Expression of endothelial nitric oxide synthase in human and rabbit gastrointestinal smooth muscle cells
Expression of endothelial nitric oxide synthase in human and rabbit gastrointestinal smooth muscle cells
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DOI：
10.1152/ajpgi.1998.275.2.g342
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发表时间：
1998-08-01
影响因子：
4.5
通讯作者：
Makhlouf, GM
中科院分区：
文献类型：
--
作者：
Teng, BQ;Murthy, KS;Makhlouf, GM
本研究的目的是确定一氧化氮合酶（NOS）的异构体表达在新鲜分散的兔胃平滑肌细胞和培养的兔胃，人肠，和豚鼠带结肠平滑肌细胞。预测大小（354 bp）的RT-PCR产物获得内皮型一氧化氮合酶（eNOS）特异性引物，但不是神经元型一氧化氮合酶（nNOS）或诱导型一氧化氮合酶（iNOS）特异性引物，在所有平滑肌制剂，除了豚鼠结肠带。对照RT-PCR研究表明，缺乏内皮标志物，血小板内皮细胞粘附分子-1（PECAM-1）和血管内皮生长因子受体（VEGFR），和间质细胞标志物，c-hit，从平滑肌细胞培养物。克隆和序列分析表明，预测的氨基酸序列（117个残基），在兔和人平滑肌细胞不同，从人eNOS只有一个残基。以PCR扩增和克隆的兔和人eNOS cDNA为探针，进行北方杂交分析，结果表明两种动物均有eNOS mRNA（4.4 kb）的表达。eNOS，但不是nNOS或iNOS，转录本定位在单一的，新鲜分散的兔胃平滑肌细胞原位RT-PCR表达是明显的，在每个制剂中的大多数细胞。我们的结论是，eNOS选择性表达在兔胃和人肠平滑肌细胞。结果证实了功能的证据存在的组成型一氧化氮合酶在不同物种的肠道平滑肌细胞，除了豚鼠结肠带。
The aim of this study was to identify the nitric oxide synthase (NOS) isoform expressed in freshly dispersed rabbit gastric smooth muscle cells and in cultured rabbit gastric, human intestinal, and guinea pig taenia coli smooth muscle cells. RT-PCR products of the predicted size (354 bp) were obtained with endothelial NOS (eNOS)-specific primers, but not neuronal NOS (nNOS)- or inducible NOS (iNOS)-specific primers, in all smooth muscle preparations except guinea pig taenia coli. Control RT-PCR studies showed absence of the endothelial markers, platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial growth factor receptor (VEGFR), and the interstitial cell marker, c-hit, from cultures of smooth muscle cells. Cloning and sequence analysis showed that the predicted amino acid sequence (117 residues) in rabbit and human smooth muscle cells differed by only one residue from that of human eNOS. Northern blot analysis, using the PCR-generated and cloned eNOS cDNA from rabbits and humans as probes, demonstrated the expression of eNOS mRNA (4.4 kb) in both species. eNOS, but not nNOS or iNOS, transcripts were localized by in situ RT-PCR in single, freshly dispersed rabbit gastric smooth muscle cells; expression was evident in the majority of cells in each preparation. We conclude that eNOS is selectively expressed in rabbit gastric and human intestinal smooth muscle cells. The results confirm functional evidence for the existence of a constitutive NOS in smooth muscle cells of the gut in different species, except for guinea pig taenia coli.