Restriction of a peptide turn conformation and conformational analysis of guanidino group using arginine-proline fused amino acids: application to mini atrial natriuretic peptide on binding to the receptor.

Compounds (2S,4S)- and (2S,4R)-4-(2'-guanidinoethyl)proline have been synthesized as a conformationally restricted arginine. Their backbones fit the i + 1 position in a turn, and the side chains are restricted compared to that of arginine. These analogues were incorporated into mini atrial natriuretic polypeptide, which has an important turnlike conformation at Gly(6)-Arg(7)()-Met(8)-Asp(9). Structural analysis revealed that the size of the conformational space of Arg(7) on binding to the receptor was approximately one-third of the entire conformational space.

Hemodynamic, hormonal, and renal effects of atrial natriuretic peptide in untreated congestive cardiac failure.

We report the effects of intravenous infusion of the atrial natriuretic peptide analogue, met-ANP-26 (2 micrograms/min for 2 to 4 hours), in four patients with cardiomyopathy and severe congestive cardiac failure who had not received any previous cardiac therapy. The average cardiac index before infusion was 1.8 L/min/m2. Severe sodium and water retention was confirmed by high levels of total body water and extracellular liquid, whereas renal blood flow and glomerular filtration rate were reduced. Plasma concentration of ANP, norepinephrine, cortisol, and growth hormone were significantly increased before infusion. The infusion had no significant hemodynamic effect. After 2 hours urine volume had increased significantly from 51 to 76 ml/hr, urinary concentration of sodium from 72 to 90 mmol/L, and sodium excretion from 4.5 to 8.2 mmol/hr. The infusion was accompanied by a significant increase in plasma ir-ANP from 193 to 980 pg/ml. There were no significant effects on the plasma concentrations of norepinephrine, epinephrine, aldosterone, vasopressin, cortisol, growth hormone, or prolactin and no significant change in plasma renin activity. After 2 hours of infusion one patient had a severe sinus tachycardia and another had a sinus bradycardia. Both arrhythmias disappeared without harmful effects soon after the infusion was stopped.

A dicarba analog of beta-atrial natriuretic peptide (beta-ANP) inhibits guanosine 3',5'-cyclic monophosphate production induced by alpha-ANP in cultured rat vascular smooth muscle cells.

The synthesis and biological properties are described of [Asu7,23']-beta-ANP-(7-28) (Asu, L-alpha-aminosuberic acid), a dicarba analog of beta-atrial natriuretic peptide (beta-ANP, an antiparallel dimer of human alpha-ANP with the chains linked by 7-23' and 7'-23 disulfide bonds). This Asu-analog (referred to as analog III) displaced 125I-alpha-ANP specifically bound to cultured rat vascular smooth muscle cells (VSMC) with an apparent Ki of 2.1 x 10(-8) M, but did not stimulate formation of intracellular cGMP at 10(-8) -10(-5) M. Analog III inhibited the alpha-ANP-stimulated cGMP production in VSMC competitively with a pA2 value of 7.45 and behaved as an antagonist of alpha-ANP in rat aorta smooth muscle relaxation. In addition, beta-ANP was also shown to inhibit the alpha-ANP-induced cGMP production in a dose-dependent manner. The mechanism of action of beta-ANP is also discussed.

Identification of structural requirements for analogues of atrial natriuretic peptide (ANP) to discriminate between ANP receptor subtypes.

The structure-activity relationships for affinity and selective binding of atrial natriuretic peptide (ANP) and analogues to guanylate cyclase coupled (CC) and non-cyclase coupled (NC) receptors in rabbit lung membranes are described. We have designed a series of peptides to try to identify the minimal sequence involved in specific recognition of each receptor subtype. The affinity of the peptides was determined from competitive binding experiments. Several peptides derived from the rat ANP sequence, e.g., des-[Phe106, Gly107, Ala115, Gln116]ANP-(103-125)NH2 (4), des-[Cys105,121]ANP-(104-126) (5), and [Acm-Cys105]ANP-(105-114)NH2 (9) have high affinity and selectivity for the noncoupled site. Peptide 4 was the most selective ligand with an affinity superior to that of ANP-(103-126). This compound does not displace the radiolabeled ligand from the guanylate cyclase coupled receptor at the highest concentration tested (100 nM). The structure-activity relationship for affinity and selectivity is discussed. Comparison of the peptide sequences suggests that the structural feature responsible for recognition of the NC site resides in a single sequence of seven contiguous amino acids from the cyclic core of the hormone. The corresponding heptapeptide retains affinity to the guanylate cyclase uncoupled binding site and is proposed to encompass the minimal sequence for specific recognition of the non-guanylate cyclase coupled ANP receptor.

Human atrial natriuretic factor (ANF). Characterisation of a monoclonal antibody panel and its use in radioimmunoassay.

The production of nine monoclonal antibodies to human atrial natriuretic factor (ANF 1-28) is described. All possible combinations of two antibodies failed to reveal any which could simultaneously bind ANF. Studies with ANF analogues and the antibodies having the three highest affinity values (KD = 5, 25 and 21 pM) indicated that the antibodies are directed to the central portion of the antigen molecule. The highest affinity antibody was able to replace polyclonal antisera in the radioimmunoassay of ANF in extracts of plasma.

Clearance and early hydrolysis of atrial natriuretic factor in vivo. Structural analysis of cleavage sites and design of an analogue that inhibits hormone cleavage.

This study examines the clearance and early hydrolysis of atrial natriuretic factor (ANF) in vivo. Radiolabeled ANF was cleared from the circulation of the rat with biphasic kinetics; the majority (90%) of ANF cleared with a t1/2 of 15 s, the remaining peptide was cleared with a t1/2 of 5 min. Microsequence analysis of ANF peptides recovered from the circulation of rats revealed five major degradation products of the intact hormone. The first cleavage occurred between amino acids 12 and 13 of the hormone and would inactivate ANF. Over time, additional fragments of the hormone were generated, including fragments of 6, 7, 21, and 24 amino acids in length. Whole body radioautography of rats injected with [123I]-ANF revealed the kidney as a predominant organ involved in clearance of ANF. Subsequent amino acid sequence analyses of radiolabeled ANF exposed to the kidney in vivo indicated that this organ generated four of the five major hydrolysis products observed in circulation, namely, the 6, 7, 16, and 21 amino acid fragments of the hormone. In an attempt to stabilize ANF in vivo, a synthetic analogue of the hormone was prepared that contained the amino acid analogue, aminoisobutyric acid, substituted at position 13. This analogue completely abolished the in vivo cleavage of ANF at this site. These studies demonstrate the usefulness of a protein chemistry approach in characterizing hormone metabolism in vivo and designing analogues with enhanced in vivo stability to cleavage.

Atrial natriuretic peptides: the role of phenylalanine on biological activity.

Previous studies have shown that atrial natriuretic peptides (ANPs) inhibit the secretion of aldosterone by isolated rat adrenal glomerulosa cells stimulated by angiotensin II, ACTH, and potassium. Structure-function studies have concentrated on the significance of the C- and N-terminal chains for the biological activity of the peptide. We investigated the role of phenylalanine at positions 8 and 26 by using [Ala8]human (h) ANP or [Ala26]hANP analogs to inhibit potassium-stimulated aldosterone secretion in granulosa cells. hANP-(1-28) inhibited potassium-stimulated aldosterone secretion with an IC50 of 0.48 nM. Synthetic [Ala26]hANP inhibited the aldosterone response to potassium with an inhibitory curve relative to hANP-(1-28) (rIC50) of 6.0 nM, which was significantly greater than that for hANP (P less than 0.001). Synthetic [Ala8]hANP was markedly less effective as an inhibitor, with an estimated rIC50 of 3.0 microM (P less than 0.0001). To determine whether the analogs act as competitive antagonists to hANP-(1-28), experiments were performed in which a fixed concentration (0.1 microM) of the analog was incubated in the presence of increasing concentrations of hANP-(1-28). When hANP-(1-28) was incubated with [Ala8]hANP, the rIC50 (0.2 nM) was significantly less than that for hANP-(1-28) alone (P less than 0.02). When hANP-(1-28) was incubated with [Ala26]hANP, the rIC50 was 0.1 nM. In summary, [Ala8]hANP and [Ala26]hANP were significantly less potent than hANP-(1-28) as inhibitors of aldosterone production from granulosa cells. Both analogs shifted the hANP-(1-28) dose-response curve to the left. Neither analog functioned as a competitive antagonist to hANP-(1-28). Our results indicate that the hydrophobic phenyl groups at these two positions are required for full biological potency of ANP as an inhibitor of aldosterone production.

Functional multiplicity of atrial natriuretic peptide receptors on cultured rat Leydig tumor cells.

Native rat atrial natriuretic peptide (NANP) was shown to bind with high affinity and to increase intracellular levels of cGMP in cultured rat Leydig tumor cells. A linear analog of NANP which lacks the disulfide-linked bridge structure also bound with high affinity but did not increase levels of intracellular cGMP or antagonize the increase of this cyclic nucleotide by NANP. These data are consistent with the existence of two functional subpopulations of ANP receptors on cultured rat Leydig tumor cells; one which is capable of activating guanylate cyclase and one which is not linked to this enzyme.

Phosphorylation of high- and low-molecular-mass atrial natriuretic peptide analogs by cyclic AMP-dependent protein kinase.

Synthetic high- and low-molecular-mass atrial peptides were phosphorylated in vitro by cyclic AMP-dependent protein kinase and [32P]ATP. From a series of atrial peptide analogs, it was deduced that the amino acid sequence, Arg101-Ser104 of atriopeptin was required for optimal phosphorylation. Phosphorylated AP(99-126) was less potent than the parent atriopeptin in vasorelaxant activity and receptor-binding properties. These results indicate that the presence of a phosphate group at the N-terminus of AP(99-126) decreases the interaction of the peptide with its receptor and, as a consequence, decreases bioactivity. These observations are in contrast to those of Rittenhouse et al. [(1986) J. Biol. Chem. 261, 7607-7610] who reported that phosphorylation of AP(101-126) enhanced the stimulation of Na/K/Cl cotransport in cultured vascular smooth muscle cells.

Physiological role of silent receptors of atrial natriuretic factor.

A ring-deleted analog of atrial natriuretic factor--des[Gln18, Ser19, Gly20, Leu21, Gly22] ANF4-23-NH2 (C-ANF4-23)--binds with high affinity to approximately 99% of ANF receptors in the isolated perfused rat kidney. In this preparation, C-ANF4-23 is devoid of detectable renal effects and does not antagonize any of the known renal hemodynamic and natriuretic actions of biologically active ANF1-28. In contrast, both C-ANF4-23 and ANF1-28 increase sodium excretion and decrease blood pressure in intact anesthetized rats. This apparent contradiction is resolved by the finding that the ring-deleted analog markedly increases plasma levels of endogenous immunoreactive ANF in the rat. The results show that the majority of the renal receptors of ANF are biologically silent. This new class of receptors may serve as specific peripheral storage-clearance binding sites, acting as a hormonal buffer system to modulate plasma levels of ANF.

Superactive analogs of the atrial natriuretic peptide (ANP).

Three analogs of the atrial natriuretic peptide ANP(105-126), lacking the N-terminal exocyclic peptide segment and containing 2-mercaptoacetic acid, 3-mercaptopropionic acid or 4-mercaptobutyric acid in place of the cysteine residue in position 105 of the peptide sequence, were synthesized by the solid-phase method. The resulting des-amino analogs showed 2 to 4 times higher diuretic/natriuretic activity than the most active natural ANP and displayed a potent hypotensive effect as well. All three analogs were relatively less potent in various in vitro bioassays and in a binding assay, indicating that their high activities in vivo may be due to resistance to enzymatic degradation and to reduced non-specific tissue adsorption. These compounds not only will serve as useful pharmacologic tools but also represent prototypes for the development of further reduced-size ANP analogs.

Disappearance of atrial natriuretic factor from circulation in the rat.

The rate of disappearance of radioiodinated forms of 3 different atrial natriuretic factors (ANF (Ser 99-Tyr 126), ANF (Arg 101-Tyr 126), ANF (Ser 103-Tyr 126)) from circulation in the rat was studied. Before proceeding to study the half-life of these peptides, the biological activity of their cold iodinated forms was examined. Upon incorporation of iodine into the ANF molecule, there was a 2 to 5-fold loss in their binding affinities to mesenteric arteries and adrenal capsules as compared to their respective uniodinated forms. A similar loss in their potency to inhibit basal aldosterone release from adrenal zona glomerulosa cells was observed. The rate of disappearance of the radioiodinated peptides from plasma was very fast; the half-life of ANF (Ser 99-Tyr 126) was 16.8 +/- 0.9 sec. Similar values were also obtained for ANF (Arg 101-Tyr 126) and ANF (Ser 103-Tyr 126). The in vivo disappearance of ANF from plasma is probably due to the binding to receptors in the cells since in vitro incubation of ANF (Ser 99-Tyr 126) with rat plasma caused only a slight loss in its immunoreactivity in the first 5 minutes. Hepatectomy and nephrectomy did not cause any major prolongation of the disappearance rate suggesting that these two organs may not be the primary sites involved in the removal of this peptide from circulation.