Okamoto H, Yayama K, Shibata H, Nagaoka M, Takano M
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Ikawadani-cho, Nishi-ku, Kobe 651-2180, Japan.
Biochim Biophys Acta. 1998 Sep 16;1404(3):329-37. doi: 10.1016/s0167-4889(98)00074-3.
To identify the presence of a local kallikrein-kinin system in vascular wall, we have studied whether rat vascular smooth muscle cells (VSMC) express kininogen in vitro and in vivo. Western blots using anti-T-kininogen antibody revealed the presence of T-kininogen in conditioned medium of cultured VSMC. T-Kininogen secretion by VSMC was markedly enhanced by the addition of lipopolysaccharide (LPS), angiotensin II (AII) and phorbol 12-myristate 13-acetate (PMA) to the culture. Experiments using specific inhibitors for protein kinases and on the PMA-induced down-regulation of protein kinase C suggested that a protein kinase C-dependent or unidentified pathway is involved in AII or LPS action, respectively. The intravenous injection of LPS (0.5 mg/kg) resulted in an increase in T-kininogen mRNA levels in the vascular smooth muscle of rat aorta, peaking at 16 h. Polyacrylamide gel electrophoresis of cDNA products generated by reverse transcription-polymerase chain reaction (RT-PCR) from aortic mRNA using primers specific for either T- or low-molecular-weight kininogen revealed that rat vascular smooth muscle expressed T-kininogen gene but not low-molecular-weight kininogen gene, and that LPS exclusively stimulated T-kininogen expression. The mRNA for high-molecular-weight kininogen was undetectable in either aortic smooth muscle or cultured VSMC by means of RT-PCR analysis. RT-PCR using specific primers for rat tissue kallikrein genes showed that aortic smooth muscle expressed KLK1 (true kallikrein) mRNA, but not KLK10 (T-kininogenase) mRNA. These results demonstrated that rat VSMC are a source of T-kininogen but not of low-molecular-weight- or high-molecular-weight kininogen, in contrast to the expression of true kallikrein but not of T-kininogenase by these cells.
为了确定血管壁中是否存在局部激肽释放酶 - 激肽系统，我们研究了大鼠血管平滑肌细胞（VSMC）在体外和体内是否表达激肽原。使用抗T - 激肽原抗体的蛋白质免疫印迹法显示，在培养的VSMC条件培养基中存在T - 激肽原。通过向培养物中添加脂多糖（LPS）、血管紧张素II（AII）和佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA），VSMC的T - 激肽原分泌显著增强。使用蛋白激酶特异性抑制剂的实验以及关于PMA诱导的蛋白激酶C下调的实验表明，蛋白激酶C依赖性或未明确的途径分别参与了AII或LPS的作用。静脉注射LPS（0.5 mg/kg）导致大鼠主动脉血管平滑肌中T - 激肽原mRNA水平升高，在16小时达到峰值。使用针对T - 或低分子量激肽原的特异性引物，通过逆转录 - 聚合酶链反应（RT - PCR）从主动脉mRNA生成的cDNA产物进行聚丙烯酰胺凝胶电泳显示，大鼠血管平滑肌表达T - 激肽原基因但不表达低分子量激肽原基因，并且LPS专门刺激T - 激肽原表达。通过RT - PCR分析，在主动脉平滑肌或培养的VSMC中均未检测到高分子量激肽原的mRNA。使用大鼠组织激肽释放酶基因特异性引物的RT - PCR表明，主动脉平滑肌表达KLK1（真激肽释放酶）mRNA，但不表达KLK10（T - 激肽原酶）mRNA。这些结果表明，与这些细胞表达真激肽释放酶但不表达T - 激肽原酶相反，大鼠VSMC是T - 激肽原的来源，但不是低分子量或高分子量激肽原的来源。
