High zinc sensitivity and pore formation in an invertebrate P2X receptor

To investigate fast purinergic signaling in invertebrates, we examined the functional properties of a P2X receptor subunit cloned from the parasitic platyhelminth Schistosoma mansoni. This purinoceptor (SmP2X) displays unambiguous homology of primary sequence with vertebrate P2X subunits. SmP2X subunits assemble into homomeric ATP-gated channels that exhibit slow activation kinetics and are blocked by suramin and PPADS but not TNP-ATP. SmP2X mediates the uptake of the dye YO-PRO-1 through the formation of large pores and can be blocked by submicromolar concentrations of extracellular Zn²⁺ ions (IC₅₀=0.4 μM). The unique receptor phenotype defined by SmP2X suggests that slow kinetics, modulation by zinc and the ability to form large pores are ancestral properties of P2X receptors. The high sensitivity of SmP2X to zinc further reveals a zinc regulation requirement for the parasite's physiology that could potentially be exploited for therapeutic purposes.     

Involvement of de novo ceramide biosynthesis in macrophage death induced by activation of ATP-sensitive P2X7 receptor

Macrophage ionotropic P2X7 receptors regulate cell-death through ill-defined signaling pathways. Here, we investigated the role of ceramide, an apoptogenic sphingolipid and showed that ATP stimulated ceramide accumulation in macrophages. Benzoylbenzoyl-ATP, a potent P2X7 agonist, was able to mimic the effects of ATP on ceramide accumulation while oxidized ATP had the opposite effect. Ceramide accumulation was blocked by de novo ceramide biosynthesis inhibitors. Interestingly, ATP-induced caspase-3/7 activation was dependent on ceramide generation. Finally, we showed that de novo ceramide biosynthesis is involved in ATP-induced macrophage death in a caspase-dependent manner. Our results indicate a novel role of ceramide in P2X7-regulated cell-death.     

The 2'-O- and 3'-O-Cy3-EDA-ATP(ADP) complexes with myosin subfragment-1 are spectroscopically distinct

Ribose-modified highly-fluorescent sulfoindocyanine ATP and ADP analogs, 2'(3')-O-Cy3-EDA-AT(D)P, with kinetics similar to AT(D)P, enable myosin and actomyosin ATPase enzymology with single substrate molecules. Stopped-flow studies recording both fluorescence and anisotropy during binding to skeletal muscle myosin subfragment-1 (S1) and subsequent single-turnover decay of steady-state intermediates showed that on complex formation, 2'-O- isomer fluorescence quenched by 5%, anisotropy increased from 0.208 to 0.357, and then decayed with turnover rate k(cat) 0.07 s(-1); however, 3'-O- isomer fluorescence increased 77%, and anisotropy from 0.202 to 0.389, but k(cat) was 0.03 s(-1). Cy3-EDA-ADP.S1 complexes with vanadate (V(i)) were studied kinetically and by time-resolved fluorometry as stable analogs of the steady-state intermediates. Upon formation of the 3'-O-Cy3-EDA-ADP.S1.V(i) complex fluorescence doubled and anisotropy increased to 0.372; for the 2'-O- isomer, anisotropy increased to 0.343 but fluorescence only 6%. Average fluorescent lifetimes of 2'-O- and 3'-O-Cy3-EDA-ADP.S1.V(i) complexes, 0.9 and 1.85 ns, compare with approximately 0.7 ns for free analogs. Dynamic polarization shows rotational correlation times higher than 100 ns for both Cy3-EDA-ADP.S1.V(i) complexes, but the 2'-O-isomer only has also a 0.2-ns component. Thus, when bound, 3'-O-Cy3-EDA-ADP's fluorescence is twofold brighter with motion more restricted and turnover slower than the 2'-O-isomer; these data are relevant for applications of these analogs in single molecule studies.     

Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2

Background

Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation.

Methods

J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases.

Results

ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP.

Conclusions

Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin.

General significance

ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.     

Calmidazolium selectively inhibits exocytotic glutamate release evoked by P2X7 receptor activation

We previously observed that activation of presynaptic P2X7 receptors located on rat cerebrocortical nerve terminals induced the release of glutamate through different modes: the channel conformation allowing Ca(2+) entry triggered exocytotic release, while the receptor itself functioned as a permeation pathway for the non-exocytotic glutamate release. Considering that exocytotic and non-exocytotic glutamate release evoked by the activation of P2X7 receptors might play a role in the control of glutamatergic synapses, we investigated whether calmidazolium (which has been found to inhibit small cation currents through recombinant P2X7 receptors, but not organic molecule permeation) could distinguish between P2X7-related exocytotic and non-exocytotic modes of glutamate release. We found that calmidazolium inhibited the intrasynaptosomal Ca(2+) response to P2X7 receptor activation and the Ca(2+)-dependent exocytotic glutamate release from rat cerebrocortical nerve terminals, but was ineffective against the Ca(2+)-independent glutamate release. The P2X7 competitive antagonist A-438079 eliminated both exocytotic and non-exocytotic P2X7 receptor-evoked glutamate release. Selective inhibition of exocytotic glutamate release indicates that calmidazolium inhibits events dependent on the function of native rat P2X7 receptors as Ca(2+) channels, and suggests that it can be used as a tool to dissociate P2X7-evoked exocytotic from non-exocytotic glutamate release.     

Escherichia coli SecA truncated at its termini is functional and dimeric

Terminal residues in SecA, the dimeric ATPase motor of bacterial preprotein translocase, were proposed to be required for function and dimerization. To test this, we generated truncation mutants of the 901aa long SecA of Escherichia coli. We now show that deletions of carboxy-terminal domain (CTD), the extreme CTD of 70 residues, or of the N-terminal nonapeptide or of both, do not compromise protein translocation or viability. Deletion of additional C-terminal residues upstream of CTD compromised function. Functional truncation mutants like SecA9-861 are dimeric, conformationally similar to SecA, fully competent for nucleotide and SecYEG binding and for ATP catalysis. Our data demonstrate that extreme terminal SecA residues are not essential for SecA catalysis and dimerization.     

P2X7 receptor activation induces cell death and microparticle release in murine erythroleukemia cells

Extracellular ATP induces cation fluxes in and impairs the growth of murine erythroleukemia (MEL) cells in a manner characteristic of the purinergic P2X7 receptor, however the presence of P2X7 in these cells is unknown. This study investigated whether MEL cells express functional P2X7. RT-PCR, immunoblotting and immunofluorescence staining demonstrated the presence of P2X7 in MEL cells. Cytofluorometric measurements demonstrated that ATP induced ethidium⁺ uptake into MEL cells in a concentration-dependent fashion and with an EC₅₀ of ∼ 154 μM. The most potent P2X7 agonist 2′- and 3′-0(4-benzoylbenzoyl) ATP, but not ADP or UTP, induced ethidium⁺ uptake. ATP-induced ethidium⁺ and YO-PRO-1²⁺ uptake were impaired by the P2X7 antagonist, A-438079. A colourmetric assay demonstrated that ATP impaired MEL cell growth. A cytofluorometric assay showed that ATP induced MEL cell death and that this process was impaired by A-438079. Finally, cytofluorometric measurements of Annexin-V binding and bio-maleimide staining demonstrated that ATP could induce rapid phosphatidylserine exposure and microparticle release in MEL cells respectively, both of which were impaired by A-438079. These results demonstrate that MEL cells express functional P2X7, and indicate that activation of this receptor may be important in the death and release of microparticles from red blood cells in vivo.     

P2X ion channel receptors and inflammation

Neuroinflammation limits tissue damage in response to pathogens or injury and promotes repair. There are two stages of inflammation, initiation and resolution. P2X receptors are gaining attention in relation to immunology and inflammation. The P2X7 receptor in particular appears to be an essential immunomodulatory receptor, although P2X1 and P2X4 receptors also appear to be involved. ATP released from damaged or infected cells causes inflammation by release of inflammatory cytokines via P2X7 receptors and acts as a danger signal by occupying upregulated P2X receptors on immune cells to increase immune responses. The purinergic involvement in inflammation is being explored for the development of novel therapeutic strategies.     

Ependymal cells along the lateral ventricle express functional P2X7 receptors

Ependymal cells line the cerebral ventricles and are located in an ideal position to detect central nervous system injury and inflammation. The signaling mechanisms of ependymal cells, however, are poorly understood. As extracellular adenosine 5′-triphosphate is elevated in the context of cellular damage, experiments were conducted to determine whether ependymal cells along the mouse subventricular zone (SVZ) express functional purinergic receptors. Using whole-cell patch clamp recording, widespread expression of P2X₇ receptors was detected on ependymal cells based on their antagonist sensitivity profile and absence of response in P2X₇ ^−/− mice. Immunocytochemistry confirmed the expression of P2X₇ receptors, and electron microscopy demonstrated that P2X₇ receptors are expressed on both cilia and microvilli. Ca²⁺ imaging showed that P2X₇ receptors expressed on cilia are indeed functional. As ependymal cells are believed to function as partner cells in the SVZ neurogenic niche, P2X₇ receptors may play a role in neural progenitor response to injury and inflammation.     

Potato tuber isoapyrases: substrate specificity, affinity labeling, and proteolytic susceptibility

Apyrase/ATP-diphosphohydrolase hydrolyzes di- and triphosphorylated nucleosides in the presence of a bivalent ion with sequential release of orthophosphate. We performed studies of substrate specificity on homogeneous isoapyrases from two potato tuber clonal varieties: Desiree (low ATPase/ADPase ratio) and Pimpernel (high ATPase/ADPase ratio) by measuring the kinetic parameters K(m) and k(cat) on deoxyribonucleotides and fluorescent analogues of ATP and ADP. Both isoapyrases showed a broad specificity towards dATP, dGTP, dTTP, dCTP, thio-dATP, fluorescent nucleotides (MANT-; TNP-; ethene-derivatives of ATP and ADP). The hydrolytic activity on the triphosphorylated compounds was always higher for the Pimpernel apyrase. Modifications either on the base or the ribose moieties did not increase K(m) values, suggesting that the introduction of large groups (MANT- and TNP-) in the ribose does not produce steric hindrance on substrate binding. However, the presence of these bulky groups caused, in general, a reduction in k(cat), indicating an important effect on the catalytic step. Substantial differences were observed between potato apyrases and enzymes from various animal tissues, concerning affinity labeling with azido-nucleotides and FSBA (5'-p-fluorosulfonylbenzoyl adenosine). PLP-nucleotide derivatives were unable to produce inactivation of potato apyrase. The lack of sensitivity of both potato enzymes towards these nucleotide analogues rules out the proximity or adequate orientation of sulfhydryl, hydroxyl or amino-groups to the modifying groups. Both apyrases were different in the proteolytic susceptibility towards trypsin, chymotrypsin and Glu-C.     

Alkaloids from Galanthus nivalis

Phytochemical studies on Galanthus nivalis of Bulgarian origin resulted in the isolation of five compounds: 11-O-(3'-hydroxybutanoyl)hamayne, 3,11-O-(3',3'-dihydroxybutanoyl)hamayne, 3-O-(2'-butenoyl)-11-O-(3'-hydroxybutanoyl)hamayne, 3,11,3'-O-(3',3',3'-trihydroxybutanoyl)hamayne, and 2-O-(3'-acetoxybutanoyl)lycorine, together with five known alkaloids: ungeremine, lycorine, tazettine, hamayne, and ismine. Their structures were determined by (1)H and (13)C NMR spectroscopy and two-dimensional (1)H-(1)H and (1)H-(13)C chemical shift correlation experiments.     

Functional evidence for presynaptic P2X7 receptors in adult rat cerebrocortical nerve terminals

The presynaptic P2X₇ receptor (P2X₇R) plays an important role in the modulation of transmitter release. We recently demonstrated that, in nerve terminals of the adult rat cerebral cortex, P2X₇R activation induced Ca²⁺-dependent vesicular glutamate release and significant Ca²⁺-independent glutamate efflux through the P2X₇R itself. In the present study, we investigated the effect of the new selective P2X₇R competitive antagonist 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine (A-438079) on cerebrocortical terminal intracellular calcium (intrasynaptosomal calcium concentration;[Ca²⁺]_i signals and glutamate release, and evaluated whether P2X₇R immunoreactivity was consistent with these functional tests. A-438079 inhibited functional responses. P2X₇R immunoreactivity was found in about 45% of cerebrocortical terminals, including glutamatergic and non-glutamatergic terminals. This percentage was similar to that of synaptosomes showing P2X₇R-mediated [Ca²⁺]_i signals. These findings provide compelling evidence of functional presynaptic P2X₇R in cortical nerve terminals.     

Functional interaction between purinergic receptors: effect of ligands for A2A and P2Y12 receptors on P2Y1 receptor function

A_2A adenosine receptor (A_2AR), P2Y₁ receptor (P2Y₁R) and P2Y₁₂ receptor (P2Y₁₂R) are predominantly expressed on human platelets. The individual role of each of these receptors in platelet aggregation has been actively reported. Previously, hetero-oligomerization between these three receptors has been shown to occur. Here, we show that Ca²⁺ signaling evoked by the P2Y₁R agonist, 2-methylthioladenosine 5’ diphosphate (2MeSADP) was significantly inhibited by the A_2AR antagonist (ZM241385 and SCH442416) and the P2Y₁₂R antagonist (ARC69931MX) using HEK293T cells expressing the three receptors. It was confirmed that inhibition of P2Y₁R signaling by A_2AR and P2Y₁₂R antagonists was indeed mediated through A_2AR and P2Y₁₂R using 1321N1 human astrocytoma cells which do not express P2Y receptors. We expect that intermolecular signal transduction and specific conformational changes occur among components of hetero-oligomers formed by these three receptors.     

Role of sodium in mitochondrial membrane depolarization induced by P2X7 receptor activation in submandibular glands

The effect of ATP on mitochondrial membrane depolarization in rat submandibular glands was investigated. Exposure of the cell suspension to high concentrations of ATP induced a sustained depolarization of mitochondrial membrane. This effect was blocked in the presence of magnesium and reproduced by low concentrations of 2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP), suggesting the implication of the P2X(7) purinergic receptor. This point was confirmed by comparison of the response to ATP by wild-type and P2X(7) knock-out (P2X(7)R(-/-)) mice. Mitochondria took up calcium after ATP stimulation but the depolarization of the mitochondrial membrane by ATP was not affected by the removal of calcium from the extracellular medium. It was nearly fully suppressed in the absence of sodium and partially blocked by the mitochondrial Na/Ca exchanger inhibitor 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP-37157). Both ATP and monensin increased the uptake of extracellular sodium (as shown by the depolarization of the plasma membrane) but the sodium ionophore did not affect the mitochondrial membrane potential. It is concluded that the activation of P2X(7) receptors depolarizes the mitochondrial membrane. The uptake of extracellular sodium is necessary but not sufficient to induce this response.     

Role of S-adenosylhomocysteine hydrolase in adenosine-induced apoptosis in HepG2 cells

Adenosine has been shown to initiate apoptosis through different mechanisms: (i) activation of adenosine receptors, (ii) intracellular conversion to AMP and stimulation of AMP-activated kinase, (iii) conversion to S-adenosylhomocysteine (AdoHcy), which is an inhibitor of S-adenosylmethionine (AdoMet)-dependent methyltransferases. Since the pathways involved are still not completely understood, we further investigated the role of AdoHcy hydrolase in adenosine-induced apoptosis. In HepG2 cells, adenosine induced caspase-like activity and DNA fragmentation, a marker of apoptosis. These effects were potentiated by co-incubation with homocysteine or adenosine deaminase inhibitor, pentostatin, and were mimicked by inhibition of AdoHcy hydrolase by adenosine-2',3'-dialdehyde (Adox). Adenosine-induced effects were significantly inhibited by dipyridamole, an inhibitor of adenosine transporter, whereas inhibitors of adenosine kinase did not affect adenosine-induced changes. Various adenosine receptor agonists and AICAR, an activator of AMP-activated kinase, did not mimic the effect of adenosine. Thus, adenosine-induced apoptosis is likely due to intracellular action of AdoHcy and independent of AMP-activated kinase and adenosine receptors. Because elevated AdoHcy levels are associated with reduced mRNA methylation, we studied mRNA expression in Adox-treated cells by microarray analysis. Since several p53-target genes and other apoptosis-related genes were up-regulated by Adox, we conclude that AdoHcy is involved in adenosine-induced apoptosis by altering gene expression.     

Identification and characterisation of a novel splice variant of the human CB1 receptor

Cannabinoid ligands are implicated in many physiological processes and to date two receptors have been identified. However, a growing body of evidence exists that suggests the presence of additional receptors. Whilst cloning the previously described hCB1a, we have identified a novel variant that we call hCB1b. Characterising these two splice variants demonstrates that they have a unique pharmacological profile and that their RNA's are expressed at low levels in a variety of tissues.     

GABA transporters mediate glycine release from cerebellum nerve endings: roles of Ca(2+)channels, mitochondrial Na(+)/Ca(2+) exchangers, vesicular GABA/glycine transporters and anion channels

GABA transporters accumulate GABA to inactivate or reutilize it. Transporter-mediated GABA release can also occur. Recent findings indicate that GABA transporters can perform additional functions. We investigated how activation of GABA transporters can mediate release of glycine. Nerve endings purified from mouse cerebellum were prelabeled with [(3)H]glycine in presence of the glycine GlyT1 transporter inhibitor NFPS to label selectively GlyT2-bearing terminals. GABA was added under superfusion conditions and the mechanisms of the GABA-evoked [(3)H]glycine release were characterized. GABA stimulated [(3)H]glycine release in a concentration-dependent manner (EC(50) = 8.26 μM). The GABA-evoked release was insensitive to GABA(A) and GABA(B) receptor antagonists, but it was abolished by GABA transporter inhibitors. About 25% of the evoked release was dependent on external Ca(2+) entering the nerve terminals through VSCCs sensitive to ω-conotoxins. The external Ca(2+)-independent release involved mitochondrial Ca(2+), as it was prevented by the Na(+)/Ca(2+) exchanger inhibitor CGP37157. The GABA uptake-mediated increases in cytosolic Ca(2+) did not trigger exocytotic release because the [(3)H]glycine efflux was insensitive to clostridial toxins. Bafilomycin inhibited the evoked release likely because it reduced vesicular storage of [(3)H]glycine so that less [(3)H]glycine can become cytosolic when GABA taken up exchanges with [(3)H]glycine at the vesicular inhibitory amino acid transporters shared by the two amino acids. The GABA-evoked [(3)H]glycine efflux could be prevented by niflumic acid or NPPB indicating that the evoked release occurred essentially by permeation through anion channels. In conclusion, GABA uptake into GlyT2-bearing cerebellar nerve endings triggered glycine release which occurred essentially by permeation through Ca(2+)-dependent anion channels. Glial GABA release mediated by anion channels was proposed to underlie tonic inhibition in the cerebellum; the present results suggest that glycine release by neuronal anion channels also might contribute to tonic inhibition.     

3-[(±)-2-Carboxypiperazin-4-yl]propyl-1-Phosphonic Acid Recognizes Two N-Methyl-D-Aspartate Binding Sites in Rat Cerebral Cortex Membranes

Binding of 3-[(+-)-2-carboxypiperazin-4-yl][3H]-propyl-1-phosphonic acid ([3H]CPP), a competitive inhibitor of N-methyl-D-aspartate (NMDA), has been studied in synaptic plasma membranes from rat cerebral cortex. Computer analysis of saturation and homologous displacement isotherms deriving from these plasma membranes indicated the existence of two binding sites: a specific, saturable, high-affinity binding site with a pKD value of 7.53 +/- 0.03 (29.5 nM) and a maximum binding value (Bmax) of 2.25 +/- 0.36 pmol/mg of protein, and a low-affinity site with a KD of approximately 600 nM and a Bmax of 7.0 pmol/mg of protein. It is argued that, in the light of current literature evidence, the low-affinity binding site may represent an agonist-dependent receptor, linked to physiological processes such as neurotransmitter release and channel regulation, whereas the high-affinity binding site may be linked to an antagonist-preferred receptor, for which no function has yet been reported.