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P2X7 purinoceptors contribute to the death of Schwann cells transplanted into the spinal cord.

Luo J, Lee S, Wu D, Yeh J, Ellamushi H, Wheeler AP, Warnes G, Zhang Y, Bo X - Cell Death Dis (2013)

Bottom Line: Furthermore, ATP did not cause death of SCs isolated from P2X7R-knockout mice.All these results suggest that P2X7R is responsible for ATP-induced SC death in vitro.These results indicate that targeting P2X7R on SCs could be a potential strategy to improve the survival of transplanted cells.

View Article: PubMed Central - PubMed

Affiliation: 1] Centre for Neuroscience and Trauma, Blizard Institute, Queen Mary University of London, London E1 2AT, UK [2] Department of Physiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

ABSTRACT
The potential to use Schwann cells (SCs) in neural repair for patients suffering from neurotrauma and neurodegenerative diseases is well recognized. However, significant cell death after transplantation hinders the clinical translation of SC-based therapies. Various factors may contribute to the death of transplanted cells. It is known that prolonged activation of P2X7 purinoceptors (P2X7R) can lead to death of certain types of cells. In this study, we show that rat SCs express P2X7R and exposure of cultured SCs to high concentrations of ATP (3-5 mM) or a P2X7R agonist, 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP) induced significant cell death rapidly. High concentrations of ATP and BzATP increased ethidium uptake by SCs, indicating increased membrane permeability to large molecules, a typical feature of prolonged P2X7R activation. SC death, as well as ethidium uptake, induced by ATP was blocked by an irreversible P2X7R antagonist oxidized ATP (oxATP) or a reversible P2X7R antagonist A438079. oxATP also significantly inhibits the increase of intracellular free calcium induced by minimolar ATP concentrations. Furthermore, ATP did not cause death of SCs isolated from P2X7R-knockout mice. All these results suggest that P2X7R is responsible for ATP-induced SC death in vitro. When rat SCs were treated with oxATP before transplantation into uninjured rat spinal cord, 35% more SCs survived than untreated SCs 1 week after transplantation. Moreover, 58% more SCs isolated from P2X7R-knockout mice survived after being transplanted into rat spinal cord than SCs from wild-type mice. This further confirms that P2X7R is involved in the death of transplanted SCs. These results indicate that targeting P2X7R on SCs could be a potential strategy to improve the survival of transplanted cells. As many other types of cells, including neural stem cells, also express P2X7R, deactivating P2X7R may improve the survival of other types of transplanted cells.

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ATP increases [Ca2+]i level in SCs. (a) Sequential images of Fluo-4 fluorescence captured by a time-lapse microscope over a period of 44 s in SCs pretreated with 350 μM oxATP and then exposed to 30 μM ATP. (b) Representative time course of [Ca2+]i levels indicated by Fluo-4 fluorescence intensities in SCs after exposure to different concentrations of ATP. (c) Representative time course of [Ca2+]i levels in SCs pretreated with oxATP (350 μM) and then exposed to different concentrations of ATP. (d) Quantification of Fluo-4 fluorescence intensities in SCs in the first 100 s (peak phase) after exposure to different concentrations of ATP with or without oxATP treatment. *P<0.05, **P<0.01 (compared between groups exposed to the same concentration of ATP with and without oxATP), single factor ANOVA, n=3
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fig4: ATP increases [Ca2+]i level in SCs. (a) Sequential images of Fluo-4 fluorescence captured by a time-lapse microscope over a period of 44 s in SCs pretreated with 350 μM oxATP and then exposed to 30 μM ATP. (b) Representative time course of [Ca2+]i levels indicated by Fluo-4 fluorescence intensities in SCs after exposure to different concentrations of ATP. (c) Representative time course of [Ca2+]i levels in SCs pretreated with oxATP (350 μM) and then exposed to different concentrations of ATP. (d) Quantification of Fluo-4 fluorescence intensities in SCs in the first 100 s (peak phase) after exposure to different concentrations of ATP with or without oxATP treatment. *P<0.05, **P<0.01 (compared between groups exposed to the same concentration of ATP with and without oxATP), single factor ANOVA, n=3

Mentions: ATP and other P2 purinoceptor agonists have been reported to evoke the increase of free intracellular Ca2+ ([Ca2+]i) in dissociated or myelinating SCs.26, 27 We tested a wider range of ATP concentrations for a longer time (15 min) on SCs with and without pretreatment with oxATP. From 1 to 300 μM ATP evoked a rapid [Ca2+]i increase and the transient rise gradually declined to and maintained at the baseline level (Figure 4b). However, at 1, 3 and 5 mM ATP, after the peak phase [Ca2+]i level gradually elevated again over the recording period. Quantification of the intensity and duration of the peak [Ca2+]i rise by combining the Fluo-4 fluorescence intensities during the first 100 s after ATP application shows that the [Ca2+]i increase is generally concentration-dependent (Figure 4d). However, the peak phase of [Ca2+]i rise at 5 mM ATP was lower than those at 1 and 3 mM, a phenomenon that we are unable to explain at the moment. Pretreatment with oxATP did not affect the peak phase of [Ca2+]i rise evoked by ATP concentrations lower than 300 μM but reduced the peak phases for 1 and 3 mM ATP (Figures 4c and d). Another obvious difference between the two groups is that oxATP pretreatment prevented the gradual [Ca2+]i rise after the peak response at 1, 3 and 5 mM ATP (Figure 4c). Therefore, it is postulated that the gradual [Ca2+]i rise after the peak may be due to the Ca2+ influx through the pores formed on the membrane.


P2X7 purinoceptors contribute to the death of Schwann cells transplanted into the spinal cord.

Luo J, Lee S, Wu D, Yeh J, Ellamushi H, Wheeler AP, Warnes G, Zhang Y, Bo X - Cell Death Dis (2013)

ATP increases [Ca2+]i level in SCs. (a) Sequential images of Fluo-4 fluorescence captured by a time-lapse microscope over a period of 44 s in SCs pretreated with 350 μM oxATP and then exposed to 30 μM ATP. (b) Representative time course of [Ca2+]i levels indicated by Fluo-4 fluorescence intensities in SCs after exposure to different concentrations of ATP. (c) Representative time course of [Ca2+]i levels in SCs pretreated with oxATP (350 μM) and then exposed to different concentrations of ATP. (d) Quantification of Fluo-4 fluorescence intensities in SCs in the first 100 s (peak phase) after exposure to different concentrations of ATP with or without oxATP treatment. *P<0.05, **P<0.01 (compared between groups exposed to the same concentration of ATP with and without oxATP), single factor ANOVA, n=3
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824653&req=5

fig4: ATP increases [Ca2+]i level in SCs. (a) Sequential images of Fluo-4 fluorescence captured by a time-lapse microscope over a period of 44 s in SCs pretreated with 350 μM oxATP and then exposed to 30 μM ATP. (b) Representative time course of [Ca2+]i levels indicated by Fluo-4 fluorescence intensities in SCs after exposure to different concentrations of ATP. (c) Representative time course of [Ca2+]i levels in SCs pretreated with oxATP (350 μM) and then exposed to different concentrations of ATP. (d) Quantification of Fluo-4 fluorescence intensities in SCs in the first 100 s (peak phase) after exposure to different concentrations of ATP with or without oxATP treatment. *P<0.05, **P<0.01 (compared between groups exposed to the same concentration of ATP with and without oxATP), single factor ANOVA, n=3
Mentions: ATP and other P2 purinoceptor agonists have been reported to evoke the increase of free intracellular Ca2+ ([Ca2+]i) in dissociated or myelinating SCs.26, 27 We tested a wider range of ATP concentrations for a longer time (15 min) on SCs with and without pretreatment with oxATP. From 1 to 300 μM ATP evoked a rapid [Ca2+]i increase and the transient rise gradually declined to and maintained at the baseline level (Figure 4b). However, at 1, 3 and 5 mM ATP, after the peak phase [Ca2+]i level gradually elevated again over the recording period. Quantification of the intensity and duration of the peak [Ca2+]i rise by combining the Fluo-4 fluorescence intensities during the first 100 s after ATP application shows that the [Ca2+]i increase is generally concentration-dependent (Figure 4d). However, the peak phase of [Ca2+]i rise at 5 mM ATP was lower than those at 1 and 3 mM, a phenomenon that we are unable to explain at the moment. Pretreatment with oxATP did not affect the peak phase of [Ca2+]i rise evoked by ATP concentrations lower than 300 μM but reduced the peak phases for 1 and 3 mM ATP (Figures 4c and d). Another obvious difference between the two groups is that oxATP pretreatment prevented the gradual [Ca2+]i rise after the peak response at 1, 3 and 5 mM ATP (Figure 4c). Therefore, it is postulated that the gradual [Ca2+]i rise after the peak may be due to the Ca2+ influx through the pores formed on the membrane.

Bottom Line: Furthermore, ATP did not cause death of SCs isolated from P2X7R-knockout mice.All these results suggest that P2X7R is responsible for ATP-induced SC death in vitro.These results indicate that targeting P2X7R on SCs could be a potential strategy to improve the survival of transplanted cells.

View Article: PubMed Central - PubMed

Affiliation: 1] Centre for Neuroscience and Trauma, Blizard Institute, Queen Mary University of London, London E1 2AT, UK [2] Department of Physiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

ABSTRACT
The potential to use Schwann cells (SCs) in neural repair for patients suffering from neurotrauma and neurodegenerative diseases is well recognized. However, significant cell death after transplantation hinders the clinical translation of SC-based therapies. Various factors may contribute to the death of transplanted cells. It is known that prolonged activation of P2X7 purinoceptors (P2X7R) can lead to death of certain types of cells. In this study, we show that rat SCs express P2X7R and exposure of cultured SCs to high concentrations of ATP (3-5 mM) or a P2X7R agonist, 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP) induced significant cell death rapidly. High concentrations of ATP and BzATP increased ethidium uptake by SCs, indicating increased membrane permeability to large molecules, a typical feature of prolonged P2X7R activation. SC death, as well as ethidium uptake, induced by ATP was blocked by an irreversible P2X7R antagonist oxidized ATP (oxATP) or a reversible P2X7R antagonist A438079. oxATP also significantly inhibits the increase of intracellular free calcium induced by minimolar ATP concentrations. Furthermore, ATP did not cause death of SCs isolated from P2X7R-knockout mice. All these results suggest that P2X7R is responsible for ATP-induced SC death in vitro. When rat SCs were treated with oxATP before transplantation into uninjured rat spinal cord, 35% more SCs survived than untreated SCs 1 week after transplantation. Moreover, 58% more SCs isolated from P2X7R-knockout mice survived after being transplanted into rat spinal cord than SCs from wild-type mice. This further confirms that P2X7R is involved in the death of transplanted SCs. These results indicate that targeting P2X7R on SCs could be a potential strategy to improve the survival of transplanted cells. As many other types of cells, including neural stem cells, also express P2X7R, deactivating P2X7R may improve the survival of other types of transplanted cells.

Show MeSH
Related in: MedlinePlus