Procedures to characterize and study P2Z/P2X7 purinoceptor: flow cytometry as a promising practical, reliable tool

Nihei, Oscar Kenji; Savino, Wilson; Alves, Luiz Anastácio

doi:10.1590/s0074-02762000000300019

Cited by 12 publications

(12 citation statements)

References 114 publications

(62 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It has unique properties compared to the other P2X receptors (3,4). The P2X7 receptor requires concentrations of ATP (>0.3 mM) much higher than the other P2X (P2X 1 -6 ) receptors for activation (5).…”

mentioning

confidence: 99%

Inhibitory Effects of Metals on ATP-Induced Current Through P2X7 Receptor in NG108-15 Cells

Watano

Matsuoka

Kimura

2002

Japanese Journal of Pharmacology

View full text Add to dashboard Cite

ABSTRACT-We investigated the effects of heavy metal ions on the ATP-induced nonselective cation current through P2X7 receptor (INS·P2X7) in NG108-15 cells using the whole-cell patch-clamp technique. Cu 2+ inhibited the INS·P2X7 most potently among the metal ions investigated. Other metals such as Ni 2+ , Cd 2+ , Zn 2+ and Co 2+ also inhibited the INS·P2X7 in concentration-dependent manners. The order of potency was Cu 2+ >> Ni 2+ > Cd 2+ > Zn 2+ > Co 2+ with IC50 values of 16 nM, 0.79 mM, 1.2 m M, 3.0 mM and 4.6 mM, respectively. Fe 3+ (10 and 100 mM) and Mn 2+ (10 mM) did not affect the I NS · P2X7 . A high concentration of Mn 2+ (100 m M) slightly inhibited the I NS · P2X7 . When the concentration-response curve of ATP was obtained in the presence of 3 and 10 nM Cu 2+ , the maximal response but not the EC 50 value appeared to be reduced, suggesting that the inhibition is not competitive. These results suggest that under physiological and toxicological conditions, metal ions, such as Cu 2+ , Ni 2+ , Cd 2+ , Zn 2+ and Co 2+ , may modulate P2X 7 receptor channels as inhibitors. Keywords: ATP, P2X7 receptor, Nonselective cation current, Metal ion, NG108-15 cellThe P2X7 receptor is one of seven subtypes (P2X1 -7) of P2X receptors, which are ligand-gated nonselective cation channels (1, 2). It has unique properties compared to the other P2X receptors (3, 4). The P2X7 receptor requires concentrations of ATP (>0.3 mM) much higher than the other P2X (P2X 1 -6 ) receptors for activation (5). The effective form of ATP for the P2X7 receptor is the free base (ATP 4-) (6), and benzoylbenzoic ATP (BzATP) is a more potent agonist than ATP (7 -9).NG108-15 cells are hybrids of mouse neuroblastoma N18TG-2 and rat glioma C6Bu-1 cells and possess the mouse P2X 7 receptor originating from N18TG-2 cells (10). The cDNA sequence of P2X 7 receptor in NG108-15 cells was almost identical (ref. 10; I. Matsuoka, unpubished result of the full length sequence) to that of mouse P2X7 receptor first cloned by Chessell et al. (11) in NTW8 mouse microglial cells. A high concentration (>0.3 mM), but not a low concentration, of ATP activates a nonselective cation current, indicating that the only functional P2X receptor is the P2X7 receptor in NG108-15 cells (7,12,13). Rat and human P2X7 receptors transform themselves from channels to large pores by prolonged application of ATP (8,14), but this does not occur in mouse P2X7 receptors in NG108-15 cells (15). Therefore, the P2X7 receptors in NG108-15 cells are different from those in rat and human macrophages and glial cells.Metals such as vanadium (V), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn) and molybdenum (Mo) are essential trace elements utilized as cofactors of various enzymes (16). Metal ions are known to affect various ion channels (17) MATERIALS AND METHODS Cell cultureNG108-15 cells were grown in high-glucose Dulbecco's modified Eagle's medium supplemented with 7% fetal bovine serum, 100 mM hypoxanthine, 0.4 mM aminopterin and 16 mM thymidine...

show abstract

mentioning

confidence: 99%

Inhibitory Effects of Metals on ATP-Induced Current Through P2X7 Receptor in NG108-15 Cells

Watano

Matsuoka

Kimura

2002

Japanese Journal of Pharmacology

View full text Add to dashboard Cite

show abstract

“…It was shown that cells expressing the P2 X7 purinergic cell surface receptor, also known as the P2 z receptor, could be permeabilized when the receptor binds to ATP 4-. The interaction with ATP resulted in the formation of a non-selective pore that allows molecules up to ~900 Daltons to pass through (Nihei et al, 2000). The P2 X7 receptor selectively binds to only ATP 4-whose presence in solution is dependent on temperature, pH, and the concentration of divalent cations such as Mg 2+ .…”

Section: Discussionmentioning

confidence: 99%

“…Closure of the pore after activation by ATP is achieved by simply removing ATP from the system or adding exogenous Mg 2+ that has a high affinity for the active form of ATP, ATP 4-. The P2 z receptor is found on a number of cell types including cells of hematopoietic origin (Nihei et al, 2000). There were several factors that likely affected the cell viability and survival of cells after ATP poration.…”

Section: Discussionmentioning

confidence: 99%

Cryopreservation of Adherent Smooth Muscle and Endothelial Cells with Disaccharides

Campbell¹,

Brockbank²

2012

Current Frontiers in Cryopreservation

View full text Add to dashboard Cite

“…In addition, sepsis induces endothelial activation (8) with release of ATP and ADP from platelets and vasculature (9). ATP and ADP are released by cell lysis, selective permeabilization of the plasma membrane and secretion from platelet dense bodies in response to specific mediators (10)(11)(12)(13) potential to modulate endothelial cells, platelet activation, leukocyte migration, and cytokine release (14). NO has been reported to regulate the mitochondrial respiratory cascade and to increase superoxide production with a lower production of ATP, an important event in sepsis (15,16).…”

Section: Introductionmentioning

confidence: 99%

Purine nucleotides reduce superoxide production by nitric oxide synthase in a murine sepsis model

Barbeiro

Debbas

et al. 2009

Braz J Med Biol Res

View full text Add to dashboard Cite

Procedures to characterize and study P2Z/P2X7 purinoceptor: flow cytometry as a promising practical, reliable tool

Cited by 12 publications

References 114 publications

Inhibitory Effects of Metals on ATP-Induced Current Through P2X7 Receptor in NG108-15 Cells

Inhibitory Effects of Metals on ATP-Induced Current Through P2X7 Receptor in NG108-15 Cells

Cryopreservation of Adherent Smooth Muscle and Endothelial Cells with Disaccharides

Purine nucleotides reduce superoxide production by nitric oxide synthase in a murine sepsis model

Contact Info

Product

Resources

About