Inhibition of atrial natriuretic peptide (ANP) C receptor expression by antisense oligodeoxynucleotides in A10 vascular smooth-muscle cells is associated with attenuation of ANP-C-receptor-mediated inhibition of adenylyl cyclase

摘要

pAtrial 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-ANPsub4-23/sub [des(Glnsup18/sup, Sersup19/sup, Glnsup20/sup, Leusup21/sup, Glysup22/sup)ANPsub4-23/sub-NHsub2/sub]-mediated inhibition of adenylyl cyclase, whereas sense and missense oligomers did not affect the inhibition of adenylyl cyclase by C-ANPsub4-23/sub. 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-ANPsub4-23/sub-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 iμ/iM. 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./p