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1 J. Exp. Biomed. Sci. 11 (2005) Expression of Nociceptin within Dura Mater in Response to Electrical Trigeminal Ganglion Stimulation in Rats Jeong Hee Kim and Won Suk Lee Department of Pharmacology, College of Medicine, Pusan National University, Busan , Korea This study aimed to investigate whether nociceptin is implicated in the trigeminovascular responses to electrical stimulation of trigeminal ganglion in rats. An open cranial window was prepared on the right parietal bone of male Sprague-Dawley rats. Trigeminovascular system was stimulated by electrical stimulation of trigeminal ganglion (ETS; 5 ms, 5 Hz, 3 V). Neonatal capsaicin treatment was performed with subcutaneous administration of capsaicin (50 mg /kg) within the first 24 hours after birth. Changes in regional cerebral blood flow were continuously measured through the cranial window by laser-doppler flowmetry, and the expression of nociceptin-like immunoreactivity was determined by immunohistochemistry. ETS caused increases in regional blood flow of pial arteriole in a voltage-dependent manner. ETS markedly and voltage-dependently increased the expression of nociceptin-like immunoreactivity in dura mater ipsilateral rather than contralateral to ETS. The nociceptin-like immunoreactivity was markedly reduced by pretreatments with calcitonin gene-related peptide(8-37) (CGRP 8-37, a CGRP 1 receptor antagonist), L (a NK 1 receptor antagonist), and [Nphe 1 ]nociceptin(1-13)nh 2 (a selective and competitive nociceptin receptor antagonist) as well as by neonatal capsaicin treatment. These results suggest that the electrical stimulation of trigeminal ganglion causes prominent expression of nociceptin within dura mater, in which not only neuropeptides inclucing substance P and CGRP but also nociceptin are implicated in the trigeminovascular responses to electrical trigeminal ganglion stimulation. Key Words: Trigeminovascular system, Nociceptin, Neuropeptide, Dura mater 서 삼차신경혈관계 (trigeminovascular system) 는삼차신경의감각신경세포와두개내혈관사이를통합하여일컫는부위로서자극을받게되면뇌막혈관주위에있는통각수용성들신경섬유에서 substance P와 calcitonin gene-related peptide (CGRP) 같은신경펩티드가유리되고이들에의하여뇌경막내에혈관확장과염증이유발된다고알려져있다 (Moskowitz, 1992; Williamson et al., 1997a, b). 뇌경막 (dura mater) 은동통에민감한수용체를고밀도로가지고있으며, 여기에존재하는동통-민감성신경섬유는동통신호를뇌내의통각중추로보낼뿐만아니라신경성염증발생을매개하기도한다. 뇌경막은천공성모세혈관과내피세포를가진혈관들을가지고있고 (Andres et al., 1987), * 논문접수 : 2005년 8월 23일수정재접수 : 2005년 9월 20일 교신저자 : 이원석 ( 우 ) 부산광역시서구아미동 1가 10, 부산대학교의과대학약리학교실 Tel: , Fax: wonslee@pusan.ac.kr 론 신경펩티드를함유하는삼차신경과위목감각신경섬유의분포를받고있으며, 비만세포를많이가지고있어 (Steiger et al., 1982; Mayberg et al., 1984) 신경성염증의발생에필요한최소한의요건을모두갖추고있으므로두통의중요한원천지라고여겨지고있다 (Moskowitz and Buzzi, 1991; Edvinsson, 2001). 한편최근에발견된내인성 opioid 펩티드인 nociceptin은다른 opioid 펩티드와는달리 N-말단의첫아미노산이 tyrosine 대신에 phenylalanine으로시작하고 (Meunier et al., 1995), µ-, δ- 그리고 κ-opioid 수용체에대한친화력이매우낮을뿐만아니라 (Reinscheid et al., 1995) 체내분포와효과도다르므로새로운펩티드로인식되고있다 (Fukuda et al., 1994; Lachowicz et al., 1995; Mollereau et al., 1996). Nociceptin과그수용체는중추신경계와말초신경계에널리분포하고있으면서다양한반응에관여하는데 (Anton et al., 1996; Ikeda et al., 1998; Calo' et al., 2000) 그중에서도통각전달에매우중요한역할을하는것으로알려졌다. 즉 nociceptin을뇌실내로척수상부에투여하면 opioid와는달리통각과민 (hyperalgesia) 이초래되고 (Grisel and Mogil, 2000), 척수강내또는말초에투여하면저용량에서는통각과민을

2 일으키지만고용량에서는진통효과를나타낸다고한다 (Yamamoto et al., 1999; Xu et al., 2000). 최근에 Hou et al. (2003) 은사람의삼차신경절내에서 nociceptin-유사면역반응 (nociceptin-like immunoreactivity) 과 nociceptin 수용체의 mrna가발현되고, nociceptin은 CGRP, substance P, nitric oxide synthase 및 pituitary adenylate cyclase activating peptide와공존함을보고하였다. 이와같이 nociceptin이삼차신경절내에서발현될뿐만아니라삼차신경혈관계의반응에있어서중요한매개물질인 CGRP 및 substance P와공존하고있다는것으로부터 nociceptin은삼차신경성감각전달에있어서추축과같은역할을할것으로여겨지고있다. 그러나삼차신경절의자극에의한삼차신경혈관계의반응에있어서 nociceptin의관여에대하여는아직자세히밝혀져있지않은실정이다. 따라서본연구에서는삼차신경절전기자극에의한삼차신경혈관계의반응에있어서 nociceptin의관여를구명하는연구의일환으로서삼차신경절전기자극에의한뇌경막내 nociceptin의발현을살펴보고자하였다. 재료및방법 1. 실험동물체중 250~300 g의 Sprague-Dawley 계숫쥐를사육장 ( 실내온도 20~25 ) 내에서물과사료를마음껏섭취하게하면서사육하였다가실험에사용하였다. 본실험에사용된실험동물의수는대조군 6 마리, 삼차신경절전기자극군 19 마리, 신생기 capsaicin 처리군 5 마리, CGRP 1 수용체차단제인 CGRP 8-37 전처치군 6 마리, NK 1 수용체차단제인 L 전처치군 5 마리및 nociceptin 수용체에선택적이면서경쟁적차단제인 [Nphe 1 ]nociceptin(1-13)nh 2 처치군 6 마리로서총 47 마리이었다. 이들중 6 마리는뇌혈류측정에사용되었고, 나머지 41 마리는 nociceptin-유사면역반응측정에사용되었다. 2. 신생기 capsaicin 처치일차들신경섬유를전반적으로손상시키기위하여 Jancso 등 (1977) 의방법에따라출생 24 시간이내의신생백서에게 capsaicin 50 mg/kg을피하주사하여전신의민말이집감각신경섬유를파괴하고약 2 개월후실험에사용하였다. 실험을시작하기전에소량의 capsaicin (1 µm) 을각막에국소투여하여봄으로써이자극에대한반응의소실유무를확인하고동공변화가있을때그백서는실험에서제외시켰다. 3. 일반수술조작실험동물을 urethane (1 g/kg, i.p.) 으로마취시키고소동물용 자동보온전기방석 (Homeothermic Blanket System, Harvard Apparatus, Edenbridge, Kent, UK) 위에바로눕힌채고정하여체온을 37 로유지시켰다. 기관절개술을시행하여 14G catheter로기도를확보하고 gallamine (3~5 mg/kg, i.m.) 을투여하여골격근을마비시킨다음동물용호흡기 (683, Harvard Apparatus, South Natick, MA, USA) 를사용하여체중에따라분당 50~60 회의호흡을유지시켰다. 양측대퇴동맥에 PE- 50 polyethylene 관을삽관하고이를통하여동맥혈내 O 2 분압과 CO 2 분압및 ph를혈액가스분석기 (i-stat Portable Clinical Analyzer, i-stat Corporation, East Windsor, NJ, USA) 로측정하여전실험기간동안 PaCO 2 의변화가기저치의 3~4% 이내에서이루어지도록호흡을조절하여유지시켰다. 4. 개방두개창설치일반적인수술전조작이끝난후실험동물의체위를복와위로변경시키고머리를정위기구 (900, David Kopf Instruments, Tujunga, CA, USA) 에고정시켰다. 두피를정중선을따라절개하여양측으로벌린후입체현미경 (Stemi SV6, Carl Zeiss, Göttingen, Germany) 하에서관상봉합의미측에위치한우측두정골에 saline-cooled drill (395, Dremel, Racine, WI, USA) 을사용하여개두술 (5 5 mm) 을시행하고뇌경막과지주막을온전하게보존시켰다. 그후미리 37 로가온한인공뇌척수액을관류시켰다. 사용한인공뇌척수액의조성은다음과같다 : Na meq/l, K meq/l, Ca meq/l, Mg meq/l, Cl meq/l, HCO meq/l, dextrose 66.5 mg/dl, urea 40.2 mg/dl (ph 7.4). 5. 삼차신경절전기자극실험동물을정위기구 (900, David Kopf Instruments) 의수평선에서 1.5 mm 하방에 incisor bar로고정하고, 두피를정중선을따라절개하여양측으로벌린후두개관을노출시켰다. 정수리점 (bregma) 으로부터 3.7 mm 후방의위치이면서시상봉합 (sagittal suture) 으로부터양측 3.2 mm인지점에직경 2 mm의머리뼈뚫개구멍을뚫고, 여기를통하여두극전극 (50 mm shaft, Rhodes Medical Instruments, Woodland Hills, CA, USA) 를 9.5 mm 깊이에있는삼차신경절에설치하였다. 오른쪽삼차신경절을전기자극기 (SI-10, Narco Bio-Systems, Houston, Texas, USA) 를사용하여 5 ms, 5 Hz, 3 V로 5 분동안자극하였다. 이때왼쪽삼차신경절은전기자극을하지않고그대로두었다. 6. 혈압변동측정전신혈압의변동을관찰하기위하여실험동물의대퇴동맥에삽입되어있는 polyethylene 관을압력변환기 (pressure transducer: Statham P23D, Gould, Cleveland, OH, USA) 에연결

3 하고, MacLab (4s, ADInstruments, Castle Hill, Australia) 과 Macintosh computer (Power Macintosh 7500/100, Seoul, Korea) 로구성된자료획득시스템 (data acquisition system) 에기록하였다. 7. 국소뇌혈류변동측정실험동물의머리를정위기구에고정시키고, 개방두개창을통하여 laser-doppler flowmeter (BLF21, Transonic Systems Inc., Ithaca, NY, USA) 용 needle probe (type NS) 를정위미세조작장치 (stereotactic micromanipulator) 를사용하여대뇌겉질표면에수직이되도록뇌연막동맥에조심스럽게접근시켰다. 일정시간동안안정시킨후실험 protocol에따라국소뇌혈류량을측정하였다. Laser-Doppler flowmeter에의하여측정된국소뇌혈류량의변동은 MacLab과 Macintosh computer로구성된자료획득시스템에기록하였다. 8. 관류고정및조직처리삼차신경절전기자극종료 1 시간후에실험동물의가슴을열어심장을노출시켰다. 좌심실에주사침을삽입하고혈액이흘러나오도록우심방을절개한후주사침에주입펌프를연결하여분당 4 ml의속도로약 200 ml 정도의 0.02 M phosphate-buffered saline (PBS) 를관류시켜혈액을제거한다음 250~300 ml의 4% paraformaldehyde/pbs로관류고정을시행하였다. 머리전체를적출한후동일고정액에넣고 4 냉장고에 12 시간동안후고정 (postfixation) 을하였으며뇌경막을분리한후 6 시간동안한번더고정하였다 M PBS로 15 분간 3 회세척하고 20% sucrose/pbs에 8 시간동안침전시켜동결시일어나는결빙과립 (ice crystal) 을방지하였다. 이후뇌경막을 silane이입혀져있는슬라이드글라스 (Muto Pure Chemicals Ltd, Tokyo, Japan) 위에부착시킨후상온에서 1 일간말린후 -20 에서 15 분동안차가운 acetate에담구어 slide를고정시켰다. 상온에서 2 시간동안말린후염색전까지 -20 냉동고에보관하였다. 9. 면역조직화학내인성과산화효소의반응을제거하기위해조직을 0.3% 과산화수소수에넣어 20 분간실온에방치하였다. 이후조직을 0.02 M PBS로 15 분간 3 회세척한후에비특이면역반응을방지하기위해 2% bovin serum albumin ( 차단항체 ) 을사용하여가습실에서 30 분동안반응시켰다. 차단항체를제거한뒤일차항체인 rabbit anti-nociceptin (Chemicon international, Temecula, CA, USA) 을 1:1500으로희석한후, 4 에서 12 시간동안반응시켰다. 이때희석액은 0.02 M PBS에 0.5% bovin serum albumin 이섞여있는것을사용하였다. 그후실온에서 0.02 M PBS로 15 분간 3 회세척하였다. 이차항체인 anti-rabbit Igs, biotinylated goat (InnoGenex, San Ramon, CA, USA) 를 1:1000으로희석하여실온에서 1 시간동안반응시켰다. 이차항체반응이끝난후에 0.02 M PBS로 5 분간 3 회세척한후삼차반응용액인 avidin-biotin complex (ABC, Vectastain Elite, Vector Laboratories, Burlingame, CA, USA) 용액으로넣어실온에서 1 시간반응시킨후 0.02 M PBS로 15 분간 3 회세척하고 diaminobenzidine (DAB) substrate kit (Vector Laboratories) 를이용하여광학현미경 (Axioskop 40, Carl Zeiss) 하에서확인하면서착색반응을시행하였다. 반응이끝난후 0.02 M PBS로 15 분간 3 회세척하여반응을중지시키고, 알코올과 xylene으로탈수및투명화과정을거친후 Entellan (Merck, Darmstadt, Germany) 으로봉입하여 cover glass를씌운후광학현미경으로 nociceptin-유사면역반응을관찰하였다. Nociceptin-유사면역반응의발현에대한분석은 Video Test-Master (Video Test Ltd, St. Petersburg, Russia) 를사용하여측정하였다. 10. 사용약물본연구에서사용한주요약물은 capsaicin, CGRP 8-37 ( 이상 Sigma Chemical Co., St. Louis, MO, USA), L-733,060 및 [Nphe 1 ]nociceptin(1-13)nh 2 ; 이상 Tocris Cookson Ltd., Langford, Bristol, UK) 등이었다. Capsaicin 은에탄올 : Tween 80 : 0.9% 생리식염수 (1 : 1 : 8 v/v) 혼합용액에용해하여 0.9% 생리식염수로희석하였다. 11. 통계처리모든측정치는평균 ± 평균의표준오차로표시하였다. 실험성적들은통계프로그램인 Prism (version 3.03, GraphPad Software Inc., San Diego, CA, USA) 을사용하여 Student's unpaired t-test로통계학적유의성을검정하거나, 또는분산분석법으로분석한후각군사이의유의성을검정하기위한사후검정법으로 Dunnett의다중비교법을사용하였다. P 값이 0.05 미만인것을유의하다고판정하였다. 결과 1. 뇌연막동맥혈류변동삼차신경절전기자극에있어서적정한전기자극조건을찾기위하여두개창이설치된같은쪽의삼차신경절을 5 ms, 5 Hz의상태에서 1, 3, 5 및 10 V의전압으로각각 2 분간전기자극하였다. 삼차신경절전기자극에의한뇌연막동맥의국소혈류는 Fig. 1에서보는바와같이전압-의존적으로증가하였고, 전기자극을마친후에는서서히기저치수준으로회복되었다

Fig. 1. Changes in regional cerebral blood flow (rcbf) and mean arterial blood pressure (MABP) in response to electrical stimulation of trigeminal ganglion (ETS).

4 Fig. 1. Changes in regional cerebral blood flow (rcbf) and mean arterial blood pressure (MABP) in response to electrical stimulation of trigeminal ganglion (ETS). The right trigeminal ganglion was stimulated consecutively at an interval of 2 min (5 ms, 5 Hz, 1, 3, 5 and 10 V, respectively). The number of animal is 6. Fig. 3. Expression of nociceptin-like immunoreactivity in dura mater by electrical trigeminal ganglion stimulation. The numbers in parentheses represent the numbers of animals. NS, no significance. # P<0.01 vs. corresponding unstimulated side of sham group. * P<0.01 vs. corresponding stimulated side of sham group. Fig. 2. Representative microphotographs showing the expression of nociceptin-like immunoreactivity in dura mater. The right trigeminal ganglion was stimulated for 5 min (5 ms, 5 Hz) with different voltage of 0, 3 or 10 V. Scale bar = 50 µm. 2. 뇌경막내 nociceptin- 유사면역반응 1) 삼차신경절전기자극효과삼차신경절전기자극에의한삼차신경혈관계의반응에대한 nociceptin의관여를보기위하여뇌경막내의 nociceptin- 유사면역반응의발현을면역조직화학법으로관찰하였다. 삼차신경절전기자극 (0, 3 및 10 V) 에의한뇌경막내 nociceptin-유사면역반응의발현은 Fig. 2와 Fig. 3에서보는바와같이전기자극과같은쪽에서전기자극반대쪽에비하여현저히증가하였다 (3 V, P=0.0001; 10 V, P<0.0001). 이러한 nociceptin- 유사면역반응의발현은전기자극과같은쪽및반대쪽모두에서전기자극의전압에의존적으로증가하였지만 ( 같은쪽, F (2,16) = , P<0.0001; 반대쪽, F (2,16) = , P<0.0001), 전기자극과같은쪽에서더욱현저하게증가하였다 (P<0.0001). 2) 신생기 capsaicin 처치및각종펩티드수용체차단제전처치효과삼차신경절을 3 V로전기자극하였을때전기자극과같은쪽의뇌경막내에초래되는 nociceptin-유사면역반응의발현은신생기에 capsaicin 처치를하거나 (P<0.01), CGRP 1 수용체차단제인 CGRP 8-37 (1 µm, P<0.01), NK 1 수용체차단제인 L (0.1 µm, P<0.01), 그리고 nociceptin 수용체차단제인 [Nphe 1 ]nociceptin(1-13)nh 2 (1 nm, P<0.01) 의전처치에의하여현저히약화되었다 (F (4,22) = , P<0.0001, Fig. 4, Fig. 5). 한편, 전기자극반대쪽의뇌경막내에서의 nociceptin-유사면역반응의발현은신생기 capsaicin 처치, CGRP 8-37, L 등의전처치에아무런영향을받지아니하였으나, [Nphe 1 ]nociceptin(1-13)nh 2 전처치에의하여유의하게약화되었다 (P<0.01). 고찰본연구를통하여삼차신경절전기자극은뇌경막내 noci

Fig. 5. Expression of nociceptin-like immunoreactivity in dura mater by electrical trigeminal ganglion stimulation. * P<0.01 vs. corresponding stimulated side of vehicle group. # P<0.01 vs. corresponding unstimulated side of vehicle group.

5 Fig. 5. Expression of nociceptin-like immunoreactivity in dura mater by electrical trigeminal ganglion stimulation. * P<0.01 vs. corresponding stimulated side of vehicle group. # P<0.01 vs. corresponding unstimulated side of vehicle group. Fig. 4. Representative microphotographs showing the expression of nociceptin-like immunoreactivity in dura mater. The right trigeminal ganglion was stimulated for 5 min (5 ms, 5 Hz, 3 V). ncaps, Rats were treated with capsaicin (50 mg/kg, s.c.) within the first 24 hours of life. CGRP 8-37 (1 µm), L (0.1 µm) and [Nphe 1 ] nociceptin(1-13)nh 2 (1 nm) were topically administered on the dura mater 5 min before and during electrical trigeminal ganglion stimulation, respectively. ceptin의발현을증가시키고, 여기에는삼차신경혈관계내의신경펩티드가중요하게관여함을관찰하였다. 두개강내의통증에민감한조직들은삼차신경절과일부위목신경절 (superior cervical ganglion) 로부터의신경지배를받고있는데, 삼차신경이자극을받으면삼차신경말단부위에있는신경전달물질인 substance P, CGRP, neurokinin A 등의신경펩티드가유리되어혈관을확장시키고혈장단백질을혈관밖으로유출시켜무균성염증을초래함으로써감각신경자극으로인한통증을야기한다 (Moskowitz, 1992; Williamson et al., 1997a, b). 이러한신경성염증 (neurogenic inflammation) 의발생은편두통 (migraine) 이나군발두통 (cluster headache) 같은혈관성두통의발병기전에중요한역할을한다고알려져있다 (Moskowitz et al., 1989). 혈관성두통의통각과정에있어서뇌혈류가관련있는것으로알려져있다. 즉, 편두통발작시뇌연막혈관의관류량의증가에의존하여국소뇌혈류가증가하고 (Olesen et al., 1990), 고양이 (Goadsby and Duckworth, 1987) 와사람 (Tran- Dinh et al., 1992) 의삼차신경절자극시뇌혈류가증가한다고한다. 본연구에서도흰쥐의삼차신경절을전기자극하였을때전압에의존하여국소뇌혈류가증가하고동시에평균동맥혈압도증가함을관찰할수있었다. 본연구에서삼차신경 절전기자극에의하여뇌혈류가증가한것이혈압의증가에의한관류량의증가때문인것으로생각할수도있으나혈류와혈압간의관계로부터혈관저항을계산하여보았을때전기자극의전압에의존적으로혈관저항이감소하였음을알수있었다 ( 미제시성적 ). 따라서삼차신경절전기자극에의한뇌혈류의증가반응은삼차신경절전기자극시삼차신경으로부터유리된신경펩티드가뇌연막동맥을확장시킴으로써초래된것으로생각된다. 그러나본연구에서여러신경펩티드수용체들에대한차단제를전처치하거나신경펩티드생성을억제한후삼차신경절전기자극을해보지않아현재로서는단언하기어렵다. Nociceptin은최근에발견된내인성 opioid 펩티드로서그생리학적기능과병적인상태에서의역할에관한연구가활발히진행되고있다. Nociceptin은전신투여시항불안작용을나타내고 (Jenck et al., 1997), 학습과기억에장애를초래할뿐만아니라 (Nabeshima et al., 1999; Noda et al., 2000), 통각전달과정에다양한효과를일으켜통각과민, 진통, 무해자극통증 (allodynia) 을일으킨다고알려져있다 (Henderson and McKnight, 1997; Meunier, 1997; Taylor and Dickenson, 1998). 통각전달에있어서 nociceptin은중추신경계에서는동통을유발하여 opioid 효현제와는반대로작용하지만, 말초신경계에서는 nociceptin 수용체와 opioid 수용체에모두작용함으로써그효과가상당히중복되어나타난다고한다 (Taylor and Dickenson, 1998; Giuliani et al., 2000). 말초에서 nociceptin 에의하여유도되는통증은일차들통각신경의신경말단에서의 substance P의유리에의존하고 (Inoue et al., 1998), nociceptin을피내주사하면비만세포에서의 histamine 유리를

6 Fig. 6. Proposed relationship between neuropeptides and nociceptin in trigeminovascular system. Solid arrow, increase; dotted arrow, decrease. 증강시키고혈관투과성을증가시키는것으로알려져있다 (Kimura et al., 2000). 그러나사람의들감각신경혹은일차염증세포에대한 nociceptin의작용방식에관한보고는없다. 한편, nociceptin은 capsaicin이나 bradykinin의투여 (Helyes et al., 1997; Habler et al., 1999) 또는뇌막혈관주위의전기자극 (Bartsch et al., 2002) 에의한들감각신경말단으로부터의 substance P와 CGRP의유리를억제함으로써역방향성 (antidromic) 혈관확장을억제하고신경성염증을억제한다고한다 (Helyes et al., 1997; Nmeth et al., 1998). 최근 Hou et al. (2003) 의보고에의하면사람의삼차신경절내에서 nociceptin-유사면역반응과 nociceptin 수용체 mrna가발현되고, CGRP, substance P, nitric oxide synthase 및 pituitary adenylate cyclase activating peptide와함께 nociceptin이공존한다고한다. 이러한보고들로부터 nociceptin과삼차신경혈관계의신경펩티드사이에는긴밀한상호관련이있을것으로추측할수있다. 따라서본연구에서는삼차신경절을전기자극하기전에신생기 capsaicin 처치를하거나, CGRP 1 수용체차단제또는 NK 1 수용체차단제를전처치한후삼차신경절전기자극에의한뇌경막내 nociceptin-유사면역반응의발현을관찰해보았다. 본연구의결과삼차신경절전기자극에의한뇌경막내 nociceptin-유사면역반응의발현이신생기 capsaicin 처치군에서대조군에비하여현저히줄었을뿐만아니라, CGRP 8-37 와 L 전처치에의하여도유의하게줄었음을관찰하였다. 이러한사실은삼차신경절전기자극에의한뇌경막내 nociceptin-유사면역반응의발현에있어서삼차신경혈관계의 CGRP와 substance P 같은신경펩티드가매우긴밀하게관여함을시사하는것으로생각된다. 즉, 삼차신경절전기자극은삼차신경혈관계에신경펩티드를유리시켜뇌경막내신경성염증반응을매개함으로써통증을야기하는데 (Moskowitz, 1992; Williamson et al., 1997a, b), 이때유리된신경펩티드가보상적으로 nociceptin의유리를일으키게되고, 유리된 nociceptin은회귀성으로신경펩티드의유리를억제하게하는기전을추측할수있다 (Fig. 6). 물론삼차신경절전기자극자체에의하여 nociceptin이직접유리될가능성을배제할수는없지만, 본연구에서신생기 capsaicin 처치, CGRP 8-37 그리고 L 전처치에의하여 nociceptin-유사면역반응의발현이모두유의하게감소된결과를감안하여볼때삼차신경혈관계에서의신경펩티드의유리가 nociceptin-유사면역반응의발현에필수적이라고생각되고, 삼차신경절전기자극자체에의한직접적인 nociceptin의유리가능성은희박한것으로생각된다. 나아가본연구에서삼차신경절전기자극시뇌경막내에서 nociceptin-유사면역반응의발현이전압-의존적으로증가하고, [Nphe 1 ]nociceptin(1-13)nh 2 전처치에의하여 nociceptin-유사면역반응의발현이현저히감소됨을관찰할수있었다. 이러한사실은삼차신경혈관계의반응에 nociceptin이밀접하게관여한다는것을시사하는것으로생각할수있다. 그러나삼차신경혈관계에서의 nociceptin 생합성과정을억제하거나 nociceptin을 knockout시킨실험동물을사용하여더연구해볼필요가있다. 뿐만아니라삼차신경혈관계의반응을나타내기위한 nociceptin 수용체가뇌막혈관에구성성으로존재하고있는지또는삼차신경혈관계의자극에의하여 nociceptin 수용체 mrna가발현되는지에관하여는본실험의결과만으로는알수없고앞으로더연구해보아야할과제로남는다. 이상을종합하여보면삼차신경절전기자극은뇌경막내 nociceptin의발현을현저히증가시키며, 여기에는삼차신경혈관계내의신경펩티드가중요하게관여하는것으로생각된다. Acknowledgment This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (R )

7 REFERENCES Andres KH, von During M, Muszynski K, Schmidt RF. Nerve fibres and their terminals of the dura mater encephali of the rat. Anat Embryol (Berl) : Anton B, Fein J, To T, Li X, Silberstein L, Evans CJ. Immunohistochemical localization of ORL-1 in the central nervous system of the rat. J Comp Neurol : Bartsch T, Akerman S, Goadsby PJ. The ORL-1 (NOP1) receptor ligand nociceptin/orphanin FQ (N/OFQ) inhibits neurogenic dural vasodilatation in the rat. Neuropharmacology : Calo' G, Guerrini R, Rizzi A, Salvadori S, Regoli D. Pharmacology of nociceptin and its receptor: a novel therapeutic target. Br J Pharmacol : Edvinsson L. Sensory nerves in man and their role in primary headaches. Cephalalgia : Fukuda K, Kato S, Mori K, Nishi M, Takeshima H, Iwabe N, Miyata T, Houtani T, Sugimoto T. cdna cloning and regional distribution of a novel member of the opioid receptor family. FEBS Lett : Giuliani S, Lecci A, Maggi CA. Nociceptin and neurotransmitter release in the periphery. Peptides : Goadsby PJ, Duckworth JW. Effect of stimulation of trigeminal ganglion on regional cerebral blood flow in cats. Am J Physiol : R270-R274. Grisel JE, Mogil JS. Effects of supraspinal orphanin FQ/nociceptin. Peptides : Habler H, Timmermann L, Stegmann J, Janig W. Effects of nociceptin and nocistatin on antidromic vasodilatation in hairless skin of the rat hindlimb in vivo. Br J Pharmacol : Helyes Z, Németh J, Pintér E, Szolcsányi J. Inhibition by nociceptin of neurogenic inflammation and the release of SP and CGRP from sensory nerve terminals. Br J Pharmacol : Henderson G, McKnight AT. The orphan opioid receptor and its endogenous ligand - nociceptin/orphanin FQ. Trends Pharmacol Sci : Hou M, Uddman R, Tajti J, Edvinsson L. Nociceptin immunoreactivity and receptor mrna in the human trigeminal ganglion. Brain Res : Ikeda K, Watanabe M, Ichikawa T, Kobayashi T, Yano R, Kumanishi T. Distribution of prepro-nociceptin/orphanin FQ mrna and its receptor mrna in developing and adult mouse central nervous systems. J Comp Neurol : Inoue M, Kobayashi M, Kozaki S, Zimmer A, Ueda H. Nociceptin/ orphanin FQ-induced nociceptive responses through substance P release from peripheral nerve endings in mice. Proc Natl Acad Sci U S A : Jancso G, Kiraly E, Jancso-Gabor A. Pharmacologically induced selective degeneration of chemosensitive primary sensory neurones. Nature : Jenck F, Moreau JL, Martin JR, Kilpatrick GJ, Reinscheid RK, Monsma FJ Jr, Nothacker HP, Civelli O. Orphanin FQ acts as an anxiolytic to attenuate behavioral responses to stress. Proc Natl Acad Sci U S A : Kimura T, Kitaichi K, Hiramatsu K, Yoshida M, Ito Y, Kume H, Yamaki K, Suzuki R, Takagi K. Intradermal application of nociceptin increases vascular permeability in rats: the possible involvement of histamine release from mast cells. Eur J Pharmacol : Lachowicz JE, Shen Y, Monsma FJ Jr, Sibley DR. Molecular cloning of a novel G protein-coupled receptor related to the opiate receptor family. J Neurochem : Mayberg MR, Zervas NT, Moskowitz MA. Trigeminal projections to supratentorial pial and dural blood vessels in cats demonstrated by horseradish peroxidase histochemistry. J Comp Neurol : Meunier JC. Nociceptin/orphanin FQ and the opioid receptor-like ORL1 receptor. Eur J Pharmacol : Meunier JC, Mollereau C, Toll L, Suaudeau C, Moisand C, Alvinerie P, Butour JL, Guillemot JC, Ferrara P, Monsarrat B, Mazarguil H, Vassart G, Parmentier M, Costentin J. Isolation and structure of the endogenous agonist of opioid receptorlike ORL1 receptor. Nature : Mollereau C, Simons MJ, Soularue P, Liners F, Vassart G, Meunier JC, Parmentier M. Structure, tissue distribution, and chromosomal localization of the prepronociceptin gene. Proc Natl Acad Sci U S A : Moskowitz MA. Neurogenic versus vascular mechanisms of sumatriptan and ergot alkaloids in migraine. Trends Pharmacol Sci : Moskowitz MA, Buzzi MG. Neuroeffector functions of sensory fibres: implications for headache mechanisms and drug actions. J Neurol : S18-S22. Moskowitz MA, Buzzi MG, Sakas DE, Linnik MD. Pain mechanisms underlying vascular headaches. Progress Report Rev Neurol (Paris) :

8 Németh J, Helyes Z, Oroszi G, Thán M, Pintér E, Szolcsányi J. Inhibition of nociceptin on sensory neuropeptide release and mast cell-mediated plasma extravasation in rats. Eur J Pharmacol : Nabeshima T, Noda Y, Mamiya T. The role of nociceptin in cognition. Brain Res : Noda Y, Mamiya T, Manabe T, Nishi M, Takeshima H, Nabeshima T. Role of nociceptin systems in learning and memory. Peptides : Olesen J, Friberg L, Olsen TS, Iversen HK, Lassen NA, Andersen AR, Karle A. Timing and topography of cerebral blood flow, aura, and headache during migraine attacks. Ann Neurol : Reinscheid RK, Nothacker HP, Bourson A, Ardati A, Henningsen RA, Bunzow JR, Grandy DK, Langen H, Monsma FJ Jr, Civelli O. Orphanin FQ: a neuropeptide that activates an opioidlike G protein-coupled receptor. Science : Steiger HJ, Tew JM Jr, Keller JT. The sensory representation of the dura mater in the trigeminal ganglion of the cat. Neurosci Lett : Taylor F, Dickenson A. Nociceptin/orphanin FQ. A new opioid, a new analgesic? Neuroreport : R65-R70. Tran-Dinh YR, Thurel C, Cunin G, Serrie A, Seylaz J. Cerebral vasodilation after the thermocoagulation of the trigeminal ganglion in humans. Neurosurgery : Williamson DJ, Hargreaves RJ, Hill RG, Shepheard SL. Intravital microscope studies on the effects of neurokinin agonists and calcitonin gene-related peptide on dural vessel diameter in the anaesthetized rat. Cephalalgia. 1997a. 17: Williamson DJ, Hargreaves RJ, Hill RG, Shepheard SL. Sumatriptan inhibits neurogenic vasodilation of dural blood vessels in the anaesthetized rat - intravital microscope studies. Cephalalgia. 1997b. 17: Xu X, Grass S, Hao J, Xu IS, Wiesenfeld-Hallin Z. Nociceptin/ orphanin FQ in spinal nociceptive mechanisms under normal and pathological conditions. Peptides : Yamamoto T, Nozaki-Taguchi N, Sakashita Y, Kimura S. Nociceptin/ orphanin FQ: role in nociceptive information processing. Prog Neurobiol :

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(01) hwp Journal of Life Science 2013 Vol. 23. No. 2. 157~166 ISSN (Print) 1225-9918 ISSN (Online) 2287-3406 DOI : http://dx.doi.org/10.5352/jls.2013.23.2.157 α μ δ κ 158 생명과학회지 2013, Vol. 23. No. 2 Journal of