Ovine atrial natriuretic factor: sequence of circulating forms and metabolism in plasma.

The sequence of ovine ANF is not known, yet sheep have been used extensively for ANF studies. We sequenced the circulating form of ovine ANF from coronary sinus plasma of sheep in paced heart failure. The main circulating form was identical to human ANF(99-126). Small amounts of ANF identical to human ANF(103-126) and ANF(101-126) peptides were also found. Incubation of labeled ANF in ovine serum suggested ANF(103-126) could be a degradation product of ANF(99-126). The endopeptidase-24.11 degradation product ANF(99-105/106-126) was not found in ovine plasma, in contrast to human plasma where it was a minor component. These results show that while the main circulating forms are similar in sheep and humans, there are differences in the minor peptides.     

Competitive peptide antagonists of ANF-induced cyclic guanosine monophosphate production

Atrial natriuretic factor (isoleucine ANF 101-126), cleaved ANF (isoleucine ANF 101-105/106-126) and des (Gln 18, Ser 19, Gly 20, Leu 21, Gly 22) ANF 4-23-NH2 (C-ANF 4-23) stimulated cyclic guanosine monophosphate production (cGMP) by rat aortic vascular smooth muscle cells (VSMC) in culture. Cleaved ANF and ANF C4-23 also antagonised or diminished the response to ANF 101-126. Agonist and antagonist actions of both peptides were dose-related. In contrast, prepro ANF (104-123), an ANF precursor fragment, exhibited no agonist or antagonist effect on cGMP production.     

Endopeptidase-24.11 in human plasma degrades atrial natriuretic factor (ANF) to ANF(99-105/106-126)

We previously demonstrated the presence of ANF(99-126), and ANF(99-126) cleaved between Cys105 and Phe106 (cleaved ANF), in human coronary sinus plasma. We now report that cleaved ANF is formed when synthetic ANF(99-126) is added to human plasma. When synthetic ANF(99-126) was incubated in heparinized human plasma, HPLC analysis showed two degradation products. The main product was shown by amino acid and sequence analysis to be cleaved ANF. Degradation of ANF was inhibited by EDTA and phosphoramidon. These findings are consistent with the action of endopeptidase EC 3.4.24.11, which may play an important part in the biological inactivation of ANF.     

Synthesis and biological activity profiles of atrial natriuretic factor (ANF) analogs

Several analogs of the atrial natriuretic factor (ANF) were synthesized by the solid-phase method using the acetamidomethyl (Acm) group for sulfhydryl protection. The compounds were tested in a receptor binding assay using bovine adrenal zona glomerulosa cell membranes and in the rat diuresis/natriuresis assay. Substitution of tyrosine in position 116 of ANF(101-126) and of the analog [3-Mpr105]ANF(105-126)(3-Mpr = 3-mercaptopropionic acid) did not alter the biological activity profiles and, therefore, these two analogs in radioiodinated form will be useful for enzymatic degradation and clearance studies. Replacement of 3-mercaptopropionic acid with 2-mercaptopropionic acid in [3-Mpr105]ANF(105-126) resulted in an analog with very low potency in both assay systems, presumably as a consequence of the steric bulk and/or local conformational restriction produced by the methyl group attached to the alpha-carbon in position 105. The analog [3-Mpr105,Nva109]ANF(105-126)(Nva = norvaline) showed very low affinity in the receptor binding assay but displayed considerable diuretic/natriuretic activity. The obtained biological activity profiles suggest that in comparison with other ANF peptides the des-amino ANF(105-126) analogs may have a somewhat longer half-life in vivo, or alternatively, may indicate a more complex situation of ANF receptor or binding site heterogeneity.     

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.     

Effect of atrial natriuretic factor on plasma vasopressin in conscious rats

An intravenous (IV) bolus injection (10 μg) of synthetic rat atrial natriuretic factor [ANF (Arg 101-Tyr 126)] into normal conscious Sprague-Dawley rats produced a significant decrease of plasma arginine vasopressin (AVP) while 1-, 2-, and 5-μg doses exerted no such effect. Mean arterial blood pressure (MAP) was lowered about 15 mmHg by an IV 10 μg bolus injection of ANF. When plasma AVP rose significantly in rats exposed to such osmotic stimuli as 600 mM NaCl and 900 mM mannitol intraperitoneally (IP), subsequent IV injection of ANF (10 μg) markedly depressed this parameter. Lower doses of ANF were ineffective against 600 mM NaCl IP. The significant elevation of plasma AVP levels by hypertonic sucrose 900 mM IP was not modified by ANF (10 μg). Blood pressure remained unchanged after IP administration of various osmotic stimuli, except mannitol, and in all these experiments an IV bolus of ANF exerted a lowering effect on MAP. Seventy-two hr water deprivation (mixed osmotic and volume stimulus) resulted in elevated plasma AVP levels which were unaffected by an IV bolus injection of ANF at doses of 0.06–10 μg. Immunoreactive ANF (IR-ANF) rose in plasma to 39.3±13 ng/ml 1 min after an IV bolus injection of 10 μg ANF, dropping to 1.01±0.2 ng/ml after 5 min and to 0.32±0.01 ng/ml after 10 min (when ANF and AVP interactions were studied), but still remained approximately six times higher than in control rats. These results suggest that, in the conscious rat, only pharmacological levels of ANF observed after an IV bolus infusion may influence both resting and osmotically-stimulated AVP levels.     

The responses of atrial natriuretic factor concentrations to acute volume changes in conscious rats

A rapid and sensitive radioimmunoassay has been developed for measurements of atrial natriuretic factor (ANF) in rat plasma. The antiserum, raised to rat ANF (99-126), cross-reacts with rat ANF (103-123), ANF (103-125), ANF (103-126) but not with smaller fragments, human ANF (99-126), angiotensin II, bradykinin or vasopressin. The plasma ANF concentration is 181 +/- 24 pg/ml (N = 24) in the unstressed conscious rats (Charles River CD, male). The ANF immunoreactivity in the plasma extracts was verified by HPLC analysis, which displayed one major immunoreactive peak of ANF corresponding to rat ANF (99-126) and some smaller fragments. Intravenous injection of saline elevated circulating ANF, whereas acute volume depletion by hemorrhage, water deprivation and furosemide diuresis greatly lowered plasma ANF. The prompt response of plasma ANF levels to acute changes in volume status is consistent with the proposed role of ANF as a volume-regulatory hormone.     

Differential Cleavage of Urodilatin and Atrial Natriuretic Factor by Thrombin and Protease 3.4.24.11

Abstract

Human urodilatin (residues 95–126) and atrial natriuretic factor (residues 99–126, based on ANF prohor-mone sequence) were incubated separately with three proteases, thrombin, angiotensin converting enzyme (ACE), and neutral endopeptidase 3.4.24.11 (NEP). Thrombin cleaved urodilatin on the carboxyl side of arginine⁹⁸ to yield ANF but under the same conditions did not cleave h-ANF. Neither urodilatin nor ANF was cleaved by ACE. ANF was rapidly degraded by NEP resulting in a major product cleaved between amino acid residues Cys^l05 and Phe¹⁰⁶. Urodilatin was also cleaved by NEP and the amino acid sequencing of the cleaved product revealed the site of cleavage to be the same Cys¹⁰⁵-Phe¹⁰⁶ site as for ANF with a second cleavage site at Gly¹¹⁸-Leu¹¹⁹. However, cleavage of urodilatin by NEP proceeded much more slowly when compared to ANF. A comparison of the affinities of ANF and urodilatin for purified NEP from rabbit kidney revealed K_m values of 11.7 and 3.1 μM, respectively. The turnover rates (k_cat/K_m) for urodilatin and h-ANF with NEP were 4.6 and 37.3 min^?1 μM^?1, respectively. Thus, urodilatin is much less efficiently hydrolyzed by purified NEP than is ANF. The four residue extension at the N-terminus of urodilatin may be important for protection against rapid biological inactivation.     

A two-site immunoradiometric assay of proatrial natriuretic factor. Application to tissue extracts

A "two-site" immunoradiometric assay (IRMA) was developed to specifically measure ANF (1-126), the precursor of ANF. This assay is based on the simultaneous use of antibodies against two different antigenic determinants: murine monoclonal antibody (2H2), which recognizes positions 101 through 103 of ANF, is linked to Immunobeads and employed to extract any ANF C-terminal; a second antibody, which is directed against positions 11 through 37, is radioiodinated and allows binding to any C-terminal-2H2-Immunobead material which bears the N-terminal antigenic site. A curvilinear relationship was obtained between radioactivity and the amount of proANF (1.5 to 400 fmol) added. Optimisation of IRMA was determined by the amount of 2H2-Immunobeads and labeled antibody used, incubation time as well as possible interference by both ANF (99-126) and ANF (1-98). Tissue extracts were used to validate the assay. proANF was detected in decreasing amounts in heart atria, heart ventricles, lungs, kidneys and adrenal glands. Its presence was further confirmed by reverse-phase HPLC followed by radioimmunoassay. IRMA is a simple and rapid method for the direct measurement of proANF in tissue extracts and chromatographic fractions. The presence of proANF in tissues strongly suggests local synthesis.     

Chronic infusion of low doses of atrial natriuretic factor (ANF Arg 101-Tyr 126) reduces blood pressure in conscious SHR without apparent changes in sodium excretion

Conscious SHR and WKY rats were infused during 7 days with synthetic ANF (Arg 101-Tyr 126), 100 ng/hr/rat (35 pmol/hr/rat) by means of miniosmotic pumps. The SHR initial blood pressure of 177 +/- 5 mmHg gradually dropped to 133 +/- 3 and 142 +/- 4 mmHg the last two days of infusion. No significant change in blood pressure was observed in the ANF-infused WKY group. No apparent difference in natriuresis or diuresis was observed in ANF-infused SHR and WKY when compared with non-infused control groups. A slight but significant lower immunoreactive ANF concentration was found in the atria of SHR than in their normotensive controls. No difference in cardiac weight was found between infused and non-infused rats. It is suggested that the hypotensive response observed in SHR and not in WKY is due to a decrease in vascular peripheral resistance. Whether ANF is involved in the development and maintenance of high blood pressure in SHR remains to be elucidated.     

Body fluids and plasma atrial peptide after its chronic infusion in hypertensive rats

To determine the role of body fluid volume in the chronic hypotensive effect of atrial natriuretic factor (ANF), spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats were infused with the peptide (Arg 101-Tyr 126) at a rate of 100 ng/h/rat for 5 days. Blood pressure (BP) was decreased from 176 +/- 4 to 133 +/- 3 mmHg in the SHR group 4 days after ANF infusion was initiated, whereas no changes were observed in ANF-infused WKY animals. Starting 5 days after the infusion began, body fluid measurements revealed no differences in plasma, blood and extracellular fluid volumes or in interstitial spaces. BP and plasma ANF concentrations were determined in another set of experiments before, during and after chronic ANF infusion. BP declined from 169 +/- 3 to 133 +/- 5 mmHg in SHR 5 days after the infusion commenced, but returned to basal values by day 10 or 11. Plasma ANF was significantly higher in SHR than in WKY rats throughout the observation period. However, there were no discernible changes in this parameter in ANF-infused SHR compared to non-infused SHR. A 3-fold rise in plasma ANF was noted in infused WKY rats at day 3 only. It is concluded that the chronic hypotensive effect of ANF in hypertensive animals is not related to changes in either body fluid volume or distribution. Moreover, the finding that chronic ANF infusion reduces BP in SHR without altering its plasma levels suggests a rapid ANF turnover.     

Comparative biological activities of ANF (Arg 101-Tyr 126) and the synthetic form of circulating ANF (Ser 99-Tyr 126)

The biological activities of ANF (Arg 101-Tyr 126) and of the circulating form, ANF (Ser 99-Tyr 126), were compared in the following assays: precontracted rabbit aortic strip and chick rectum, rat natriuresis, inhibition of aldosterone secretion and receptor affinity in bovine and rat adrenal zona glomerulosa cells, and receptor affinity in rabbit aorta and rat mesenteric artery cells. The results demonstrate that both peptides share the same biological activities. It is concluded that the addition of two amino acids to the N-terminal of ANF (Arg 101-Tyr 126) does not modify its biological characteristics, validating thus previous research employing this peptide.     

Specificity of serine proteases for cleavage sites on proatrial natriuretic factor

An immunological approach was used to investigate the specificity of protease cleavage sites on proANF. Cleavage of ³⁵S-cysteine biosynthetically-labeled proANF by whole serum, thrombin and kallikrein was examined. Reaction products were immunoprecipitated with two antibodies directed to different epitopes: a previously characterized antibody directed toward the carboxy-terminus of ANF_103–126, which cross reacts with proANF, ANF_99–126 and ANF_103–126, and a newly prepared antisera to synthetic ANF_99–105, which uniquely recognizes ANF_99–126, but not proANF or ANF_103–126. With increasing time of incubation with rat serum, proANF is sequentially cleaved at the C-terminus of a monobasic Pro-Arg dipeptide sequence to form ANF_99–126, and then at the C-terminus of a dibasic Arg-Arg dipeptide sequence to yield ANF_103–126. This cleavage activity of serum is blocked by leupeptin (40 μg/ml), but not by hirudin (100 nM), a specific inhibitor of thrombin, or by aprotinin (200 KIU/ml), a kallikrein inhibitor. When 100-fold purified serum cleavage enzyme was used in place of crude serum, similar results were obtained. Thrombin cleaves proANF only at the monobasic site to produce ANF_99–126 while kallikrein cleaves only at the dibasic site to produce ANF_103–126. As expected, the generation of these cleavage products can be inhibited by hirudin or aprotinin respectively. These data indicate that the substrate specificity of the serum cleavage activity is broader than that of thrombin or kallikrein, and that cleavage of proANF by serum proteases may be influenced by conformational restraints. The methods developed here should help in the future characterization of the physiological proANF cleaving enzyme.     

Rat atrial natriuretic factor (ANP-III) inhibits phosphate transport in brush border membrane from superficial and juxtamedullary cortex

Previous studies have shown that administration of synthetic atrial natriuretic factor (ANF, 101-126) decreases sodium-dependent phosphate transport across renal brush border membrane vesicles (BBMV) in rats fed a normal or low phosphate diet. In the present study, infusion of rat ANF (atriopeptin III (ANP-III), 103-126 rat ANF) to rats fed a normal phosphate diet caused natriuretic and phosphaturic effects similar to those of ANF (101-126), but unlike ANF (101-126) did not increase the glomerular filtration rate. The effect of ANP-III infusion on sodium-dependent transport of phosphate was also determined in BBM vesicles isolated from the superficial cortex (BBMV-SC) and juxtamedullary cortex (BBMV-JM). The results indicate that ANP-III decreases phosphate transport across BBMV-SC and BBMV-JM similarly (20-24%). However, it had no effect on sodium-dependent transport of proline in these vesicles. The infusion of ANP-III to rats fed a normal phosphate diet inhibits phosphate uptake both in BBMV-SC and BBMV-JM and causes phosphaturia without increments in glomerular filtration rate.     

Bioactive atrial natriuretic factor-like peptides in rat anterior pituitary

The presence of biologically active atrial natriuretic factor (ANF)-like peptides was demonstrated in rat anterior pituitary. ANF-like immunoreactivity was detected in rat anterior pituitary by specific radioimmunoassay and was extracted from rat anterior pituitary homogenates by heat-activated Vycor glass beads; extracts were purified by reverse-phase high performance liquid chromatography. Two peaks containing ANF immunoreactive material were obtained. The first peak was eluted from the C18 mu Bondapak column at a position similar to the 28-amino acid carboxy terminal peptide (Ser99-Tyr126)-ANF of prohormone. The second peak had the same pattern of elution as the 126-amino acid prohormone, (Asn1-Tyr126)-ANF. The biological activity of the smaller molecular weight peptide (28 amino acid) was assessed by its inhibitory effect on 10(-8) M ACTH-stimulated aldosterone secretion in rat zona glomerulosa cell suspension. This ANF-like material also displaced I125-labelled ANF from rat glomerular receptors with a potency similar to synthetic (Arg101-Tyr126)-ANF. Immunocytochemical localization revealed a distribution of ANF-stained cells similar in pattern and location to that of gonadotrophs. These results suggest the existence of biologically active ANF-like peptides and ANF prohormone within the anterior pituitary. However, their role remains to be elucidated.     

Reduction of blood pressure and increased diuresis and natriuresis during chronic infusion of atrial natriuretic factor (ANF Arg 101-Tyr 126) in conscious one-kidney, one-clip hypertensive rats

Conscious one-kidney, one-clip hypertensive rats and their normotensive controls were infused during 7 days with synthetic ANF (Arg 101-Tyr 126) at 100 ng/hr/rat (35 pmol/hr/rat) by means of osmotic minipumps. The basal blood pressure of 193 +/- 6 mmHg gradually declined to 145 +/- 6 mmHg at day 4 after the infusion was started. No changes in blood pressure were observed in ANF-infused normotensive rats. A significantly higher diuresis and natriuresis was observed in ANF-infused hypertensive rats when compared to the non-treated hypertensive group. No such changes were observed in ANF-treated normotensive animals. No differences in PRA were seen in any group. Atrial immunoreactive ANF was significantly lower in one-kidney, one-clip rats than in the normotensive animals, but whether this is the reflection of an increased release in the circulation remains to be elucidated. It is suggested that the hypotensive response of one-kidney, one-clip animals to ANF may be secondary to a dual mechanism, vasodilatation and volume depletion.     

Characterization of glucagon-like peptide-I(7–36)amide receptors of rat lung membranes by covalent cross-linking

¹²⁵I-labelled GLP-I(7–36)amide was cross-linked to a specific binding protein in rat lung membranes using disuccinimidyl suberate. A single radio-labelled band at M_r 66000 was identified by SDS-PAGE after solubilization of the ligand-binding protein complex which is consistent with the presence of a single class of binding sites on rat lung membranes. The band was undetectable when 1 μmol/1 GLP-I(7–36)amide was included in the binding assay. No change in the mobility of the band was observed under reducing conditions suggesting that the binding protein in the receptor is not part of a larger disulphide-liked protein. The intensity of the radiolabelled protein band was reduced when the incubation with ¹²⁵I-labelled GLP-I(7–36)amide was carried out in the presence of guanine nucleotides suggesting that the GLP-I(7–36)amide receptor is coupled to the adenylate cyclase system.     

Localization and characterization of atrial natriuretic factor (ANF)-like peptide in the frog atrium

The distribution of ANF was studied in the heart of the frog (Rana ridibunda) using indirect immunofluorescence. ANF-like immunoreactivity was localized mainly in the right and left atrium, most of cardiocytes being intensively labelled. At the electron microscopic level, all secretory granules present in atrial cardiocytes contained ANF immunoreactive material. Using a specific radioimmunoassay, we found higher concentrations of ANF in the left atrium (208 +/- 25 ng/mg protein) than in the right atrium (120 +/- 16 ng/mg protein) whilst in the rat, the right atrium contains the highest ANF concentration. The concentration of ANF in the ventricle was 10 times lower than in the whole atrium (32 +/- 4 ng/mg protein). Sephadex G-50 gel filtration of atrial extracts showed that ANF-like immunoreactivity eluted in three peaks. Most of the immunoreactivity corresponded to high molecular weight material eluting at the void volume while 20% of the material co-eluted with synthetic (Arg 101-Tyr 126) ANF. These results indicate that frog cardiocytes synthetize a peptide which is immunologically and biochemically related to mammalian ANF.     

Identification of a biologically active circulating form of rat atrial natriuretic factor

An atrial natriuretic peptide has been isolated from plasma of morphine treated rats by means of glass beads extraction, immunoaffinity chromatography, and reverse phase HPLC. 1.3 micrograms of immunoreactive material was obtained. The biological activity of this material was found comparable to that of ANF (Arg 101 - Tyr 126) on the inhibition of basal aldosterone secretion by rat adrenal zona glomerulosa cells and the displacement curve of iodinated ANF from ANF receptors in a mesenteric artery preparation. Gas phase amino acid sequencing indicated that it is related to ANF (Ser 99 - Tyr 126). These results suggest that the maturation of ANF may require a tryptic-like cleavage after a single Arg residue.     

The effects of intracerebroventricular infusion of atrial natriuretic factor in conscious sheep

The effects of 24-hour intracerebroventricular infusion of human atrial natriuretic factor (ANF) and two related fragments were studied in conscious sheep. ANF (1-28) had no effect on either mean arterial pressure (MAP) or heart rate (HR) when infused at 3 or 10 micrograms/hr, however a small diuresis and an increase in urinary sodium (Na) excretion was observed. The smaller fragment, ANF (5-27) infused at 10 micrograms/hr, increased MAP, HR and body temperature, although the same rate of infusion of ANF (5-28) was without effect. All peptides increased plasma sodium concentration and plasma osmolality. None of the peptides affected plasma ACTH, glucose or renin concentration. ANF (1-28) had no effect on either Na intake or water intake in Na-depleted sheep. These studies suggest that members of the ANF family can influence a number of physiological functions following central administration.