Wang X, McGregor C G, Miller V M
Division of Cardiothoracic Surgery, Departments of Surgery and Physiology and Biophysics, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Am J Physiol. 1998 Oct;275(4):H1122-9. doi: 10.1152/ajpheart.1998.275.4.H1122.
An inducible isoform of nitric oxide synthase (type II, iNOS) is expressed in cardiac and vascular smooth muscle in response to inflammatory cytokines. The dog is an important large animal used for cardiovascular research including effects of exercise, heart failure, and allograft rejection. However, molecular probes for iNOS developed in other mammals have not been reliable for the study of iNOS induction in canine vascular smooth muscle. Experiments were designed to develop a molecular probe for canine iNOS. Smooth muscle cells were isolated from canine aortas. The cells (passages 3-10) were incubated for 1, 3, 6, 12, 24, 48, or 72 h in the absence and presence of Escherichia coli lipopolysaccharide (LPS) to induce iNOS. Total RNA was isolated from the cells using standard techniques. RT-PCR with primers against conserved regions of all known iNOS enzyme was used to clone the iNOS cDNA. RT-PCR showed a single band only from cells treated with LPS. Cloned cDNA from cultured canine aortic smooth muscle cells has 84% homology to human, 81% to rat, and 81% to mouse iNOS gene. Identification of the cDNA for canine iNOS will be useful in the study of differential, transcriptional regulation of inducible (type II) compared with constitutive endothelial (type III) NOS in canine studies of allograft rejection and cardiovascular disease.
一氧化氮合酶的一种可诱导同工型（II型，诱导型一氧化氮合酶）在心脏和血管平滑肌中表达，以响应炎性细胞因子。犬是用于心血管研究的重要大型动物，包括运动、心力衰竭和同种异体移植排斥反应的影响。然而，在其他哺乳动物中开发的诱导型一氧化氮合酶分子探针在犬血管平滑肌中诱导型一氧化氮合酶的研究中并不可靠。设计实验以开发犬诱导型一氧化氮合酶的分子探针。从犬主动脉分离平滑肌细胞。在不存在和存在大肠杆菌脂多糖（LPS）的情况下，将细胞（传代3至10代）孵育1、3、6、12、24、48或72小时以诱导诱导型一氧化氮合酶。使用标准技术从细胞中分离总RNA。使用针对所有已知诱导型一氧化氮合酶保守区域的引物进行逆转录聚合酶链反应（RT-PCR）以克隆诱导型一氧化氮合酶cDNA。RT-PCR仅显示来自用LPS处理的细胞的单一条带。从培养的犬主动脉平滑肌细胞克隆的cDNA与人类诱导型一氧化氮合酶基因有84%的同源性，与大鼠有81%的同源性，与小鼠有81%的同源性。在犬同种异体移植排斥反应和心血管疾病研究中，鉴定犬诱导型一氧化氮合酶的cDNA将有助于研究诱导型（II型）与组成型内皮型（III型）一氧化氮合酶的差异转录调控。
