[Extracellular Ca(2+) influx and NO generation are inhibited by small interference RNA targeting extracellular Ca(2+)-sensing receptor in human umbilical vein endothelial cells].
To investigate the effect of  Ca(2+)-sensing receptor (CaR) on Spermine-induced extracellular Ca(2+)  influx and NO generation in human umbilical vein endothelial cells (HUVEC), the small interference RNA (siRNA) specifically targeting CaR gene was designed, synthesized and transfected into HUVEC according to the cDNA sequence of human CaR gene in GenBank. The transfection efficiency and the interference efficiency of CaR protein were determined by laser scanning confocal microscopy and Western blot, respectively. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by Fura-2/AM loading. The production of NO and the activity of endothelial nitric oxide synthase (eNOS) were determined by the DAF-FM diacetate (DAF-FM DA). Western blot results demonstrated that siRNA targeting the CaR specifically decreased the expression of CaR protein in CaR siRNA group 48 h after transfection (P < 0.05). At the same time, the Spermine-induced [Ca(2+)](i), eNOS activity and NO generation were also significantly reduced (P < 0.05) in CaR siRNA group compared with those in the untransfected or negative siRNA transfected group. In conclusion, the present study suggests that the CaR plays an important role in the Spermine-evoked process of extracellular Ca(2+) influx and NO generation in HUVEC.
Department of Pathophysiology, Medical College of Shihezi University, Shihezi, China.
Xiao
X
Liang
Xiao-Lin
XL
Luo
Hua
H
Zhong
Qing-Hua
QH
Hu
Fang
F
He
D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D061307:Human Umbilical Vein Endothelial Cells / Q000502:physiology*; D006801:Humans; D009569:Nitric Oxide / Q000502:physiology*; D052250:Nitric Oxide Synthase Type III / Q000502:physiology; D034741:RNA, Small Interfering; D044169:Receptors, Calcium-Sensing / Q000235:genetics / Q000502:physiology*; D013096:Spermine / Q000494:pharmacology; D014162:Transfection
D034741:RNA, Small Interfering; D044169:Receptors, Calcium-Sensing; D013096:Spermine; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D002118:Calcium