Journal of Korean Medicine Rehabilitation (한방재활의학과학회지)

The society of Korean Medicine Rehabilitation (한방재활의학과학회)
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Effect of Intracellular Calcium Chelator on Phosphorylation of Spinal N-Methyl-D-Aspartate Receptor following Electroacupuncture Stimulation in Rats

칼슘 저해제가 전침자극에 의한 척수 N-Methyl-D-Aspartate 수용체 인산화에 미치는 영향

  • Jung, Taek-Guen (Dept. of Oriental Rehabilitation Medicine, College of Oriental Medicine, Dong-Eui University) ;
  • Cho, Sung-Woo (Dept. of Oriental Rehabilitation Medicine, College of Oriental Medicine, Dong-Eui University) ;
  • Kang, Yeon-Kyeong (Dept. of Oriental Rehabilitation Medicine, College of Oriental Medicine, Dong-Eui University) ;
  • Chang, Dong-Ho (Dept. of Oriental Rehabilitation Medicine, College of Oriental Medicine, Dong-Eui University) ;
  • Lee, In-Seon (Dept. of Oriental Rehabilitation Medicine, College of Oriental Medicine, Dong-Eui University)
  • 정택근 (동의대학교 한의과대학 한방재활의학과교실) ;
  • 조성우 (동의대학교 한의과대학 한방재활의학과교실) ;
  • 강연경 (동의대학교 한의과대학 한방재활의학과교실) ;
  • 장동호 (동의대학교 한의과대학 한방재활의학과교실) ;
  • 이인선 (동의대학교 한의과대학 한방재활의학과교실)
  • Received : 2009.12.22
  • Accepted : 2010.01.12
  • Published : 2010.01.31
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Abstract

Objectives : We investigated the role of intracellular calcium chelator, bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid(BAPTA), on the modulation of phosphorylation of the spinal N-methyl-D-aspartate receptor(NMDAR) NR1 and NR2B subunits following electroacupuncture(EA). Methods : Bilateral 2 Hz EA stimulation with 1.0 mA was delivered at those acupoints corresponding to Zusanli(ST36) and Sanyinjiao(SP6) in man via needles for 30min. Results : EA analgesia was reduced by intra-peritoneal injection at a higher dose of BAPTA from termination of EA stimulation. At 60 min after EA treatment, the total number of c-fos-immunostained neurons in each regions of the dorsal horn in the $L_{4-5}$ segments was decreased by BAPTA injection, especially in nucleus proprius. The mean integrated optical density (IOD) of NR1 and NR2B subunits were increased only in superficial laminae of EA-treated rats when compared with normal rats. However, the mean IOD of pNR1 was significantly decreased by BAPTA injection in both the superficial laminae and neck region and pNR2B in the superficial laminae. Western blot analyses confirmed the decreased expression of pNR1 and pNR2B. Conclusions : We concluded that intracellular calcium may well play an important role in EA analgesia by modulating the phosphorylation state of spinal NMDAR subunits.

목 적 : 본 연구는 전침 자극에 의한 척수내 N-methyl-D-aspartate(NMDA) 수용체의 NR1 및 NR2B subunit 인산화에 미치는 세포내 칼슘 저해제 bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraaceticacid(BAPTA)의 영향을 조사하였다. 방 법 : 인체의 족삼리(足三里)(ST36)와 삼음교(三陰交)(SP6)에 해당하는 혈자리에 2 Hz전침 자극을 1.0mA 세기로 30분 동안 자극하였다. 결 과 : 전침 무통각을 측정한 결과 높은 농도의 BAPTA 복강내주사 처리군에서 저하가 관찰되었다. 전침 처치 후 60분 후 분리한 $L_{4-5}$ 척수 분절에서 C-fos 발현 신경세포 수는 BAPTA 처리에 의해 감소하는데 특히 고유핵에서 저하가 현저하였다. 평균 integrated optical density로 비교한 NR1과 NR2B subunit에 대한 면역조직화학적 발현을 보면 전침 자극은 정상군에 비해 얕은 층판에서 증가하였다. NR1과 NR2B subunit의 인산화형에 대한 발현을 보면 NR1 인산화형은 척수 배각 전 부위에서, NR2B 인산화형은 얕은 층판에서 증가하였으며 BAPTA 처리에 의해 NR1 인산화형은 얕은 층판과 목 부위에서 NR2B 인산화형은 얕은 층판에서 현저한 감소를 보였다. western blot로 살펴본 BAPTA 처리에 의한 NR1 및 NR2B 인산화형 변화는 면역조직화학적 방법과 유사한 결과를 보여 주었다. 결 론 : 전침 무통각은 세포내 칼슘에 의한 척수 내 NMDA 수용체의 인산화가 중요한 요인으로 작용할 가능성이 있다.

Keywords

References

  1. Han JS. Acupuncture: neuropeptide release produced by electrical stimulation of different frequencies. Trend. Neurosci. 2003;26:17-22. https://doi.org/10.1016/S0166-2236(02)00006-1
  2. Zhang YQ, Ji GC, Wu GC, Zhao ZQ. Excitatory amino acid receptor antagonists and electroacupuncture synergetically inhibit carrageenan-induced behavioral hyperalgesia and spinal fos expression in rats. Pain. 2002;99:525-35. https://doi.org/10.1016/S0304-3959(02)00268-3
  3. Choi BT, Lee JH, Wan Y, Han HJ. Involvement of ionotropic glutamate receptors in low frequency electroacupuncture analgesia in rats. Neurosci. Lett. 2005;377:185-8. https://doi.org/10.1016/j.neulet.2004.11.095
  4. Gao X, Kim HK, Chung JM, Chung K. Enhancement of NMDA receptor phosphorylation of the spinal dorsal horn and nucleus gracilis neurons in neuropathic rats. Pain. 2005;116:62-72. https://doi.org/10.1016/j.pain.2005.03.045
  5. Hatt H. Modification of glutamate receptor channels: molecular mechanisms and functional consequences. Naturwissenschaften. 1999;86:177-86. https://doi.org/10.1007/s001140050593
  6. Cull-Candy S, Brickley S, Farrant M. NMDA receptor subunits: diversity, development and disease. Curr. Opin. Neurobiol. 2001;11:327-35. https://doi.org/10.1016/S0959-4388(00)00215-4
  7. Lieberman DN, Mody I. Regulation of NMDA channel function by endogenous Ca(2+)-dependent phosphatase. Nature. 1994;369:235-9. https://doi.org/10.1038/369235a0
  8. Sabina Lim, Hyung-Taeck Lim, Hi-joon Park, Ji-Ryeon Jang, Il-Hwan Choi, Seok-Chan Lee, Dae-Soo Kim, Hee-sup Shin. The analgestic mechanism of acupuncture at $ST_{36}$ in the abdominal pain of the mouse. The Korean Journal of Meridian & Acupoint. 2004;21(2):69-79.
  9. Ternov K, Nilsson M, Lofberg L, Algotsson L, Akeson J. Acupuncture for pain relief during childbirth. Acupunct Electrothery Res.1998;23:19-26. https://doi.org/10.3727/036012998816356599
  10. Jones TL, Sorkin LS. Activated PKA and PKC, but not CaMKIIalpha, are required for AMPA/Kainate-mediated pain behavior in the thermal stimulus model. Pain. 2005;117:259-70. https://doi.org/10.1016/j.pain.2005.06.003
  11. Yoshimura M, Jessell T. Amino acid-mediated EPSPs at primary afferent synapses with substantia gelatinosa neurones in the rat spinal cord. J. Physiol. 1990;430:315-35. https://doi.org/10.1113/jphysiol.1990.sp018293
  12. Hartell NA. Strong activation of parallel fibers produces localized calcium transients and a form of LTD that spreads to distant synapses. Neuron.1996;16:601-10. https://doi.org/10.1016/S0896-6273(00)80079-3
  13. Neveu D, Zucker RS. Postsynaptic levels of [Ca2+]i needed to trigger LTD and LTP. Neuron. 1996;16:619-29. https://doi.org/10.1016/S0896-6273(00)80081-1
  14. Jaremy R. Acupuncture Point Combinations.: the key to Clinical Success. 1st ed. London: Churchill Livingstone. 2004:213,231.
  15. Sandkuhler J. Long-lasting analgesia following TENS and acupuncture: Spinal mechanisms beyond gate control. In M Dever, MC Rowbotham, Z Wiesenfeld-Hallin (Eds.), Proc. 9th World Congress on Pain, Progress in Pain Research and Management, Vol.16, Seattle WA:IASP. 2000:359-69.
  16. Willis WD. Role of neurotransmitters in sensitization of pain responses. Ann. N .Y. Acad. Sci. 2001;933:142-56.
  17. Hu JY, Zhao ZQ. Differential contributions of NMDA and non-NMDA receptors to spinal Fos expression evoked by superficia ltissue and muscle inflammation in therat. Neuroscience. 2001;106:823-31. https://doi.org/10.1016/S0306-4522(01)00299-8
  18. Chan SF, Sucher NJ. An NMDA receptor signaling complex with protein phosphatase 2A. J. Neurosci. 2001;21:7985-92.
  19. Tan PH, Yang LC, Shih HC, Lan KC, Cheng JT. Gene knockdown with intrathecal siRNA of NMDA receptor NR2B subunit reduces formalin-induced nociception in the rat. Gene Ther. 2005;12:59-66. https://doi.org/10.1038/sj.gt.3302376
  20. Zhang X, Wu J, Lei Y, Fang L, Willis WD. Protein phosphatase modulates the phosphorylation of spinal cord NMDA receptors in rats following intradermal injection of capsaicin. Brain Res. Mol. Brain Res. 2005;138:264-72. https://doi.org/10.1016/j.molbrainres.2005.05.001