一氧化氮(NO)是一個半衰期只有幾秒的小分子，其合成是經由三種不同類型的一氧化氮合成酶所催化合成的，分別為內皮型一氧化氮合成酶(eNOS)、神經型一氧化氮合成酶(nNOS)及誘導型一氧化氮合成酶(iNOS)三種。正常生理狀態下內皮型及神經型一氧化氮合成酶都會持續性的表達產生一氧化氮。而正常生理濃度的一氧化氮對於血管功能的調節扮演著重要的角色。相反的，當病人受到感染後會促使誘導型一氧化氮合成酶表達而產生大量的一氧化氮，造成嚴重的低血壓。因此，專一性的抑制誘導型一氧化氮合成酶的活性，可做為治療敗血症休克的策略之一。所以，我們的研究實驗目的即是從經由N-nitro-L-arginine (NLA)所衍生合成得到的一氧化氮抑制劑中，找出對於誘導型一氧化氮合成酶具有選擇性的抑制劑。
我們利用以lipopolysaccharide (LPS)活化的RAW264.7巨噬細胞細胞株來檢測合成的物質對於誘導型一氧化氮合成酶活性的抑制效果。這些一氧化氮酶的合成抑制劑依結構分為兩類: NEx (x=2~4)與NH2Ex (x=1~10)。結果顯示NE2、NE3及NE4的IC50分別為19.37、14.93及12.37 microM，這一類的抑制劑也會阻斷經由電刺激所誘導的成豬腦血管擴張作用，其IC50分別為2.12、0.93及1.03 microM。而在NH2Ex型的合成抑制劑中，NH2E7~10對誘導型一氧化氮合成酶的活性有較強的抑制作用，其IC50為9.18~25.21 microM，與NE2~NE4的IC50相近。NH2Ex類的抑制劑對於電刺激所誘導產生的血管擴張沒有很強的抑制作用，其IC50都大於100 microM。我們更進一步取用NE4, NH2E7, NH2E10檢測其對於eNOS活性的作用，結果顯示，此三種藥物在濃度100 microM時對eNOS活性並沒有顯著性影響。
我們的實驗結果顯示NEx為非專一性一氧化氮合成酶抑制劑，而NH2E7~10為選擇性的誘導型一氧化氮合成酶抑制劑，其中以NH2E7及NH2E10的選擇性最好。

Synthesis of Nitric oxide (NO), a gas molecule with a half-life of few seconds, is catalyzed by three NOS isoforms: eNOS, nNOS and iNOS. The neuronal (nNOS) and endothelial (eNOS) isoforms are constitutively expressed. The physiological concentration of NO is important in regulating normal vascular function. In contrast, iNOS, which is induced following infection, generates large amount of NO with severe hypotension. In patients with sepsis, specific inhibition of iNOS activity is therefore an alternative strategy in treating septic shock. The purpose of this study is to search for preferential iNOS inhibitors derived from nitro-L-arginine (NLA). We examined the inhibitory effects of these synthetic NLA derivatives on iNOS activity in activated RAW264.7 macrophages. One group of derivative with modification made on hydroxyl moiety of NLA (Nω-nitro-L-arginine alkyl ester, NEx) and the second group with modification made on hydroxyl and nitro moieties of NLA (Nω-amino-L-arginine alkyl ester, NH2Ex). The results indicated that these compounds inhibited NO synthesis. The IC50 values of NE2 and NE4 were 12.37 and 19.37 �慆icroM, respectively. This NEx group of inhibitors also blocked transmural nerve stimulation (TNS)-induced relaxation of isolated porcine basilar arterial rings with IC50 values of 0.93~2.12 �慆icroM. The NH2Ex group, NH2E7~10, was more potent inhibitors than NEx group for iNOS activity with IC50 value of 9.18~25.21 �慆icroM. The NH2Ex inhibitor, however, was rather insensitive in blocking TNS-induced relaxation of the porcine basilar arteries with the IC50 values of greater than 100 �慆icroM, suggesting that the derivatives of the NH2Ex have significantly less effect than the NEx derivatives on nNOS activity. We further observed that NE4, NH2E7, and NH2E10 did not affect endothelium-dependent, eNOS-mediated relaxation, suggesting that these inhibitors did not affect eNOS activity. These results indicate that the NEx derivatives are nonspecific NOS inhibitors, while NH2E7~10 are preferential iNOS inhibitors.

Contents
Abbreviations------------------------------------------------------2
中文摘要-----------------------------------------------------------3
Abstract-----------------------------------------------------------5
Introduction-------------------------------------------------------7
Nitric oxide-------------------------------------------------------7
Constitutive NOS enzymes and their physiological role--------------8
Inducible NOS and its role in pathology----------------------------9
NOS inhibitors-----------------------------------------------------11
Therapeutic use of NOS inhibitors during sepsis--------------------11
Purpose------------------------------------------------------------13
Material and Methods-----------------------------------------------14
Results------------------------------------------------------------22
Discussion---------------------------------------------------------28
Conclusion---------------------------------------------------------31
Reference----------------------------------------------------------32
Figures------------------------------------------------------------35

References
Avontuur JA, Bruining HA and Ince C (1995) Inhibition of nitric oxide synthesis causes myocardial ischemia in endotoxemic rats. Circ Res 76:418-425.
Babu BR, Frey C and Griffith OW (1999) L-arginine binding to nitric-oxide synthase. The role of H-bonds to the nonreactive guanidinium nitrogens. J Biol Chem 274:25218-25226.
Baeuerle PA and Baltimore D (1996) NF-kappa B: ten years after. Cell 87:13-20.
Baldwin AS, Jr. (1996) The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu Rev Immunol 14:649-683.
Barouch LA, Harrison RW, Skaf MW, Rosas GO, Cappola TP, Kobeissi ZA, Hobai IA, Lemmon CA, Burnett AL, O'Rourke B, Rodriguez ER, Huang PL, Lima JA, Berkowitz DE and Hare JM (2002) Nitric oxide regulates the heart by spatial confinement of nitric oxide synthase isoforms. Nature 416:337-339.
Cohen RI, Shapir Y, Chen L and Scharf SM (1998) Right ventricular overload causes the decrease in cardiac output after nitric oxide synthesis inhibition in endotoxemia. Crit Care Med 26:738-747.
Coleman JW (2001) Nitric oxide in immunity and inflammation. Int Immunopharmacol 1:1397-1406.
Crane BR, Arvai AS, Ghosh DK, Wu C, Getzoff ED, Stuehr DJ and Tainer JA (1998) Structure of nitric oxide synthase oxygenase dimer with pterin and substrate. Science 279:2121-2126.
De Sanctis GT, MacLean JA, Hamada K, Mehta S, Scott JA, Jiao A, Yandava CN, Kobzik L, Wolyniec WW, Fabian AJ, Venugopal CS, Grasemann H, Huang PL and Drazen JM (1999) Contribution of nitric oxide synthases 1, 2, and 3 to airway hyperresponsiveness and inflammation in a murine model of asthma. J Exp Med 189:1621-1630.
Fedorov R, Hartmann E, Ghosh DK and Schlichting I (2003) Structural basis for the specificity of the nitric-oxide synthase inhibitors W1400 and Nomega-propyl-L-Arg for the inducible and neuronal isoforms. J Biol Chem 278:45818-45825.
Fedorov R, Vasan R, Ghosh DK and Schlichting I (2004) Structures of nitric oxide synthase isoforms complexed with the inhibitor AR-R17477 suggest a rational basis for specificity and inhibitor design. Proc Natl Acad Sci U S A 101:5892-5897.
Fleming WW, Westfall DP, De la Lande IS and Jellett LB (1972) Log-normal distribution of equiefective doses of norepinephrine and acetylcholine in several tissues. J Pharmacol Exp Ther 181:339-345.
Griffith OW and Stuehr DJ (1995) Nitric oxide synthases: properties and catalytic mechanism. Annu Rev Physiol 57:707-736.
Guzik TJ, West NE, Pillai R, Taggart DP and Channon KM (2002) Nitric oxide modulates superoxide release and peroxynitrite formation in human blood vessels. Hypertension 39:1088-1094.
Hibbs JB, Jr., Taintor RR, Vavrin Z and Rachlin EM (1988) Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem Biophys Res Commun 157:87-94.
Ignarro LJ (2002) Nitric oxide as a unique signaling molecule in the vascular system: a historical overview. J Physiol Pharmacol 53:503-514.
Ischiropoulos H and al-Mehdi AB (1995) Peroxynitrite-mediated oxidative protein modifications. FEBS Lett 364:279-282.
Kirkeboen KA and Strand OA (1999) The role of nitric oxide in sepsis--an overview. Acta Anaesthesiol Scand 43:275-288.
Kobzik L, Reid MB, Bredt DS and Stamler JS (1994) Nitric oxide in skeletal muscle. Nature 372:546-548.
Lee TJ, Su C and Bevan JA (1976) Neurogenic sympathetic vasoconstriction of the rabbit basilar artery. Circ Res 39:120-126.
Lee TJ, Zhang W and Sarwinski S (2000) Presynaptic beta(2)-adrenoceptors mediate nicotine-induced NOergic neurogenic dilation in porcine basilar arteries. Am J Physiol Heart Circ Physiol 279:H808-816.
Li H and Poulos TL (2005) Structure-function studies on nitric oxide synthases. J Inorg Biochem 99:293-305.
Marletta MA (1994) Nitric oxide synthase: aspects concerning structure and catalysis. Cell 78:927-930.
Meyer J, Lentz CW, Stothert JC, Jr., Traber LD, Herndon DN and Traber DL (1994) Effects of nitric oxide synthesis inhibition in hyperdynamic endotoxemia. Crit Care Med 22:306-312.
Michel T and Feron O (1997) Nitric oxide synthases: which, where, how, and why? J Clin Invest 100:2146-2152.
Napoli C and Ignarro LJ (2001) Nitric oxide and atherosclerosis. Nitric Oxide 5:88-97.
Okamoto I, Abe M, Shibata K, Shimizu N, Sakata N, Katsuragi T and Tanaka K (2000) Evaluating the role of inducible nitric oxide synthase using a novel and selective inducible nitric oxide synthase inhibitor in septic lung injury produced by cecal ligation and puncture. Am J Respir Crit Care Med 162:716-722.
Parratt JR (1997) Nitric oxide. A key mediator in sepsis and endotoxaemia? J Physiol Pharmacol 48:493-506.
Proskuryakov SY, Konoplyannikov AG, Skvortsov VG, Mandrugin AA and Fedoseev VM (2005) Structure and activity of NO synthase inhibitors specific to the L-arginine binding site. Biochemistry (Mosc) 70:8-23.
Raman CS, Li H, Martasek P, Babu BR, Griffith OW, Masters BS and Poulos TL (2001a) Implications for isoform-selective inhibitor design derived from the binding mode of bulky isothioureas to the heme domain of endothelial nitric-oxide synthase. J Biol Chem 276:26486-26491.
Raman CS, Li H, Martasek P, Kral V, Masters BS and Poulos TL (1998) Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center. Cell 95:939-950.
Raman CS, Li H, Martasek P, Southan G, Masters BS and Poulos TL (2001b) Crystal structure of nitric oxide synthase bound to nitro indazole reveals a novel inactivation mechanism. Biochemistry 40:13448-13455.
Rand MJ (1992) Nitrergic transmission: nitric oxide as a mediator of non-adrenergic, non-cholinergic neuro-effector transmission. Clin. Exp. Pharmacol. Physiol. 19:147~169.
Salvemini D, Ischiropoulos H and Cuzzocrea S (2003) Roles of nitric oxide and superoxide in inflammation. Methods Mol Biol 225:291-303.
Xie Q and Nathan C (1994) The high-output nitric oxide pathway: role and regulation. J Leukoc Biol 56:576-582.
Zhang HQ, Fast W, Marletta MA, Martasek P and Silverman RB (1997) Potent and selective inhibition of neuronal nitric oxide synthase by N omega-propyl-L-arginine. J Med Chem 40:3869-3870.
Zhang W, Edvinsson L and Lee TJ (1998) Mechanism of nicotine-induced relaxation in the porcine basilar artery. J Pharmacol Exp Ther 284:790-797.