Concentrations Of UTP Research Articles

Formation of dsRNA by-products during in vitro transcription can be reduced by using low steady-state levels of UTP.

Introduction: Exogeneous messenger ribonucleic acid (mRNA) can be used as therapeutic and preventive medication. However, during the enzymatic production process, commonly called in vitro transcription, by-products occur which can reduce the therapeutic efficacy of mRNA. One such by-product is double-stranded RNA (dsRNA). We therefore sought to limit the generation of dsRNA by-products during in vitro transcription. Materials and methods: In vitro transcription was performed with a DNA template including a poly(A)-tail-encoding region, dinucleotide or trinucleotide cap analogs for cotranscriptional capping, and relevant nucleoside triphosphates. Concentrations of UTP or modified UTP (m1ΨTP) and GTP were reduced and fed over the course of the reaction. mRNA was analyzed for dsRNA contamination, yield of the reaction, RNA integrity, and capping efficiency before translational activity was assessed. Results: Limiting the steady-state level of UTP or m1ΨTP during the enzymatic reaction reduced dsRNA formation, while not affecting mRNA yield or RNA integrity. Capping efficiency was optimized with the use of a combined GTP and UTP or m1ΨTP feed, while still reducing dsRNA formation. Lower dsRNA levels led to higher protein expression from the corresponding mRNAs. Discussion: Low steady-state concentrations of UTP and GTP, fed in combination over the course of the in vitro transcription reaction, produce mRNA with high capping and low levels of dsRNA formation, resulting in high levels of protein expression. This novel approach may render laborious purification steps to remove dsRNA unnecessary.

A molting chemical cue (N-acetylglucosamine-6-phosphate) contributes to cannibalism of Chinese mitten crab Eriocheir sinensis

Under high-density culture, cannibalism occurs frequently during the molting of the Chinese mitten crabs Eriocheir sinensis, resulting in a large reduction in production. We found that the leakage of molting fluid from sexually immature crabs informs conspecifics that they are in a molting process. This hypothesis was verified through metabolomics analyses combined with behavioral experiments. The GlcNAc-6-P was identified as a molting biomarker from the differential metabolites by non-targeted metabolomics. In addition, we found that the concentration of GlcNAc-6-P in the molting fluid was significantly higher than other molting metabolites at different molting stages, reaching 5.84 μmol L−1, indicating that the molting fluid was the source of GlcNAc-6-P. Moreover, the behavioral experiments showed that crabs were actively approached to high concentrations of GlcNAc-6-P (1 μmol L−1), but had no obvious choice tendency at different concentrations of UTP, 20-HE and low concentrations of GlcNAc-6-P (0.1 μmol L−1, 0.01 μmol L−1) compared with the control groups. In conclusion, that E. sinensis by sensing the concentration change of GlcNAc-6-P can locate the source of GlcNAc-6-P release and actively approach the high concentration GlcNAc-6-P area and attack the molting crab, causing cannibalism.Blocking the reception pathway of molting chemical cues in E. sinensis, thereby preventing the perception of signals originating from conspecifics' molting in the vicinity, could lead to a reduction in cannibalistic behavior and an increase in overall production. Additionally, this method presents a prospective solution for addressing cannibalism in other crustacean species where such behavior is prevalent.

Signaling Through Purinergic Receptor P2Y2 Enhances Macrophage IL-1β Production.

The release of nucleotides during necrosis or apoptosis has been described to have both proinflammatory and anti-inflammatory effect on the surrounding cells. Here we describe how low concentrations of UTP and ATP applied during macrophage priming enhance IL-1β production when subsequently the NLRP3 inflammasome is activated in murine resident peritoneal macrophages. Deficiency or pharmacological inhibition of the purinergic receptor P2Y2 reverted the increase of IL-1β release induced by nucleotides. IL-1β increase was found dependent on the expression of Il1b gene and probably involving JNK activity. On the contrary, nucleotides decreased the production of a different proinflammatory cytokines such as TNF-α. These results suggest that nucleotides could shape the response of macrophages to obtain a unique proinflammatory signature that might be relevant in unrevealing specific inflammatory conditions.

Opportunities of high-technology phenotyping by the NMR spectroscopy method by example of the metabolic response of the rat liver to the effect of alcohol and Reishi

The metabolic profiling of the rat liver was carried out in order to estimate the efficiency of proton nuclear magnetic resonance (1H NMR) spectroscopy as a method of metabolomic phenotyping and a method of detecting early metabolic changes during toxic effects. The tissue samples were taken from three groups of rats: group C, control animals; group A, animals that repeatedly received 15% alcohol solution (its intake induced no pathological liver changes, but stimulated the proliferation of the hepatocytes); and group A + R, alcohol in combination with the plant hepatoprotective Reishi preparation (Ganoderma lucidum). As follows from the results obtained, the changes in the metabolite concentration in liver (observed in group A) reflected the typical metabolic reaction to the alcohol intake and were expressed in a decrease in the content of glycine, leucine, isoleucine, valine, choline, and lactate, as well as in an increase in the TMAO level. The concentration of glycine, valine, and TMAO was restored in the rats of group A + R during the Reishi intake. In addition, an increase in the concentrations of NAD, ATP, UTP, succinate, pyranose, and acetate was registered in group A + R relative to the control. Individual variations in the content of valine, isoleucine, leucine, lactate, choline, and pyranose is correlated to the number of binuclear hepatocytes (as an indicator of proliferative activity). Thus, the study of liver tissues by 1H NMR spectroscopy method allows us to detect early deviations in the level of metabolites during chronic consumption of small alcohol doses and can be a promising approach both for diagnostic detection of alcoholic liver damage and for estimation of the efficiency of the applied drug therapy.

NMR metabolic profiling of the liver following administrationof alcohol andthemushroom Ganoderma lucidum in rats

We have evaluated the efficiency of a metabonomic approach to metabolic phenotyping and detection of early metabolic changes under a toxic influence. For this purpose, a metabolic profiling of rat liver was performed with 1H NMR spectroscopy. Rat tissues from animals in three groups were analyzed. Group C consisted of control animals; animals in group A received alcohol repeatedly (15 % ethanol); and animals in group A+ R received alcohol in combination with a hepatoprotective herbal medicine (Reishi, Ganoderma lucidum) repeatedly. Noteworthy, alcohol consumption did not cause pathological changes, but stimulated hepatocyte proliferation. Our data suggest that changes in metabolite concentrations in A represent a typical metabolic response to alcohol consumption, namely decrease in glycine, leucine, isoleucine, valine, choline and lactate content, and increase in TMAO content. Treatment with Reishi (A+ R) had positive effects, in that it restored the levels of glycine, valine and TMAO. Furthermore, increase in NAD, ATP, UTP, succinate, pyranose, and acetate concentrations was observed in A+ R. A correlation was found between the valine, isoleucine, lactate, cho­line, and pyranose content and the num­ber of binuclear hepatocytes. Binuclear hepatocytes indicate proliferative activity, and the concentration of the metabolites participating in the formation of new hepatic cells decreases. Thus, the study of liver tissues by 1H NMR spectroscopy allows for detection of early changes in metabolite concentra­tions following chronic consumption of alcohol at insignificant doses. Consequently, 1H NMR spectro­scopy can serve as a promising approach to detecting alcohol-related liver pathologies and assessing the efficiency of the therapy used.

Abstract 4144: Extracellular nucleotides and purinergic signaling as novel, underappreciated, pro-metastatic factors for human lung cancer cells

Abstract Background: One of the challenging problems of current radio-chemotherapy is recurrence and metastasis of cancer cells that survive initial treatment. We have proposed that one of the unwanted effects of radio-chemotherapy is release of phospho-sphingolipids in response to treatment and thus induction of pro-metastatic microenvironment in several organs (Mol Cancer Res 2013; 11:793). However, at the same time also intracellular nucleotides such as ATP and UTP are released from damaged (“leaky”) cells exerting chemotactic functions. Hypothesis: Based on these observations that, in addition to phospho-sphingolipids, also nucleotides (e.g., ATP and UTP) are released from damaged cells we hypothesized that these molecules if released into extracellular microenvironment may also direct chemotaxis and metastasis of cancer cells - alone or in synergy with phospho-sphingolipids. Materials and Methods: Several complementary in vitro and in vivo approaches were employed to demonstrate a novel role of extracellular nucleotides (EXN) in a model of metastasis of lung cancer cells. In our studies we employed four large cell and two small cell lung cancer cell lines and a set of nucleotides including ATP, ADP, AMP, UTP, TTP, CTP, and GTP. Real time RT-PCR analysis of the expression of purinergic P1 and P2 receptors as well as chemotaxis, adhesion, proliferation, intracellular calcium flux and cell signaling studies in response to EXN stimulation were performed. Concentrations of ATP and UTP in several organs before and after radio-chemotherapy were measured by colorimetric kits. Purinergic receptor agonists and antagonists, inhibiting all or selected subtypes, were assayed in vitro and in vivo in pro-metastatic assays. Results: We observed that EXN accumulate in in several organs in response to radio-chemotherapy. RT-PCR analysis indicated that most of the P2X, P2Y and adenosine receptor subtypes are expressed in tested lung cancer cell lines. By employing in vitro migration assays, we found that, out of all the nucleotides tested, ATP, AMP and UTP have the strongest chemotactic activity for most of the human lung cancer cell lines correlating with phosphorylation of MAPKp42/44 and AKT. We also observed increased adhesion to fibronectin after stimulation with tested EXN, whereas proliferation was not affected by of EXN. More important, metastasis of lung cancer cells could be inhibited in immune-deficient mice in a presence of specific small molecule inhibitors of nucleotide receptors. Conclusions: Both systemic and local radio-chemotherapy leads to upregulation of EXN release in damaged tissues, and the side effect of such treatment is induction of an unwanted pro-metastatic microenvironment in different organs. Based on this data, EXN are novel pro-metastatic factors and inhibition of their pro-metastatic effects could become an important part of anti-metastatic treatment. Citation Format: Gabriela Schneider, Talita Glaser, Ahmed Abdelbaset Ismail, Henning Ulrich, Mariusz Z. Ratajczak. Extracellular nucleotides and purinergic signaling as novel, underappreciated, pro-metastatic factors for human lung cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4144. doi:10.1158/1538-7445.AM2015-4144

Regulation of Human Cytidine Triphosphate Synthetase 2 by Phosphorylation*

Cytidine triphosphate synthetase (CTPS) is the rate-limiting enzyme in de novo CTP synthesis and is required for the formation of RNA, DNA, and phospholipids. This study determined the kinetic properties of the individual human CTPS isozymes (hCTPS1 and hCTPS2) and regulation through substrate concentration, oligomerization, and phosphorylation. Kinetic analysis demonstrated that both hCTPS1 and hCTPS2 were maximally active at physiological concentrations of ATP, GTP, and glutamine, whereas the K(m) and IC(50) values for the substrate UTP and the product CTP, respectively, were close to their physiological concentrations, indicating that the intracellular concentrations of UTP and CTP may precisely regulate hCTPS activity. Low serum treatment increased hCTPS2 phosphorylation, and five probable phosphorylation sites were identified in the hCTPS2 C-terminal domain. Metabolic labeling of hCTPS2 with [(32)P]H(3)PO(4) demonstrated that Ser(568) and Ser(571) were two major phosphorylation sites, and additional studies demonstrated that Ser(568) was phosphorylated by casein kinase 1 both in vitro and in vivo. Interestingly, mutation of Ser(568) (S568A) but not Ser(571) significantly increased hCTPS2 activity, demonstrating that Ser(568) is a major inhibitory phosphorylation site. The S568A mutation had a greater effect on the glutamine than ammonia-dependent activity, indicating that phosphorylation of this site may influence the glutaminase domain of hCTPS2. Deletion of the C-terminal regulatory domain of hCTPS1 also greatly increased the V(max) of this enzyme. In summary, this is the first study to characterize the kinetic properties of hCTPS1 and hCTPS2 and to identify Ser(568) as a major site of CTPS2 regulation by phosphorylation.

Dietary Na+inhibits the open probability of the epithelial sodium channel in the kidney by enhancing apical P2Y2‐receptor tone

Apical release of ATP and UTP can activate P2Y(2) receptors in the aldosterone-sensitive distal nephron (ASDN) and inhibit the open probability (P(o)) of the epithelial sodium channel (ENaC). Little is known, however, about the regulation and physiological relevance of this system. Patch-clamp studies in freshly isolated ASDN provide evidence that increased dietary Na(+) intake in wild-type mice lowers ENaC P(o), consistent with a contribution to Na(+) homeostasis, and is associated with increased urinary concentrations of UTP and the ATP hydrolytic product, ADP. Genetic deletion of P2Y(2) receptors in mice (P2Y(2)(-/-); littermates to wild-type mice) or inhibition of apical P2Y-receptor activation in wild-type mice prevents dietary Na(+)-induced lowering of ENaC P(o). Although they lack suppression of ENaC P(o) by dietary NaCl, P2Y(2)(-/-) mice do not exhibit NaCl-sensitive blood pressure, perhaps as a consequence of compensatory down-regulation of aldosterone levels. Consistent with this hypothesis, clamping mineralocorticoid activity at high levels unmasks greater ENaC activity and NaCl sensitivity of blood pressure in P2Y(2)(-/-) mice. The studies indicate a key role of the apical ATP/UTP-P2Y(2)-receptor system in the inhibition of ENaC P(o) in the ASDN in response to an increase in Na(+) intake, thereby contributing to NaCl homeostasis and blood pressure regulation.

Mysteries of Renal Autoregulation

Autoregulation is an important renal regulatory mechanism that provides an important protective role in glomerular hemodynamics.1 The phenomenon of autoregulatory behavior has been recognized for many decades, but a clear understanding of the underlying mechanisms involved in mediating resistance adjustments remains to be established. As illustrated in Figure 1, pressure-mediated autoregulatory behavior operates by recognizing a change in renal perfusion pressure, or renal vascular transmural pressure, and initiating induction of autoregulatory resistance changes intended to stabilize renal blood flow and/or glomerular filtration rate. The net result is that renal blood flow and glomerular filtration rate remain relatively stable over a wide range of renal perfusion pressures. Autoregulatory behavior is exquisitely sensitive in healthy normal kidneys but significantly less efficient in pathological settings, such as diabetes mellitus and some forms of hypertension. Complete loss of autoregulatory control results in renal blood flow that behaves more passively, thus increasing variability and rendering flow more directly determined by renal perfusion pressure.1,2 Figure 1. Outline of the pressure-induced signaling scheme postulated for autoregulatory adjustments in the afferent arteriolar diameter. Whole kidney autoregulation reflects regulatory input from the myogenic and tubuloglomerular feedback mechanisms at the very least.3–7 Recently, contributions of a third and possibly a fourth mechanism have been suggested.6,7 Consequently, with continued study, the mechanisms underlying renal autoregulatory resistance adjustments are slowly becoming clearer. In addition to the P1 receptor hypothesis put forth to explain signal transduction for tubuloglomerular feedback-dependent resistance changes, we have explored the hypothesis that pressure-mediated autoregulatory responses are mediated by extracellular ATP and activation of P2X1 receptors.3,8,9 Conclusive identification of the signaling molecules that mediate autoregulation remains a point of debate, but it appears clear that the release of a purine-based moiety is required for autoregulatory adjustments in afferent arteriolar resistance.3–5,8–10 This …

Nitric Oxide–mediated Modulation of Synaptic Activity by Astrocytic P2Y Receptors

We investigated the mechanism of synaptic suppression by P2Y receptors in mixed hippocampal cultures wherein networked neurons exhibit synchronized Ca2+ oscillations (SCO) due to spontaneous glutamatergic synaptic transmission. Pharmacological studies suggested that SCO suppression was mediated by P2Y2/P2Y4 receptors. Immunostaining studies and characterization of ATP/UTP-stimulated Ca2+ responses in solitary neurons and astrocytes revealed that the SCO attenuation was effectuated by astrocytes. We demonstrate that nitric oxide released from activated astrocytes causes synaptic suppression by inhibiting neurotransmitter release. Physiological concentrations of ATP and UTP evoked NO production in astrocytes. SCO suppression was considerably diminished by removal of extracellular NO by membrane-impermeable scavenger c-PTIO or by pretreatment of cells with nitric oxide synthase inhibitor L-NAME. The nitric oxide donor DETA/NO effectively suppressed the SCO. ATP/UTP inhibited KCl-induced exocytosis at presynaptic terminals in an NO-dependent manner. In the absence of exogenously added ATP/UTP, both the NO scavenger and NOS inhibitor enhanced the frequency of SCO, implying that astrocytes release NO during spontaneous synaptic activity and exert a suppressive effect. We report for the first time that under physiological conditions astrocytes use NO as a messenger molecule to modulate the synaptic strength in the networked neurons.

Poliovirus cis -Acting Replication Element-Dependent VPg Uridylylation Lowers the K m of the Initiating Nucleoside Triphosphate for Viral RNA Replication

Initiation of RNA synthesis by RNA-dependent RNA polymerases occurs when a phosphodiester bond is formed between the first two nucleotides in the 5' terminus of product RNA. The concentration of initiating nucleoside triphosphates (NTPi) required for RNA synthesis is typically greater than the concentration of NTPs required for elongation. VPg, a small viral protein, is covalently attached to the 5' end of picornavirus negative- and positive-strand RNAs. A cis-acting replication element (CRE) within picornavirus RNAs serves as a template for the uridylylation of VPg, resulting in the synthesis of VPgpUpU(OH). Mutations within the CRE RNA structure prevent VPg uridylylation. While the tyrosine hydroxyl of VPg can prime negative-strand RNA synthesis in a CRE- and VPgpUpU(OH)-independent manner, CRE-dependent VPgpUpU(OH) synthesis is absolutely required for positive-strand RNA synthesis. As reported herein, low concentrations of UTP did not support negative-strand RNA synthesis when CRE-disrupting mutations prevented VPg uridylylation, whereas correspondingly low concentrations of CTP or GTP had no negative effects on the magnitude of CRE-independent negative-strand RNA synthesis. The experimental data indicate that CRE-dependent VPg uridylylation lowers the K(m) of UTP required for viral RNA replication and that CRE-dependent VPgpUpU(OH) synthesis was required for efficient negative-strand RNA synthesis, especially when UTP concentrations were limiting. By lowering the concentration of UTP needed for the initiation of RNA replication, CRE-dependent VPg uridylylation provides a mechanism for a more robust initiation of RNA replication.

Synthesis of Genomic and Subgenomic RNA in Mosquito Cells Infected with Two Sindbis Virus nsP4 Mutants: Influence of Intracellular Nucleoside Triphosphate Concentrations

Cells infected with Sindbis virus (SV) make two positive-strand RNAs, a genomic-length RNA (G) RNA and a subgenomic (SG) RNA. In cells infected with SVstd, and in general in cells infected with wt alphaviruses, more SG RNA is made than G RNA. How the balance between synthesis of G RNA and SG RNA is regulated is not known. SVpzf and SVcpc are nsP4 mutants of SV which, in mosquito cells, make more G RNA than SG RNA. When low concentrations of pyrazofurin (inhibits the synthesis of UTP and CTP) were added to SVpzf-infected cells, the yield of virus was increased, and the ratio of SG/G RNA was changed from <1 to >1. These effects were reversed by uridine. In SVcpc-infected cells, but not in SVstd-infected cells, synthesis of viral RNA was inhibited by the addition of either uridine or cytidine, and viral yields were lowered. Our findings suggest that the activities of the viral RNA-synthesizing complexes in cells infected with SVpzf or SVcpc, in contrast to those in SVstd-infected cells, are sensitive to high concentrations of UTP or CTP. Using a cell-free system that synthesizes both SG and G RNA, we measured viral RNA synthesis as a function of the UTP/CTP concentrations. The results indicated that the presence of the SVpzf mutations in nsP4 and the SG promoter produced a pattern quite different from that seen with the SVstd nsP4 and SG promoter. As the UTP/CTP concentrations were increased, the SVpzf system, in contrast to the SVstd system, made more G RNA than SG RNA, reflecting the situation in cells infected with SVpzf.

Structural Requirements for the Activation of Escherichia coli CTP Synthase by the Allosteric Effector GTP Are Stringent, but Requirements for Inhibition Are Lax

Cytidine 5'-triphosphate synthase catalyzes the ATP-dependent formation of CTP from UTP using either NH(3) or l-glutamine (Gln) as the source of nitrogen. GTP acts as an allosteric effector promoting Gln hydrolysis but inhibiting Gln-dependent CTP formation at concentrations of >0.15 mM and NH(3)-dependent CTP formation at all concentrations. A structure-activity study using a variety of GTP and guanosine analogues revealed that only a few GTP analogues were capable of activating Gln-dependent CTP formation to varying degrees: GTP approximately 6-thio-GTP > ITP approximately guanosine 5'-tetraphosphate > O(6)-methyl-GTP > 2'-deoxy-GTP. No activation was observed with guanosine, GMP, GDP, 2',3'-dideoxy-GTP, acycloguanosine, and acycloguanosine monophosphate, indicating that the 5'-triphosphate, 2'-OH, and 3'-OH are required for full activation. The 2-NH(2) group plays an important role in binding recognition, whereas substituents at the 6-position play an important role in activation. The presence of a 6-NH(2) group obviates activation, consistent with the inability of ATP to substitute for GTP. Nucleotide and nucleoside analogues of GTP and guanosine, respectively, all inhibited NH(3)- and Gln-dependent CTP formation (often in a cooperative manner) to a similar extent (IC(50) approximately 0.2-0.5 mM). This inhibition appeared to be due solely to the purine base and was relatively insensitive to the identity of the purine with the exception of inosine, ITP, and adenosine (IC(50) approximately 4-12 mM). 8-Oxoguanosine was the best inhibitor identified (IC(50) = 80 microM). Our findings suggest that modifying 2-aminopurine or 2-aminopurine riboside may serve as an effective strategy for developing cytidine 5'-triphosphate synthase inhibitors.

Characterization of the Metabolic Activation of Hepatitis C Virus Nucleoside Inhibitor β-d-2′-Deoxy-2′-fluoro-2′-C-methylcytidine (PSI-6130) and Identification of a Novel Active 5′-Triphosphate Species

beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) replication in the subgenomic HCV replicon system, and its corresponding 5'-triphosphate is a potent inhibitor of the HCV RNA polymerase in vitro. In this study the formation of PSI-6130-triphosphate was characterized in primary human hepatocytes. PSI-6130 and its 5'-phosphorylated derivatives were identified, and the intracellular concentrations were determined. In addition, the deaminated derivative of PSI-6130, beta-d-2'-deoxy-2'-fluoro-2'-C-methyluridine (RO2433, PSI-6026) and its corresponding phosphorylated metabolites were identified in human hepatocytes after incubation with PSI-6130. The formation of the 5'-triphosphate (TP) of PSI-6130 (PSI-6130-TP) and RO2433 (RO2433-TP) increased with time and reached steady state levels at 48 h. The formation of both PSI-6130-TP and RO2433-TP demonstrated a linear relationship with the extracellular concentrations of PSI-6130 up to 100 mum, suggesting a high capacity of human hepatocytes to generate the two triphosphates. The mean half-lives of PSI-6130-TP and RO2433-TP were 4.7 and 38 h, respectively. RO2433-TP also inhibited RNA synthesis by the native HCV replicase isolated from HCV replicon cells and the recombinant HCV polymerase NS5B with potencies comparable with those of PSI-6130-TP. Incorporation of RO2433-5'-monophosphate (MP) into nascent RNA by NS5B led to chain termination similar to that of PSI-6130-MP. These results demonstrate that PSI-6130 is metabolized to two pharmacologically active species in primary human hepatocytes.

P2 Purinoceptor-Mediated Cardioprotection in Ischemic-Reperfused Mouse Heart

P2 purinoceptor modulation of injury during ischemia-reperfusion was studied in murine hearts. Effects of P2 agonism or antagonism, and interstitial accumulation of P2 agonists (UTP, ATP, and ADP), were assessed in Langendorff perfused hearts during 20 min of ischemia and 45 min of reperfusion. In control hearts, ventricular pressure development recovered to 68 +/- 4 mm Hg (63 +/- 3% baseline), diastolic pressure remained elevated (23 +/- 2 mm Hg), and 26 +/- 4 U/g lactate dehydrogenase (LDH) was released during reperfusion, evidencing necrosis. Treatment with 250 nM UTP improved pressure development (85 +/- 5 mm Hg, or 77 +/- 2%) and reduced diastolic contracture (by approximately 70%, to 7 +/- 1 mm Hg) and LDH loss (by approximately 60%, to 11 +/- 2 U/g). In contrast, P2Y1 agonism with 50 nM 2-methyl-thio-ATP (2-MeSATP) was ineffective. In the presence of the P2Y antagonist suramin (10 or 200 microM), UTP no longer improved postischemic outcomes. Ischemia also substantially elevated interstitial [UTP], [ATP], and [ADP], potentially activating P2 receptors. This was supported in part by effects of antagonists: 200 microM suramin worsened LDH efflux (53 +/- 9 IU/g) and contractile dysfunction (41 +/- 2 mm Hg diastolic pressure; 28 +/- 3 mm Hg developed pressure), as did P2Y antagonism with either 10 or 100 microM reactive blue 2. However, a 10 microM concentration of suramin failed to alter outcome. P2X antagonism with 10 microM pyridoxal phosphate-6-azo-(benzene-2,4-disulfonic acid and P2X1-selective pyridoxal-alpha5-phosphate-6-phenylazo-4'-carboxylic acid (MRS2159) (30 microM) was ineffective. Data collectively support cardioprotection with low concentrations of UTP, and they are consistent with P2Y2 involvement. Endogenous nucleotides may also play a protective role, as evidenced by effects of P2 antagonists, although this warrants further investigation.

H 2O 2 modulates purinergic-dependent calcium signalling in osteoblast-like cells

Reactive oxygen species (ROS) have long been considered as toxic by-products of aerobic metabolism and appear involved in the pathogenesis of degenerative diseases. The physiological role of ROS as second messengers in cell signal transduction is, on the other hand, increasingly recognized. Here we investigated the effects of H 2O 2 and extracellular nucleotides on calcium signalling in four osteoblastic cell lines. In the highly differentiated HOBIT cells, sensitive to nanomolar concentrations of ADP and UTP, millimolar H 2O 2 induced oscillatory increases of the cytosolic calcium concentration followed by a steady and sustained calcium increase. Long lasting rhythmic calcium activity was induced by micromolar H 2O 2 doses. The H 2O 2-induced calcium signals, due to both release from intracellular stores and influx from the extracellular milieu, were totally prevented by incubating the cells with the P2 receptor antagonist suramin or with the ATP/ADP hydrolyzing enzyme apyrase. In the osteosarcoma SaOS-2 cells micromolar H 2O 2 failed to evoke calcium signals and millimolar H 2O 2 induced a slowly developing calcium influx which was unaffected by suramin and apyrase. These cells responded to micromolar concentrations of ATP and ADP, but were largely insensitive to UTP. ROS 17/2.8 osteosarcoma cells were totally insensitive to ATP, ADP and UTP in keeping with the evidence that these cells lack functional purinergic receptors. In these cells, H 2O 2 up to 1 mM did not increase the cytosolic calcium concentration. In ROS/P2Y 2 cells, stably expressing the P2Y 2 receptor, spontaneous calcium oscillations were observed in 38% of the population and nanomolar concentration of extracellular ATP or UTP activated oscillations in quiescent cells. Spontaneous calcium signals were inhibited by suramin and apyrase. In these cells H 2O 2 induced oscillatory calcium activity that was blocked by suramin and apyrase. The sensitivity of ROS/P2Y 2 cells to UTP decreased significantly in the presence of DTT, which was effective also in inhibiting spontaneous calcium oscillations. On the other hand, the membrane-impermeant thiol oxidant DTNB induced calcium oscillations that were inhibited by incubating the cells with suramin or apyrase. Since peroxide did not increase extracellular ATP in these cell lines, we propose that, in osteoblasts, mild oxidative conditions could activate purinergic signalling through the sensitization of P2Y 2 receptor.

Liver-specific increase of UTP and UDP-sugar concentrations in rats induced by dietary vitamin B6-deficiency and its relation to complex N-glycan structures of liver membrane-proteins

This is the first known report on the influence of vitamin B6-deficiency on the concentration of UDP-sugars and other uracil nucleotides in rats. Animals aged 3 weeks or 2 months were fed a vitamin B6-free diet for periods varying from 3 days to 7 weeks. Nucleotides were quantified by enzymatic-photometry and by SAX-high precision liquid chromatography. In 3 week-old rats, vitamin B6-deficiency resulted in an up to 6.3-fold increase in the concentrations of UTP, UDP, UMP and UDP-sugars and less of CTP in rat liver, while no changes were observed in older rats. In young rats, the concentration of uracil nucleotides started to increase after 1 week diet, with a maximum after 2 weeks. After 5 weeks, the concentrations returned to normal values. In heart, lungs, kidney and brain, concentrations were measured after 2 weeks diet in young rats. In contrast to liver, the heart muscle uracil nucleotide concentrations were decreased by 40%. In kidney, the sum of UTP, UDP and UMP showed a decrease of 40%, whereas UDP-sugars were increased 1.4-fold. In the lungs, nucleotide concentrations were mostly unaffected by vitamin B6-deficiency, despite a 70% increase of UDP-GA. In brain, UDP-Glc, UDP-Gal and the sum of CTP and CDP showed an increase of 30-50%. We became surprised that the increased UDP-sugar concentrations did not influence the structure of liver plasma membrane-N-glycans. Despite the 4 to 6-fold increase of UTP and UDP-sugars, no changes in the complexity or sialylation of these N-glycans could be detected. This study demonstrates that, especially in liver, pyridoxal phosphate is closely involved in the control of uracil nucleotides during a defined period of development. In contrast to in vitro experiments, in vivo N-glycan biosynthesis in liver is regulated by a more complex or higher mechanism than substrate concentrations.

Knock-out Mice Reveal the Contributions of P2Y and P2X Receptors to Nucleotide-induced Ca2+ Signaling in Macrophages

Immune cell function is modulated by changes in extracellular nucleotide levels. Here we used reverse transcription-PCR analyses, single cell Ca2+ imaging, and knock-out mice to define the receptors mediating nucleotide-induced Ca2+ signaling in resident peritoneal macrophages. In Ca2+-free buffer, the potent (K0.5<1 microm) stimulatory effect of UTP (or ATP) on endoplasmic reticulum (ER) Ca2+ release was abolished in cells isolated from P2Y2/P2Y4 double knock-out mice. Moreover, P2Y4(0/-), but not P2Y2-/-, macrophages responded to UTP. In P2Y2-/- macrophages, we could elicit Ca2+ responses to "pure" P2X receptor activation by applying ATP in buffer containing Ca2+. Purified UDP and ADP were ineffective agonists, although modest UDP-induced Ca2+ responses could be elicited in macrophages after "activation" with lipopolysaccharide and interferon-gamma. Notably, in Ca2+-free buffer, UTP-induced Ca2+ transients decayed within 1 min, and there was no response to repeated agonist challenge. Measurements of ER [Ca2+] with mag-fluo-4 showed that ER Ca2+ stores were depleted under these conditions. When extracellular Ca2+ was available, ER Ca2+ stores refilled, but Ca2+ increased to only approximately 40% of the initial value upon repeated UTP challenge. This apparent receptor desensitization persisted in GRK2+/- and GRK6-/- macrophages and after inhibition of candidate kinases protein kinase C and calmodulin-dependent kinase II. Initial challenge with UTP also reduced Ca2+ mobilization by complement component C5a (and vice versa). In conclusion, homologous receptor desensitization is not the major mechanism that rapidly dampens Ca2+ signaling mediated by P2Y2, the sole Gq-coupled receptor for UTP or ATP in macrophages. UDP responsiveness (P2Y6 receptor expression) increases following macrophage activation.

Cytotoxic and biochemical effects of 3,3′,4,4′,5,5′-hexahydroxystilbene, a novel resveratrol analog in HL-60 human promyelocytic leukemia cells

Resveratrol (3,4',5,-trihydroxystilbene, RV), an ingredient of wine, is an inhibitor of the proliferation-linked enzyme ribonucleotide reductase (RR) and shows a broad spectrum of cytotoxic effects against human cancer cells. In order to enhance these effects, we introduced additional hydroxyl moieties into the molecule. In the present study, the activity of a novel RV analog, 3,3',4,4',5,5'-hexahydroxystilbene (M8), was investigated in HL-60 human promyelocytic leukemia cells. Cytotoxicity of M8 alone or in combination with Ara-C was assessed employing growth inhibition assays. Effects of M8 on nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) were examined by HPLC. The apoptotic potential of M8 and RV was compared using a specific double-staining method and inhibition of TNF-alpha-induced activation of NF-kappaB was studied. Cell-cycle distribution was analyzed by FACS. Addition of ascorbic acid decreased the IC(50) value of M8 from 6.25 microM to 2 microM. M8 depleted dATP and dTTP pools to 41% and 21% of control values, whereas dCTP pools increased to 199% of untreated controls. In addition, TTP, ATP, CTP, and GTP concentrations were decreased while UTP concentrations increased. M8 induced apoptosis at concentrations significantly lower than RV and could remarkably inhibit the activation of NF-kappaB. M8 arrested cells in the S phase of the cell cycle while depleting cells in the G2-M phase and exhibited synergistic combination effects when applied simultaneously with Ara-C. Due to these promising results, this novel polyhydroxylated stilbene derivative might become an additional option for the treatment of leukemia and therefore deserves further preclinical and in vivo testing.

P2Y2 Receptor Up-Regulation Induced by Guanosine or UTP in Rat Brain Cultured Astrocytes

Among P2 metabotropic ATP receptors, P2Y2 subtype seems to be peculiar as its upregulation triggers important biological events in different cells types. In non-stimulated cells including astrocytes, P2Y2 receptors are usually expressed at levels lower than P2Y1 sites, however the promoter region of the P2Y2 receptors has not yet been studied and little is known about the mechanisms underlying the regulation of the expression of this ATP receptor. We showed that not only UTP and ATP are the most potent and naturally occurring agonist for P2Y2 sites, but also guanosine induced an up-regulation of astrocyte P2Y2 receptor mRNA evaluated by Northern blot analysis. We also focused our attention on this nucleoside since in our previous studies it was reported to be released by cultured astrocytes and to exert different neuroprotective effects. UTP and guanosine-evoked P2Y2 receptor up-regulation in rat brain cultured astrocytes was linked to an increased P2Y2-mediated intracellular calcium response, thus suggesting an increased P2Y2 activity. Actinomycin D, a RNA polymerase inhibitor, abrogated both UTP and guanosine-mediated P2Y2 up-regulation, thus indicating that de novo transcription was required. The effect of UTP and guanosine was also evaluated in astrocytes pretreated with different inhibitors of signal transduction pathways including ERK, PKC and PKA reported to be involved in the regulation of other cell surface receptor mRNAs. The results show that ERK1-2/MAPK pathway play a key role in the P2Y2 receptor up-regulation mediated by either UTP or guanosine. Moreover, our data suggest that PKA is also involved in guanosine-induced transcriptional activation of P2Y2 mRNA and that increased intracellular calcium levels and PKC activation may also mediate P2Y2 receptor up-regulation triggered by UTP. The extracellular release of ATP under physiological and pathological conditions has been widely studied. On the contrary, little is known about the release of pyrimidines and in particular of UTP. Here we show that astrocytes are able to release UTP, either at rest or during and following hypoxia/hypoglycemia obtained by submitting the cells to glucose-oxygen deprivation (OGD). Interestingly, also P2Y2 receptor mRNA increased by about two-fold the control values when the cultures were submitted to OGD. It has been recently reported that P2Y2 receptors can play a protective role in astrocytes, thus either guanosine administration or increased extracellular concentrations of guanosine and UTP reached locally following CNS injury may increase P2Y2-mediated biological events aimed at promoting a protective astrocyte response.