Identification and characterization of splice variants of the human P2X7 ATP channel

Cheewatrakoolpong, Boonlert; Gilchrest, Helen; Anthes, John C.; Greenfeder, Scott

doi:10.1016/j.bbrc.2005.04.087

Cited by 182 publications

(234 citation statements)

References 37 publications

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“…The expression of purinergic receptors P2X [1][2][3][4][5][6][7] in Leydig cells was detected by Western blot analysis. Since a large number of high purity Leydig cells are needed for these experiments, we used 20 testes in each preparation.…”

Section: Western Blotsmentioning

confidence: 99%

“…Proteins were then transferred to nitrocellulose membranes (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) by Bio-Rad Trans-Blot SD semidry transfer cell (Bio-Rad Laboratories Inc., Richmond, CA, USA). Membranes were blocked with 5% BSA in TBST (100 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20) for 1 h, and then incubated for 1 h with anti-P2X [1][2][3][4][5][6][7] (1:400) polyclonal antibodies (Alomone Labs Ltd., Jerusalem, Israel) diluted in 5% BSA in TBST. After washing, membranes were incubated for 1 h with goat anti-rabbit IGg conjugated to horseradish peroxidase, diluted in TBST.…”

Section: Western Blotsmentioning

confidence: 99%

“…The coverslips were incubated 120 min with the same primary antibodies anti-P2X [1][2][3][4][5][6][7] (1:200; rabbit) and anti-calreticulin (Chemicon International, Temecula, California, USA; 1:200; chicken) diluted with 0.01 M PBS, 0.5% Triton X-100, 1% BSA. Then, the cells were incubated with secondary antibodies Alexa Fluor 488 (goat, anti-rabbit) and 594 (goat, anti-chicken; Molecular Probes Inc., Eugene, USA) for 60 min, both in a dilution of 1:800.…”

Section: Immunofluorescencementioning

confidence: 99%

“…This is supported by the fact that the same band is also disclosed in lysates of macrophages, used as positive control. Nevertheless, smaller molecular weight bands (around 45 and 53 kDa) are also present, probably representing truncated splice variants of P2X 7 . Alternatively, Leydig P2X 7 may have undergone partial degradation.…”

Section: Molecular Identification Of P2x Receptors In Mouse Leydig Cellsmentioning

confidence: 99%

“…Each new receptor thus formed has distinct properties when compared with homomeric assemblies [49,9]. Until now splice variants have been described for P2X 1 , P2X 2 , P2X 4 , P2X 5, and P2X 7 purinergic receptor subunits [20,9,12,13,7].…”

Section: Introductionmentioning

confidence: 99%

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Mouse Leydig cells express multiple P2X receptor subunits

Antonio

Costa

Gomes

et al. 2008

Purinergic Signalling

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ATP acts on cellular membranes by interacting with P2X (ionotropic) and P2Y (metabotropic) receptors. Seven homomeric P2X receptors (P2X 1 -P2X 7 ) and seven heteromeric receptors (P2X 1/2 , P2X 1/4 , P2X 1/5 , P2X 2/3 , P2X 2/6 , P2X 4/6 , P2X 4/7 ) have been described. ATP treatment of Leydig cells leads to an increase in [Ca 2+ ] i and testosterone secretion, supporting the hypothesis that Ca 2+ signaling through purinergic receptors contributes to the process of testosterone secretion in these cells. Mouse Leydig cells have P2X receptors with a pharmacological and biophysical profile resembling P2X 2 . In this work, we describe the presence of several P2X receptor subunits in mouse Leydig cells. Western blot experiments showed the presence of P2X 2 , P2X 4 , P2X 6 , and P2X 7 subunits. These results were confirmed by immunofluorescence. Functional results support the hypothesis that heteromeric receptors are present in these cells since 0.5 μM ivermectin induced an increase (131.2±5.9%) and 3 μM ivermectin a decrease (64.2±4.8%) in the whole-cell currents evoked by ATP. These results indicate the presence of functional P2X 4 subunits. P2X 7 receptors were also present, but they were non-functional under the present conditions because dye uptake experiments with Lucifer yellow and ethidium bromide were negative. We conclude that a heteromeric channel, possibly P2X 2/4/6 , is present in Leydig cells, but with an electrophysiological and pharmacological phenotype characteristic of the P2X 2 subunit.

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Section: Western Blotsmentioning

confidence: 99%

Section: Western Blotsmentioning

confidence: 99%

Section: Immunofluorescencementioning

confidence: 99%

Section: Molecular Identification Of P2x Receptors In Mouse Leydig Cellsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Mouse Leydig cells express multiple P2X receptor subunits

Antonio

Costa

Gomes

et al. 2008

Purinergic Signalling

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P2X₇ receptors: Properties and relevance to CNS function

Duan

Neary

2006

Glia

123

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Among seven members of P2X ionotropic receptors activated by extracellular ATP, the P2X(7) subtype is unique in that it can function as a cation channel, a nonselective pore, or even a signaling complex coupled with multiple downstream components. Several roles of P2X(7) receptors have been described in CNS cells in the past decade, including release of cytokines and transmitters, modulation of presynaptic transmitter release, and activation of multiple signaling pathways. The finding that P2X(7) pores may directly mediate efflux of cytosolic glutamate, GABA, and ATP in glial cells is particularly interesting, as it provides a novel mechanism of glial transmitter release that may play important roles not only in physiological intercellular communication but also in pathological neural injury.

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ATP-induced IL-1β secretion is selectively impaired in microglia as compared to hematopoietic macrophages

Burm

Zuiderwijk‐Sick

Weert

et al. 2016

Glia

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Under stressful conditions nucleotides are released from dying cells into the extracellular space, where they can bind to purinergic P2X and P2Y receptors. High concentrations of extracellular ATP in particular induce P2X7-mediated signaling, which leads to inflammasome activation. This in turn leads to the processing and secretion of pro-inflammatory cytokines, like interleukin (IL)-1β. During neurodegenerative diseases, innate immune responses are shaped by microglia and we have previously identified microglia-specific features of inflammasome-mediated responses. Here, we compared ATP-induced IL-1β secretion in primary rhesus macaque microglia and bone marrow-derived macrophages (BMDM). We assessed the full expression profile of P2 receptors and characterized the induction and modulation of IL-1β secretion by extracellular nucleotides. Microglia secreted significantly lower levels of IL-1β in response to ATP when compared to BMDM. We demonstrate that this is not due to differences in sensitivity, kinetics or expression of ATP-processing enzymes, but rather to differences in purinergic receptor expression levels and usage. Using a combined approach of purinergic receptor agonists and antagonists, we demonstrate that ATP-induced IL-1β secretion in BMDM was fully dependent on P2X7 signaling, whereas in microglia multiple purinergic receptors were involved, including P2X7 and P2X4. These cell type-specific features of conserved innate immune responses may reflect adaptations to the vulnerable CNS microenvironment. GLIA 2016;64:2231-2246.

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Identification and characterization of splice variants of the human P2X7 ATP channel

Cited by 182 publications

References 37 publications

Mouse Leydig cells express multiple P2X receptor subunits

Mouse Leydig cells express multiple P2X receptor subunits

P2X₇ receptors: Properties and relevance to CNS function

ATP-induced IL-1β secretion is selectively impaired in microglia as compared to hematopoietic macrophages

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Identification and characterization of splice variants of the human P2X7 ATP channel

Cited by 182 publications

References 37 publications

Mouse Leydig cells express multiple P2X receptor subunits

Mouse Leydig cells express multiple P2X receptor subunits

P2X7 receptors: Properties and relevance to CNS function

ATP-induced IL-1β secretion is selectively impaired in microglia as compared to hematopoietic macrophages

Contact Info

Product

Resources

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P2X₇ receptors: Properties and relevance to CNS function