Biomedical subjects

Susanna Amadio

Publications and source records attributed to Susanna Amadio.

9 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↗

PubMed2006

P2X7 receptor modulation on microglial cells and reduction of brain infarct caused by middle cerebral artery occlusion in rat.

Adenosine 5'-triphosphate outflow increases after an ischemic insult in the brain and may induce the expression of P2X7 receptors in resting microglia, determining its modification into an activated state. To assess the effects of P2X7 receptor blockade in preventing microglia activation and ameliorating brain damage and neurological impairment, we delivered the P2 unselective antagonist Reactive Blue 2 to rats after middle cerebral artery occlusion. In sham-operated animals, devoid of brain damage, double immunofluorescence verified the absence of P2X7 immunoreactivity on resting microglia, astrocytes, and neurons, identified, respectively, by OX-42, glial fibrillary acid protein, and neuronal nuclei (NeuN) immunoreactivity. After ischemia, vehicle-treated rats showed monolateral sensorimotor deficit and tissue damage in striatum and frontoparietal cortex. Moreover, P2X7 immunoreactivity was de novo expressed on activated microglia in infarcted and surrounding areas, as well as on a reactive form of microglia, resting in shape but P2X7 immunoreactive, present in ipsi- and contralateral cingulate and medial frontal cortex. Reactive Blue 2 improved sensorimotor deficit and restricted the volume of infarction, without preventing the expression of P2X7, but inducing it in the microglia of contralateral frontal and parietal cortex and striatum, which had lost reciprocal connections with the remote infarct area. De novo expression of P2X7 occurred in both activated and reactive microglia, suggesting their differentiated roles in the area of infarct and in remote regions. Reactive Blue 2 reduced ischemic brain damage, likely blocking the function of activated microglia in the infarct area, but in the remote brain regions promoted the expression of P2X7 on reactive microglia, developing defense and reparative processes.

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

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

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↗