Downregulation of Atrial Natriuretic Peptide ANP-C Receptor Is Associated with Alterations in G-Protein Expression in A10 Smooth Muscle Cells^†

Abstract

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.

Ligand-regulated Internalization, Trafficking, and Down-regulation of Guanylyl Cyclase/Atrial Natriuretic Peptide Receptor-A in Human Embryonic Kidney 293 Cells

“…Our recent data in COS-7 cells transiently expressing NPRA have provided the evidence that its internalization seems to be controlled by sequences located within the carboxyl-terminal domain of this receptor protein (28). The phenomena of receptor-mediated internalization and metabolic processing of ANP through the non-guanylyl cyclasecontaining receptor, NPRC, have been investigated in a number of laboratories utilizing vascular smooth muscle cells, which contain a predominantly high density of endogenous NPRC (35)(36)(37)(38)(39)(40)(41)(42)(43). Early studies of the post-binding events of NPRC were greatly facilitated because of its predominant presence in vascular smooth muscle cells, which are among the important target cells for ANP.…”

Natriuretic Peptide Receptor-C Attenuates Hypertension in Spontaneously Hypertensive Rats

“…NPR-A and NPR-B are membrane guanylyl cyclase receptors, whereas NPR-C does not possess guanylyl cyclase activity. Two different subtypes of NPR-C with molecular mass of 67 and 77 kDa have been identified with a broad range of ligands, including ANP, brain natriuretic peptide, C-type natriuretic peptide, and C-ANP [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] [des(Gln, 18 Ser, 19 Gly, 20 Leu, 21 ; however, C-ANP 4-23 is specific for NPR-C and has no affinity for NPR-A or NPR-B. 8,9 The 77-kDa protein is implicated in ligand internalization as a clearance receptor, 10 whereas 67-kDa protein is coupled to adenylyl cyclase inhibition through inhibitory guanine nucleotide regulatory protein Gi 7,11 or to activation of phospholipase C (PLC).…”

“…8,9 The 77-kDa protein is implicated in ligand internalization as a clearance receptor, 10 whereas 67-kDa protein is coupled to adenylyl cyclase inhibition through inhibitory guanine nucleotide regulatory protein Gi 7,11 or to activation of phospholipase C (PLC). 12 However, we showed that NPR-C activation by C-ANP [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] and resultant decrease in cAMP levels contribute to the activation of phosphatidyl inositol turnover signaling and suggested a cross-talk between NPR-C-mediated adenylyl cyclase inhibition and PLC signaling pathways. 13 The activity of adenylyl cyclase is regulated by 2 guanine nucleotide regulatory proteins (G proteins): Gs (stimulatory) and Gi (inhibitory).…”

“…C-ANP [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] that specifically interacts with NPR-C has been reported to decrease the levels of Giα proteins in A10 vascular smooth muscle cell (VSMC) 23 and in VSMC from SHRs. 24 In addition, C-ANP [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] was also shown to attenuate the enhanced oxidative stress in VSMC from SHRs 24 that contributes to the enhanced expression of Giα proteins in SHRs.…”

“…24 In addition, C-ANP [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] was also shown to attenuate the enhanced oxidative stress in VSMC from SHRs 24 that contributes to the enhanced expression of Giα proteins in SHRs. 25 Taken together, it may be possible that C-ANP 4-23 treatment of SHRs could also attenuate the high BP in SHRs.…”