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Shou-Bin Ning, Zhong-Bing Zhang, Wei-Fen Xie, Xiu-Jiang Yang, Department of Gastroenterology, Changzheng Hospital, The Second Military Medical University 200003, Shanghai, China
Qian Sheng, Department of Laboratory Diagnosis, Changhai Hospital, The Second Military Medical University 200433, Shanghai, China
Xin Zhao, Department of Vascular Surgery, Changhai Hospital, The Second Military Medical University 200433, Shanghai, China
Shuan-Li Xin, Department of cardiology, Changzheng Hospital, The Second Military Medical University 200003, Shanghai, China
Correspondence to: Zhong-Bing Zhang, Department of Gastroenterology, Changzheng Hospital, The Second Military Medical University 200003, Shanghai, China. zhongbingzhang@hotmail.com
Received: October 9, 2002 Revised: October 20, 2002 Accepted: October 30, 2002 Published online: July 15, 2003
AIM
To generate an adenoviral vector carrying endothelial nitric-oxide synthase (eNOS) gene in order to mediate the expression of eNOS gene in gastrointestinal smooth muscle cells (SMC) and assess the enzyme activity of eNOS.
METHODS
A recombinant adenovirus (Ad-eNOS) containing the bovine eNOS cDNA fragment was generated by homologous recombination in bacteria. The SMC of distal part of esophagus and gastric fundus of cat were isolated and cultured in vitro and infected with Ad-eNOS. The expression of eNOS gene was detected by Western blot and RT-PCR. The enzyme activity of NOS and the output of NO in SMC were measured by NOS and NO assay kit, furthermore, the different effects of given factors on the enzyme activity and the yield of NO were studied.
RESULTS
The Ad-eNOS can infect the cultured SMC efficiently (MOI = 50, infection rate = 74%). Western blot and RT-PCR confirmed the expression of eNOS in those infected cells. After the cells had been infected with Ad-eNOS, the basal activity of NOS significantly increased from 47±13 nkat/L to 93±13/L (P<0.05), and the level of NO in cell culture supernatants increased by 3 fold (45±13 vs 16±7 μmol/L). In the presence of L-arginine (NOS enzyme substrate), calcium, EGTA (calcium chelating agent), and L-NAME (NOS inhibitor), NOS activity was 94±8, 173±25, 29±6, 58±11 nkat/L and NO level was 48±14, 106±18, 6±2, 17±11 μmol/L, respectively.
CONCLUSION
The constructed recombinant adenovirus, Ad-eNOS, can efficiently mediate the expression of eNOS gene in cultured SMC of digestive tract. The activity of eNOS can be regulated by the concentration of calcium. L-arginine is Not the rate-limiting step for nitric oxide generation from endothelial nitric oxide synthase.
Key Words: N/A
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