2-amino-4-methylpyridine as a potent inhibitor of inducible NO synthase activity in vitro and in vivo
2-amino-4-methylpyridine as a potent inhibitor of inducible NO synthase activity in vitro and in vivo
复制标题
DOI：
10.1111/j.1476-5381.1996.tb16010.x
复制
发表时间：
1996-11-01
影响因子：
7.3
通讯作者：
Pettipher, ER
中科院分区：
文献类型：
--
作者：
Faraci, WS;Nagel, AA;Pettipher, ER
1在体外和体内研究了2-氨基-4-甲基吡啶抑制诱导型一氧化氮合酶（NOS II）催化活性的能力。2在体外，2-氨基-4-甲基吡啶抑制小鼠RAW 264.7细胞的NOS II活性，IC 50为6 nM。酶动力学研究表明，抑制是竞争性的精氨酸。2-氨基-4-甲基吡啶对人重组NOS II的效力较低（IC 50 =40 nM），对人重组NOS I和NOS III的效力仍然较低（IC 50 =100 nM）。N-G-单甲基-L-精氨酸（L-NMMA）、N-6-亚氨基乙基-L-赖氨酸（L-NIL）和氨基胍对小鼠NOS II的抑制作用比2-氨基-4-甲基吡啶弱得多，但与2-氨基-4-甲基吡啶不同，它们对人重组NOS II的活性相似。L-NMMA以相似的效力抑制所有三种NOS亚型（IC（50）为3-7 μ M）。相反，与对人重组NOS III的活性相比，L-NIL对小鼠NOS II的选择性为10倍，对人重组NOS II的选择性为11倍，而氨基胍对小鼠NOS II的选择性为7.3倍，对人重组NOS II的选择性为3.7倍。3小鼠RAW 264.7巨噬细胞在脂多糖（LPS）存在下培养过夜时产生高水平的亚硝酸盐。和干扰素-γ。添加2-氨基-4-甲基吡啶的同时，LPS和IFN-γ，剂量依赖性地降低亚硝酸盐的水平（IC 50 =1.5 μ M），而不影响诱导的NOS II蛋白。增加精氨酸的细胞外浓度降低了2-氨基-4-甲基吡啶的效力，但在浓度高达10 μ M时，2-氨基-4-甲基吡啶并不抑制[H-3]-精氨酸摄取到细胞中。在酶诱导后加入2-氨基-4-甲基吡啶也剂量依赖性地抑制亚硝酸盐的产生。总之，这些数据表明，2-氨基-4-甲基吡啶通过竞争性抑制NOS II的催化活性来减少NO的细胞产生，这与体外酶动力学研究获得的结果一致。2-amino-4-methylpyridine抑制腹腔注射LPS引起的血浆硝酸盐升高（ID 50 =0.009 mg kg（-1）min（-1））。在未治疗的清醒大鼠中，需要更大剂量的2-氨基-4-甲基吡啶来升高平均动脉压（艾德（50）=0.060 mg kg（-1）min（-1）），这表明体内NOS II的选择性是NOS III的6.9倍。在相同条件下，L-NMMA无选择性，而L-NIL和氨基胍的选择性分别为5.2倍和8.6倍。所有这些化合物在高于抑制体内NOS II活性的ID 50的剂量下均引起平均动脉压显著增加。5 2-氨基-4-甲基吡啶也抑制皮下注射后LPS诱导的血浆硝酸盐升高。（ID 50 =0.3 mg kg（-1））或口服（ID 50 =20.8 mg kg（-1））给药。6这些数据表明，甲基吡啶是一种有效的NOS II活性抑制剂，在体外和体内具有类似程度的同功酶选择性的L-NIL和氨基胍在啮齿类动物。
1 The ability of 2-amino-4-methylpyridine to inhibit the catalytic activity of the inducible NO synthase (NOS II) enzyme was characterized in vitro and in vivo.2 In vitro, 2-amino-4-methylpyridine inhibited NOS II activity derived from mouse RAW 264.7 cells with an IC50 Of 6 nM. Enzyme kinetic studies indicated that inhibition is competitive with respect to arginine. 2-Amino-4-methylpyridine was less potent on human recombinant NOS II (IC50=40 nM) and was still less potent on human recombinant NOS I and NOS III (IC50=100 nM). N-G-monomethyI-L-arginine (L-NMMA), N-6-iminoethyl-L-lysine (L-NIL) and aminoguanidine were much weaker inhibitors of murine NOS II than 2-amino-4-methylpyridine but, unlike 2-amino-4-methylpyridine, retained similar activity on human recombinant NOS II. L-NMMA inhibited all three NOS isoforms with similar potency (IC(50)s 3-7 mu M). In contrast, compared to activity on human recombinant NOS III, L-NIL displayed 10x selectivity for murine NOS II and 11x selectivity for human recombinant NOS II while aminoguanidine displayed 7.3x selectivity for murine NOS II and 3.7x selectivity for human recombinant NOS II.3 Mouse RAW 264.7 macrophages produced high levels of nitrite when cultured overnight in the presence of lipopolysaccharide (LPS) and interferon-gamma. Addition of 2-amino-4-methylpyridine at the same time as the LPS and IFN-gamma, dose-dependently reduced the levels of nitrite (IC50=1.5 mu M) without affecting the induction of NOS II protein. Increasing the extracellular concentration of arginine decreased the potency of 2-amino-4-methylpyridine but at concentrations up to 10 mu M, 2-amino-4-methylpyridine did not inhibit the uptake of [H-3]-arginine into the cell. Addition of 2-amino-4-methylpyridine after the enzyme was induced also dose-dependently inhibited nitrite production. Together, these data suggest that 2-amino-4-methylpyridine reduces cellular production of NO by competitive inhibition of the catalytic activity of NOS II, in agreement with results obtained from in vitro enzyme kinetic studies.4 When infused i.v. in conscious unrestrained rats, 2-amino-4-methylpyridine inhibited the rise in plasma nitrate produced in response to intraperitoneal injection of LPS (ID50=0.009 mg kg(-1) min(-1)). Larger doses of 2-amino-4-methylpyridine were required to raise mean arterial pressure in untreated conscious rats (ED(50)=0.060 mg kg(-1) min(-1)) indicating 6.9x selectivity for NOS II over NOS III in vivo. Under the same conditions, L-NMMA was nonselective while L-NIL and aminoguanidine displayed 5.2x and 8.6x selectivity respectively. All of these compounds caused significant increases in mean arterial pressure at doses above the ID50 for inhibition of NOS II activity in vivo.5 2-Amino-4-methylpyridine also inhibited LPS-induced elevation in plasma nitrate after either subcutaneous (ID50=0.3 mg kg(-1)) or oral (ID50=20.8 mg kg(-1)) administration.6 These data indicate that 2-amino-4-methylpyridine is a potent inhibitor of NOS II activity in vitro and in vivo with a similar degree of isozyme selectivity to that of L-NIL and aminoguanidine in rodents.