Huang Yong, Lu Min-Qiang, Li Hua, Xu Chi, Yi Shu-Hong, Chen Gui-Hua
Liver Transplantation Centre, the Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510630, Guangdong Province, China.
World J Gastroenterol. 2006 Sep 14;12(34):5483-9. doi: 10.3748/wjg.v12.i34.5483.
To examine the existence of Nitric oxide/cGMP sensitive store-operated Ca(2+) entry in mouse fibroblast NIH/3T3 cells and its influence on matrix metalloproteinase (MMP) production and adhesion ability of fibroblasts.
NIH/3T3 cells were cultured. Confocal laser scanning microscopy was used to examine the existence of thapsigargin-induced store-operated Ca(2+) entry in fibroblasts. Gelatin zymography and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) were employed to detect the involvement of [Ca(2+)]i and NO/cGMP in MMP secretion. The involvement of NO/cGMP-sensitive Ca(2+) entry in adhesion was determined using matrigel-coated culture plates.
8-bromo-cGMP inhibited the thapsigargin-induced Ca(2+) entry in 3T3 cells. The cGMP-induced inhibition was abolished by an inhibitor of protein kinase G, KT5823 (1 micromol/L). A similar effect on the Ca(2+) entry was observed in 3T3 cells in response to a NO donor, (+/-)-S-nitroso-N-acetylpenicillamine (SNAP). The inhibitory effect of SNAP on the thapsigargin-induced Ca(2+) entry was also observed, indicating NO/cGMP-regulated Ca(2+) entry in 3T3 cells. Results of gelatin zymography assay showed that addition of extracellular Ca(2+) concentration induced MMP release and activation in a dose-dependent manner. RT-PCR also showed that cGMP and SNAP reduced the production of MMP mRNA in 3T3 cells. Experiments investigating adhesion potentials demonstrated that cGMP and SNAP could upgrade 3T3 cell attachment rate to the matrigel-coated culture plates.
NO/cGMP sensitive store-operated Ca(2+) entry occurs in fibroblasts, and attenuates their adhesion potentials through its influence on MMP secretion.
研究一氧化氮/环磷酸鸟苷（NO/cGMP）敏感的钙库操纵性钙离子内流在小鼠成纤维细胞NIH/3T3细胞中的存在情况及其对成纤维细胞基质金属蛋白酶（MMP）产生和黏附能力的影响。
培养NIH/3T3细胞。采用共聚焦激光扫描显微镜检测毒胡萝卜素诱导的成纤维细胞钙库操纵性钙离子内流的存在情况。用明胶酶谱法和半定量逆转录聚合酶链反应（RT-PCR）检测细胞内钙离子浓度（[Ca(2+)]i）和NO/cGMP在MMP分泌中的作用。使用基质胶包被的培养板确定NO/cGMP敏感的钙离子内流在黏附中的作用。
8-溴环磷酸鸟苷（8-bromo-cGMP）抑制毒胡萝卜素诱导的3T3细胞钙离子内流。蛋白激酶G抑制剂KT5823（1 μmol/L）可消除cGMP诱导的抑制作用。在3T3细胞中，一氧化氮供体（±）-S-亚硝基-N-乙酰青霉胺（SNAP）对钙离子内流也有类似作用。也观察到SNAP对毒胡萝卜素诱导的钙离子内流的抑制作用，表明3T3细胞中存在NO/cGMP调节的钙离子内流。明胶酶谱分析结果显示，增加细胞外钙离子浓度以剂量依赖方式诱导MMP释放和激活。RT-PCR也显示cGMP和SNAP可降低3T3细胞中MMP mRNA的产生。研究黏附潜能的实验表明，cGMP和SNAP可提高3T3细胞在基质胶包被培养板上的附着率。
成纤维细胞中存在NO/cGMP敏感的钙库操纵性钙离子内流，其通过影响MMP分泌减弱成纤维细胞的黏附潜能。
