大鼠三叉神经节痛觉神经元上ATP引起的胞内钙信号传导途径
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摘要
目的
三磷酸腺苷(ATP)除了在细胞能量代谢中起着极为重要的作用外,现已发现ATP也是一种重要的神经递质,参与体内多种功能活动的调控。ATP含量的增高和局部注射ATP还可以造成面部和身体皮肤的疼痛。然而ATP是如何参与痛觉的调控以及其调控的机制,尤其对三叉神经痛的调控机制目前还不清楚。本实验应用钙离子技术研究神经递质ATP通过何种途径引起大鼠三叉神经节痛觉神经元胞内游离Ca2+浓度([Ca2+]i)升高并探讨其细胞信号的转导途径。
方法
1.三叉神经节神经元的分离将SD大鼠乙醚麻醉后断头处死,打开颅骨,取出双侧三叉神经节置于氧饱和的DMEM液中,pH值7.4,溶液渗透压为340 mosm/L。在体视显微镜下游离三叉神经节,然后用弹簧剪将神经节尽可能地剪碎。在37℃条件下,用胰蛋白酶(0.5 mg/ml)、胶原酶(1.0 mg/ml)消化液消化35 min,每隔十分钟左右吹打一次,然后用1000转低速离心机离心5 min,去除上层消化液,加入小牛血清终止消化。最后将三叉神经节细胞打散,细胞悬液转移至培养皿中静置30 min,待细胞贴壁后加细胞外液,实验在10小时内完成。
2.钙成像记录所用仪器为德国TILLvision成像系统。荧光指示剂为Fura-2/AM,先将Fura-2/AM溶于DMSO制成高浓度的母液,实验时再用细胞外液稀释至终浓度为2μmol/L。在37 oC条件下,用含有Fura-2/AM外液孵育三叉神经节神经元30 min,然后用正常外液洗去Fura-2/AM。实验中选择贴壁良好,细胞膜完整,小直径的神经元(< 30μm)用于钙离子成像实验。Fura-2/AM是一种可以穿透细胞膜的荧光染料,Fura-2/AM进入细胞后可以和游离Ca2+结合,结合Ca2+后主要在340 nm激发光下发荧光,未结合状态主要在380 nm激发光下发荧光,发射波长均为510 nm,这样就可以使用340 nm和380 nm这两个荧光的比值(F340/F380)来检测细胞内游离Ca2+浓度的变化,利用钙离子成像分析软件(TILL vision软件),可实时监测[Ca2+]i的变化,整个实验过程在室温下进行。
3.统计学分析采用Igor Pro(美国)软件分析数据。实验结果以x±SEM表示,组间均数比较用非配对t检验,p < 0.05时差异有显著性。
结果
1.在痛觉三叉神经节(TG)神经元上,用正常外液或去除细胞外Ca2+灌流细胞,分别给予thapsigargin(1μmol/L),咖啡因(20 mmol/L)和ATP(100μmol/L)均能够引起[Ca2+]i不同程度地升高,这说明在痛觉TG神经元中同时存在有IP3敏感钙库和Ryanodine敏感钙库。
2.在去除细胞外Ca2+条件下,ATP引起的TG神经元[Ca2+]i升高可被thapsigargin可逆性地抑制(n = 8,p < 0.001),而不能被咖啡因抑制(n = 6, p > 0.05)。由此可以推测出,在无钙的环境中,ATP引起的TG神经元[Ca2+]i升高是通过细胞内的IP3敏感钙库释放出引起的,而不是通过Ryanodine敏感钙库释放。
3.在无钙的细胞外液里,ATP引起的TG神经元[Ca2+]i升高可被P2受体阻断剂Suramin (100μmol/ L, 60 s)明显的抑制,由此可以推测出ATP是通过激动P2Y受体激动IP3敏感钙库引起的钙释放。
4.在正常细胞外液条件下,thapsigargin(1μM)引起的TG神经元[Ca2+]i升高,在thapsigargin引起[Ca2+]i升高达到最大后再次给予ATP(100μmol/L),此时ATP仍然能引起[Ca2+]i进一步的升高。因此我们可以推测,此时ATP引起的TG神经元[Ca2+]i升高是通过激动P2X受体引起的外钙内流所引起。
结论
在大鼠痛觉TG神经元中,存在有IP3敏感钙库和ryanodine敏感钙库。ATP可通过其两种途径引起细胞内[Ca2+]i升高,一种途径是激动P2Y受体而引起IP3敏感钙库的Ca2+释放,另一种途径是激动P2X受体引起细胞外Ca2+内流。
Objective: Adenosine 5′-triphosphate (ATP), in addition to its function as an intracellular energy donor, is now recognized as an important neurotransmitter or cotransmitter in both the central and peripheral nervous systems, which may mediate a variety of function activities in the body. According to the previous study, ATP plays a prominent role in nociception when ATP increased or injected, which could cause facial and skin pain. However, the mechanism of ATP modulating pain is not clearly till now. Particularly, the origins of the cytoplasmic Ca2+ rise induced by ATP remain unknown in the trigeminal ganglion. In this experiment, we used Fura-2-based microfluorimetry techniques to measure [Ca2+]i and pharmacological tools to determine the nature of Ca2+ stores in order to clarify the intracellular calcium signal transduction pathway in nociceptive TG neurons.
Methods: 1. Sprague-Dawley rats (80-120 g) were anesthetized with ether and rapidly decapitated, in accordance with the Animal Care and Use Committee of Anhui Medical University. The TGs were dissected out and transferred immediately into Dulbecco's modified Eagle's medium (DMEM) at pH 7.4. After the removal of the surrounding connective tissues, the TGs were minced with fine spring scissors and the ganglion fragments were placed in a flask containing 5 mL of DMEM supplemented with trypsin (0.5 mg/mL, type II-S) and collagenase (1.0 mg/mL, type I-A) and incubated at 37°C in a shaking water bath for 35 min. While incubating, TGs were triturated by fine fire-polished Pasteur pipettes three times (10 min each). After incubation, TG neurons were separated by centrifugation (1000 rpm, 5 min). Enzymatic digestion was stopped by addition of phosphate buffered saline with 10% normal cattle serum. Cell suspensions were then plated onto culture dishes and studied within 10 hrs.
2. Calcium imaging: [Ca2+]i was measured using TILLvision Imaging System with a Ca2+-sensitive dye Fura-2 acetoxymethyl ester (Fura-2/AM). The Fura-2/AM was prepared as a stock solution and diluted with extracellular solution to the final concentration (2μmol/L) immediately before use. Isolated TG neurons were incubated for 30 min in a bath solution containing Fura-2/AM at 37°C, then washout Fura-2/AM with extracellular solution. The well Fura-2/AM-loaded small neurons were selected to do calcium image experiments. The Fura-2/AM fluorescence, which can go through cell membrane, was measured with a 10 Hz alternating wavelength time scanning with excitation wavelengths of 340 nm and 380 nm, and an emission wavelength of 510 nm. F340 and F380 representing the fluorescence intensity elicited by 340 nm and 380 nm excitation light, respectively. The results were expressed as the ratio of F340/F380 intensity to estimate the change in [Ca2+]i by TILL vision software. All experiments were performed at room temperature.
3. Statistical analysis: Data were analyzed with Igor Pro software (Wavemetrics, Inc. Oregon, USA). Statistical data are presented as means±SEM. Comparisons between means were performed using Student’s unpaired t-test. Differences were considered to be significant when p < 0.05.
Results
1.In small TG neurons, the application of thapsigargin (1μmol/L), caffeine (20 mmol/L) or ATP (100μmol/L) induced a transient [Ca2+]i increase under the condition of normal extracellular solution or Ca2+-free condition. Those results indicate that there exist both IP3- and ryanodine-sensitive Ca2+ stores in rat nociceptive TG neurons.
2.In the extracellular Ca2+-free condition, the ATP-induced [Ca2+]i transient rise was reversibly inhibited by thapsigargin (n = 8,p < 0.001), but were not affected by caffeine in extracellular Ca2+-free condition (n = 6, p > 0.05). Those results indicate that ATP-induced [Ca2+]i rise in Ca2+-free conditions occurs is the calcium released from IP3-sensitive Ca2+ stores and not from ryanodine-sensitive Ca2+ stores.
3.In the extracellular Ca2+-free condition, ATP-induced [Ca2+]i transient rise was reversibly inhibited by suramin (100μmol/ L, 60 s), an antagonist against P2 receptors, indicating that ATP induced the [Ca2+]i rise via acting on P2Y purinoreceptors. 4.In normal extracellular solution, ATP (100μM) could still induced a transient [Ca2+]i rise after pretreated with thapsigargin (1μM) which had induced [Ca2+]i rise to a high platform in small TG neurons. These results indicate that besides P2YRs, P2XRs activation may cause extracellular calcium to flow into intracellular and induce [Ca2+]i rise.
Conclusion
There exist inositol-1,4,5-triphosphate (IP3) - and ryanodine-sensitive Ca2+ stores in rat nociceptive TG neurons. Two pathways are involved in the purinoreceptor-mediated [Ca2+]i rise in nociceptive TG neurons. One pathway involved the metabotropic P2Y receptors, associated with the IP3 Ca2+ sensitive store; the second being coupled to the ionotropic P2X receptors inducing Ca2+ influx.
三磷酸腺苷(ATP)除了在细胞能量代谢中起着极为重要的作用外,现已发现ATP也是一种重要的神经递质,参与体内多种功能活动的调控。ATP含量的增高和局部注射ATP还可以造成面部和身体皮肤的疼痛。然而ATP是如何参与痛觉的调控以及其调控的机制,尤其对三叉神经痛的调控机制目前还不清楚。本实验应用钙离子技术研究神经递质ATP通过何种途径引起大鼠三叉神经节痛觉神经元胞内游离Ca2+浓度([Ca2+]i)升高并探讨其细胞信号的转导途径。
方法
1.三叉神经节神经元的分离将SD大鼠乙醚麻醉后断头处死,打开颅骨,取出双侧三叉神经节置于氧饱和的DMEM液中,pH值7.4,溶液渗透压为340 mosm/L。在体视显微镜下游离三叉神经节,然后用弹簧剪将神经节尽可能地剪碎。在37℃条件下,用胰蛋白酶(0.5 mg/ml)、胶原酶(1.0 mg/ml)消化液消化35 min,每隔十分钟左右吹打一次,然后用1000转低速离心机离心5 min,去除上层消化液,加入小牛血清终止消化。最后将三叉神经节细胞打散,细胞悬液转移至培养皿中静置30 min,待细胞贴壁后加细胞外液,实验在10小时内完成。
2.钙成像记录所用仪器为德国TILLvision成像系统。荧光指示剂为Fura-2/AM,先将Fura-2/AM溶于DMSO制成高浓度的母液,实验时再用细胞外液稀释至终浓度为2μmol/L。在37 oC条件下,用含有Fura-2/AM外液孵育三叉神经节神经元30 min,然后用正常外液洗去Fura-2/AM。实验中选择贴壁良好,细胞膜完整,小直径的神经元(< 30μm)用于钙离子成像实验。Fura-2/AM是一种可以穿透细胞膜的荧光染料,Fura-2/AM进入细胞后可以和游离Ca2+结合,结合Ca2+后主要在340 nm激发光下发荧光,未结合状态主要在380 nm激发光下发荧光,发射波长均为510 nm,这样就可以使用340 nm和380 nm这两个荧光的比值(F340/F380)来检测细胞内游离Ca2+浓度的变化,利用钙离子成像分析软件(TILL vision软件),可实时监测[Ca2+]i的变化,整个实验过程在室温下进行。
3.统计学分析采用Igor Pro(美国)软件分析数据。实验结果以x±SEM表示,组间均数比较用非配对t检验,p < 0.05时差异有显著性。
结果
1.在痛觉三叉神经节(TG)神经元上,用正常外液或去除细胞外Ca2+灌流细胞,分别给予thapsigargin(1μmol/L),咖啡因(20 mmol/L)和ATP(100μmol/L)均能够引起[Ca2+]i不同程度地升高,这说明在痛觉TG神经元中同时存在有IP3敏感钙库和Ryanodine敏感钙库。
2.在去除细胞外Ca2+条件下,ATP引起的TG神经元[Ca2+]i升高可被thapsigargin可逆性地抑制(n = 8,p < 0.001),而不能被咖啡因抑制(n = 6, p > 0.05)。由此可以推测出,在无钙的环境中,ATP引起的TG神经元[Ca2+]i升高是通过细胞内的IP3敏感钙库释放出引起的,而不是通过Ryanodine敏感钙库释放。
3.在无钙的细胞外液里,ATP引起的TG神经元[Ca2+]i升高可被P2受体阻断剂Suramin (100μmol/ L, 60 s)明显的抑制,由此可以推测出ATP是通过激动P2Y受体激动IP3敏感钙库引起的钙释放。
4.在正常细胞外液条件下,thapsigargin(1μM)引起的TG神经元[Ca2+]i升高,在thapsigargin引起[Ca2+]i升高达到最大后再次给予ATP(100μmol/L),此时ATP仍然能引起[Ca2+]i进一步的升高。因此我们可以推测,此时ATP引起的TG神经元[Ca2+]i升高是通过激动P2X受体引起的外钙内流所引起。
结论
在大鼠痛觉TG神经元中,存在有IP3敏感钙库和ryanodine敏感钙库。ATP可通过其两种途径引起细胞内[Ca2+]i升高,一种途径是激动P2Y受体而引起IP3敏感钙库的Ca2+释放,另一种途径是激动P2X受体引起细胞外Ca2+内流。
Objective: Adenosine 5′-triphosphate (ATP), in addition to its function as an intracellular energy donor, is now recognized as an important neurotransmitter or cotransmitter in both the central and peripheral nervous systems, which may mediate a variety of function activities in the body. According to the previous study, ATP plays a prominent role in nociception when ATP increased or injected, which could cause facial and skin pain. However, the mechanism of ATP modulating pain is not clearly till now. Particularly, the origins of the cytoplasmic Ca2+ rise induced by ATP remain unknown in the trigeminal ganglion. In this experiment, we used Fura-2-based microfluorimetry techniques to measure [Ca2+]i and pharmacological tools to determine the nature of Ca2+ stores in order to clarify the intracellular calcium signal transduction pathway in nociceptive TG neurons.
Methods: 1. Sprague-Dawley rats (80-120 g) were anesthetized with ether and rapidly decapitated, in accordance with the Animal Care and Use Committee of Anhui Medical University. The TGs were dissected out and transferred immediately into Dulbecco's modified Eagle's medium (DMEM) at pH 7.4. After the removal of the surrounding connective tissues, the TGs were minced with fine spring scissors and the ganglion fragments were placed in a flask containing 5 mL of DMEM supplemented with trypsin (0.5 mg/mL, type II-S) and collagenase (1.0 mg/mL, type I-A) and incubated at 37°C in a shaking water bath for 35 min. While incubating, TGs were triturated by fine fire-polished Pasteur pipettes three times (10 min each). After incubation, TG neurons were separated by centrifugation (1000 rpm, 5 min). Enzymatic digestion was stopped by addition of phosphate buffered saline with 10% normal cattle serum. Cell suspensions were then plated onto culture dishes and studied within 10 hrs.
2. Calcium imaging: [Ca2+]i was measured using TILLvision Imaging System with a Ca2+-sensitive dye Fura-2 acetoxymethyl ester (Fura-2/AM). The Fura-2/AM was prepared as a stock solution and diluted with extracellular solution to the final concentration (2μmol/L) immediately before use. Isolated TG neurons were incubated for 30 min in a bath solution containing Fura-2/AM at 37°C, then washout Fura-2/AM with extracellular solution. The well Fura-2/AM-loaded small neurons were selected to do calcium image experiments. The Fura-2/AM fluorescence, which can go through cell membrane, was measured with a 10 Hz alternating wavelength time scanning with excitation wavelengths of 340 nm and 380 nm, and an emission wavelength of 510 nm. F340 and F380 representing the fluorescence intensity elicited by 340 nm and 380 nm excitation light, respectively. The results were expressed as the ratio of F340/F380 intensity to estimate the change in [Ca2+]i by TILL vision software. All experiments were performed at room temperature.
3. Statistical analysis: Data were analyzed with Igor Pro software (Wavemetrics, Inc. Oregon, USA). Statistical data are presented as means±SEM. Comparisons between means were performed using Student’s unpaired t-test. Differences were considered to be significant when p < 0.05.
Results
1.In small TG neurons, the application of thapsigargin (1μmol/L), caffeine (20 mmol/L) or ATP (100μmol/L) induced a transient [Ca2+]i increase under the condition of normal extracellular solution or Ca2+-free condition. Those results indicate that there exist both IP3- and ryanodine-sensitive Ca2+ stores in rat nociceptive TG neurons.
2.In the extracellular Ca2+-free condition, the ATP-induced [Ca2+]i transient rise was reversibly inhibited by thapsigargin (n = 8,p < 0.001), but were not affected by caffeine in extracellular Ca2+-free condition (n = 6, p > 0.05). Those results indicate that ATP-induced [Ca2+]i rise in Ca2+-free conditions occurs is the calcium released from IP3-sensitive Ca2+ stores and not from ryanodine-sensitive Ca2+ stores.
3.In the extracellular Ca2+-free condition, ATP-induced [Ca2+]i transient rise was reversibly inhibited by suramin (100μmol/ L, 60 s), an antagonist against P2 receptors, indicating that ATP induced the [Ca2+]i rise via acting on P2Y purinoreceptors. 4.In normal extracellular solution, ATP (100μM) could still induced a transient [Ca2+]i rise after pretreated with thapsigargin (1μM) which had induced [Ca2+]i rise to a high platform in small TG neurons. These results indicate that besides P2YRs, P2XRs activation may cause extracellular calcium to flow into intracellular and induce [Ca2+]i rise.
Conclusion
There exist inositol-1,4,5-triphosphate (IP3) - and ryanodine-sensitive Ca2+ stores in rat nociceptive TG neurons. Two pathways are involved in the purinoreceptor-mediated [Ca2+]i rise in nociceptive TG neurons. One pathway involved the metabotropic P2Y receptors, associated with the IP3 Ca2+ sensitive store; the second being coupled to the ionotropic P2X receptors inducing Ca2+ influx.
引文
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34.Ceruti S, Fumagalli M, Villa G, Verderio C, Abbracchio MP. Purinoceptor-mediated calcium signaling in primary neuron-glia trigeminal cultures. Cell Calcium, 2008,43(6):576-590
35.Hoyle CH, Knight GE, Burnstock G. Suramin antagonizes responses to P2-purinoceptor agonists and purinergic nerve stimulation in the guinea-pig urinary bladder and taenia coli. Br J Pharmacol,1990,99(3):617-621
36.Blachier F, Malaisse W J. Effect of exogenous ATP upon inositol phosphate production cationic fluxes and insulin release in pancreatic islet cells. Biochim Biophys Acta,1988,970(2):222-229
1.Bradbury ES, Burnstock G, McMahon SB. The expression of P2X3 purinoceptors in sensory neurons effects of axotomy and glial-derived neurotrophic factor. Mol Cell Neurosci,1998,12(4-5):256-268
2.Bertrand PP. ATP and Sensory Transduction in the Enteric Nervous System. Neuroscientist,2003,9(4):243-260
3.Abbracchio MP, Boeynaems JM, Barnard EA, Boyer JL, Kennedy C, Miras-Portugal MT, King BF, Gachet C, Jacobson KA, Weisman GA, Burnstock G. Characterization of the UDP-glucose receptor(re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family. Trends Pharmacol Sci,2003,24(2):52-55
4.Nihei OK, de Carvalho AC, Savino W, Alves LA. Pharmacologic properties of P(2Z)/P2X(7) receptor characterized in murine dendritic cells: role on the induction of apoptosis. Blood,2000,96(3):996-1005
5.Shimizu I, Iida T, Guan Y, Zhao C, Raja SN, Jarvis MF, Cockayne DA, Caterina MJ. Enhanced thermal avoidance in mice lacking the ATP receptor P2X3. Pain, 2005,116(1-2):96-108
6.Trang T, Beggs S, Salter MW. Purinoceptors in microglia and neuropathic pain. Pflugers Arch,2006,452(5):645-652
7.Sawynok J, Downie JW, Reid AR, Cahill CM, White TD. ATP release from dorsal apinal cord synaptosomes: characterization and neuronal origin. Brain Res,1993, 610(1):32-38
8.Burnstock G. A unifying purinergic hypothesis for the initiation of pain. Lancet, 1996,347(9015):1604-1610
9.Vulchanova L, Riedl MS, Shuster SJ, Stone LS, Hargreaves KM, Buell G, Surprenant A, North RA, Elde R. P2X3 is expressed by DRG neurons that terminate in innerlamina II. Eur J Neurosci,1998,10(11):3470-3478
10.Hamilton SG, McMahon SB, Lewin GR. Selective activation of nociceptors by P2X receptor agonists in normal and inflamed rat skin. J Physiol,2001,534(2):437-459
11.Cockayne DA, Hamilton SG, Zhu QM, Dunn PM, Zhong Y, Novakovic S, Malmberg AB, Cain G, Berson A, Kassotakis L, Hedley L, Lachnit WG, Burnstock G, McMahon SB, Ford AP. Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice. Nature,2000,407(6807):1011-1015
12.Masamichi Shinoda, Noriyuki Ozaki, Hideaki Asai, Kenjiro Nagamine, Yasuo Sugiura. Changes in P2X3 receptor expression in the trigeminal ganglion following monoarthritis of the temporomandibular joint in rats. Pain,2005,116(1-2):42-51
13.Xu Gy, Huang LY. Peripheral inflammation sensitizes P2X receptor-mediated responses in rat dorsal root ganglion neurons. J Neurosci,2002,22(1):93-102
14.Paukert M, Osteroth R, Geisler HS, Brandle U, Glowatzki E, Ruppersberg JP, Gründer S. Inflammory mediaters potentiate ATP-gated channels through the P2X3 subunit. J Biol Chem,2001,276(24):21077-21082
15.D’Arco M, Giniatullin R, Simonetti M, Fabbro A, Nair A, Nistri A, Fabbretti E. Neutralization of nerve growth factor induces plasticity of ATP-sensitive P2X3 receptors of nociceptive trigeminal ganglion neurons. J Neurosci,2007,27(31): 8190-8201
16.Shinoda M, Kawashima K, Ozaki N, Asai H, Nagamine K, Sugiura Y. P2X3 receptor mediates heat hyperalgesia in a rat model of trigeminal neuropathic pain. J Pain,2007,8(7):588-597
17.Kennedy C, Assis S, Currie AJ, Rowan EG. Crossing the pain barrier: P2 receptors as targets for novel analgesics. J Physiol,2003,553(Pt 3):683-694
18.Gu YZ, Yin GF, Guan BC, Li ZW. Characteristics of P2X purinoceptors in the membrane of rat trigeminal ganglion neurons. Research paper,2006,58(2):164-170
19.Li C, Peoples RW, Weight FF. Enhancement of ATP-activated current by protons in dorsal root ganglion neurons. Pflugers Arch,1997,433(4):446-454
20.Honore P, Mikusa J, Bianchi B, McDonald H, Cartmell J, Faltynek C, Jarvis MF. TNP-ATP, a potent P2X3 receptor antagonist, blocks acetic acid-induced abdominal constriction in mice: comparison with reference analgesics. Pain,2002,96(1-2): 99-105
21.Fredholm BB, Abbracchio MP, Burnstock G, Daly JW, Harden TK, Jacobson KA, Leff P, Williams M. Nomenclature and classification of purinoceptors. Pharmacol Rev,1994,46(2):143-156
22.Li C, Peoples RW, Li Z, Weight FF. Zn2+ potentiates excitatory action of ATP on mammalian neurons. Proc Natl Acad Sci U S A,1993,90(17):8264-82677
23.Hu HZ, Li ZW. Substance P potentiates ATP-activated currents in rat primary sensory neurons. Brain Res,1996,739(1-2):163-168
24.Davies DL, Machu TK, Guo Y, Alkana RL. Ethanol sensitivity in ATP-gated P2X recepors is subunit dependent. Alcohol Clin Exp Res,2002,26(6):773-778
25.Charles K. P2X Receptors: Targets for Novel Analgesics? Neuroscientist,2005, 11(4):345-356
26.Di Virgilio F, Chiozzi P, Ferrari D, Falzoni S, Sanz JM, Morelli A, Torboli M, Bolognesi G, Baricordi OR. Nucleotide receptors: an emerging family of regulatory molecules in blood cells. Blood,2001,97(3):587-600
27.Perez-Medrano A, Donnelly-Roberts DL, Honore P, Hsieh GC, Namovic MT, Peddi S, Shuai Q, Wang Y, Faltynek CR, Jarvis MF, Carroll WA. Discovery and biological evaluation of novel cyanoguanidine P2X(7) antagonists with analgesic activity in a rat model of neuropathic pain. J Med Chem,2009,52(10):3366-3376
28.Labasi JM, Petrushova N, Donovan C, McCurdy S, Lira P, Payette MM, Brissette W, Wicks JR, Audoly L, Gabel CA. Absence of the P2X7 receptor alters leukocytefunction and attenuates an inflammatory response. J Immunol,2002,168(12): 6436-6464
29.王元银,黄擎,唐慧,王烈成.莫达非尼对大鼠海马神经元IGABA的抑制作用机制.中国药理学通报,2008,24(6):727-730
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10.Hamilton SG, McMahon SB, Lewin GR. Selective activation of nociceptors by P2X receptor agonists in normal and inflamed rat skin. J Physiol,2001,534(2):437-445
11.Cockayne DA, Hamilton SG, Zhu QM, Dunn PM, Zhong Y, Novakovic S, Malmberg AB, Cain G, Berson A, Kassotakis L, Hedley L, Lachnit WG, Burnstock G, McMahon SB, Ford AP. Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice. Nature,2000,407(6807):1011-1015
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20.Dussor G, Zylka MJ, Anderson DJ, McCleskey EW. Cutaneous sensory neurons expressing the Mrgprd receptor sense extracellular ATP and are putative nociceptors. J Neurophysiol,2008,99(4):1581-1589
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26.Gu YZ, Yin GF, Guan BC, Li ZW. Characteristics of P2X purinoceptors in the membrane of rat trigeminal ganglion neurons. Sheng Li Xue Bao,2006,58(2): 164-170
27.Egan TM, Khakh BS. Contribution of calcium ions to P2X channel responses. J Neurosci,2004,24(13):3413-3420
28.Abbracchio MP, Boeynaems JM, Barnard EA, Boyer JL, Kennedy C, Miras-Portugal MT, King BF, Gachet C, Jacobson KA, Weisman GA, Burnstock G. Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family. Trends Pharmacol Sci,2003,24(2):52-55
29.Lewis C, Neidhart S, Holy C, North RA, Buell G, Surprenant A. Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons. Nature,1995,377(6548):432-435
30.Ruan HZ, Burnstock G. Localisation of P2Y1 and P2Y4 receptors in dorsal root, nodose and trigeminal ganglia of the rat. Histochem Cell Biol,2003,120(5):415-426
31.Gerevich Z, Illes P. P2Y receptors and pain transmission. Purinergic Signalling, 2004,1(1):3-10
32.Chen CC, Akopian AN, Sivilotti L, Colquhoun D, Burnstock G, Wood JN. A P2X purinoceptor expressed by a subset of sensory neurons. Nature,1995,377(6548): 428-431
33.Shinoda M, Kawashima K, Ozaki N, Asai H, Nagamine K, Sugiura Y. P2X3 receptor mediates heat hyperalgesia in a rat model of trigeminal neuropathic pain. J Pain,2007,8(7):588-597
34.Ceruti S, Fumagalli M, Villa G, Verderio C, Abbracchio MP. Purinoceptor-mediated calcium signaling in primary neuron-glia trigeminal cultures. Cell Calcium, 2008,43(6):576-590
35.Hoyle CH, Knight GE, Burnstock G. Suramin antagonizes responses to P2-purinoceptor agonists and purinergic nerve stimulation in the guinea-pig urinary bladder and taenia coli. Br J Pharmacol,1990,99(3):617-621
36.Blachier F, Malaisse W J. Effect of exogenous ATP upon inositol phosphate production cationic fluxes and insulin release in pancreatic islet cells. Biochim Biophys Acta,1988,970(2):222-229
1.Bradbury ES, Burnstock G, McMahon SB. The expression of P2X3 purinoceptors in sensory neurons effects of axotomy and glial-derived neurotrophic factor. Mol Cell Neurosci,1998,12(4-5):256-268
2.Bertrand PP. ATP and Sensory Transduction in the Enteric Nervous System. Neuroscientist,2003,9(4):243-260
3.Abbracchio MP, Boeynaems JM, Barnard EA, Boyer JL, Kennedy C, Miras-Portugal MT, King BF, Gachet C, Jacobson KA, Weisman GA, Burnstock G. Characterization of the UDP-glucose receptor(re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family. Trends Pharmacol Sci,2003,24(2):52-55
4.Nihei OK, de Carvalho AC, Savino W, Alves LA. Pharmacologic properties of P(2Z)/P2X(7) receptor characterized in murine dendritic cells: role on the induction of apoptosis. Blood,2000,96(3):996-1005
5.Shimizu I, Iida T, Guan Y, Zhao C, Raja SN, Jarvis MF, Cockayne DA, Caterina MJ. Enhanced thermal avoidance in mice lacking the ATP receptor P2X3. Pain, 2005,116(1-2):96-108
6.Trang T, Beggs S, Salter MW. Purinoceptors in microglia and neuropathic pain. Pflugers Arch,2006,452(5):645-652
7.Sawynok J, Downie JW, Reid AR, Cahill CM, White TD. ATP release from dorsal apinal cord synaptosomes: characterization and neuronal origin. Brain Res,1993, 610(1):32-38
8.Burnstock G. A unifying purinergic hypothesis for the initiation of pain. Lancet, 1996,347(9015):1604-1610
9.Vulchanova L, Riedl MS, Shuster SJ, Stone LS, Hargreaves KM, Buell G, Surprenant A, North RA, Elde R. P2X3 is expressed by DRG neurons that terminate in innerlamina II. Eur J Neurosci,1998,10(11):3470-3478
10.Hamilton SG, McMahon SB, Lewin GR. Selective activation of nociceptors by P2X receptor agonists in normal and inflamed rat skin. J Physiol,2001,534(2):437-459
11.Cockayne DA, Hamilton SG, Zhu QM, Dunn PM, Zhong Y, Novakovic S, Malmberg AB, Cain G, Berson A, Kassotakis L, Hedley L, Lachnit WG, Burnstock G, McMahon SB, Ford AP. Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice. Nature,2000,407(6807):1011-1015
12.Masamichi Shinoda, Noriyuki Ozaki, Hideaki Asai, Kenjiro Nagamine, Yasuo Sugiura. Changes in P2X3 receptor expression in the trigeminal ganglion following monoarthritis of the temporomandibular joint in rats. Pain,2005,116(1-2):42-51
13.Xu Gy, Huang LY. Peripheral inflammation sensitizes P2X receptor-mediated responses in rat dorsal root ganglion neurons. J Neurosci,2002,22(1):93-102
14.Paukert M, Osteroth R, Geisler HS, Brandle U, Glowatzki E, Ruppersberg JP, Gründer S. Inflammory mediaters potentiate ATP-gated channels through the P2X3 subunit. J Biol Chem,2001,276(24):21077-21082
15.D’Arco M, Giniatullin R, Simonetti M, Fabbro A, Nair A, Nistri A, Fabbretti E. Neutralization of nerve growth factor induces plasticity of ATP-sensitive P2X3 receptors of nociceptive trigeminal ganglion neurons. J Neurosci,2007,27(31): 8190-8201
16.Shinoda M, Kawashima K, Ozaki N, Asai H, Nagamine K, Sugiura Y. P2X3 receptor mediates heat hyperalgesia in a rat model of trigeminal neuropathic pain. J Pain,2007,8(7):588-597
17.Kennedy C, Assis S, Currie AJ, Rowan EG. Crossing the pain barrier: P2 receptors as targets for novel analgesics. J Physiol,2003,553(Pt 3):683-694
18.Gu YZ, Yin GF, Guan BC, Li ZW. Characteristics of P2X purinoceptors in the membrane of rat trigeminal ganglion neurons. Research paper,2006,58(2):164-170
19.Li C, Peoples RW, Weight FF. Enhancement of ATP-activated current by protons in dorsal root ganglion neurons. Pflugers Arch,1997,433(4):446-454
20.Honore P, Mikusa J, Bianchi B, McDonald H, Cartmell J, Faltynek C, Jarvis MF. TNP-ATP, a potent P2X3 receptor antagonist, blocks acetic acid-induced abdominal constriction in mice: comparison with reference analgesics. Pain,2002,96(1-2): 99-105
21.Fredholm BB, Abbracchio MP, Burnstock G, Daly JW, Harden TK, Jacobson KA, Leff P, Williams M. Nomenclature and classification of purinoceptors. Pharmacol Rev,1994,46(2):143-156
22.Li C, Peoples RW, Li Z, Weight FF. Zn2+ potentiates excitatory action of ATP on mammalian neurons. Proc Natl Acad Sci U S A,1993,90(17):8264-82677
23.Hu HZ, Li ZW. Substance P potentiates ATP-activated currents in rat primary sensory neurons. Brain Res,1996,739(1-2):163-168
24.Davies DL, Machu TK, Guo Y, Alkana RL. Ethanol sensitivity in ATP-gated P2X recepors is subunit dependent. Alcohol Clin Exp Res,2002,26(6):773-778
25.Charles K. P2X Receptors: Targets for Novel Analgesics? Neuroscientist,2005, 11(4):345-356
26.Di Virgilio F, Chiozzi P, Ferrari D, Falzoni S, Sanz JM, Morelli A, Torboli M, Bolognesi G, Baricordi OR. Nucleotide receptors: an emerging family of regulatory molecules in blood cells. Blood,2001,97(3):587-600
27.Perez-Medrano A, Donnelly-Roberts DL, Honore P, Hsieh GC, Namovic MT, Peddi S, Shuai Q, Wang Y, Faltynek CR, Jarvis MF, Carroll WA. Discovery and biological evaluation of novel cyanoguanidine P2X(7) antagonists with analgesic activity in a rat model of neuropathic pain. J Med Chem,2009,52(10):3366-3376
28.Labasi JM, Petrushova N, Donovan C, McCurdy S, Lira P, Payette MM, Brissette W, Wicks JR, Audoly L, Gabel CA. Absence of the P2X7 receptor alters leukocytefunction and attenuates an inflammatory response. J Immunol,2002,168(12): 6436-6464
29.王元银,黄擎,唐慧,王烈成.莫达非尼对大鼠海马神经元IGABA的抑制作用机制.中国药理学通报,2008,24(6):727-730