1,721,071 research outputs found
Detecting adenosine triphosphate in the pericellular space
No full textRelease of adenosine triphosphate (ATP) into the extracellular space occurs in response to a multiplicity of physiological and pathological stimuli in virtually all cells and tissues. A role for extracellular ATP has been identified in processes as different as neurotransmission, endocrine and exocrine secretion, smooth muscle contraction, bone metabolism, cell proliferation, immunity and inflammation. However, ATP measurement in the extracellular space has proved a daunting task until recently. To tackle this challenge, some years ago, we designed and engineered a novel luciferase probe targeted to and expressed on the outer aspect of the plasma membrane. This novel probe was constructed by appending to firefly luciferase the N-terminal leader sequence and the C-terminal glycophosphatidylinositol anchor of the folate receptor. This chimeric protein, named plasma membrane luciferase, is targeted and localized to the outer side of the plasma membrane. With this probe, we have generated stably transfected HEK293 cell clones that act as an in vitro and in vivo sensor of the extracellular ATP concentration in several disease conditions, such as experimentally induced tumours and inflammation
Purinergic signaling in giant cell formation
No full textCell fusion into multinucleated giant cells (MGC) is an essential process that contributes to many important biological mechanisms in mammalians. In the bone and immune system, macrophages are endowed with a remarkable potential for cell fusion events as evidenced by their propensity to fuse with other cells and between themselves during both normal processes and disease. Macrophage fusion is critical for the normal development of multinucleated osteoclasts, the cells responsible for bone resorption. Macrophages from various tissue compartments also undergo fusion into MGC, a hallmark of granulomatous inflammation. To date, the mechanisms underlying macrophage fusion remain poorly understood. Receptor-ligand interactions are thought to mediate this process and several lines of evidence implicate purinergic receptors in both osteoclast and MGC formation. Notably, the P2X7 receptor for extracellular ATP is expressed in osteoclasts and in many types of granulomas associated with infection, foreign body response and sterile inflammation. Through their ability to sense extracellular cues and ATP, a messenger of intercellular communication, purinergic receptors likely contribute to cell-cell interactions that result in macrophage fusion
Expression of the P2X7 receptor and metabolic adaptation in serum and glucose deprivation
No full textOne of the main features in cancers is the adaptation to stressing conditions. This adaptation confers them the ability to
grow under reduced nutrient availability shape. Several findings point to the PI3K/Akt pathway as the main pathway
involved. Lately, adaptation to glucose deprivation, as well as to serum starvation, is considered a crucial event in tumour
progression. Recently, in our laboratory, we have demonstrated a close relationship between expression of the P2X7
receptor and increased proliferation in the absence of serum (Adinolfi et al. 2009). Here, we analyze the adaptation of
HEK293 cells stably expressing the P2X7 receptor (HEK293-P2X7) to two key metabolic stress factors: glucose
deprivation and serum starvation. In the absence of glucose (4 mM), HEK293-P2X7 have a higher growth rate compared
with HEK293 cells transfected with the empty vector (HEK293-mock). In addition, HEK293-P2X7 are shown to produce
twofold more ATP and release twice as much lactate (p<0.05) than HEK293-mock. Moreover, in the absence of serum,
PKM2 and PDHK1 are overexpressed in HEK293-P2X7 and are hypermodulated by the glucose deprivation. GLUT1 is
overexpressed in HEK293-P2X7. Akt is activated in HEK293-P2X7. These observations suggest a higher efficiency in
glucose uptake and employment by HEK293-P2X7, explaining a possible role for the P2X7 receptor in cancer cell survival
Expression of the P2X7 receptor add metabolic adaptation in serum and glucose deprivation
No full textCHIMERIC PROTEINS FOR MEASURING ATP CONCENTRATIONS IN PERICELLULAR SPACE AND RELATED SCREENING METHOD
No full textThe invention relates to luminescent chimeric proteins comprising a first N-terminal protein sequence, a second protein sequence and a third C-terminal protein sequence wherein: (i) said first and said third protein sequence are a leader sequence and an anchor sequence belonging to at least a receptor localized on a plasma membrane site; (ii) said second protein sequence encodes for the full-length or partial sequence of a photoprotein and is inserted in frame between said first and said third sequence (i); said chimeric protein being addressed to said plasma membrane site of the cell wherein it is expressed
ATP receptors and giant cell formation
No full textWe have investigated the role of the purinergic P2X7 receptor in the formation of multinucleated giant cells in human monocyte/macrophage cultures stimulated with either concanavalin A or phytohemagglutinin. Macrophage fusion can be blocked by a P2X7-selective pharmacological antagonist or by a mAb directed against the extracellular P2X7 domain. Furthermore, macrophage cell clones expressing high P2X7 levels spontaneously fuse in culture, whereas macrophage clones lacking P2X7 are unable to fuse. Our findings suggest that the newly identified purinergic P2X7 receptor plays a central role in the complex chain of events leading to generation of macrophage-derived giant cells
P2 receptors in cancer progression and metastatic spreading
No full textTumor microenvironment is nucleoside and nucleotide rich. Adenosine is a key determinant of the highly immunosuppressive tumor interstitium. Extracellular ATP also affects anti-tumor immunity, albeit its effects on host-tumor interaction are incompletely understood. We give here an overview of recent literature covering the role of nucleotide-selective (P2) plasma membrane receptors in tumor growth and progression. P2 receptors are expressed on both host and cancer cells, where depending on the receptor subtype, the inflammatory infiltrate and the tumor cell type they may drive an anti-tumor response or promote tumor progression. It is anticipated that knowledge of the pharmacology, biochemistry and functional activity of the P2 receptors will allow a better understanding of host-tumor interaction and the development of innovative anti-cancer therapy
Assessing Extracellular ATP as Danger Signal In Vivo: The pmeLuc System
No full textInflammation is the key pathophysiological response triggered by noxious agents in multicellular organisms. Central to inflammation is detection of exogenous or endogenous danger signals by immune cells. Extracellular ATP is a ubiquitous danger signal released during septic or sterile inflammation. The development of reliable techniques to measure extracellular ATP in vivo has become an urgent need in inflammation studies after the discovery that the most potent plasma membrane receptor responsible for NLRP3 inflammasome activation is an ATP-activated receptor, P2RX7. Here we describe an easy bioluminescence technique for the measurement of extracellular ATP in vivo
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