血清素受體2B調控鈣離子變化影響機械性痛覺敏感

5-HT2B regulated calcium response mediating mechanical hyperalgesia

林詩媛(Shih-yuan Lin)

指導教授 ： 孫維欣

國立中央大學/理學院/生命科學系/碩士(2011年)

摘要

血清素（Serotonin, 5-Hydroxytryptamine, 5-HT）是一種重要的發炎調節因子，會參與疼痛以及產生痛覺敏感現象。藉由周邊組織的肥大細胞或是血小板釋放血清素，可以產生兩種調節下游機制的反應：第一，可以直接作用在血清素離子通道（5-HT3）上，使通道開起改變細胞的離子通透性，並且會使可以痛覺神經興奮；第二，血清素與細胞膜上的血清素G蛋白偶合受體 （5-HT1, 2, 4, 5, 6, 7）結合，活化下游G蛋白，進而產生一連串訊號傳遞機制。先前研究發現血清素受體2B主要表現在中小直徑（10-34 μm）的小鼠背根神經節細胞，且血清素2B/2C抑制劑可以抑制由血清素引發的機械性痛覺敏感。然而血清素受體2B在此所扮演的角色以及作用機制依舊不清楚。因此我利用注射血清素或血清素受體2B/2C的抑制劑於小鼠的後腳掌內，並培養背根神經節細胞，之後以血清素刺激觀察細胞內鈣離子含量的變化情形。研究發現血清素刺激引發三種細胞內鈣離子含量變化：短暫快速上升、延遲以及混合型。注射血清素後，細胞對血清素刺激的敏感性提升2.6倍（5.1% vs. 13.3%），IB4 (-)細胞內鈣離子含量增加2.4倍，IB4 (+)細胞則無顯著差異。在注射血清素受體2B/2C抑制劑後，細胞對血清素的敏感性下降至1.3%，短暫快速上升的鈣離子曲線亦被完全抑制，此外在IB4 (-)的細胞中，血清素可增強辣椒素引發的細胞內鈣離子含量變化，並且可經由血清素受體2B/2C抑制劑所抑制。值得注意的是注射血清素無法增強血清素引發的細胞內鈉離子變化，但是細胞內鈉離子含量變化可以受血清素受體2B/2C抑制劑所抑制，綜合以上結果顯示血清素受體2B確實參與機械性痛覺敏感現象。

關鍵字

背根神經節 ； 機械性痛覺敏感 ； 血清素受體2B

並列摘要

Serotonin (5-Hydroxytryptamine, 5-HT) is one of the important inflammatory mediators in pain and hyperalgesia. 5-HT released from mast cells or platelets in peripheral tissues, can directly act on 5-HT-gated ion channel (5-HT3) to change sodium and calcium permeability and excitability of nociceptors. 5-HT can also induce chemical interaction through the activation of 5-HT G-protein coupled receptors (5-HT1, 2, 4, 5, 6, 7). Previous studies have found that 5-HT2B is highly localized in small-to-medium diameter (10-34 μm) dorsal root ganglion (DRG) neurons but not 5-HT2C. The antagonist of 5-HT2B/2C inhibits 5-HT-induced mechanical hyperalgesia. However, it remains unclear whether 5-HT2B-mediated 5-HT signaling is involved in 5-HT-induced mechanical hyperalgesia and detailed mechanisms. To address this question, I have injected 5-HT and 5-HT2B/2C antagonist into mouse hind paw and DRG were taken from injected mice to culture. Cultured neurons were stimulated by 5-HT, followed by calcium imaging. Three patterns of 5-HT-induced intracellular calcium increase were found: transient, sustained and mixed. After 5-HT-injection, the number of 5-HT-responding neurons had 2.6-fold increase (5.1% vs. 13.3%) and the 5-HT-induced calcium increase was 2.4-fold increased on ipsilateral IB4-negative neurons but not on IB4-positive neurons. 5-HT2B/2C antagonist injection reduced the number of 5-HT-responding neurons (1.3%) and inhibited the transient [Ca2+]i rise in patterns 1 and 2. I also found that capsaicin-induced calcium influx was increased on IB4-negative neurons after 5-HT-injection and the increased calcium influx was inhibited by 5-HT2B/2C antagonist. Interestingly, 5-HT-induced sodium current was not enhanced by 5-HT injection but reduced by injection of 5-HT2B/2C antagonist. These results suggest that 5-HT2B-mediated calcium response is involved in 5-HT-induced mechanical hyperalgesia.

並列關鍵字

DRG ； 5-HT2B ； mechanical hyperalgesia

參考文獻

Aley KO, Levine JD (1999) Role of protein kinase a in the maintenance of inflammatory pain. The Journal of Neuroscience 19 (6):2181-2186.

Amaya F, Wang H, Costigan M, Allchorne AJ, Hatcher JP, Egerton J, Stean T, Morisset V, Grose D, Gunthorpe MJ, Chessell IP, Tate S, Green PJ, Woolf CJ (2006) The Voltage-Gated Sodium Channel Nav1.9 Is an Effector of Peripheral Inflammatory Pain Hypersensitivity. The Journal of Neuroscience 26 (50): 12852-12860.

Baker MD (2005) Protein kinase C mediates up-regulation of tetrodotoxin-resistant, persistent Na+ current in rat and mouse sensory neurones. J Physiol 567: 851-867.

Bennett DLH, Michael GJ, Ramachandran N, Munson JB, Averill S, Yan Q, McMahon SB, Priestley JV (1998) A distinct subgroup of small DRG cells express GDNF receptor components and GDNF is protective for these neurons afternerve injury. The Journal of Neuroscience 18 (8): 3059-3072.

Benson CJ, Xie J, Wemmie JA, Price MP, Henss JM, Welsh MJ, Snyder PM (2002) Heteromultimers of DEG/ENaC subunits form H+-gated channels in mouse sensory neurons. Proc. Natl. Acad. Sci. 99 (4): 2338-2343.