Effects of microinjection of antisense nNOS oligonucleotide into the nucleus tractus solitarii on circulation

孤束核显微注射反义 nNOS 寡核苷酸对循环的影响

• 批准号: 10670074
• 负责人: MAEDA Masanobu
• 金额: $ 1.34万
• 依托单位: University of Occupational and Environmental Health
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670074/
• 关键词: nucleus tractus solitarii; nitric oxide; nitric oxide synthase; antisense; oligonucleotide; brain; circulation; molecular biology

We examined the cardiovascular effects of bilateral microinjection of antisense oligodeoxynucleotides (AS-ODNs) to neuronal nitric oxide synthase (nNOS) into the nucleus tractus solitalii (NTS) of Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), and compared the results of SHR with those of WKY to investigate the mechanisms of hypertension of SHR. The AS-ODN 5'-CTTCCATGGTATCTGTG-3' was designed to target an initiation codon of rat nNOS mRNA ; the complementary sense sequence was 5'-CACAGATACCATGGAAG-3' ; the scrambled sequence with the same GC content was 5'-CGTAGTAGGCATACGTA-3'. The ODNs were diluted in artificial cerebrospinal fluid and mixed with cationic lipids for transfection. In urethane-anesthetized, paralyzed WKY, bilateral microinjection of nNOS AS-ODNs (20 pmole in a 50 nl volume) into the NTS produced a significant increase in mean arterial blood pressure (MABP) 30-60 min after injection as compared with rats injected with nNOS sense or scrambled ODNs. Similarly, nNOS AS-ODNs produced a significant increase in heart rate 30 min after microinjection compared with nNOS sense ODNs. In SHR, bilateral microinjection of nNOS AS-ODNs into the NTS produced a significant increase in MABP 15 min after injection compared with rats injected with nNOS sense or scrambled ODNs. The pressor responses in SHR were produced earlier than those in WKY. Immunohistochemical study demonstrated that nNOS-immunoreactivity in the rat NTS was suppressed by nNOS AS-ODNs. These results suggest that expression of the nNOS gene in the NTS of WKY and SHR regulates blood pressure.

研究了Wistar-Kyoto大鼠（WKY）和自发性高血压大鼠（SHR）双侧微注射反义寡脱氧核苷酸（AS-ODNs）对神经元一氧化氮合酶（nNOS）的心血管影响，并将其与自发性高血压大鼠（SHR）的结果进行比较，探讨SHR高血压的机制。AS-ODN 5‘-CTTCCATGGTATCTGTG-3’被设计用于靶向大鼠nNOS mRNA的起始密码子；互补义序列为5′-CACAGATACCATGGAAG-3′；GC含量相同的加乱序列为5′-CGTAGTAGGCATACGTA-3′。odn在人工脑脊液中稀释后与阳离子脂质混合转染。在尿素麻醉、瘫痪的WKY中，双侧微量注射nNOS as -ODNs（20摩尔/ 50毫升）至NTS，与注射nNOS感觉或打乱ODNs的大鼠相比，注射后30-60分钟平均动脉血压（MABP）显著升高。同样，与nNOS感觉odn相比，nNOS as - odn在微注射后30分钟的心率显著增加。在SHR中，双侧显微注射nNOS AS-ODNs到NTS后15分钟，与注射nNOS感觉或打乱ODNs的大鼠相比，MABP显著增加。SHR组的压力反应比WKY组产生得早。免疫组化研究表明，nNOS AS-ODNs可抑制nNOS在大鼠NTS中的免疫反应性。提示WKY和SHR NTS中nNOS基因的表达对血压有调节作用。

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