Downregulation of atrial natriuretic peptide ANP-C receptor is associated with alterations in G-protein expression in A10 smooth muscle cells

Atrial natriuretic peptide (ANP) receptors A and B are guanylyl cyclase receptors, whereas ANP-C receptors are coupled to adenylyl cyclase through inhibitory guanine nucleotide (Gi) protein. ANP has been shown to downregulate ANP-A and -B receptors and cGMP response in various tissues. In the present studies, we have examined the regulation of ANP-C receptor-adenylyl cyclase signal transduction by ANP and des(Gln(18),Ser(19),Gln(20),Leu(21), Gly(22))ANP(4-23)-NH(2)](C-ANP(4-23)) that interacts specifically with ANP-C receptor in A10 smooth muscle cells (SMC). Treatment of the cells with C-ANP(4-23) for 24 h resulted in a reduction in ANP receptor binding activity. (125)I]ANP(99-126) bound to control and C-ANP(4-23)-treated cell membranes at a single site with dissociation constants of 33.7 +/- 6 and 35.0 +/- 4.5 pM and B(max) of 74.0 +/- 5.0 and 57.6 +/- 4.0 fmol/mg of protein, respectively. C-ANP(4-23) inhibited adenylyl cyclase activity in a concentration-dependent manner in control cells. A maximal inhibition observed was about 30-40% with an apparent K(i) of about 1 nM; however, this inhibition was completely attenuated in cells pretreated with ANP(99-126) or C-ANP(4-23) (10(-7) M). However, the inhibition of adenylyl cyclase by 17-amino acid peptide (RRNHQEESNIGKHRELR) (R17A) of cytoplasmic domain of ANP-C receptor was attenuated by about 50% but was not completely abolished by C-ANP(4-23) treatment. The attenuation of C-ANP(4-23)-mediated inhibition of adenylyl cyclase was dependent on the concentration and time of pretreatment of the cells with C-ANP(4-23). In addition, angiotensin II- (Ang II-) mediated inhibition of adenylyl cyclase ( approximately 30%) was also abolished by C-ANP(4-23) treatment, indicating that the desensitization elicited by ANP was heterologous. In addition, C-ANP(4-23) treatment decreased the expression of Gialpha-2 and Gialpha-3 proteins by about 40 and 60%, respectively, and their mRNA by 40%. However, the levels of Gi proteins were not altered when the cells were treated for shorter period of time (2-4 h) or with lower concentrations of C-ANP(4-23) (10(-10) M). On the other hand, the levels of Gsalpha but not of Gbeta were increased by about 35% by C-ANP(4-23) treatment. Furthermore, the stimulations exerted by GTPgammaS, isoproterenol, FSK, and NaF on adenylyl cyclase were also augmented in cells treated with C-ANP(4-23). These results indicate that C-ANP(4-23) treatment of A10 cells desensitizes ANP-C receptor-mediated inhibition of adenylyl cyclase which may be due to the downregulation of ANP-C receptor and decreased expression of Gialpha proteins to which these receptors are coupled.