other types of hemolytic anemia.