Cell cycle regulation of astrocytes by extracellular nucleotides and fibroblast growth factor-2

Extracellular ATP enhances the mitogenic activity of fibroblast growth factor-2 (FGF2) in astrocytes, but the molecular mechanism underlying this synergistic interaction is not known. To determine whether the potentiating effect of extracellular ATP involves cell cycle control mechanisms, we have measured the expression of cyclins that are induced in different phases of the cell cycle in primary cultures of rat cortical astrocytes. We found that ATP potentiated the ability of FGF2 to stimulate expression of cyclin D1, a regulator of cell cycle entry, as well as cyclin A, a regulator of DNA replication. Because FGF2 and P2 purinergic receptors are coupled to extracellular signal regulated protein kinase (ERK), a key member of a signaling cascade that regulates proliferation, we also investigated the role of ERK in regulating cyclin expression induced by FGF2 and ATP. We found that the potentiating effect of ATP on cyclin expression was significantly reduced by U0126, an inhibitor of MEK, the upstream activator of ERK. P2 receptor agonist studies revealed that UTP enhanced FGF2-induced cyclin expression and mitogenesis whereas 2-methylthioADP was ineffective. By contrast, 2-3-O-(4-benzoyl)-benzoyl-ATP markedly inhibited FGF2-induced mitogenesis. Consistent with opposing effects of P2Y and P2X receptors on mitogenesis, UTP stimulated a transient activation of ERK whereas BzATP stimulated a more sustained ERK signal. These findings suggest that signaling by P2Y receptors, most likely of the purine/pyrimidine subtype, enhance the ability of FGF2 to stimulate entry into a new cell cycle, as well as DNA replication, by an ERK-dependent mechanism, whereas signaling by P2X receptors, possibly the P2X7 subtype, inhibits FGF2-induced mitogenesis in astrocytes. Interactions between P2Y, P2X and polypeptide growth factor signaling pathways may have important implications for CNS development as well as injury and repair

Structure-activity relationships of anthraquinone derivatives derived from bromaminic acid as inhibitors of ectonucleoside triphosphate diphosphohydrolases (E-NTPDases)

Reactive blue 2 (RB-2) had been characterized as a relatively potent ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) inhibitor with some selectivity for NTPDase3. In search for the pharmacophore and to analyze structure-activity relationships we synthesized a series of truncated derivatives and analogs of RB-2, including 1-amino-2-sulfo-4-ar(alk)ylaminoanthraquinones, 1-amino-2-methyl-4-arylaminoanthraquinones, 1-amino-4-bromoanthraquinone 2-sulfonic acid esters and sulfonamides, and bis-(1-amino-4-bromoanthraquinone) sulfonamides, and investigated them in preparations of rat NTPDase1, 2, and 3 using a capillary electrophoresis assay. Several 1-amino-2-sulfo-4-ar(alk)ylaminoanthraquinone derivatives inhibited E-NTPDases in a concentration-dependent manner. The 2-sulfonate group was found to be required for inhibitory activity, since 2-methyl-substituted derivatives were inactive. 1-Amino-2-sulfo-4-p-chloroanilinoanthraquinone (18) was identified as a nonselective competitive blocker of NTPDases1, 2, and 3 (Ki 16–18 μM), while 1-amino-2-sulfo-4-(2-naphthylamino)anthraquinone (21) was a potent inhibitor with preference for NTPDase1 (Ki 0.328 μM) and NTPDase3 (Ki 2.22 μM). Its isomer, 1-amino-2-sulfo-4-(1-naphthylamino)anthraquinone (20), was a potent and selective inhibitor of rat NTPDase3 (Ki 1.5 μM)

Antibody reactivity against potato apyrase, a protein that shares epitopes with Schistosoma mansoni ATP diphosphohydrolase isoforms, in acute and chronically infected mice, after chemotherapy and reinfection

Schistosoma mansoni ATP diphosphohydrolase isoforms and potato apyrase share conserved epitopes. By enzyme-linked immunosorbent assays, elevated levels of IgM, IgG2a and IgG1 antibody reactivity against potato apyrase were observed in S. mansoni-infected BALB/c mice during the acute phase of infection, while only IgM and IgG1 antibody reactivity levels maintained elevated during the chronic phase of infection. Antibody reactivity against potato apyrase was monitored over an 11-month period in chronically-infected mice treated with oxamniquine. Eleven months later, the level of seropositive IgM decreased significantly (~30%) compared to the level found in untreated, infected mice. The level of seropositive IgG1 decreased significantly four months after treatment (MAT) (61%) and remained at this level even after 11 months. The IgG2a reactivity against potato apyrase, although unchanged during chronic phase to 11 MAT, appeared elevated again in re-infected mice suggesting a response similar to that found during the acute phase. BALB/c mouse polyclonal anti-potato apyrase IgG reacted with soluble egg antigens probably due to the recognition of parasite ATP diphosphohydrolase. This study, for the first time, showed that the IgG2a antibody from S. mansoni-infected BALB mice cross-reacts with potato apyrase and the level of IgG2a in infected mice differentiates disease phases. The results also suggest that different conserved-epitopes contribute to the immune response in schistosomiasis

A capillary electrophoresis method for the characterization of ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) and the analysis of inhibitors by in-capillary enzymatic microreaction

A capillary electrophoresis (CE) method for the characterization of recombinant NTPDases 1, 2, and 3, and for assaying NTPDase inhibitors has been developed performing the enzymatic reaction within the capillary. After hydrodynamic injection of plugs of substrate solution with or without inhibitor in reaction buffer, followed by a suspension of an enzyme-containing membrane preparation, and subsequent injection of another plug of substrate solution with or without inhibitor, the reaction took place close to the capillary inlet. After 5 min, the electrophoretic separation of the reaction products was initiated by applying a constant current of  μA. The method employing a polyacrylamide-coated capillary and reverse polarity mode provided baseline resolution of substrates and products within a short separation time of less than 7 min. A 50 mM phosphate buffer (pH 6.5) was used for the separations and the products were detected by their UV absorbance at 210 nm. The Michaelis–Menten constants (Km) for the recombinant rat NTPDases 1, 2, and 3 obtained with this method were consistent with previously reported data. The inhibition studies revealed pronounced differences in the potency of reactive blue 2, pyridoxalphosphate-6-azophenyl-2-4-disulfonic acid (PPADS), suramin, and N6-diethyl-β,γ-dibromomethylene-ATP (ARL67156) towards the NTPDase isoforms. Notably, ARL67156 does not inhibit all NTPDases, having only a minor inhibitory effect on NTPDase2. Dipyridamole is not an inhibitor of the NTPDase isoforms investigated. The new method is fast and accurate, it requires only tiny amounts of material (nanoliter scale), no sample pretreatment and can be fully automated; thus it is clearly superior to the current standard methods

Adjusting Phenotypes by Noise Control

Genetically identical cells can show phenotypic variability. This is often caused by stochastic events that originate from randomness in biochemical processes involving in gene expression and other extrinsic cellular processes. From an engineering perspective, there have been efforts focused on theory and experiments to control noise levels by perturbing and replacing gene network components. However, systematic methods for noise control are lacking mainly due to the intractable mathematical structure of noise propagation through reaction networks. Here, we provide a numerical analysis method by quantifying the parametric sensitivity of noise characteristics at the level of the linear noise approximation. Our analysis is readily applicable to various types of noise control and to different types of system; for example, we can orthogonally control the mean and noise levels and can control system dynamics such as noisy oscillations. As an illustration we applied our method to HIV and yeast gene expression systems and metabolic networks. The oscillatory signal control was applied to p53 oscillations from DNA damage. Furthermore, we showed that the efficiency of orthogonal control can be enhanced by applying extrinsic noise and feedback. Our noise control analysis can be applied to any stochastic model belonging to continuous time Markovian systems such as biological and chemical reaction systems, and even computer and social networks. We anticipate the proposed analysis to be a useful tool for designing and controlling synthetic gene networks

P2X7 nucleotide receptors mediate caspase-8/9/3-dependent apoptosis in rat primary cortical neurons

Apoptosis is a major cause of cell death in the nervous system. It plays a role in embryonic and early postnatal brain development and contributes to the pathology of neurodegenerative diseases. Here, we report that activation of the P2X7 nucleotide receptor (P2X7R) in rat primary cortical neurons (rPCNs) causes biochemical (i.e., caspase activation) and morphological (i.e., nuclear condensation and DNA fragmentation) changes characteristic of apoptotic cell death. Caspase-3 activation and DNA fragmentation in rPCNs induced by the P2X7R agonist BzATP were inhibited by the P2X7R antagonist oxidized ATP (oATP) or by pre-treatment of cells with P2X7R antisense oligonucleotide indicating a direct involvement of the P2X7R in nucleotide-induced neuronal cell death. Moreover, Z-DEVD-FMK, a specific and irreversible cell permeable inhibitor of caspase-3, prevented BzATP-induced apoptosis in rPCNs. In addition, a specific caspase-8 inhibitor, Ac-IETD-CHO, significantly attenuated BzATP-induced caspase-9 and caspase-3 activation, suggesting that P2X7R-mediated apoptosis in rPCNs occurs primarily through an intrinsic caspase-8/9/3 activation pathway. BzATP also induced the activation of C-jun N-terminal kinase 1 (JNK1) and extracellular signal-regulated kinases (ERK1/2) in rPCNs, and pharmacological inhibition of either JNK1 or ERK1/2 significantly reduced caspase activation by BzATP. Taken together, these data indicate that extracellular nucleotides mediate neuronal apoptosis through activation of P2X7Rs and their downstream signaling pathways involving JNK1, ERK and caspases 8/9/3

Purinergic modulation of microglial cell activation

Microglial cells are resident macrophages in the brain and their activation is an important part of the brain immune response and the pathology of the major CNS diseases. Microglial activation is triggered by pathological signals and is characterized by morphological changes, proliferation, phagocytosis and the secretion of various cytokines and inflammatory mediators, which could be both destructive and protective for the nervous tissue. Purines are one of the most important mediators which regulate different aspects of microglial function. They could be released to the extracellular space from neurons, astrocytes and from the microglia itself, upon physiological neuronal activity and in response to pathological stimuli and cellular damage. Microglial activation is regulated by various subtypes of nucleotide (P2X, P2Y) and adenosine (A1, A2A and A3) receptors, which control ionic conductances, membrane potential, gene transcription, the production of inflammatory mediators and cell survival. Among them, the role of P2X7 receptors is especially well delineated, but P2X4, various P2Y, A1, A2A and A3 receptors also powerfully participate in the microglial response. The pathological role of microglial purine receptors has also been demonstrated in disease models; e.g., in ischemia, sclerosis multiplex and neuropathic pain. Due to their upregulation and selective activation under pathological conditions, they provide new avenues in the treatment of neurodegenerative and neuroinflammatory illnesses

Dynamic purine signaling and metabolism during neutrophil–endothelial interactions

During episodes of hypoxia and inflammation, polymorphonuclear leukocytes (PMN) move into underlying tissues by initially passing between endothelial cells that line the inner surface of blood vessels (transendothelial migration, TEM). TEM creates the potential for disturbances in vascular barrier and concomitant loss of extravascular fluid and resultant edema. Recent studies have demonstrated a crucial role for nucleotide metabolism and nucleoside signaling during inflammation. These studies have implicated multiple adenine nucleotides as endogenous tissue protective mechanisms invivo. Here, we review the functional components of vascular barrier, identify strategies for increasing nucleotide generation and nucleoside signaling, and discuss potential therapeutic targets to regulate the vascular barrier during inflammation

P2X7 receptor: Death or life?

The P2X7 plasma membrane receptor is an intriguing molecule that is endowed with the ability to kill cells, as well as to activate many responses and even stimulate proliferation. Here, the authors give an overview on the multiplicity and complexity of P2X7-mediated responses, discussing recent information on this receptor. Particular attention has been paid to early and late signs of apoptosis and necrosis linked to activation of the receptor and to the emerging field of P2X7 function in carcinogenesis