Palaparti A, Li Y, Anand-Srivastava M B
Department of Physiology and Groupe de recherche sur le système nerveux autonome, University of Montreal, C.P. 6128, Succ. centre-ville, Montreal, Quebec, Canada H3C 3J7.
Biochem J. 2000 Mar 1;346 Pt 2(Pt 2):313-20.
Atrial natriuretic peptide (ANP) mediates a variety of physiological effects through its interaction with ANP-A, ANP-B or ANP-C receptors. However, controversies exist regarding the involvement of ANP-C receptor and adenylyl cyclase/cAMP signal-transduction systems to which these receptors are coupled in mediating these responses. In the present studies, we have employed an antisense approach to eliminate the ANP-C receptor and to examine the effect of this elimination on adenylyl cyclase inhibition. An 18-mer antisense phosphorothioate oligodeoxynucleotide (OH-2) targeted at the initiation codon of the ANP-C receptor was used to examine its effects on the expression of the ANP-C receptor and ANP-C-receptor-mediated inhibition of adenylyl cyclase in vascular smooth-muscle cells (A10). Treatment of the cells with antisense oligonucleotide resulted in complete attenuation of C-ANP(4-23) [des(Gln(18), Ser(19), Gln(20), Leu(21), Gly(22))ANP(4-23)-NH(2)]-mediated inhibition of adenylyl cyclase, whereas sense and missense oligomers did not affect the inhibition of adenylyl cyclase by C-ANP(4-23). In addition, the stimulatory effects of guanine nucleotides, isoproterenol, sodium fluoride and forskolin as well as the inhibitory effects of angiotensin II on adenylyl cyclase were not affected by antisense-oligonucleotide treatment. The attenuation of C-ANP(4-23)-mediated inhibition of adenylyl cyclase by antisense oligonucleotide was dose- and time-dependent. A complete attenuation of ANP-C-receptor-mediated inhibition of adenylyl cyclase was observed at 2.5 microM. In addition, treatment of the cells with antisense oligonucleotide and not with sense or missense oligomers resulted in the inhibition of the levels of ANP-C-receptor protein and mRNA as determined by immunoblotting and Northern blotting using antisera against the ANP-C receptor and a cDNA probe of the ANP-C receptor respectively. On the other hand, ANP-A/B-receptor-mediated increases in cGMP levels were not inhibited by antisense-oligonucleotide treatment. Our results demonstrate conclusively that the elimination of ANP-C receptor by antisense oligonucleotide attenuates ANP-induced inhibition of adenylyl cyclase and provide evidence that antisense oligonucleotide of the ANP-C receptor may serve as a useful pharmacological tool to elucidate the physiological functions of the ANP-C receptor.
心房利钠肽（ANP）通过与ANP - A、ANP - B或ANP - C受体相互作用介导多种生理效应。然而，关于ANP - C受体以及与其偶联的腺苷酸环化酶/cAMP信号转导系统在介导这些反应中的作用存在争议。在本研究中，我们采用反义方法消除ANP - C受体，并研究这种消除对腺苷酸环化酶抑制作用的影响。一种针对ANP - C受体起始密码子的18聚体硫代磷酸反义寡脱氧核苷酸（OH - 2）被用于研究其对血管平滑肌细胞（A10）中ANP - C受体表达以及ANP - C受体介导的腺苷酸环化酶抑制作用的影响。用反义寡核苷酸处理细胞导致C - ANP(4 - 23) [去(Gln(18), Ser(19), Gln(20), Leu(21), Gly(22))ANP(4 - 23) - NH(2)]介导的腺苷酸环化酶抑制作用完全减弱，而正义和错义寡聚物对C - ANP(4 - 23)抑制腺苷酸环化酶的作用没有影响。此外，鸟嘌呤核苷酸、异丙肾上腺素、氟化钠和福斯可林的刺激作用以及血管紧张素II对腺苷酸环化酶的抑制作用不受反义寡核苷酸处理的影响。反义寡核苷酸对C - ANP(4 - 23)介导的腺苷酸环化酶抑制作用的减弱呈剂量和时间依赖性。在2.5 microM时观察到ANP - C受体介导的腺苷酸环化酶抑制作用完全减弱。此外，用反义寡核苷酸而非正义或错义寡聚物处理细胞导致分别使用抗ANP - C受体抗血清和ANP - C受体cDNA探针通过免疫印迹和Northern印迹测定的ANP - C受体蛋白和mRNA水平受到抑制。另一方面，反义寡核苷酸处理并未抑制ANP - A/B受体介导的cGMP水平升高。我们的结果确凿地表明，反义寡核苷酸消除ANP - C受体可减弱ANP诱导的腺苷酸环化酶抑制作用，并提供证据表明ANP - C受体的反义寡核苷酸可作为阐明ANP - C受体生理功能的有用药理学工具。
