An oxidized analog of α‐human atrial natriuretic polypeptide is a selective agonist for the atrial‐natriuretic‐polypeptide clearance receptor which lacks a guanylate cyclase

Abstract

The differences in biological functions between a-human atrial natriuretic polypeptide (a-hANP) and its oxidized analog, MetSO-a-hANP, have been investigated. Analysis of the ANP receptor subtypes by affinity labeling has shown that a bovine pulmonary aortic endothelial cell line (CPAE cells) primarily expresses ANP-R1 (R, receptor) coupled to particulate guanylate cyclase, while Hela cells from human cervical carcinoma predominantly express ANP-R2, which lacks a guanylate cyclase. a-hANP could bind to both ANP receptor subtypes with high affinity, while MetSO-a-hANP showed more selective binding to ANP-R2 than to ANP-RI . The activity of MetSO-a-hANP for stimulation of guanylate cyclase coupled to ANP-R1 was about 520-fold less than that of a-hANP (median effective dose = 2.5 nM for a-hANP, 1.3 pM for MetSO-a-hANP), indicating that MetSO-a-hANP was a partial agonist for this receptor. While this oxidized analog could inhibit the cAMP production through ANP-R2, with 0.15 times the activity of a-hANP (median concentration = 0.31 nM for a-hANP, 2.0 nM for MetSO-a-hANP). In in vivo studies, the diuretic activity of MetSO-a-hANP was 25 -100-fold less than that of a-hANP. In addition, MetSO-a-hANP could potentiate the diuretic activity of a-hANP that was also caused by C-ANF4-,,, a specific agonist for ANP-R2.These results demonstrate that MetSO-a-hANP can act as an agonist more selective for ANP-R2 than for ANP-Rl, both in vivo and in vitro. The relationship between receptor selectivities and the conformation of a-hANP or MetSO-a-hANP was also discussed.Atrial natriuretic polypeptides (ANP) are peptide hormones regulating the fluid balance, electrolyte balance, blood pressure and the renin-angiotensin system [l]. ANP evoke their biological activities by interacting with receptors expressed on a cell surface. Two subclasses of ANP receptors, ANP-R1 and ANP-R2, have been isolated and their cDNA have been cloned [2-61. ANP-R1 is a single-chain glycoprotein with a molecular mass of 130 kDa containing a particulate guanylate cyclase in its intracellular domain [7]. The interaction of ANP with ANP-R1 results in the activation of its guanylate cyclase and increases the intracellular cGMP level [l, 51. It is well accepted that cGMP acts as a second messenger for the biological activity of ANP, at least for vasorelaxation, diuresis and natriuresis 11, 81. ANP-R, is a homodimer of two 65 kDa glycoproteins linked by disulfide bonds which lack a guanylate cyclase and is more abundant than ANP-R1 in various tissues 111.Correspondence to S. Koyama and M . Kobayashi, Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 1 -6, a-Chome, Kashima, Yodogawa-ku, Osaka, Japan 532Abbreviations. ANP, atrial natriuretic polypeptide; u-hANP, a-human ANP; B,,,, density of receptors; DMEM, Dulbecco's modified Eagle's medium; Gi, inhibitory guanine-nucleotide-binding regulatory protein; R, receptor; ICs0, median inhibitory concentration; ED,o, median effective dose.Maack and his co-workers have reported that ANP-R2 is biologically silent an...

Characterization of the hormone binding site of natriuretic peptide receptor‐C

“…Testing of synthetic peptide analogs on position 188 mutant receptors confirmed the role of this site in determining the binding properties of rat or human NPR-C. However, in testing synthetic peptides such as hANP(F8Y) [12], hANP(G10K,RI1S) [13], hANP(O-Met) [14] and cANF [15] on the full set of mutant receptors we have now identified a second mutant, rY205N, with an altered pharmacology which is in between the high affinity state of rat C-IgG and the lower affinity state of human C-IgG (Fig. 1).…”

Structural insight into hormone recognition and transmembrane signaling by the atrial natriuretic peptide receptor

“…8). In fact, NPRC has been shown to bind fragments of the natriuretic peptide, and some of these peptides lack the elements that can form pseudo two-fold symmetry in the peptides 31,32 . To bind to peptides lacking pseudo two-fold symmetry and to transmit its binding signal into the cell, the NPRC might require a signal transmission mechanism that does not depend on ligands having a pseudo two-fold symmetry.…”

“…12 -hANP comprises a methionine sulfoxide molecule formed through sulfur oxidation in Met12 of hANP , which inhibits its ANP receptor agonistic properties 30,31 . The crystal structure complexed with hANP (Fig.…”

Novel analog of atrial natriuretic peptide selective for receptor-A produces increased diuresis and natriuresis in rats.

“…When the NPR-C selective analog cANF [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] was coinfused with ANP in the isolated, perfused, rat kidney, these investigators observed a trend toward both increased potency (ED 50 ) and an increased maximal response for urinary sodium excretion (13). An analogous experiment has been described in vivo for coinfusion into rats of hANP and the analog hANP(0-Met) with an oxidized methionine at position 12 (33). This analog is more selective for NPR-C than NPR-A (19,22,33), and when coinfused with hANP there is a synergistic effect on diuresis (natriuresis was not measured) (33).…”

“…An analogous experiment has been described in vivo for coinfusion into rats of hANP and the analog hANP(0-Met) with an oxidized methionine at position 12 (33). This analog is more selective for NPR-C than NPR-A (19,22,33), and when coinfused with hANP there is a synergistic effect on diuresis (natriuresis was not measured) (33). For both of these experiments (13, 33) the selective blocking, or binding, of NPR-C by these ANP analogs resulted in an increased renal response during coinfusion with ANP, consistent with our proposed role for NPR-C as a decoy receptor attenuating renal NPR-A activation.…”