Clara Quintas

Microglia P2Y 6 receptors mediate nitric oxide release and astrocyte apoptosis

BackgroundDuring cerebral inflammation uracil nucleotides leak to the extracellular medium and activate glial pyrimidine receptors contributing to the development of a reactive phenotype. Chronically activated microglia acquire an anti-inflammatory phenotype that favors neuronal differentiation, but the impact of these microglia on astrogliosis is unknown. We investigated the contribution of pyrimidine receptors to microglia-astrocyte signaling in a chronic model of inflammation and its impact on astrogliosis.MethodsCo-cultures of astrocytes and microglia were chronically treated with lipopolysaccharide (LPS) and incubated with uracil nucleotides for 48 h. The effect of nucleotides was evaluated in methyl-[3H]-thymidine incorporation. Western blot and immunofluorescence was performed to detect the expression of P2Y6 receptors and the inducible form of nitric oxide synthase (iNOS). Nitric oxide (NO) release was quantified through Griess reaction. Cell death was also investigated by the LDH assay and by the TUNEL assay or Hoechst 33258 staining.ResultsUTP, UDP (0.001 to 1  mM) or PSB 0474 (0.01 to 10 μM) inhibited cell proliferation up to 43 ± 2% (n = 10, P <0.05), an effect prevented by the selective P2Y6 receptor antagonist MRS 2578 (1 μM). UTP was rapidly metabolized into UDP, which had a longer half-life. The inhibitory effect of UDP (1 mM) was abolished by phospholipase C (PLC), protein kinase C (PKC) and nitric oxide synthase (NOS) inhibitors. Both UDP (1 mM) and PSB 0474 (10 μM) increased NO release up to 199 ± 20% (n = 4, P <0.05), an effect dependent on P2Y6 receptors-PLC-PKC pathway activation, indicating that this pathway mediates NO release. Western blot and immunocytochemistry analysis indicated that P2Y6 receptors were expressed in the cultures being mainly localized in microglia. Moreover, the expression of iNOS was mainly observed in microglia and was upregulated by UDP (1 mM) or PSB 0474 (10 μM). UDP-mediated NO release induced apoptosis in astrocytes, but not in microglia.ConclusionsIn LPS treated co-cultures of astrocytes and microglia, UTP is rapidly converted into UDP, which activates P2Y6 receptors inducing the release of NO by microglia that causes astrocyte apoptosis, thus controlling their rate of proliferation and preventing an excessive astrogliosis.

Pharmacological modulation of HDAC1 and HDAC6 in vivo in a zebrafish model: Therapeutic implications for Parkinson's disease.

Histone deacetylases (HDACs) are key epigenetic enzymes and emerging drug targets in cancer and neurodegeneration. Pan-HDAC inhibitors provided neuroprotection in Parkinsons Disease (PD) models, however, the HDAC isoforms with highest neuroprotective potential remain unknown. Zebrafish larvae (powerful pharmacological testing tools bridging cellular and in vivo studies) have thus far been used in PD modelling with limited phenotypic characterization. Here we characterize the behavioural and metabolic phenotypes of a zebrafish PD model induced with MPP(+), assess the feasibility of targeting zebrafish HDAC1 and HDAC6 isoforms, and test the in vivo effects of their selective inhibitors MS-275 and tubastatin A, respectively. MPP(+) induced a concentration-dependent decrease in metabolic activity and sensorimotor reflexes, and induced locomotor impairments rescuable by the dopaminergic agonist apomorphine. Zebrafish HDAC1 and HDAC6 isoforms show high sequence identity with mammalian homologues at the deacetylase active sites, and pharmacological inhibition increased acetylation of their respective histone and tubulin targets. MS-275 and tubastatin rescued the MPP(+)-induced decrease in diencephalic tyrosine hydroxylase immunofluorescence and in whole-larvae metabolic activity, without modifying mitochondrial complex activity or biogenesis. MS-275 or tubastatin alone modulated spontaneous locomotion. When combined with MPP(+), however, neither MS-275 nor tubastatin rescued locomotor impairments, although tubastatin did ameliorate the head-reflex impairment. This study demonstrates the feasibility of pharmacologically targeting the zebrafish HDAC1 and HDAC6 isoforms, and indicates that their inhibition can rescue cellular metabolism in a PD model. Absence of improvement in locomotion, however, suggests that monotherapy with either HDAC1 or HDAC6 inhibitors is unlikely to provide strong benefits in PD. This study highlights parameters dependent on the integrity of zebrafish neuronal circuits as a valuable complement to cell-based studies. Also, the demonstrated feasibility of pharmacologically targeting HDAC1 and HDAC6 in this organism paves the way for future studies investigating HDAC inhibitors in other diseases modelled in zebrafish.

Coupling to protein kinases A and C of adenosine A2B receptors involved in the facilitation of noradrenaline release in the prostatic portion of rat vas deferens.

In the prostatic portion of rat vas deferens, the non-selective adenosine receptor agonist NECA (0.1-30 microM), but not the A(2A) agonist CGS 21680 (0.001-10 microM), caused a facilitation of electrically evoked noradrenaline release (up to 43 +/- 4%), when inhibitory adenosine A(1) receptors were blocked. NECA-elicited facilitation of noradrenaline release was prevented by the A(2B) receptor-antagonist MRS 1754, enhanced by preventing cyclic-AMP degradation with rolipram, abolished by the protein kinase A inhibitors H-89, KT 5720 and cyclic-AMPS-Rp and attenuated by the protein kinase C inhibitors Ro 32-0432 and calphostin C. The adenosine uptake inhibitor NBTI also elicited a facilitation of noradrenaline release; an effect that was abolished by adenosine deaminase and attenuated by MRS 1754, by inhibitors of the extracellular nucleotide metabolism and by blockade of alpha(1)-adrenoceptors and P2X receptors with prazosin and NF023, respectively. It was concluded that adenosine A(2B) receptors are involved in a facilitation of noradrenaline release in the prostatic portion of rat vas deferens that can be activated by adenosine formed by extracellular catabolism of nucleotides. The receptors seem to be coupled to the adenylyl cyclase-protein kinase A pathway but activation of the protein kinase C by protein kinase A, may also contribute to the adenosine A(2B) receptor-mediated facilitation of noradrenaline release.

Functional crosstalk of prejunctional receptors on the modulation of noradrenaline release in mesenteric vessels: A differential study of artery and vein

The role of angiotensin II receptors, bradykinin receptors and β-adrenoceptors in the modulation of noradrenaline release and the influence of α(2)-autoinhibition in these effects was investigated in the mesenteric artery and vein. Rings of mesenteric vessels of male Wistar rats were labelled with [(3)H]-noradrenaline and the effects of modulators on tritium overflow evoked by 100 pulses at 2Hz (marked α(2)-autoinhibition) and by 20 pulses at 50Hz or 100 pulses at 2Hz plus yohimbine (1μM; reduced α(2)-autoinhibition) were evaluated. Angiotensin II and bradykinin enhanced noradrenaline release evoked by 100 pulses at 2Hz, in a concentration-dependent manner, in both vessels. These effects were attenuated under conditions of reduced α(2)-autoinhibition. The attenuation was partially reversed by activation of adenosine A(1) receptors in both vessels and by activation of P2Y receptors in the vein. Isoprenaline and the selective β(2)-adrenoceptor agonist formoterol enhanced tritium overflow independently of α(2)-autoinhibition in the vein. In the artery, the enhancement by formoterol was only observed under reduced α(2)-autoinhibition. Pharmacological characterization of the β-adrenoceptors indicated that in the artery the effect of isoprenaline was mediated by the β(1)-subtype under marked α(2)-autoinhibition and by the β(2)-subtype under reduced α(2)-autoinhibition whereas in the vein the effect was independent of α(2)-autoinhibition. The results indicate that α(2)-autoinhibition is a key determinant of the magnitude of facilitation caused by angiotensin II and bradykinin in both types of mesenteric vessels and regulates the effects mediated by β(1)-and β(2)-adrenoceptors which co-exist in the artery.

Microglia P2Y13 Receptors Prevent Astrocyte Proliferation Mediated by P2Y1 Receptors

Cerebral inflammation is a common feature of several neurodegenerative diseases that requires a fine interplay between astrocytes and microglia to acquire appropriate phenotypes for an efficient response to neuronal damage. During brain inflammation, ATP is massively released into the extracellular medium and converted into ADP. Both nucleotides acting on P2 receptors, modulate astrogliosis through mechanisms involving microglia-astrocytes communication. In previous studies, primary cultures of astrocytes and co-cultures of astrocytes and microglia were used to investigate the influence of microglia on astroglial proliferation induced by ADPβS, a stable ADP analog. In astrocyte cultures, ADPβS increased cell proliferation through activation of P2Y1 and P2Y12 receptors, an effect abolished in co-cultures (of astrocytes with ∼12.5% microglia). The possibility that the loss of the ADPβS-mediated effect could have been caused by a microglia-induced degradation of ADPβS or by a preferential microglial localization of P2Y1 or P2Y12 receptors was excluded. Since ADPβS also activates P2Y13 receptors, the contribution of microglial P2Y13 receptors to prevent the proliferative effect of ADPβS in co-cultures was investigated. The results obtained indicate that P2Y13 receptors are low expressed in astrocytes and mainly expressed in microglia. Furthermore, in co-cultures, ADPβS induced astroglial proliferation in the presence of the selective P2Y13 antagonist MRS 2211 (3 μM) and of the selective P2Y12 antagonist AR-C66096 (0.1 μM), suggesting that activation of microglial P2Y12 and P2Y13 receptors may induce the release of messengers that inhibit astroglial proliferation mediated by P2Y1,12 receptors. In this microglia-astrocyte paracrine communication, P2Y12 receptors exert opposite effects in astroglial proliferation as a result of its cellular localization: cooperating in astrocytes with P2Y1 receptors to directly stimulate proliferation and in microglia with P2Y13 receptors to prevent proliferation. IL-1β also attenuated the proliferative effect of ADPβS in astrocyte cultures. However, in co-cultures, the anti-IL-1β antibody was unable to recover the ADPβS-proliferative effect, an effect that was achieved by the anti-IL-1α and anti-TNF-α antibodies. It is concluded that microglia control the P2Y1,12 receptor-mediated astroglial proliferation through a P2Y12,13 receptor-mediated mechanism alternative to the IL-1β suppressive pathway that may involve the contribution of the cytokines IL-1α and TNF-α.

LPS-activated microglia overexpress P2Y(6) receptors that mediate an inhibition of astroglial proliferation by inducing no mediated apoptosis

Abstracts of the Fourth Joint German-Italian Purine Club Meeting 2011s of the Fourth Joint German-Italian Purine Club Meeting 2011 Conference organization Conference chairperson Christa E. Müller Conference coordinator Heidi Zinecker Scientific coordination Italian Chapter of Purine Club German Chapter of Purine Club Maria P. Abbracchio (Milan) Joachim Deitmer (Kaiserslautern) Pier A. Borea (Ferrara) Peter Illes (Leipzig) Pier Leopoldo Capecchi (Siena) Karl-Norbert Klotz (Würzburg) Stefania Ceruti (Milan) Friedrich Koch-Nolte (Hamburg) Renata Ciccarelli (Chieti) Georg Reiser (Magdeburg) Patrizia Popoli (Rome) Günther Schmalzing (Aachen) Anna Maria Pugliese (Florence) Torsten Schöneberg (Leipzig) Letizia Trincavelli (Pisa) Jürgen Schrader (Düsseldorf) Rosaria Volpini (Camerino) Norbert Sträter (Leipzig) Organizing Committee Ivar von Kügelgen (Bonn) Christa E. Müller (Bonn) Karen Nieber (Leipzig) Alexander Pfeifer (Bonn) Anke C. Schiedel (Bonn) Christian Steinhäuser (Bonn) Herbert Zimmermann (Frankfurt) Heidi Zinecker (Bonn) Business Secretariat (University of Bonn) Michael Hemmersbach Beate Ponatowski Anke C. Schiedel Heidi Zinecker Contact: e-mail: purineclub@uni-bonn.de visit: www.purineclub.uni-bonn.de call: +49 228 732697 Purinergic Signalling (2012) 8:107–175 DOI 10.1007/s11302-011-9257-4

Inhibition of astroglia proliferation by nucleotides: contribution of P1 receptors

Third joint Italian-German Purine Club meeting: “Purinergic receptors: new frontiers for novel therapies” Invited Lectures Abstracts Purinergic signalling: some exciting new directions Geoffrey Burnstock Autonomic Neuroscience Centre, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK Presenting author: g.burnstock@ucl.ac.uk After a brief initial description of some of the important steps in the establishment of purinergic signalling, the talk will focus on some of the exciting new directions in the field, particularly those relating to purinergic pathophysiology and potential therapeutic applications. There will be discussion of the long-term trophic effects of purines and pyrimidines in blood vessel remodelling in restenosis; plasticity of purinergic cotransmission of diseased urinary bladder; hypertensive rats; spermmotility in IVF; purinergic mechanosensory transduction in visceral pain and the role of spinal microglial purinoceptors in neuropathic pain; the potential for P2X7 receptor antagonists in osteoporosis and kidney failure and the growing literature about the roles of purinergic signalling of disorders of the central nervous system; the role of ATP in the treatment of cancer; transient trophic purinergic signalling in embryological development and in stem cell activities; and studies of the evolutionary origins of purinergic receptors will also be discussed. Finally, a novel hypothesis will be presented for the involvement of purinergic signalling in acupuncture. New frontiers for ligands of adenosine and P2Y receptors Kenneth A. Jacobson National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health, Bethesda, MD, 20902, USA Presenting author: kajacobs@helix.nih.gov Selective agonists and antagonists are now available for each of the four adenosine receptor (AR) subtypes, making possible research advances leading to the implementation of new therapeutic concepts. Development of selective P2Y receptor ligands is also underway and has been aided by computer modeling and in silico screening. Nevertheless, the field of purine and pyrimidine signaling has particular challenges for medicinal chemists and pharmacologists. The bioavailability and stability of polar, charged, or hydrolyzable compounds are often limited. Effective and selective inhibitors of nucleotidases are lacking. The interplay of chemically related signaling molecules, their metabolism and formation, and time-dependent signaling, in any given cell or tissue, is to be considered as a system rather than as isolated components. The relationship of disease treatment to Gprotein-dependent and independent pathways needs to be established, and new assays are needed. The definition of agonism vs partial agonism vs antagonism may depend on the model used. Large species differences, affecting both affinity and efficacy of new compounds, exist for these receptors. Novel drug delivery or prodrug approaches are needed to overcome side effects because receptors tend to be widespread. In addition, the effects of oligomerization of receptors on the pharmacology are mainly unknown. Thus, subtype selectivity and favorable pharmacokinetics alone are insufficient to propose a new clinical candidate. To address these needs, the predictive value and limitations of homology modeling were analyzed in light of the X-ray structure of the A2AAR in support of structure-based drug design. At the A3AR, we designed and synthesized novel Purinergic Signalling (2010) 6:49–115 DOI 10.1007/s11302-009-9171-1

Microglia P2Y6 receptors mediate an inhibition of astroglia proliferation in cultures of rat brain cortex

The rodent P2X7k splice variant, which utilizes a different exon 1 and is expressed in both wild type and the Glaxo P2X7−/−mice, has at least a 10-fold greater sensitivity to agonists ATP and BzATP than the full length mouse P2X7a variant, as determined from patch clamp experiments. ThemP2X7k receptor also coupled to the rapid uptake of the cationic dye ethidium, and this process was unaffected by the SNP, P451L, that previously was shown to inhibit mP2X7a receptor-mediated dye uptake. In HEK293 cells, activation of mP2X7k also triggered the uptake of the anionic dye Lucifer yellow, the rate of which was dramatically increased by a 5-min pre-incubation with the pannexin-1 inhibitor, carbenoxolone (CBX) at 37°C. CBX also increased mP2X7k-mediated ethidium uptake but the Ca2+ signal evoked by activation of mP2X7k was unaffected, suggesting that CBX selectively affects ‘large pore’ formation. Themechanismof action of CBX is unclear but the results are not consistentwith pannexin-1 being the dye uptake pathway. P2X7k is expressed in mice spleen T-lymphocytes where NAD is reported to be a physiologically important agonist. In HEK293 cells, 10 uM NAD stimulated mP2X7k-mediated ethidium uptake whereas mP2X7a did not respond to concentrations up to 300 uM NAD. NAD also stimulated a rapid Ca2+ response in HEK293 cells expressing mP2X7k. The coexpression of mP2X7a and mP2X7k in HEK cells reduced the amplitude of the NAD response compared to cells expressing mP2X7k alone. Our results suggest the expression of P2X7k increases the diversity of P2X7 receptor signaling in rodents.

Influence of microglia on nucleotide-induced astroglia proliferation

P2X4 and P2X7 are the predominant purinergic receptor subtypes expressed in macrophages, microglia and epithelial cells, and they are potentially important therapeutic targets for treatment of pain and inflammation. For both subtypes, there is evidence that plasmamembrane expression is tightly regulated. P2X4 receptors are prominently localized to lysosomes and resist degradation by virtue of N-linked glycans decorating the intra-luminal loop of the receptor. P2X7 receptors are reported to be predominantly intracellular in monocytes and are upregulated at the plasma membrane upon differentiation of monocytes to macrophages. We have previously shown an interaction between P2X4 and P2X7 receptors, suggesting that they might form an association. The mechanisms that regulate their plasma membrane expression are not well understood, and we have used biochemical methods to look at the size and distribution of the native complexes in a variety of cell types in which they are co-expressed. We have compared the proportion of receptors expressed at the cell surface in cultured microglia and macrophages following exposure to modulators of microglial/macrophage activation. Surface expression was analysed by biotinylation of exposed proteins and by cross-linking proteins with membrane impermeant cross-linkers, followed by SDS-PAGE and western blotting. The modulators included lipopolysaccharide (LPS), ATP and phorbol esters. Cross-linking of surface receptors also provides a means of analysing the subunit composition of the complexes at the plasma membrane, based upon the size difference of P2X4 and P2X7 subunits. These results are compared with those obtained using blue native (BN)-PAGE analysis of the total P2X receptor population.Plenary Presentations The struggle to establish purinergic signalling Burnstock, Geoffrey Autonomic Neuroscience Centre Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK Early experiments in the 1960’s will be described leading to the purinergic neurotransmission hypothesis proposed in 1972. The influence of publications by key figures such as Andrew Szent-Györgyi, Pamela Holton, Robert Berne, Mike Rand and Jack Eccles is recognised. Unfortunately, the hypothesis was regarded with scepticism by many for the next 25 years and stories of this resistance will be recounted. The cloning of receptors for purines and pyrimidines in the early 1990’s was an important turning point in the acceptance of the hypothesis and the important contributions of many outstanding scientists will be related. Current strong interest in the pathophysiological roles of purinergic signalling and exploration of the therapeutic potential for a number of disease conditions will be discussed. From chemoreception to eye development -fundamental roles of ATP signalling

P2 receptor-subtypes in the modulation of reactive gliosis

P2X4 and P2X7 are the predominant purinergic receptor subtypes expressed in macrophages, microglia and epithelial cells, and they are potentially important therapeutic targets for treatment of pain and inflammation. For both subtypes, there is evidence that plasmamembrane expression is tightly regulated. P2X4 receptors are prominently localized to lysosomes and resist degradation by virtue of N-linked glycans decorating the intra-luminal loop of the receptor. P2X7 receptors are reported to be predominantly intracellular in monocytes and are upregulated at the plasma membrane upon differentiation of monocytes to macrophages. We have previously shown an interaction between P2X4 and P2X7 receptors, suggesting that they might form an association. The mechanisms that regulate their plasma membrane expression are not well understood, and we have used biochemical methods to look at the size and distribution of the native complexes in a variety of cell types in which they are co-expressed. We have compared the proportion of receptors expressed at the cell surface in cultured microglia and macrophages following exposure to modulators of microglial/macrophage activation. Surface expression was analysed by biotinylation of exposed proteins and by cross-linking proteins with membrane impermeant cross-linkers, followed by SDS-PAGE and western blotting. The modulators included lipopolysaccharide (LPS), ATP and phorbol esters. Cross-linking of surface receptors also provides a means of analysing the subunit composition of the complexes at the plasma membrane, based upon the size difference of P2X4 and P2X7 subunits. These results are compared with those obtained using blue native (BN)-PAGE analysis of the total P2X receptor population.Plenary Presentations The struggle to establish purinergic signalling Burnstock, Geoffrey Autonomic Neuroscience Centre Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK Early experiments in the 1960’s will be described leading to the purinergic neurotransmission hypothesis proposed in 1972. The influence of publications by key figures such as Andrew Szent-Györgyi, Pamela Holton, Robert Berne, Mike Rand and Jack Eccles is recognised. Unfortunately, the hypothesis was regarded with scepticism by many for the next 25 years and stories of this resistance will be recounted. The cloning of receptors for purines and pyrimidines in the early 1990’s was an important turning point in the acceptance of the hypothesis and the important contributions of many outstanding scientists will be related. Current strong interest in the pathophysiological roles of purinergic signalling and exploration of the therapeutic potential for a number of disease conditions will be discussed. From chemoreception to eye development -fundamental roles of ATP signalling