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

本实验观察了双侧孤束核内微量注射反义寡核苷酸(AS-ODN)和神经元型一氧化氮合酶(NNOS)对自发性高血压大鼠(SHR)和自发性高血压大鼠(SHR)的心血管效应，并比较了SHR和WKY的结果，以探讨SHR的高血压机制。设计了针对大鼠nNOS基因起始密码子的AS-ODN 5‘-CTTCCATGGATCTGTG-3’，互补正义序列为5‘-CAGATACCATGGAAG-3’，具有相同GC含量的加扰序列为5‘-CGTAGTAGGCATACGTA-3’。将ODN在人工脑脊液中稀释，与阳离子脂类混合后进行转染。在乌拉坦麻醉、麻痹的WKY大鼠，双侧NTS微量注射nNOS AS-ODN(50nL体积20pmole)30-60min后，平均动脉压(MABP)显著高于注射nNOS正义或杂乱ODN的大鼠。同样，与nNOS正义寡核苷酸相比，nNOS反义寡核苷酸在注射30min后心率显著增加。在自发性高血压大鼠，双侧NTS微量注射nNOS AS-ODN后15分钟MABP显著高于注射nNOS正义或杂乱ODN的大鼠。SHR的升压反应早于WKY。免疫组织化学研究表明，nNOS AS-ODN抑制了大鼠NTS内的nNOS免疫反应。上述结果提示，WKY和SHR NTS中nNOS基因的表达对血压有调节作用。

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.