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.     

Atrial natriuretic factor selectively activates particulate guanylate cyclase and elevates cyclic GMP in rat tissues

The effects on guanylate cyclase and cyclic GMP accumulation of a synthetic peptide containing the amino acid sequence and biological activity of atrial natriuretic factor (ANF) were studied. ANF activated particulate guanylate cyclase in a concentration- and time- dependent fashion in crude membranes obtained from homogenates of rat kidney. Activation of particulate guanylate cyclase by ANF was also observed in particulate fractions from homogenates of rat aorta, testes, intestine, lung, and liver, but not from heart or brain. Soluble guanylate cyclase obtained from these tissues was not activated by ANF. Trypsin treatment of ANF prevented the activation of guanylate cyclase, while heat treatment had no effect. Accumulation of cyclic GMP in kidney minces and aorta was stimulated by ANF activation of guanylate cyclase. These data suggest a role for particulate guanylate cyclase in the molecular mechanisms underlying the physiological effects of ANF such as vascular relaxation, natriuresis, and diuresis.     

An atrial natriuretic factor-like activity in rat posterior hypophysis

An atrial natriuretic factor-(ANF) like immunoreactivity (IR-ANF), is present in the posterior hypophysis of the rat. In order to obtain more direct information on the presence and biological activity of this new posterior hypophysis peptide, we applied a procedure similar to that described for rat atria, to extract an ANF-like material from the posterior hypophysis of the rat. An analysis of the tissue extracts by reverse-phase high performance liquid chromatography (RP-HPLC) suggested that, in this organ, the ANF-like peptides may be present in multiple forms: a low molecular weight peptide which had a RP-HPLC pattern similar to that of the synthetic rat 28 amino acid C-terminal (Ser 99-Tyr 126) ANF, and an unidentified higher molecular weight peptide. The partially purified low molecular weight peptide was found to have a potency similar to that of synthetic rat ANF in the inhibition of adrenocorticotropin-stimulated aldosterone secretion in dispersed zona glomerulosa cells, suggesting that the ANF-like peptide was biologically active. Immunohistochemical visualization of the ANF-like peptides revealed the distribution of the peptide within the posterior hypophysis. There was no immunohistochemical staining for ANF in the intermediate lobe. These results suggest the existence of biologically active ANF-like peptides within the posterior hypophysis of the rat. It is possible that these peptides may modulate locally the posterior hypophysis hormone secretion.     

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.     

Molecular cloning and expression of murine guanylate cyclase/atrial natriuretic factor receptor cDNA

The potent diuretic and natriuretic peptide hormone atrial natriuretic factor (ANF), with vasodilatory activity also stimulates steroidogenic responsiveness in Leydig cells. The actions of ANF are mediated by its interaction with specific cell surface receptors and the membrane-bound form of guanylate cyclase represents an atrial natriuretic factor receptor (ANF-R). To understand the mechanism of ANF action in testicular steroidogenesis and to identify guanylate cyclase/ANF-R that is expressed in the Leydig cells, the primary structure of murine guanylate cyclase/ANF-R has been deduced from its cDNA sequence. A cDNA library constructed from poly(A+) RNA of murine Leydig tumor (MA-10) cell line was screened for the membrane-bound form of ANF-R/guanylate cyclase sequences by hybridization with a rat brain guanylate cyclase/ANF-R cDNA probe. The amino acid sequence deduced from the cDNA shows that murine guanylate cyclase/ANF-R cDNA consists of 1057 amino acids with 21 amino acids comprising the transmembrane domain which separates an extracellular ligand-binding domain (469 amino acid residues) and an intracellular guanylate cyclase domain (567 amino acid residues). Upon transfection of the murine guanylate cyclase/ANF-R cDNA in COS-7 cells, the expressed protein showed specific binding to 125I-ANF, stimulation of guanylate cyclase activity and production of intracellular cGMP in response to ANF. The expression of guanylate cyclase/ANF-R cDNA transfected in rat Leydig tumor cells stimulated the production of testosterone and intracellular cGMP after treatment with ANF. The results presented herein directly show that ANF can regulate the testicular steroidogenic responsiveness in addition to its known regulatory role in the control of cardiovascular homeostasis.     

Identification of the released form of atrial natriuretic factor by the perfused rat heart

Atrial natriuretic factor (ANF), released by the isolated perfused rat heart, was extracted from the perfusates by C18 Sep-Pak cartridges and then isolated by immunoaffinity chromatography and by reverse phase HPLC. About 500 ng of immunoreactive material were so obtained and submitted to amino acid sequencing. The C-terminal Tyr was detected by radiolabelling. Identification of these residues indicated that the primary structure corresponds to ANF (Ser 99-Tyr 126) which is identical to the circulating form in the rat. These results indicate that the ANF released by the atria corresponds to a short peptide. Therefore, its maturation process may therefore take place either intracellularly or during secretion and implicates a tryptic-like cleavage after a single Arg residue in position 98.     

Synthesis of atrial natriuretic peptides and studies on structural factors in tissue specificity

Two atrial natriuretic peptides, containing 25 amino acid residues, ANF IV, and 21 amino acid residues, ANF V, were synthesized by a solid phase method and oxidized with K3Fe(CN)6 to form a disulfide bridge. Synthetic ANF IV exhibited a natriuretic activity with an ED50 70 times higher than that of synthetic ANF V, whereas the longer peptide was only 2.5 times more potent in chick rectal smooth muscle relaxant activity. Both peptides inhibited norepinephrine-induced contraction of rabbit aorta. The shorter peptide, ANF V, was 300 times less efficient than the longer peptide, ANF IV. It is proposed that the carboxy-terminal of ANF IV seems to have a modulating effect on receptor affinity in kidney and vascular tissue.     

Identification of a peptidase which processes atrial natriuretic factor precursor to its active form with 28 amino acid residues in particulate fractions of rat atrial homogenate

With the objective of identifying specific peptidase responsible for the processing of atrial natriuretic factor precursor pro-ANF to the circulating active form ANF (99-126), a fluorometric assay method was devised using synthetic fluorogenic substrate Boc-Ala-Gly-Pro-Arg-MCA(methylcoumarinamide) which contains the amino acid sequence immediately adjacent to the arginyl peptide bond which is cleaved in the natural processing of pro-ANF. A protease which selectively cleaves this bond and produces the natural circulating peptide was identified in the particulate fraction of rat atrial homogenate and was solubilized by 1.6 M KCl. It was partially purified by affinity chromatography heparin-agarose column and was shown to be a serine protease. Its reaction product with natural pro-ANF was identified as ANF (99-126) containing 28 amino acid residues.     

Atrial pronatriodilatin: a precursor for natriuretic factor and cardiodilatin. Amino acid sequence evidence

Numerous peptides isolated from rat heart atria, including two containing 33 and 73 amino acids, were isolated and shown to exhibit natriuretic activities. Here, we describe the purification and partial amino acid sequence of a 106-residue peptide containing the previously sequenced 33- and 73-amino-acid ANF peptides. The determined sequence is a novel one and is not significantly homologous to any known protein or segment thereof. In fact, this sequence shows significant homology only to another novel partial sequence obtained from sequence analysis of a porcine peptide, called cardiodilatin, also found in heart atria. This relationship is taken as evidence that ANF and cardiodilatin are part of the same precursor molecule which would contain at the very least 126 amino acids.     

Purification of rat pro-atrial natriuretic factor: a simplified scheme using reversed-phase high-performance liquid chromatography

A simple scheme for the rapid and efficient isolation of rat pro-atrial natriuretic factor (pro-ANF) has been developed. An isolated rat adrenal cell bioassay for ANF was established to optimize heart tissue extraction and chromatography conditions. This assay is based on the ability of ANF to inhibit angiotensin II-stimulated aldosterone secretion. IC50 values for ANF were approximately 320 pM. The protocol that was established consisted of extraction of rat atria in 5 N acetic acid containing protease inhibitors. The extract was lyophilized, resolubilized in 0.1% trifluoroacetic acid containing 1% (w/v) sodium chloride, and subjected to RP-HPLC. Extraction of small batches of atria (i.e., from 10 or 20 rats) resulted generally in a yield of 2 nmol per rat (i.e., approximately 30 micrograms). The identity and purity of the pro-ANF were confirmed by the determination of both the amino acid composition and the amino-terminal sequence. Purified pro-ANF was radioiodinated and the efficiency of the extraction and purification procedure was assessed by adding labeled peptide to the initial tissue extract. The structural integrity and overall recovery of radioactivity were determined by RP-HPLC. The purification scheme provides undamaged pro-ANF of high purity. Purified pro-ANF was compared with synthetic rat ANF in the rat adrenal glomerulosa cell and isolated rat aortic strip bioassays. The peptides were apparently equally active in the adrenal cell system and approximately threefold less potent in relaxing aortic strips. The apparent equipotency in the adrenal cell bioassay may be due to the conversion of pro-ANF to ANF-like peptides during the bioassay incubation.     

NH2-terminal fragment of rat pro-atrial natriuretic factor in the circulation: identification, radioimmunoassay and half-life

A radioimmunoassay was developed to measure the NH2-terminal counterpart of rat pro-atrial natriuretic factor (pro-ANF) in plasma. Synthetic rat ANF (Asp 11-Ala 37) coupled to bovine serum albumin was used to immunize New Zealand rabbits. The antiserum demonstrated good immunoreactivity towards rat ANF (Asn 1-Arg 98), (Asn 1-Tyr 126), (Asp 11-Ala 37) and even human ANF (Asn 1-Ser 30). The standard curve had an ED80 of 9.5 +/- 2.5 and ED50 of 44.0 +/- 10.5 fmol/tube. Immunoreactive ANF NH2-terminal peptide was measured directly in rat plasma without prior extraction. In fact, extraction of ANF NH2-terminal from plasma by C18 silica gel chromatography revealed inconsistent recovery and a lack of parallelism. Morphine (0.75 mg/100 g), chosen to elicit increased ANF (Ser 99-Tyr 126) secretion, elevated its plasma concentration from 54.1 +/- 3.2 to 190.8 +/- 55.8 fmol/ml after 20 min. At the same time, the immunoreactive NH2-terminal fragment rose from 378 +/- 16 to 1181 +/- 201 fmol/ml. The identity of this immunoreactive material was verified following affinity chromatography and reverse-phase high-performance liquid chromatography (HPLC) of plasma from morphine-treated rats. Molecular sieving and amino acid sequencing demonstrated that it appears to be consistent with or identical to rat ANF (Asn 1-Arg 98). The disappearance rate of ANF (Asn 1-Arg 98) was studied by injecting radioactive material into anesthetized rats. The exponential decay was analyzed by a two-compartment model in which the fast and slow components had a half-life of 2.5 +/- 0.3 and 54.8 +/- 3.9 min, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and expression of the atrial natriuretic factor gene

Atrial natriuretic factor (ANF) is a 28-amino acid peptide hormone with potent natriuretic, diuretic and vasodilator properties. Isolation and DNA sequence analysis of rat and human cDNA clones revealed that ANF is synthesized from a 126-amino acid precursor which is highly conserved in both species. Southern blot analysis indicated that the ANF gene is present in a single copy per haploid genome. Both human and rat ANF genes were isolated and showed a similar structural organization which consisted of three exons and two introns. The ANF gene was localized to the short arm of human chromosome 1 and mouse chromosome 4. While atria are the major site of expression of the ANF gene in adult heart, other tissues like ventricles, lung, anterior pituitary, hypothalamus and adrenal synthesize ANF albeit to a much lower extent. In ventricles, ANF mRNA levels are 150 times lower than in atria. However, in cardiac hypertrophy or in congestive heart failure, ventricular ANF mRNA and peptide levels are dramatically (100-fold) increased both in animal models and in humans. This suggests that ventricles are a major site of ANF gene expression in certain pathophysiological conditions and that ANF is not an exclusively atrial peptide as was originally thought.     

Truncated atrial natriuretic factor analogs retain full agonist activity.

The synthesis, receptor binding, and agonist activity of a series of truncated atrial natriuretic analogs (ANF) are described. These analogs incorporate two portions of the native 28 amino peptide, the eight amino acids C-terminal to Cys7, and two amino acids from the C-terminus (phenylalanine and arginine), into disulfide-bonded cyclic peptides. The inclusion of the C-terminal amino acids converted the ANF analogs from receptor ligands to full agonists, as measured by several methods, including the stimulation of cGMP biosynthesis in endothelial cells, inhibition of aldosterone biosynthesis in rat adrenal cells, and natriuretic-hypotensive activity in vivo. The most potent analogs have cyclohexylalanine (Cha) at position 8. The lead compound (Arg6,Cha8 ANF 6-15 Phe-Arg-Cys-NH2) is a tridecapeptide that integrates the C-terminal amino acids inside the disulfide ring. This peptide, designated as A-68828, has a binding affinity of IC50 = 120 nM, approximately 1/400 of ANF 1-28. However, this analog, in vivo, is only slightly less natriuretic (1/20-1/50) than ANF 1-28. Unlike the native peptide, A-68828 is only mildly hypotensive and at the highest concentration tested reduced blood pressure less than 15 mmHg (1 mmHg = 133.322 Pa). A-68828 inhibited ACTH-induced aldosterone release to a greater extent than ANF 1-28: 100 vs. 50%. The selective natriuretic activity of A-66828, relative to ANF, suggests clinical utility for the treatment of acute renal failure.     

Analysis of atrial natriuretic factor biosynthesis and secretion in adult and neonatal rat atrial cardiocytes

Atrial natriuretic factor (ANF) is stored in atrial cardiocytes as the 126 amino acid polypeptide, proANF, which is later cleaved to the 24-28 amino acid carboxyterminal peptides, the major circulating forms. Earlier studies have demonstrated that isolated, cultured neonatal rat cardiocytes both store and secrete proANF, which can be cleaved to the smaller circulating form(s) by a serum protease. Since differences may exist between neonatal and adult cardiocytes with respect to ANF synthesis and processing, we compared the forms of ANF stored and secreted by neonatal rat cardiocytes with those of adult cells. Using four to five day cultures of isolated atrial cardiocytes prepared from the hearts of neonatal and adult rats, pulse-chase studies were performed with 35S-cysteine and 35S-methionine. Analysis of ANF stored and secreted by these cells was performed by immunoprecipitation of cell extracts and culture media using antibodies directed to either the carboxyterminus or aminoterminus of proANF followed by SDS-PAGE and autoradiography. Cell extracts from both adult and neonatal cultures were found to contain only a 17-kDa polypeptide, previously identified as proANF. The predominant form found in the culture media was also the 17-kDa peptide, with smaller quantities of its 3-kDa carboxyterminal and 14-kDa aminoterminal cleavage products. We conclude from these studies that proANF is the major form stored and secreted by both adult and neonatal cardiocytes in culture; the activity of the protease that cleaves proANF to the smaller forms found in the circulation is either attenuated or is overwhelmed by high ANF-secretory rates in these cultures. Alternatively, the ANF processing and secretory pathways may be somehow altered in culture such that proANF escapes protease cleavage. Further studies will elucidate the nature and location of this protease.     

Purification and sequence determination of bovine atrial natriuretic factor

We report the purification and the sequence determination of bovine atrial natriuretic factor (ANF) in acid extracts of bovine atrial appendages. The monitoring of the activity along the purification steps was performed with a radio-receptor assay using bovine adrenal cortex membranes sites and 125I ANF. Bovine ANF was separated by carboxymethyl agarose gel chromatography from catecholamines and major protein contaminants. It behaved as a 3 K dalton peptide on Sephadex G-50. The active fractions were then subjected to high performance liquid chromatography (HPLC) using a sulfopropyl cation exchange column. Subsequent purification steps by reverse phase mode on Ultrapore RPSC, Vydac TP 218 and uBondapak using acetonitrile gradients led to the obtention of a pure fraction which amino acid sequence was identical to that for human ANF. This confirms the high degree of homology of ANF structure among mammalian species and advocates the use of the radio-receptor assay based on bovine adrenal receptor for measuring human ANF.     

Degradation of atrial natriuretic factor in the rat.

The biologically active circulating form of atrial natriuretic factor (ANF) in the rat is the 28-amino-acid peptide ANF-(Ser-99-Tyr-126). Degradation of this peptide in vivo as well as in vitro, in whole blood, in plasma and by the isolated mesenteric artery was investigated. Studies in vivo in the rat demonstrated that the elimination and degradation of ANF was extremely fast: within 3 min more than 95% of the injected immunoreactive material was eliminated from circulation. The production of a short C-terminal peptide was detected on injection of 125I-ANF-(Ser-99-Tyr-126) into the rat. This peptide increased proportionately with incubation time. Experiments in vitro in the presence of whole blood or plasma did not cause any major destruction of ANF even after incubation for 60 min. After this prolonged incubation in plasma, ANF-(Ser-99-Tyr-126) was partially converted into ANF-(Ser-103-Tyr-126), a less potent peptide. Isolated mesenteric-artery preparation appeared to degrade ANF in a manner very similar to the system in vivo. These results suggest that degradation of ANF may occur either after internalization in the vascular cells or by a membrane-bound enzyme in the vasculature.     

Identification of protease 3.4.24.11 as the major atrial natriuretic factor degrading enzyme in the rat kidney

We have identified a metalloendoprotease from rat kidney cortex that cleaves the cysteine-phenylalanine bond (Cys7-Phe8) within the 17 amino acid ring structure of atrial natriuretic factor (ANF). Cleavage at this site represents the major ANF degradative activity in rat kidney, and is inhibited by the known metalloendoprotease inhibitors, thiorphan, phosphoramidon and zincov with IC50 values in the nanomolar range. Since these are specific inhibitors of protease 3.4.24.11, both protease 3.4.24.11 and ANF degrading activities were monitored during purification. Both activities copurified at each chromatographic step. Furthermore, purified protease 3.4.24.11 cleaved ANF specifically at the Cys7-Phe8 bond. It is concluded from this work that the major ANF degrading enzyme in rat kidney is protease 3.4.24.11.     

Stimulation of guanylate cyclase by atrial natriuretic factor in isolated human glomeruli

A 23 amino acid synthetic peptide fragment of atrial natriuretic factor (ANF) stimulated guanylate cyclase activity in isolated human glomeruli in a concentration- and time-dependent manner. ANF activated particulate guanylate cyclase whereas it had no effect on soluble guanylate cyclase. These results demonstrate that the glomerulus is a target structure for ANF in humans. They also suggest that ANF-induced increase in glomerular filtration rate is due to a direct effect of this peptide on the glomerular cells mediated by activation of glomerular guanylate cyclase.     

Atrial natriuretic factor-like peptide and its prohormone within single cell organisms.

The present investigation was designed to determine if the atrial natriuretic peptide hormonal system is present within single cell organisms. Paramecium multimicronucleatum were examined with 3 sensitive and specific radioimmunoassays which recognize the N-terminus [amino acids 1-98; proANF(1-98)], the midportion of the N-terminus [amino acids 31-67; proANF(31-67)] and C-terminus (amino acids 99-126; ANF) of the 126 amino acid atrial natriuretic factor (ANF) prohormone. ProANF(1-98), proANF(31-67), and ANF-like peptides were all present within these unicellular organisms at concentrations of 460 +/- 19 pg/ml, 420 +/- 15 pg/ml, and 14.5 +/- 2 pg/ml, respectively. These concentrations are similar to their respective concentrations in the plasma of the rat (Rattus norvegicus). These results suggest that even single cell organisms contain the atrial natriuretic peptide-like hormonal system.     

Atrial natriuretic factor in mammalian testis: immunological detection in spermatozoa.

Immunoreactive atrial natriuretic factor (ANF) was localized by immunochemistry and radioimmunoassay in mouse and rat testes. The analyses of acid extracts of testes by gel filtration and reverse phase high pressure liquid chromatography (HPLC) revealed the presence of a processed 31-residues peptide and the precursor form of 126-residues pro-ANF molecule corresponding to a molecular weights (Mr) of 3,300 and 18,000, respectively. The concentration of ANF in mice testis averaged 12 +/- 3 ng and in rat testis 8 +/- 2 ng per g of tissue. Specific immunochemical staining was localized in the spermatids and elongating spermatozoa of mammalian testis. The demonstration of immunoreactive ANF in testis and specific localization in spermatids reveals a new site at which ANF may be actively synthesized and regulate paracrine and/or autocrine function(s) during spermiogenesis, suggesting a broader spectrum of ANF action in addition to its known regulatory role in the control of blood pressure homeostasis.