【作者】 陈筱雨；

【导师】 刘长金；

【作者基本信息】 华中科技大学 ， 生理学， 2007， 硕士

【摘要】 本实验应用全细胞膜片钳技术,在大鼠三叉神经节神经元上探讨低渗透压对ATP-激活电流的调制作用。结果显示:(1)大部分受检细胞(80.90%)对外加ATP敏感,产生一具有浓度依赖性的内向电流,且该电流可以被P2X受体拮抗剂PPADS所阻断。(2)预加低渗溶液(220 mOsm,260 mOsm,300 mOsm),对IATP产生抑制作用,该抑制作用呈可逆性,渗透压依赖性和非电压依赖性。(3)在低渗透压(260mOsm)环境下,ATP-激活电流的浓度反应曲线明显下移;阈值和最大反应浓度不变,EC50为1.19×10-4M,与单独加ATP时的1.33×10-4M基本一致;而最大反应浓度时的ATP电流幅值减小了25.26±2.41%(n=6)。(4)低渗透压对IATP的抑制作用可被TRPV4(transient receptor potential vanilloid 4)受体激动剂4ɑ-PDD(4ɑ-phorbol 12,13-didecanoate)增强,并可被TRPV4受体非特异性阻断剂钌红(ruthemium red, RR)阻断。(5)在细胞外液中加入forskolin或8-Br-cAMP预孵育,可以部分翻转低渗对ATP-激活电流的抑制作用,而H89预孵育则不影响低渗对IATP的影响。以上结果表明,低渗透压作用于TRPV4受体,可能通过直接抑制cAMP的生成而抑制P2X受体介导的电流,从而发挥对ATP激活电流的抑制作用。更多还原

【Abstract】 This experiment aimed to investigate the modulation of hypotonicity on ATP-activated currents (IATP) in primary sensory neurons. Whole cell-patch clamp recording was performed on cultured SD rat trigeminal ganglion (TG) neurons. (1) The majority of neurons examined (80.90%) were sensitive to ATP (10-5~10-3M), ATP activated an inward currents in a concentration-dependent manner, and IATP were blocked by PPADS(pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid, an antagonist of the P2X receptor. (2)When Hypotonicity(220 mOsm, 260 mOsm, 300 mOsm)was preapplied extracellularly, it reduced ATP-activated currents (IATP) significantly. This inhibitory effect was reversible, osmosis-dependent, and voltage-independent. (3) The concentration-response curves for ATP with and without preapplication of hypotonicity showed that preapplication of hypotonicity shifted the curve downward; maximal amplitude of IATP with hypotonicity decreased by 25.26±2.41%, while the threshold value remained unchanged; the EC50 value of the two curves were very close(1.19×10-4M vs 1.33×10-4M). (4) This inhibitory effect was increased by 4ɑ-PDD, a selective TRPV4 receptor agonist, and was reversed by ruthenium red (RR), a nonselective TRPV4 receptor antagonist, suggesting that the inhibition is mediated via TRPV4 receptor. (5) Preapplication of forskolin or 8-Br-cAMP partly reversed the inhibition of IATP by hypotonicity, whereas preapplication of H89 had not effect on it. These results suggested that hypotonicity inhibited ATP-actived currents via TRPV4 receptor, which was mediated partly by reducing cAMP concentration directly rather than by PKA and downstream process.更多还原