Ikeda U, Shimpo M, Ohki R, Takahashi M, Yamamoto K, Ikeda M, Minota S, Shimada K
Department of Cardiology, Jichi Medical School, Utsunomiya University, Tochigi, Japan.
J Hypertens. 2000 Nov;18(11):1597-604. doi: 10.1097/00004872-200018110-00010.
If calcium channel blockers affect nitric oxide synthesis in the vascular tissue, they could influence disease progression in coronary arteries. We investigated the effects of the calcium channel blocker amlodipine on nitric oxide synthesis by measuring the production of nitrite, a stable metabolite of nitric oxide, in vascular smooth muscle cells.
We measured the production of nitrate in cultured rat vascular smooth muscle cells with the Griess reagent Inducible nitric oxide synthase protein and mRNA expression were assayed by Western blotting and reverse transcription-polymerase chain reaction, respectively. The levels of NF-kappaB proteins in nuclear extracts were analyzed by gel retardation assay.
Incubation of cultures with interleukin-1 , (10 ng/ ml) for 24 h caused a significant increase in nitrite generation. Interleukin-1 l-induced nitrite production by vascular smooth muscle cells was significantly increased by amlodipine in a dose-dependent manner. This augmentative effect of amlodipine was completely abolished in the presence of N(G)-monomethyl-L-arginine or actinomycin D. Amlodipine-induced nitrite production was accompanied by increased inducible nitric oxide synthase mRNA and protein accumulation. Interleukin-1 , induced NF-kappaB activation in vascular smooth muscle cells, and addition of amlodipine further increased this NF-kappaB activation. The effect of amlodipine on nitrite production was maintained in the presence of the calcium channel agonist Bay K 8644.
Amlodipine enhances nitric oxide synthesis in cytokine-stimulated cultured vascular smooth muscle cells by L-type calcium channel-independent mechanisms.
如果钙通道阻滞剂影响血管组织中一氧化氮的合成，那么它们可能会影响冠状动脉疾病的进展。我们通过测量血管平滑肌细胞中一氧化氮的稳定代谢产物亚硝酸盐的生成，来研究钙通道阻滞剂氨氯地平对一氧化氮合成的影响。
我们用格里斯试剂测量培养的大鼠血管平滑肌细胞中硝酸盐的生成。分别通过蛋白质印迹法和逆转录 - 聚合酶链反应检测诱导型一氧化氮合酶蛋白和mRNA的表达。通过凝胶阻滞试验分析核提取物中NF-κB蛋白的水平。
用白细胞介素 -1（10 ng/ml）孵育培养物24小时导致亚硝酸盐生成显著增加。氨氯地平以剂量依赖的方式显著增加了白细胞介素 -1诱导的血管平滑肌细胞亚硝酸盐生成。在存在N（G）-单甲基 -L-精氨酸或放线菌素D的情况下，氨氯地平的这种增强作用完全被消除。氨氯地平诱导的亚硝酸盐生成伴随着诱导型一氧化氮合酶mRNA和蛋白积累的增加。白细胞介素 -1诱导血管平滑肌细胞中NF-κB激活，添加氨氯地平进一步增加了这种NF-κB激活。在存在钙通道激动剂Bay K 8644的情况下，氨氯地平对亚硝酸盐生成的作用得以维持。
氨氯地平通过不依赖L型钙通道的机制增强细胞因子刺激的培养血管平滑肌细胞中一氧化氮的合成。
