Biomedical subjects

Cinzia Volonté

Publications and source records attributed to Cinzia Volonté.

13 recordsLinked to original sources

PubMed2006

A novel pathway of cell growth regulation mediated by a PLA2alpha-derived phosphoinositide metabolite.

The phosphoinositides have well-defined roles in the control of cellular functions, including cytoskeleton dynamics, membrane trafficking, and cell signaling. However, the interplay among the phosphoinositides and their diffusible derivatives that originate through phospholipase A2 action (the lysophosphoinositides and glycerophosphoinositols) remains to be fully elucidated. Here we demonstrate that in PCCl3 rat thyroid cells, the intracellular levels of glycerophosphoinositol are finely modulated by ATP and norepinephrine through the P2Y metabotropic and alpha-adrenergic receptors, respectively. The enzyme involved here is phospholipase A2 IValpha (PLA2 IValpha), which in these cells specifically hydrolyzes phosphatidylinositol, forming lysophosphatidylinositol, glycerophosphoinositol, and arachidonic acid. This receptor-mediated activation of PLA2 IValpha leads to stimulation of PCCl3 cell growth. The involvement of a PLA2 IValpha-mediated pathway is demonstrated by inhibition of the increase in intracellular glycerophosphoinositol levels and cell proliferation by specific inhibitors, RNA interference, and overexpression of the dominant-negative PLA2 IValpha(1-522). Modulation of PCCl3 cell growth is not seen with inhibitors of arachidonic acid metabolism. In conclusion, these data characterize glycerophosphoinositol as a mediator of the purinergic and adrenergic regulation of PCCl3 cell proliferation, defining a novel regulatory cascade specifically involving this soluble phosphoinositide derivative and widening the involvement of the phosphoinositides in the regulation of cell function.

Animals↗

PubMed2006

P2 receptor web: complexity and fine-tuning.

The present review offers a new perspective on a family of receptors, termed P2 receptors, specific for nucleoside tri- and diphosphates of purines/pyrimidines. We emphasize here that while decoding the inputs of various related extracellular ligands, P2 receptors are a clear example of increasing biological complexity. They are represented by 7 ionotropic P2X and 8 metabotropic P2Y receptors; they have very heterogeneous ligands and binding characteristics, molecular properties, transduction mechanisms, cellular localization and protein-protein interactions. While the reason for this sophistication is unknown, a few compelling issues emerge while looking at such a rich variety. We ask, for instance, why so many different receptor subtypes are necessary for triggering biological properties and functions, and if these receptors are more than the sum of their single entities. A first possibility is that newly synthesized P2 proteins are casually located on the cell surface (stochastic hypothesis). Alternatively, distinct subunits are engaged on different cell phenotypes by genetic control (genetic determinism) and/or selective recruitment under physiopathological conditions and epigenetic stimuli (epigenetic determinism). Nevertheless, an appropriate way to both dissect the vast biological scenario and molecular complexity among P2 receptors and to integrate and upgrade their assortment is to regard them as a "combinatorial receptor web", that is, a dynamic architecture of P2 proteins demonstrating economic efficiency and involving a process of "fine-tuning", a mechanism which endorses the dynamic nature of all biological reactions. In the present analysis, we stimulate a scientific query about what contributes to such a vast P2 receptor sophistication.

Animals↗

PubMed2005

The metabotropic P2Y4 receptor participates in the commitment to differentiation and cell death of human neuroblastoma SH-SY5Y cells.

Extracellular nucleotides exert a variety of biological actions through different subtypes of P2 receptors. Here we characterized in the human neuroblastoma SH-SY5Y cells the simultaneous presence of various P2 receptors, belonging to the P2X ionotropic and P2Y metabotropic families. Western blot analysis detected the P2X1,2,4,5,6,7 and P2Y1,2,4,6, but not the P2X3 and P2Y12 receptors. We then investigated which biological effects were mediated by the P2Y4 subtype and its physiological pyrimidine agonist UTP. We found that neuronal differentiation of the SH-SY5Y cells with dibutiryl-cAMP increased the expression of the P2Y4 protein and that UTP itself was able to positively interfere with neuritogenesis. Moreover, transient transfection and activation of P2Y4 also facilitated neuritogenesis in SH-SY5Y cells, as detected by morphological phase contrast analysis and confocal examination of neurofilament proteins NFL. This was concurrent with increased transcription of immediate-early genes linked to differentiation such as cdk-5 and NeuroD6, and activity of AP-1 transcription family members such as c-fos, fos-B, and jun-D. Nevertheless, a prolonged activation of the P2Y4 receptor by UTP also induced cell death, both in naive, differentiated, and P2Y4-transfected SH-SY5Y cells, as measured by direct count of intact nuclei and cytofluorimetric analysis of damaged DNA. Taken together, our data indicate that the high expression and activation of the P2Y4 receptor participates in the neuronal differentiation and commitment to death of SH-SY5Y cells.

Brain Neoplasms↗

PubMed2005

Differences in the neurotoxicity profile induced by ATP and ATPgammaS in cultured cerebellar granule neurons.

Extracellular ATP and P2 receptors may play a crucial role in the neurodegeneration of the CNS. Here, we investigated in neuronal cerebellar granule cultures the biological effect of the quite stable P2 receptor agonist ATPgammaS and compare it to the cytotoxic action of ATP. Time-course experiments showed that 500 microM ATPgammaS causes 50-100% cell death in 15-24 h. As proved by pharmacological means, ATPgammaS toxicity apparently involves neither indirect activation of NMDA receptors, nor ectonucleotidase activities, nor nucleoside transport and intracellular purine metabolism. Moreover, ATPgammaS induces detrimental effects without modifying the expression of several P2X and P2Y receptor proteins. Cell death instead occurs after extracellular release of the cytosolic enzyme lactic dehydrogenase and inhibition of the overall activity of the intracellular dehydrogenases. Moreover, ATPgammaS causes transient outflow of cytochrome c from mitochondria (maximal 2.5-fold stimulation in 4 h), it raises the intracellular reactive oxygen species (about four-fold in 1 h) and cAMP levels (about 40% in 15 min-4 h). Among several P2 receptor antagonists, only pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium promotes 80-100% neuroprotection.

Adenosine Triphosphate↗

PubMed2004

Role of the metabotropic P2Y(4) receptor during hypoglycemia: cross talk with the ionotropic NMDAR1 receptor.

It is well established that both extracellular ATP and glutamate exert a critical role during metabolic impairment, that several P2 receptor subunits are directly involved in this action and that a strong relationship exists between glutamatergic and purinergic signals. Therefore, here we studied the molecular behavior of the purinergic metabotropic P2Y(4) and the glutamatergic ionotropic NMDAR1 receptors during hypoglycemic cell death. We find that these proteins are oppositely modulated during glucose starvation (P2Y(4) is induced, whereas NMDAR1 is inhibited) and that both P2 and NMDA antagonists can restore basal protein expression levels. Moreover, double immunofluorescence experiments with confocal laser microscopy reveal co-localization at the membrane level between the P2Y(4) and NMDAR1 receptors, in both homologous (cerebellar granule neurons) and heterologous (Hek-293) cellular systems. This is furthermore confirmed by co-immunoprecipitation experiments. Finally, when we express the P2Y(4) receptor in the heterologous SH-SY5Y neuronal cell line, hypoglycemia then causes severe cell death and simultaneous downregulation of the NMDAR1 protein. In summary, our work establishes a potential molecular interplay between P2Y(4) and NMDAR1 receptors during glucose deprivation and the causative role of the P2Y(4) during cell death.

Adenosine Triphosphate↗

PubMed2004

P2X3 receptor localizes into lipid rafts in neuronal cells.

P2X receptors are a family of seven (P2X(1-7)) cation channels gated by extracellular ATP, widely expressed in neurons and nonneuronal cells. Lipid rafts are cholesterol/sphingolipid-rich membrane domains, involved in many cellular processes, including transmembrane receptor signaling, vesicle traffic, and protein sorting. We provide direct biochemical evidence that P2X3 receptor localizes into lipid rafts, in primary cultures of cerebellar granule neurons as well as in brain and dorsal root ganglia extracts. We show that P2X3 exhibits all the characteristics distinctive of a protein associated with lipid rafts. These characteristics include resistance to detergent extraction at 4 degrees C, solubility after extraction of cholesterol from membranes with either saponin or methyl-beta-cyclodextrin, and partitioning to low buoyant density fractions after sucrose gradient centrifugation in both detergent-containing and detergent-free conditions. Furthermore, P2X3 localizes in raft-containing fractions in transiently transfected SH-SY5Y neuroblastoma cells. The present finding contributes to the characterization of the functional localization of P2X3 in neurons and provides a novel potential mechanism for correct targeting and dynamic activation of this receptor.

Actins↗

PubMed2004

2-ClATP exerts anti-tumoural actions not mediated by P2 receptors in neuronal and glial cell lines.

We investigated the effects of the ATP analogue and P2 receptor agonist 2-ClATP on growth and survival of different neuronal (PC12, PC12nnr5 and SH-SY5Y) and glial (U87 and U373) cell lines, by the use of direct count of intact nuclei, fluorescence microscopy, fluorescence-activated cell sorter analysis (FACS) and high pressure liquid chromatography (HPLC). 2-ClATP lowered the number of cultured PC12nnr5, SH-SY5Y, U87 and U373 cells to almost 5%, and of PC12 cells to about 35% after 3-4 days of treatment. EC(50) was in the 5-25 microM range, with 2-ClATP behaving as a cytotoxic or cytostatic agent. Analysis of the biological mechanisms demonstrated that pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (P2 receptor antagonist and nucleotidases inhibitor), but not Caffeine or CGS-15493 (P1 receptor antagonists) effectively prevented 2-ClATP-induced toxicity. 2-ClATP metabolic products (2-ClADP, 2-ClAMP, 2-Cladenosine) and new synthesis derivatives (2-CldAMP, 2-Cldadenosine-3',5'-bisphosphate and 2-CldATP) exerted similar cytotoxic actions. Inhibition of both serum nucleotidases and purine nucleoside transporters strongly reduced 2-ClATP-induced cell death, which was conversely increased by the nucleotide hydrolyzing enzyme apyrase. The adenosine kinase inhibitor 5-iodotubericidin totally prevented 2-ClATP or 2-Cladenosine-induced toxicity. In summary, our findings indicate that 2-ClATP exerts either cell cycle arrest or cell death, acting neither on P2 nor on P1 receptors, but being extracellularly metabolized into 2-Cladenosine, intracellularly transported and re-phosphorylated.

2-Chloroadenosine↗

PubMed2004

Pathways of survival induced by NGF and extracellular ATP after growth factor deprivation.

In a previous work we demonstrated that extracellular adenosine-5'-triphosphate (ATP), acting on P2 receptors, exerts neuritogenic and trophic effects on the phaeochromocytoma PC12 cell line. These actions are comparable to those sustained by nerve growth factor (NGF) and involve several overlapping pathways. In this work, we describe some of the mechanisms recruited by ATP and NGF in maintaining PC12 cell survival after serum deprivation. We show that both ATP and NGF upregulate the expression of the stress-induced heat shock protein HSP70 and HSP90, whilst glucose-response protein GRP75 and GRP78 are not affected. In parallel with NGF, ATP prevents the cleavage and activation of caspase-2 and inhibits the release of cytochrome c from mitochondria into the cytoplasm. Finally, neither NGF, nor ATP directly modulate the expression of P2 receptors in the induction of cell survival. Our data contribute to dissect the biological mechanisms activated by extracellular purines exerting trophic actions and to establish that survival and neurite outgrowth lie on different mechanistic pathways.

Adenosine Triphosphate↗

PubMed2004

Overexpression of superoxide dismutase 1 protects against beta-amyloid peptide toxicity: effect of estrogen and copper chelators.

Beta-amyloid peptides (Abeta) are major constituents of senile plaques in Alzheimer's disease (AD) brain and contribute to neurodegeneration, operating through activation of apoptotic pathways. It has been proposed that Abeta induces death by oxidative stress, possibly through the generation of peroxynitrite from superoxide and nitric oxide. Estrogen is thought to play a protective role against neurodegeneration through a variety of mechanisms including scavenging of reactive oxygen species (ROS). In this study, we have challenged with Abeta, either in the presence or in the absence of 17beta-estradiol, differentiated human neuroblastoma SH-SY5Y cells (named line SH) and the same line overexpressing anti-oxidant enzyme superoxide dismutase 1 (SOD1; named line WT). We have observed that: (1) WT cells are less susceptible than SH cells to Abeta insult; (2) caspase-3, but not caspase-1, is involved in Abeta-induced apoptosis in this system; (3) estrogen protects both lines, without significantly affecting SOD activity; and (4) copper chelators prevent Abeta-induced toxicity. Our results further support the notion that anti-oxidant therapy might be beneficial in the treatment of AD by preventing activation of selected apoptotic pathways.

Amyloid beta-Peptides↗

PubMed2004

Synaptic P2X7 and oxygen/glucose deprivation in organotypic hippocampal cultures.

The P2X7 receptor for extracellular ATP is the main candidate, among P2 receptors, inducing cell death in the immune system. Here, we demonstrate the direct participation of this receptor to cell damage induced by oxygen/glucose deprivation, in the ex vivo model of organotypic hippocampal cultures. By pharmacological and immunological approaches, we show that P2X7 is rapidly and transiently up regulated in hippocampal areas eliciting metabolism impairment. Moreover, the P2 antagonists 2',3',-dialdehyde ATP and reactive blue 2 prevent both up regulation of this receptor and hypoxic/hypoglycemic damage. By confocal laser microscopy, we show that P2X7 is present at the synaptic level of fibers extending from the CA1-2 pyramidal cell layer throughout the strata oriens and radiatum, but absent on oligodendrocytes, astrocytes or neuronal cell bodies. Colocalization of P2X7 is obtained with neurofilament-L protein and with synaptophysin, not with myelin basic protein, glial fibrillary acidic protein or a marker for neuronal nuclei. P2X7 up regulation and diffuse cellular damage are also induced by 3'-O-(4-benzoyl) benzoyl-ATP, an agonist selective but not exclusive for P2X7. In summary, our study demonstrates that P2X7 not only directly participates to the hypoxic/hypoglycemic process, but also owns specific phenotypic localization. We do not exclude that it might serve as a sensor of dysregulated neuronal activity and ATP release, both occurring during oxygen/glucose deprivation.

Animals↗

PubMed2003

Nucleotide-mediated calcium signaling in rat cortical astrocytes: Role of P2X and P2Y receptors.

ATP is the dominant messenger for astrocyte-to-astrocyte calcium-mediated communication. Definition of the exact ATP/P2 receptors in astrocytes and of their coupling to intracellular calcium ([Ca(2+)](i)) has important implications for brain physiology and pathology. We show that, with the only exception of the P2X(6) receptor, primary rat cortical astrocytes express all cloned ligand-gated P2X (i.e., P2X(1-5) and P2X(7)) and G-protein-coupled P2Y receptors (i.e., P2Y(1), P2Y(2), P2Y(4), P2Y(6), and P2Y(12)). These cells also express the P2Y-like UDP-glucose receptor, which has been recently recognized as the P2Y(14) receptor. Single-cell image analysis showed that only some of these receptors are coupled to [Ca(2+)](i). While ATP induced rapid and transient [Ca(2+)](i) increases (counteracted by the P2 antagonists suramin, pyridoxal-phosphate-6-azophenyl-2'-4'-disulfonic acid and oxidized ATP), the P2X(1)/P2X(3) agonist alphabetameATP produced no changes. Conversely, the P2X(7) agonist BzATP markedly increased [Ca(2+)](i); the presence and function of the P2X(7) receptor was also confirmed by the formation of the P2X(7) pore. ADP and 2meSADP also produced [Ca(2+)](i) increases antagonized by the P2Y(1) antagonist MRS2179. Some cells also responded to UTP but not to UDP. Significant responses to sugar-nucleotides were also detected, which represents the first functional response reported for the putative P2Y(14) receptor in a native system. Based on agonist preference of known P2 receptors, we conclude that, in rat astrocytes, ATP-induced calcium rises are at least mediated by P2X(7) and P2Y(1) receptors; additional receptors (i.e., P2X(2), P2X(4), P2X(5), P2Y(2), P2Y(4), and P2Y(14)) may also contribute.

Adenosine Diphosphate↗

PubMed2003

Extracellular ATP and neurodegeneration.

ATP is a potent signaling molecule abundantly present in the CNS. It elicits a wide array of physiological effects and is regarded as the phylogenetically most ancient epigenetic factor playing crucial biological roles in several different tissues. These can range from neurotransmission, smooth muscle contraction, chemosensory signaling, secretion and vasodilatation, to more complex phenomena such as immune responses, pain, male reproduction, fertilization and embryonic development. ATP is released into the extracellular space either exocytotically or from damaged and dying cells. It is often co-released with other neurotransmitters and it can interact with growth factors at both receptor- and/or signal transduction-level. Once in the extracellular environment, ATP binds to specific receptors termed P2. Based on pharmacological profiles, on selectivity of coupling to second-messenger pathways and on molecular cloning, two main subclasses with multiple subtypes have been distinguished. They are P2X, i.e. fast cation-selective receptor channels (Na+, K+, Ca2+), possessing low affinity for ATP and responsible for fast excitatory neurotransmission, and P2Y, i.e. slow G protein-coupled metabotropic receptors, possessing higher affinity for the ligand. In the nervous system, they are broadly expressed in both neurons and glial cells and can mediate dual effects: short-term such as neurotransmission, and long-term such as trophic actions. Since massive extracellular release of ATP often occurs after metabolic stress, brain ischemia and trauma, purinergic mechanisms are also correlated to and involved in the etiopathology of many neurodegenerative conditions. Furthermore, extracellular ATP per se is toxic for primary neuronal dissociated and organotypic CNS cultures from cortex, striatum and cerebellum and P2 receptors can mediate and aggravate hypoxic signaling in many CNS neurons. Conversely, several P2 receptor antagonists abolish the cell death fate of primary neuronal cultures exposed to excessive glutamate, serum/potassium deprivation, hypoglycemia and chemical hypoxia. In parallel with these detrimental effects, also trophic functions have been extensively described for extracellular purines (both for neuronal and non-neuronal cells), but these might either aggravate or ameliorate the normal cellular conditions. In summary, extracellular ATP plays a very complex role not only in the repair, remodeling and survival occurring in the nervous system, but even in cell death and this can occur either after normal developmental conditions, after injury, or acute and chronic diseases.

Adenosine Triphosphate↗

PubMed2002

Extracellular ATP and nerve growth factor intensify hypoglycemia-induced cell death in primary neurons: role of P2 and NGFRp75 receptors.

In this study, we monitored the direct expression of P2 receptors for extracellular ATP in cerebellar granule neurons undergoing metabolism impairment. Glucose deprivation for 30-60 min inhibited P2Y1 receptor protein, only weakly modulated P2X1, P2X2 and P2X3, and up-regulated by about two-fold P2X4, P2X7 and P2Y4. The P2X/Y antagonist basilen blue, protecting cerebellar neurons from hypoglycemic cell death, maintained within basal levels only the expression of P2X7 and P2Y4 proteins, but not P2X4 or P2Y1. Glucose starvation transiently increased (up to three-fold) the expression of NGFRp75 receptor protein and strongly stimulated the extracellular release of nerve growth factor (NGF; about 10-fold). Exogenously added NGF then augmented hypoglycemic neuronal death by about 60%, increasing the percentage of Höechst-positive nuclei (from approximately 62 to 95%), reducing lactate dehydrogenase (LDH) release (from about 50 to 14%) and significantly overstimulating the hypoglycemia-induced expression of P2X7 and P2Y4. Conversely, extracellular ATP augmented hypoglycemic neuronal death by about 80%, reducing the number of Höechst-positive nuclei (from approximately 62% to 14%), augmenting LDH outflow (by about 30%) and further increasing the hypoglycemia-induced expression of NGFRp75. Our results indicate that P2 and NGFRp75 receptors are modulated during glucose starvation and that extracellular ATP and NGF drive features of, respectively, necrotic and apoptotic hypoglycemic cell death, aggravating the consequences of metabolism impairment in cerebellar primary neurons.

Adenosine Triphosphate↗