Xu Z, Li P, Tong C, Figueroa J, Tobin J R, Eisenach J C
Perinatal Research Laboratories, Wake Forest University Medical Center, Winston-Salem, North Carolina, USA.
Anesthesiology. 1996 Apr;84(4):890-9. doi: 10.1097/00000542-199604000-00017.
Nitric oxide synthase is located in the spinal cord dorsal horn and intermediolateral cell column, where it may modulate sensory and sympathetic neuronal activity. However, the biochemical characteristics of this enzyme have not been examined in these different areas in the spinal cord. Although alpha(2)-adrenergic agonists, muscarinic agonists, and nitric oxide may interact in the spinal cord to produce antinociception, these interactions have not been characterized.
Sheep spinal cord tissue was homogenized ad centrifuged at high sped to separate soluble and membrane-bound fractions. Nitric oxide synthase activity was determined by conversion of [(14)C]-L-arginine to [(14)C]-L-citrulline and its kinetic characteristics, dependency on cofactors, and sensitivity to inhibitors determined. Sheep spinal cord was stained for nicotinamide adenine dinucleotide phosphate diaphorase as a marker for nitric oxide synthase. Antinociception to a mechanical stimulus from intrathecal clonidine alone and with neostigmine was determined and the effects of L-arginine and n-methyl-L-arginine were determined.
More than 85% of nitric oxide synthase activity was present in the soluble form and its kinetic, cofactor, and antagonist properties were similar to those of the neuronal isoform of nitric oxide synthase. Biochemical and histochemical studies localized nitric oxide synthase to the superficial dorsal horn and the intermediolateral cell column. Clonidine antinociception was enhanced by L-arginine and neostigmine, but not by D-arginine. Neostigmine's enhancement of clonidine antinociception was blocked by n-methyl-L-arginine.
These results confirm those of previous studies demonstrating localization of nitric oxide synthase to superficial dorsal horn and intermediolateral cell column of mammalian spinal cord, and suggesting its identity as the neuronal isoform. Spinal alpha(2)-adrenergic agonist antinociception may be partly dependent on cholinergic and nitric oxide mechanisms.
一氧化氮合酶位于脊髓背角和中间外侧细胞柱，在这些部位它可能调节感觉和交感神经神经元活动。然而，尚未在脊髓的这些不同区域研究该酶的生化特性。尽管α₂ - 肾上腺素能激动剂、毒蕈碱激动剂和一氧化氮可能在脊髓中相互作用以产生镇痛作用，但这些相互作用尚未得到明确描述。
将绵羊脊髓组织匀浆并高速离心以分离可溶性和膜结合部分。通过将[¹⁴C] - L - 精氨酸转化为[¹⁴C] - L - 瓜氨酸来测定一氧化氮合酶活性，并确定其动力学特性、对辅因子的依赖性以及对抑制剂的敏感性。对绵羊脊髓进行烟酰胺腺嘌呤二核苷酸磷酸黄递酶染色作为一氧化氮合酶的标志物。测定鞘内注射可乐定单独及与新斯的明联合使用时对机械刺激的镇痛作用，并确定L - 精氨酸和N - 甲基 - L - 精氨酸的作用。
超过85%的一氧化氮合酶活性以可溶形式存在，其动力学、辅因子和拮抗剂特性与一氧化氮合酶的神经元同工型相似。生化和组织化学研究将一氧化氮合酶定位于脊髓背角浅层和中间外侧细胞柱。L - 精氨酸和新斯的明增强了可乐定的镇痛作用，但D - 精氨酸没有。新斯的明对可乐定镇痛作用的增强被N - 甲基 - L - 精氨酸阻断。
这些结果证实了先前的研究结果，即一氧化氮合酶定位于哺乳动物脊髓背角浅层和中间外侧细胞柱，并表明其为神经元同工型。脊髓α₂ - 肾上腺素能激动剂的镇痛作用可能部分依赖于胆碱能和一氧化氮机制。
