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1 J. Exp. Biomed. Sci. 11 (2005) Cerebroprotective Effect of Nociceptin on Transient Focal Cerebral Ischemia in Rats Seung Yoon Lee 1, Won Suk Lee 2 and Chang Hwa Choi 1 1 Department of Neurosurgery, College of Medicine, Pusan National University, Busan , Korea. 2 Department of Pharmacology, College of Medicine, Pusan National University, Busan , Korea This study aimed to investigate the cerebroprotective effect of nociceptin on transient focal cerebral ischemia in Sprague-Dawley rats by determining the changes in regional cerebral blood flow (rcbf) and the infarct size. Right middle cerebral artery (MCA) was occluded for 2 hours, and thereafter was followed by reperfusion by an intraluminal monofilament technique. An open cranial window was made on the right parietal bone for determination of continuous changes in rcbf by laser-doppler flowmetry. The infarct size was morphometrically determined using the 2,3,5- triphenyltetrazolium chloride technique. In normal rats, nociceptin (0.01~100 nmol/kg, i.v.) increased rcbf and decreased cerebral arterial resistance in a dose-dependent manner. Systemic arterial blood pressure was little affected by nociceptin at the doses of 0.01 and 0.1 nmol/kg, but dose-dependently reduced at the doses of 1 nmol/kg or more. In transient cerebral ischemic rats, nociceptin (0.01~0.1 nmol/kg, i.p.) significantly attenuated the postischemic cerebral hyperemia, and progressively increased rcbf. The improving effect of nociceptin on the postischemic rcbf response was markedly blocked by pretreatment with [Nphe 1 ]nociceptin(1-13)nh 2 (1 nmol/kg, i.p.), a selective nociceptin receptor antagonist, but not by naloxone (3 µmol/kg, i.p.), a selective opioid receptor antagonist. The cerebral infarct size was significantly reduced by nociceptin (0.01~0.1 nmol/kg) administered i.p. 5 min after MCA occlusion in transient cerebral ischemia of 2-hour MCA occlusion and 22-hour reperfusion. It is suggested that nociceptin improves the postischemic cerebral hemodynamics and thereby has a cerebroprotective effect in transient focal cerebral ischemia. Key Words: Nociceptin, Transient focal cerebral ischemia, Rat 서 Nociceptin은 prepronociceptin이라는전구물질에서유래되는 17개의아미노산으로구성되어있는근래에발견된펩티드로서그생리학적기능과병적인상태에서의역할에대한연구가활발히진행되고있다. Prepronociceptin은 enkephalin 과 dynorphin 등의내인성아편유사펩티드의전구물질인 preproenkephalin 및 preprodynorphin과그구조가유사하고 (Houtani et al., 1996; Mollereau et al., 1996; Nothacker et al., 1996; Ikeda et al., 1998; Neal et al., 1999), dynorphin 또는 α-neoendorphin 등과아미노산배열이유사하다고밝혀졌다 (Meunier et al., 1995; Reinscheid et al., 1995; Mollereau et al., * 논문접수 : 2005 년 5 월 3 일수정재접수 : 2005 년 5 월 19 일 교신저자 : 최창화, ( 우 ) 부산광역시서구아미동 1 가 10 번지, 부산대학교의과대학신경외과학교실 Tel: , Fax: chwachoi@pusan.ac.kr 론 1996). 또한 nociceptin이결합하는수용체역시 µ, δ 그리고 κ 수용체와 C-말단부위에서약 60% 의동질성을갖고있어서아편유사제와비슷한작용을할것으로기대되었다 (Fukuda et al., 1994; Mollereau et al., 1994; Wick et al., 1994; Lachowicz et al., 1995). 그러나 N-말단의첫아미노산이 tyrosine으로시작하는다른아편유사펩티드와는달리 phenylalanine으로시작되고, µ, δ 그리고 κ 수용체에대하여친화력이아주낮으며 (Fukuda et al., 1994; Lachowicz et al., 1995), 그분포와생체효과에있어서아편유사제와다르므로새로운펩티드로인식되고있다. Nociceptin은척수상부동통과민또는통증유발 (Reinscheid et al., 1995; Hara et al., 1997), 척수진통 (King et al., 1997), 자발운동감소 (Reinscheid et al., 1995), 항불안 (Jenck et al., 2000), 학습억제 (Yu et al., 1997; Noda et al., 2000) 등을유발한다고알려져있지만, nociceptin과 nociceptin 수용체의분포가대뇌와척수에광범위하게이루어지고있어보다다양한생리작용을가지고있을것으로여겨지고있다 (Henderson and McKnight, 1997; Meunier, 1997; Taylor and Dic

2 kenson, 1998). 한편대뇌허혈에의한뇌손상에있어서 β-endorphin이나 dynorphin 등의아편유사제가관련되어있음은잘알려져있다. 즉인간의뇌경색시뇌척수액내에 β-endorphin이증가되어있고 (Avery et al., 1983; Baskin et al., 1986), 선택적인아편유사물질수용체길항제인 naloxone에의하여뇌혈류증진 (Baskin and Hosobuchi, 1981; Faden et al., 1982; Turner et al., 1984; Choi and Rothman, 1990; Baskin et al., 1994), 신경학적기능개선 (Hosobuchi et al., 1982; Levy et al., 1982; Furui et al., 1984; Hariri et al., 1986; Hara et al., 1993), 경색부위의크기감소 (Miller and Perry, 1989) 및생존율의향상 (Zabramski et al., 1984) 등과같은효과를얻을수있음이보고되었다. 또한 U50,488과같은 κ-수용체효능약물은허혈을일으키기전뿐만아니라일으킨후에투여하여도대뇌허혈손상을감소시킨다고보고되었다 (Baskin et al., 1994). 대뇌허혈에있어서 nociceptin이유의한역할을할것이라는가능성으로는 nociceptin이허혈성뇌손상유발의주된물질인 glutamate의유리를억제하고 (Nicol et al., 1996), glutamate에의한흥분성신경전도를억제하며 (Faber et al., 1996; Liebel et al., 1997; Yu et al., 1997; Shu et al., 1998; Yu and Xie, 1998) 뇌손상에직접관여하는전압-의존성 Ca 2+ 통로를억제한다 (Connor et al., 1996; Abdulla and Smith, 1997; Connor and Christie, 1998; Morikawa et al., 1998) 는점들을바탕으로추론할수있다. 그러나 nociceptin이국소뇌허혈시어떠한역할을하는지에관하여는보고된바가거의없다. 따라서본연구에서는실험동물에게일과성국소뇌허혈을일으키고그이후에초래되는뇌혈류역학의변동과뇌조직손상에대한 nociceptin의효과를검토함으로써대뇌허혈시 nociceptin의역할을관찰하고자하였다. 재료및방법 1. 실험동물체중 300~350 g의 Sprague-Dawley계숫쥐를사육장 ( 실내온도, 20~25 ) 내에서물과사료를마음대로섭취하게하면서사육하였다가실험에사용하였다. 2. 일반수술조작실험동물을 urethane (1 g/kg) 으로복강내주사하여마취시키고, 체온을 37 로유지시키기위하여소동물용자동보온전기방석 (homeothermic blanket system, Harvard Apparatus, Edenbridge, Kent, UK) 위에놓고앙와위로고정하였다. 기관절개술을행하여 14G catheter로기도를확보하고 gallamine (3~5 mg/kg, i.m.) 을투여하여골격근을마비시켰다. 동물용호흡기 (model 683, Harvard Apparatus, South Natick, MA, USA) 를사용하여분당 50~60회의호흡을유지시켰다. 양측대퇴동맥에 PE-50 polyethylene 관을삽관하고이를통하여혈액 gas 및 ph를측정하여 (STAT Profile 3, Nova Biochemicals, Boston, MA, USA) PaCO 2 가 33~45 mmhg가되도록호흡을조절한후이를유지시켰다. 3. 혈압측정전신혈압의변동을관찰하기위하여실험동물의대퇴동맥에삽입된 polyethylene tube에연결된 pressure transducer (Statham P23D, Gould, Cleveland, OH, USA) 를통하여혈압을측정하여 MacLab (4s, ADInstruments, Castle Hill, Australia) 과 Macintosh computer (Power Macintosh 7500/100, Seoul, Korea) 로구성된 data acquisition system에기록하였다. 4. 개방두개창설치일반적인수술조작이끝난후실험동물의체위를복와위로변경시키고두부를뇌정위기구 (stereotaxic apparatus, model 900, David Kopf Instruments, Tujunga, CA, USA) 에고정시켰다. 정중선을따라두피를절개한후우측두정골에 saline-cooled drill (model 395, Dremel, Racine, WI, USA) 을사용하여두개절제술 (5 5 mm) 을행하고미리가온한인공뇌척수액 (37 ) 을관류하였다. 사용한인공뇌척수액의조성은다음과같다 : Na meq/l, K meq/l, Ca meq/l, Mg meq/l, Cl meq/l, HCO meq/l, dextrose 66.5 mg/dl, 그리고 urea 40.2 mg/dl (ph 7.4). 5. 국소뇌혈류및뇌동맥저항측정실험동물의두부를 stereotaxic apparatus에고정시키고, 개방두개창을통하여 laser-doppler flowmeter (BLF21, Transonic Systems Inc., Ithaca, NY, USA) 용 needle probe (type NS, Transonic Systems Inc., Ithaca, NY, USA) 를대뇌피질표면에수직이되도록 stereotactic micromanipulator를사용하여뇌연막동맥에조심스럽게접근시켰다. 일정시간동안안정시킨후국소뇌혈류량을측정하였다. Laser-Doppler flowmeter에의하여측정된국소뇌혈류량의변동은 MacLab과 Macintosh computer로구성된 data acquisition system에기록하였다. 한편뇌동맥의저항은평균동맥압을국소뇌혈류로나누어줌으로써계산하였다. 6. 일과성국소뇌허혈뇌경색의실험동물모델로서널리사용하는중간대뇌동맥 (middle cerebral artery, MCA) 폐쇄는 Longa 등 (1989) 의방법에따라시행하였다. 실험동물의복측정중선을따라경부를절개하고, 우측외경동맥의분지들을전기소작시켜절단하고, 내경동맥의분지인익구개동맥의기시부를 7~0 나

3 일론봉합사로결찰하여총경동맥의분지중두개외에존재하는것은오로지내경동맥만이남아있도록하였다. 그후외경동맥의기시부주위를 6~0 견봉합사로느슨하게묶은다음총경동맥을 6~0 견봉합사로결찰하고내경동맥에미세혈관용클립을장치하여혈류를차단시키고, 미세수술용가위로써외경동맥에작은구멍을만들어그내강을통하여말단부를둥글게만든길이약 2 cm의 3~0 단선조나일론봉합사를내경동맥내로삽입하였다. 출혈을방지하기위하여혈관내의나일론봉합사주위를외경동맥기부주위에느슨하게있던견봉합사로졸라맨후미세혈관용클립을제거하였다. 나일론봉합사를천천히진입시켜그끝이 MCA 의기시부에도달되도록하여 MCA를폐쇄하였다. MCA 폐쇄를 2시간동안유지시켰다가내경동맥내에삽입되어있는단선조나일론봉합사를제거해줌으로써혈액재관류를시행하였다. MCA의폐쇄및혈액재관류의성공여부는검안경 (ophthalmoscope) 을사용하여실험동물의허혈동측의안저동맥을관찰함으로써확인하였다. 그후경부의절개부위를봉합하고마취가깰때까지보온상자내에서회복토록한후일반사육상자내로옮겨실온에서사료와물을마음대로섭취하도록하였다. 7. 뇌경색크기측정 MCA 폐쇄 24시간후에과량의마취제를투여한후단두치사시키고, 전체뇌를두개골로부터적출하였다. 적출한뇌를뇌주형 (RBM-4000C, ASI, USA) 위에놓고, 전두엽말단에서부터후방으로 2 mm 간격으로절단하여 7개의연속관상뇌절편을만들고, Bederson 등 (1986) 의방법에따라 2% 2,3,5-triphenyltetrazolium chloride (TTC) 용액에담구어실온에서 30분간처치한후 10% 중성 formalin 완충액으로고정시켰다. 고정된뇌조직절편의배측면을실체현미경 (SV6, Carl Zeiss, Germany) 에연결된비디오칼러프린터 (Model CVP-G7, Sony, Japan) 로사진을찍고, 각절편에생긴뇌경색부위의면적을면적계 (KP-21, Koizumi, Japan) 로써측정하였다. 8. 사용약물본연구에사용된약물은 nociceptin (Phoenix Pharmaceuticals, Mountain View, CA, USA), [Nphe 1 ]nociceptin(1-13)nh 2 (Tocris Cookson Ltd, Avonmouth, Bristol, UK) 및 naloxone (Sigma, Natick, MA, USA) 이다. 모든시약들은일단 0.9% 생리식염수에용해시켰다가투여경로에따라 0.9% 생리식염수또는인공뇌척수액으로희석하여사용하였다. 9. 통계처리모든측정치는평균 ± 평균의표준오차로표시하였다. 각 Fig. 1. Effects of nociceptin on the cerebral hemodynamics in normal rat. Nociceptin (0.01~100 nmol/kg) was administered i.v. at a 30-min interval. The number of animals was five. Arrow heads indicate the time of bolus injection of nociceptin. rcbf, regional cerebral blood flow; MABP, mean arterial blood pressure. 군사이의유의성검정은반복측정이원배치분산분석법으로분석한후 Dunnett의다중비교법으로사후검정하여 P 값이 0.05 미만인것을유의하다고판정하였다. 결 과 1. Nociceptin 이뇌혈류역학에미치는효과 정상실험동물에게 nociceptin (0.01~100 nmol/kg) 을 30분간격으로정맥내투여한후시간경과에따라초래되는국소뇌혈류, 평균동맥압및뇌동맥저항등의변동을관찰하였다. Fig. 1에서보는바와같이 nociceptin은국소뇌혈류를용량- 의존적으로증가시켰다 (P<0.0001). 반면 nociceptin은 0.1 nmol/kg 이하의용량에서는평균동맥압 ( 기저치평균동맥압, 86.30±8.92 mmhg) 에별영향을미치지아니하였지만, 1 nmol/kg 이상의용량에서는평균동맥압을용량- 의존적으로감소시켰다 (P=0.001). 한편평균동맥압을국소뇌혈류로나누어줌으로써얻은뇌동맥의저항은 nociceptin 0.1 nmol/kg 에서부터용량-의존적으로감소되었다 (P<0.001). 따라서본연구에서는 nociceptin 0.01 및 0.1 nmol/kg을적정용량으로선택하여실험에사용하였다. 2. 일과성국소뇌허혈로인한국소뇌혈류변동에미치는효과 뇌허혈 -재관류동안의국소뇌혈류의변동을 laser-doppler flowmetry 법으로측정한결과대조군의경우국소뇌혈류는뇌허혈기간동안거의차단되었고, 혈액재관류와함께초래된반응성뇌충혈상태를약 1.5시간동안보이다가시간경과에따라점차감소되었다 (Fig. 2). 그러나 nociceptin

4 처치군에서는혈액재관류직후의반응성뇌충혈상태를현저히억제시켰고 (P<0.0001; nociceptin 0.01 nmol/kg의경우 P<0.0001; nociceptin 0.1 nmol/kg의경우 P<0.0001), 그이후 nociceptin 0.01 nmol/kg의경우더이상의유의한변동을보이지않다가시간경과에따라점차감소되었고, nociceptin 0.01 nmol/kg의경우완만한증가를보이다가재관류 2시간이후부터는증가된상태를유지하였다. 3. Nociceptin 에의한허혈성국소뇌혈류반응개선에대한아편유사물질길항제전처치효과일과성국소뇌허혈로인한국소뇌혈류의변동이 nociceptin 0.1 nmol/kg에의하여호전되는효과에관여하는수용체를구명하기위하여 µ-, δ- 및 κ-수용체에대하여모두상경 Fig. 2. Effect of nociceptin on the alterations of regional cerebral blood flow (rcbf) following transient focal cerebral ischemia. Nociceptin (NC) was administered i.p. 5 min after right middle cerebral artery (MCA) occlusion. Numbers in parentheses represent the numbers of animals., MCA occlusion;, reperfusion. Fig. 3. Blockade by pretreatment with opioid receptor family antagonists of the nociceptin-induced improvement of postischemic rcbf response. Pretreatment with [Nphe 1 ]nociceptin(1-13)nh 2 (1 nmol/kg), naloxone (3 µmol/kg), and vehicle (0.9% normal saline) was undertaken i.p. 15 min before MCA occlusion, respectively. Nociceptin (0.1 nmol/ kg) was administered i.p. 5 min after MCA occlusion. Numbers in parentheses represent the numbers of animals., MCA occlusion;, reperfusion. Fig. 4. Effect of nociceptin on the cerebral infarct size (left, infarct area; right, infarct volume) induced by transient focal cerebral ischemia. Right MCA was occluded for 2 hours and followed by reperfusion for 22 hours. Nociceptin (NC) was administered i.p. 5 min after MCA occlusion. Numbers in parentheses and columns indicate the numbers of animals. *P< 0.05; **P<0.01 vs. corresponding vehicle group

5 적으로길항작용을하는 naloxone과 orphan opioid 수용체 (nociceptin 수용체 ) 에선택적으로길항작용을하는 [Nphe 1 ] nociceptin(1-13)nh 2 의전처치효과를비교관찰하였다 (Fig. 3). Nociceptin 0.1 nmol/kg에의한허혈성국소뇌혈류반응호전효과는 naloxone 3 µmol/kg 전처치에의하여하등의영향을받지아니하였으나, [Nphe 1 ]nociceptin(1-13)nh 2 1 nmol/ kg 전처치에의하여거의완전히차단되었다 (P<0.0001). A B C 4. 뇌경색형성에미치는효과 일과성국소뇌허혈을실험동물에게일으키고, 전체뇌를적출하여 2% TTC 용액으로염색을해본결과허혈동측대뇌반구의 MCA 분포영역에육안적으로뚜렷이구별되는뇌경색이형성되었다. 전체뇌를전두엽말단에서부터후방으로 2 mm간격으로절단하여얻은 7 개의연속관상절편각각의경색면적은 Fig. 4 및 Fig. 5에서보는바와같이 nociceptin 0.1 nmol/kg 처치에의하여 1번과 7번절편을제외한나머지절편에서유의하게감소되어 (2, 3 및 6번, 각각 P<0.05; 4 및 5번, P< 0.01) 뇌절편전반에걸쳐유의하게감소되었다 (P<0.0001). 나아가뇌전체에형성된뇌경색의크기를비교하였을때 nociceptin 0.1 nmol/kg 처치군 (182.42±13.71 mm 3 ) 의뇌경색크기는대조군 (291.80±22.30 mm 3, 반대측대뇌반구의 ±0.05%) 에비하여현저히감소되었다 (P<0.01). Fig. 5. Representative photographs of cerebral infarction in 2-mm thick slices of the cerebrum of transient MCA occlusion. Vehicle (A) or nociceptin (B, 0.1 nmol/kg) was administered i.p. 5 min after MCA occlusion. C, Pretreatment with [Nphe 1 ]nociceptin (1-13)NH 2 (1 nmol/kg, i.p.) 15 min before MCA occlusion and treatment with nociceptin (0.1 nmol/kg, i.p.) 5 min after MCA occlusion. Fig. 6. Blockade by [Nphe 1 ]nociceptin(1-13)nh 2 of nociceptin effect on the cerebral infarct size (left, infarct area; right, infarct volume) in transient focal cerebral ischemia. [Nphe 1 ]nociceptin(1-13)nh 2 ([Nphe 1 ]NC, 1 nmol/kg) was administered i.p. 15 min before MCA occlusion. Nociceptin (NC, 0.1 nmol/kg) was admi- nistered i.p. 5 min after MCA occlusion. Numbers in parentheses and columns indicate the numbers of animals. V, vehicle. *P< 0.05; **P<0.01 vs. corresponding vehicle group. #P<0.05 vs. V+NC group

6 5. Nociceptin 에의한뇌경색크기감소에대한 nociceptin 수용체길항제전처치효과일과성국소뇌허혈로인한뇌경색형성이 nociceptin에의하여감소되는효과가허혈성국소뇌혈류변동에대한실험 (Fig. 3) 에서본바와같이 [Nphe 1 ]nociceptin(1-13)nh 2 전처치에의하여차단되는지를관찰하였다. MCA를폐쇄시키기 15 분전에 [Nphe 1 ]nociceptin(1-13)nh 2 1 nmol/kg을복강내처치하였을때 nociceptin 0.1 nmol/kg에의한각뇌절편의경색면적및뇌전체경색의크기감소효과는 Fig. 5 및 Fig. 6에서보는바와같이 [Nphe 1 ]nociceptin (1-13)NH 2 처치에의하여유의하게차단되었다 ( 경색면적 P<0.01; 경색크기 P<0.05). 고찰본연구에서는일과성국소뇌허혈을일으킨실험동물에서 nociceptin이뇌허혈후에초래되는뇌혈류역학의변동을개선시키고뇌경색의크기를감소시킴이관찰되었다. Nociceptin이혈관계에미치는효과에관하여는몇몇보고들이나와있는바 Gumusel 등 (1997) 은고양이의적출콩팥동맥, 목동맥, 넙다리동맥에대한 in vitro 실험에서 nociceptin은저농도 (pmol/l 범위 ) 에서부터농도-의존적으로혈관긴장도를감소시켰다고하였고, Hugghins 등 (2000) 도흰쥐의적출대동맥의긴장도가 nociceptin의농도에의존적으로감소하였다고보고하였다. 이들의보고를바탕으로볼때 nociceptin은말초장기에대하여어떤중요한생물학적작용을가지고있으며, 전신혈압과국소혈류의조절에관여할것이라고추측되고있다. 사람의혈장내 nociceptin의농도 ( 약 10 pg/ml) 는매우낮아서 (Brooks et al., 1998) 생리적조건에서는순환혈중의 nociceptin은심혈관계에대하여말초적으로거의작용을일으키지않으나, 말초에염증이발생되면일차지각신경특히후근신경절에서 prepronociceptin mrna의발현과 nociceptin 의유리가현저히증가하여염증반응으로인한저혈압발생에관여한다고한다 (Andoh et al., 1997). 본연구에서 nociceptin (1 nmol/kg 이상 ) 을정맥내로투여하였을때용량-의존적으로혈압하강이초래되었다. 뿐만아니라동맥혈관의저항도용량 -의존적으로감소되었다. Nociceptin은화학구조상많은양전하를띠고있어혈액-뇌장벽을통과하지못하므로외부에서정맥내로투여한 nociceptin에의한혈압하강현상은중추성기전에의한다기보다는말초적으로직접혈관에작용한결과라고생각된다. 본연구에서 nociceptin 정맥투여에의하여국소뇌혈류변동이용량 -의존적으로일어남을관찰할수있었다. 저용량의 nociceptin (0.1 nmol/kg 미만 ) 은전신동맥압과혈관저항그리고국소뇌혈류에유의한변동을일으키지못하였지만, 0.1~1 nmol/kg에서는전신동맥압과혈관저항에큰변동을일으키지않으면서국소뇌혈류를증가시켰고, 그이상의용량에서는더이상의국소뇌혈류증가를일으키지는아니하였다. 이와같이고용량의 nociceptin에서국소뇌혈류의증가가더이상일어나지않은것은 nociceptin에의하여혈관확장과전신혈압하강작용이강하게초래되었기때문으로생각된다. 이결과에서알수있듯이 nociceptin은전신동맥압에는큰영향을미치지않으면서국소뇌혈류에영향을미칠수있는치료용량의범위가매우좁기때문에향후의보다깊은연구와임상적응용을위해서는전신혈압에는거의영향을미치지않는 nociceptin 유도체의개발이필요할것으로여겨진다. Champion 등 (1998; 1999; 2002) 의보고에따르면혈관내피세포를제거하거나 nitric oxide 합성효소억제제인 N ω - nitro-l-arginine을처치하여도 nociceptin의혈관이완작용이여전히존재하므로 nociceptin의혈관이완작용에는내피세포에서유래된 nitric oxide의관련성이배제되고, muscarine성수용체나 naloxone-민감성아편유사물질수용체또는 calcitonin gene-related peptide 수용체등의활성과도관계없고, 아드레날린성신경기능의억제와도관련이없으며, 혈관이완성 prostaglandin이나 ATP-민감성 K + 통로의개구와도관련이없다고한다. 본연구의결과 nociceptin은허혈-재관류에의한국소뇌혈류변동을개선시켜주었는데, 특히 nociceptin 0.1 nmol/kg은혈액재관류와함께초래되는반응성뇌충혈상태를현저히억제시키고점진적으로국소뇌혈류를증가시켜주었다. 이러한 nociceptin의작용은 µ-, δ- 및 κ-수용체모두에대하여상경적길항제인 naloxone 전처치에의하여는아무런영향을받지아니하였지만 nociceptin 수용체에대하여효현작용이없으면서선택적으로길항작용을나타내는 [Nphe 1 ]nociceptin(1-13)nh 2 (Calo' et al., 2000) 전처치에의하여차단됨을관찰할수있었다. 본연구에서 nociceptin이국소뇌혈류를증가시키는데관여한기전에대하여앞에서 Champion 등 (1998; 1999) 이배제시켰던여러기전과의연관성유무를확인하지는못하였지만 nociceptin 수용체에매개된기전이라는것을찾아낸점은의의가있다고생각되며앞으로세포분자수준에서의자세한기전까지구명하여야할과제로남는다. 뇌허혈은뇌조직혈류량의감소로인해세포에산소와포도당의공급이원활치못함으로써세포내 ATP의고갈을유발하고, 이에따라 Na + -K + pump, Ca 2+ -ATPase 등이억제됨으로써세포내 Ca 2+ 과 Na + 농도를증가시키고, 반면세포외 K + 농도를증가시켜세포의탈분극을일으키게된다. 이탈분극은 glutamate성신경세포에서 glutamate의급격한유리를

7 일으켜흥분독성 (excitotoxicity) 을유발하게된다 (Choi and Rothman, 1990). 유리된 glutamate는다시연접후신경에존재하는 glutamate 수용체에작용하여연접후신경세포내 Ca 2+ 과 Na + 농도증가에따른탈분극을일으켜그신경세포가함유하고있는신경전달물질을유리시키고, 한편세포내 Ca 2+ 농도의증가는 protein kinase C, protein kinase A, phospholipases, Ca 2+ -activated neutral peptidase 또는 DNAse 등을활성화하여이로인해세포막의손상, DNA의손상, prostaglandins의유리에따른 oxygen free radicals의증가가일어나고, mitochondria내 Ca 2+ 과부하로인하여 oxidative phosphorylation이억제됨으로써 ATP 고갈이심화되어결국세포의사망이초래되게된다 (Hara et al., 1993). 이러한일련의허혈성뇌손상의병태생리학적기전에있어서 nociceptin이어떤역할을수행하는지는아직자세히밝혀지지않은채 nociceptin에의한 glutamate의유리억제 (Nicol et al., 1996), glutamate에의한흥분성신경전도억제 (Faber et al., 1996; Liebel et al., 1997; Yu et al., 1997; Shu et al., 1998; Yu and Xie, 1998) norepinephrine의유리감소 (Schlicker et al., 1998), K + 전도도의증가 (Vaughan and Christie, 1996; Vaughan et al., 1997; Ikeda et al., 1997) 및전압-의존성 Ca 2+ 통로억제 (Connor et al., 1996; Abdulla and Smith, 1997; Connor and Christie, 1998; Morikawa et al., 1998) 등의작용들을바탕으로대뇌허혈에있어서유의한역할을할것이라는가능성만제시되어있는실정이다. 본연구에서 nociceptin이뇌경색의크기를유의하게감소시키고, 이효과는 nociceptin 수용체길항제인 [Nphe 1 ]nociceptin(1-13)nh 2 에의하여길항된다는것을처음으로밝힌결과는매우고무적이라고할수있다. 비록 nociceptin 수용체가 G 단백질에연계되어있고 (Chen et al., 1994; Fukuda et al., 1994; Mollereau et al., 1994; Lachowicz et al., 1995), 뇌내에도존재하며 (Meunier et al., 1995; Reinscheid et al., 1995), 흥분시 cyclic AMP의축적을억제한다고는알려져있지만 (Meunier et al., 1995; Reinscheid et al., 1995; Wu et al., 1997; Okawa et al., 1998) nociceptin이어떤기전을통하여뇌보호효과를나타내었는지본연구의결과만으로는확실히는알수없으나아마도혈액재관류직후에일어나는반응성뇌충혈을억제시켜줌으로써이때동반되어초래되는다량의세포독성물질의생성을감소시켜주었기때문으로생각된다. 그러나이러한가설을증명하기위하여는앞으로더욱자세한연구가뒤따라야할과제로남는다. 본연구를통하여 nociceptin이뇌허혈로인한뇌혈류역학의변동을개선시키고뇌경색을감소시킨다는것이밝혀짐으로써향후허혈성뇌손상의진행을억제하고뇌순환개선효과가큰신약개발과적용등에대한새로운방향을제시할수있을것으로기대되는바가크다. REFERENCES Abdulla FA, Smith PA. Nociceptin inhibits T-type Ca 2+ channel current in rat sensory neurons by a G-protein-independent mechanism. J Neurosci : Andoh T, Itoh M, Kuraishi Y. Nociceptin gene expression in rat dorsal root ganglia induced by peripheral inflammation. Neuroreport : Avery SF, Crockard HA, Russell RW. Improved survival following severe cerebral ischemia using naloxone. J Cereb Blood Flow Metab : S331-S332. Baskin DS, Hosobuchi Y. Naloxone reversal of ischemic neurological deficits in man. Lancet : Baskin DS, Hosobuchi Y, Grevel JC. Treatment of experimental stroke with opiate antagonists: Effects on neurological function, infarct size and survival. J Neurosurg : Baskin DS, Widmayer MA, Browning JL, Heizer ML, Schmidt WK. Evaluation of delayed treatment of focal cerebral ischemia with three selective κ-opioid agonists in cats. Stroke : Bederson JB, Pitts LH, Germano SM, Nishimura MC, Davis RL, Bartkowski HM. Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke : Brooks H, Elton CD, Smart D, Rowbotham DJ, McKnight AT, Lambert DG. Identification of nociceptin in human cerebrospinal fluid: comparison of levels in pain and non-pain states. Pain : Calo' G, Guerrini R, Bigoni R, Rizzi A, Marzola G, Okawa H, Bianchi C, Lambert DG, Salvadori S, Regoli D. Characterization of [Nphe1]nociceptin(1-13)NH 2, a new selective nociceptin receptor antagonist. Br J Pharmacol : Champion HC, Bivalacqua TJ, Zadina JE, Kastin AJ, Hyman AL, Kadowitz PJ. Role of nitric oxide in mediating vasodilator responses to opioid peptides in the rat. Clin Exp Pharmacol Physiol : Champion HC, Bivalacqua TJ, Zadina JE, Kastin AJ, Kadowitz PJ. Vasodilator responses to the endomorphin peptides, but not nociceptin/ofq, are mediated by nitric oxide release. Ann N Y Acad Sci : Champion HC, Pierce RL, Kadowitz PJ. Nociceptin, a novel endogenous ligand for the ORL1 receptor, dilates isolated resistance arteries from the rat. Regul Pept :

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