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)     

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.     

The cosecretional maturation of atrial natriuretic factor by primary atrial myocytes.

Cardiac myocytes store the 126-amino acid precursor of atrial natriuretic factor (pro-ANF), yet the mature, bioactive 28-amino acid peptide, ANF-(99-126), and the resulting N-terminal product, ANF-(1-98), are the forms of the hormone that are released by the heart and found in the circulation. Although previous studies have shown that the maturation of ANF takes place in the heart, it is not known whether it occurs in or on the myocyte concurrently with secretion, or whether cleavage takes place postsecretionally on either the myocyte surface or the surface of a nonmuscle cardiac cell. To address these questions, experiments were carried out in the present study using primary atrial cultures that had been prepared such that greater than 90% of the cells were myocytes. Reversed-phase and ion-exchange HPLC, coupled with immunoprecipitation of biosynthetically labeled ANF, showed that the stored peptide, pro-ANF, was cleaved between residues 98 and 99 such that ANF-(1-98) and (99-126) accumulated in the medium. Coupling biosynthetic labeling with timed secretion experiments showed that the extent of ANF processing was not dependent on the time after secretion; maximal levels of processing were observed at all secretion times examined. Additionally, the processing-competent myocyte-enriched cultures were unable to cleave exogenously added pro-ANF. These results indicate that the myocyte is the cell type responsible for pro-ANF maturation and that this cleavage event takes place cosecretionally.     

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.     

Bovine adrenal chromaffin granules are a site of synthesis of atrial natriuretic factor

We have previously reported the existence of a peptide factor in the adrenal medulla which inhibits aldosterone secretion in cultured bovine zona glomerulosa cells. The acid extracts of chromaffin granules from bovine adrenal medulla were purified by a four step high performance liquid chromatography procedure. Two active fractions exhibited sequence homology with bovine atrial natriuretic factor ANF (Ser99-Tyr126) and its polypeptide precursor (Asn1-Tyr126). The occurrence of both precursor and mature forms of ANF within chromaffin granules indicates the endogenous character of ANF in the adrenal medulla and suggests the potential usefulness of cultured adrenal chromaffin cells for investigating the synthesis, maturation and secretion of atrial peptides.     

Atrioactivase, a specific peptidase in bovine atria for the processing of pro-atrial natriuretic factor. Purification and characterization

A seryl protease which catalyzes conversion of proatrial natriuretic factor (ANF) to the active circulating form, ANF(99-126), was purified from a particulate fraction of bovine atria. The enzyme was solubilized with 1.6 M KCl. The molecular mass of the purified enzyme was 580 kDa on gel filtration, whereas by sodium dodecyl sulfate-polyacrylamide gel electrophoresis a cluster of six bands with molecular masses around 30 kDa was observed. The purified enzyme produced ANF(99-126) from partially purified bovine pro-ANF by the selective cleavage of the arginyl peptide bond in the -Pro97-Arg98-Ser99-sequence in pro-ANF. The enzyme was localized mainly in the microsomal fraction rather than the granule fraction. It is likely that the enzyme selectively cleaves the Arg98-Ser99 peptide bond in pro-ANF during the process of secretion.     

Molecular heterogeneity of pro-atrial natriuretic factor

Analysis by two-dimensional gel electrophoresis and Western blotting of the atrial natriuretic factor (ANF) content of atrial granules revealed the presence of at least 15 immunoreactive spots whose molecular mass distribution ranged from 16.8 to 35 kDa and their pI values from 5.12 to 5.98. About 90% of the immunoreactive ANF material was contained within four spots (spot 1: 34.8 kDa, pI 5.67; spot 5: 16.8 kDa, pI 5.50; spot 6: 16.8 kDa, pI 5.67; spot 7: 16.8 kDa, pI 5.98). Investigation of the molecular nature of spot 1 indicated that it is a dimer of pro-ANF since it possesses the same immunoreactivity, the same charge, double its mass, and can be converted with dithiothreitol into a 16.8-kDa pro-ANF form. Alkaline phosphatase and protein kinase A treatments indicated that spots 5, 6, and 7 are probably not phosphorylated forms of pro-ANF. Carboxypeptide A and B treatments in conjunction with amino acid analysis suggested that spot 7 is ANF-(1-128); spot 6, the major one, ANF-(1-126); and spot 5, ANF-(1-123) or ANF-(1-124). Water deprivation or morphine injection, two maneuvers which are known to influence ANF secretion and atrial ANF content, failed to affect the molecular heterogeneity of pro-ANF except for spot 1. The formation of the dimer appeared to be time-dependent. These results emphasize the heterogeneity of the pro-ANF molecule stored in atrial granules. We suggest that this heterogeneity may be due, in part, to the action of some proteases, such as carboxypeptidase E or a tripeptidyl carboxyhydrolase.     

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.     

Conversion factors of high- to low-molecular-weight forms of atrial natriuretic factor

The atrial natriuretic factor (ANF) is comprised of a 126-amino-acid precursor (pro-ANF) and its biologically active fragments. Partially purified pro-ANF and its larger fragments (greater than 10,000 daltons) have been referred to as high-molecular-weight (Mr) ANF, the partially purified smaller fragments (less than 10,000 daltons) as low Mr ANF. In vitro, mild proteolysis of high Mr ANF yielded low Mr ANF and enhanced biological activity. In the rat, pro-ANF was the predominant atrial form; however, low Mr ANF was largely released from isolated perfused hearts, which suggests that conversion of pro-ANF to low Mr ANF occurred immediately before or during secretion. High Mr ANF was also found in the perfusate of isolated rat hearts and in the plasma of rats, which suggests that some pro-ANF was secreted with low Mr ANF. Evidence for extraatrial conversion and activation of pro-ANF comes from two studies. 1) Intra-renal-arterial injection of high Mr ANF had little renal vascular action, whereas its i.v. injection caused renal vascular dilation, which suggests that the renal vasodilatory action of high Mr ANF became activated during circulation. 2) When high Mr ANF was incubated with rat blood or rat platelets in vitro, its natriuretic activity was converted to low Mr ANF within minutes; the platelet-induced conversion was associated with enhanced activity in relaxing aortic smooth muscle.     

The propeptide Asn1-Tyr126 is the storage form of rat atrial natriuretic factor.

Granules from rat atria were isolated by differential centrifugation and by a 53% (v/v) Percoll gradient after tissue homogenization in 0.25 M-sucrose/50 mM-Na2EDTA. About 40% of the immunoreactive ANF (atrial natriuretic factor) sedimented with the atrial granules during differential centrifugations. On the Percoll gradient, two distinct bands were observed. Cell debris, mitochondria, lysosomes, myofilaments and microsomes were mostly contained in the lightest-density (rho) (1.03-1.07 g/ml) fraction, as demonstrated by electron microscopy and by enzymic markers such as lactate dehydrogenase, monoamine oxidase, cytochrome c reductase, beta-glucuronidase and acid phosphatase. Atrial granules were mostly contained in the denser (rho 1.11-1.15 g/ml) band and were only slightly contaminated by lysosomes, as shown by beta-glucuronidase activity. Analysis of the ANF content in these isolated granules by h.p.l.c., amino acid composition and sequencing demonstrated that it was only the pro-ANF [ANF-(Asn1-Tyr126)-peptide]. The precursor was present in all granules, as demonstrated by immunocytochemistry. Since hormonal propeptides usually undergo intracellular processing, and the matured peptides are subsequently stored in the secretory granules, these results indicate that the processing pathway of ANF may be different from that of other hormonal peptides.     

The circadian rhythm of the N-terminus and C-terminus of the atrial natriuretic factor prohormone

Circadian variation in the circulating concentrations of the N-terminal and C-terminal portions of the atrial natriuretic factor prohormone (pro ANF) was evaluated in 8 men, ages 41-47, who have been followed for 19 years with respect to circadian variation in physiological variables including blood pressure and clinical chemistries. The N-terminus of the ANF prohormone contains two peptides consisting of amino acids 1-30 and 31-67 while the C-terminus contains 1 peptide (amino acids 99-126) of this 126 amino acid prohormone which lower blood pressure and have natriuretic properties. To determine if either the N-terminus and/or the C-terminus of the prohormone have a circadian variation in their circulating plasma concentrations these 8 men had blood samples obtained for radiommunoassay every 3 hr during a 24-hr period. Three radiommunoassays which immunologically recognize (1) the whole N-terminus (i.e. amino acids 1-98), (2) the midportion of the N-terminus (amino acids 31-67) and (3) the C-terminus (amino acids 99-126) of the ANF prohormone were utilized. The whole N-terminus, the midportion of the N-terminus which circulates after being proteolytically cleaved from the rest of the N-terminus, and the C-terminus each had a peak circulating concentration between 0400 and 0700 which were significantly (P less than 0.001) higher than their concentrations at any other time throughout the 24-hr period.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Rat pro-atrial natriuretic factor expression and post-translational processing in mouse corticotropic pituitary tumor cells

Atrial natriuretic factor (ANF) is stored within atrial myocyte secretory granules as pro-ANF (ANF-(1-126] and is proteolytically processed co-secretionally C-terminal to a single basic amino acid to form ANF-(1-98) and the bioactive product ANF-(99-126). Pro-ANF is also expressed in certain non-cardiac neuroendocrine cell types (e.g. brain, adrenal). Although the relatively low levels of the peptide in these cell types have precluded detailed processing and secretion studies using cultured cells, some work with tissue extracts suggests that pro-ANF is pre-secretionally processed between or C-terminal to Arg101-Arg102 in such cells. In order to assess whether cultured non-cardiac endocrine cells process pro-ANF pre- or co-secretionally, and to establish whether both paired and single basic amino acids can serve as cleavage sites, transfection studies were carried out using the adrenocorticotropic hormone (ACTH)-producing pituitary tumor cell line AtT-20/D-16v. These cells normally cleave pro-ACTH/endorphin pre-secretionally at selected, but not all, pairs of basic amino acids to a variety of product peptides. A prepro-ANF expression plasmid was constructed and transfected into the AtT-20 cells. The resulting ANF/AtT-20 cell clone selected for this study expressed ACTH at levels similar to the untransfected wild type cells and secreted immunoreactive ANF-related material at a rate of approximately 1 fmol/min/10(5) cells, which was about 10% the rate of ACTH secretion. The rates of secretion of both ANF and ACTH could be increased 3-5-fold with a variety of known AtT-20 cell secretagogues including phorbol esters and the beta-adrenergic agonist, isoproterenol, thus indicating that both peptides were routed through regulated secretory pathways. Utilizing a combination of specific antisera directed against various regions of pro-ANF, size exclusion and reversed phase high performance liquid chromatography, and peptide mapping, it was shown that the ANF/AtT-20 cells contained and secreted the bioactive peptide ANF-(103-126) and -(1-97). These results indicate that the ANF/AtT-20 cells specifically cleave pro-ANF pre-secretionally at the same single basic site used by cardiac tissue; this single basic cleavage is apparently followed by removal of Arg98 by carboxypeptidase H. It is also apparent that the cells can cleave at the sole paired basic site in pro-ANF, which is the probable cleavage site used by neurons and some other endocrine cells that express low levels of the prohormone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Atrial granules contain an amino-terminal processing enzyme of atrial natriuretic factor

At least three enzymes have been identified in atrial tissue homogenates that are capable of processing pro-atrial natriuretic factor to active atrial peptides. The atrial peptides possess potent natriuretic, diuretic, vasorelaxant, and hemodynamic properties, and their existence has implicated the mammalian heart as an endocrine organ. We have purified and characterized a serine proteinase (Mr approximately equal to 70,000) associated with atrial granules that preferentially hydrolyzes the Arg-Ser bond in the synthetic substrates Gly-Pro-Arg-Ser-Leu-Arg, benzoyl-Gly-Pro-Arg-Ser-Leu-Arg, and benzoyl-Gly-Pro-Arg-Ser-Leu-Arg-Arg-2-naphthylamide, the Arg-2-naphthylamide bond in the substrate benzoyl-Gly-Pro-Arg-2-naphthylamide, and the Arg-Ser bond in a 31-residue substrate (Gly96-Tyr126 peptide) corresponding to residues Arg98-Ser99 in pro-atrial natriuretic factor. The Gly96-Tyr126 peptide contains the putative processing site in pro-atrial natriuretic factor and the sequence for the bioactive peptides. Our results indicate that the minimum processing site sequence is -Gly-Pro-Arg-Ser-Leu-Arg-Arg- and that the Ser99-Tyr126 natriuretic peptide is the predominant hydrolytic product. After prolonged incubation or at high enzyme concentrations, the Ser103-Tyr126 natriuretic peptide may also be formed. The Ser103-Arg125 natriuretic peptide was only a very minor product. The doublet of basic amino acids is not the primary processing site in pro-atrial natriuretic factor, but their presence may influence cleavage at the single Arg residue "upstream." Our findings are consistent with the idea that the pro-protein and the processing enzymes are packaged into the secretory granule and in response to the proper stimulus, the pro-protein is processed to the active peptides, probably during the process of secretion. The processing pathway of pro-atrial natriuretic factor is discussed.     

Atrial natriuretic factor in the impulse-conduction system of rat cardiac ventricles

Summary A complex network of atrial natriuretic factor-producing cells has been delineated by biochemical and morphological techniques in the rat ventricular myocardium. The chordae tendineae spuriae (CTS; false tendons) contain ANF mRNA and the ANF propeptide (Asn 1-Tyr 126) as assessed by Northern blot analysis, high-pressure liquid chromatography and immunohisto- and -cytochemistry, using three different affinity-purified antibodies: monoclonal and polyclonal antibodies against C-terminal ANF (Arg 101-Tyr 126) and polyclonal antibodies against N-terminal ANF (Asp 11-Ala 37). Two types of cells harboring ANF-containing secretory granules constitute the CTS: the majority (Purkinje type I) have ultrastructural similarities with both atrial and classical Purkinje fibers. Purkinje type-II fibers resemble working ventricular cardiocytes. Both cell types harbor a large paranuclear Golgi complex. The subendocardial Purkinje network is also made up of these two cell types. In this location, Purkinje type-I fibers form cable-like structures while Purkinje type-II fibers are either located beneath the former or abut directly on the endocardium. The latter are not separated from adjacent working ventricular cardiocytes by connective tissue septa. Coronary arteries and arterioles, as in birds, are surrounded by a cushion of Purkinje type-II fibers which blend with the surrounding myocardium. These results indicate that, in the rat, the entire intraventricular conduction system is constituted of endocrine cells producing ANF.Supported by a Medical Research Council of Canada Group Grant to the Multidisciplinary Research Group on Hypertension, by the National Research Council of Canada, the Pfizer Company (England), Bio-Méga Inc. and the Canadian Heart Foundation     

Increased release of the N-terminal and C-terminal portions of the prohormone of atrial natriuretic factor during immersion-induced central hypervolemia in normal humans

The role of peptides from the N terminus and C terminus of the 126 amino acid atrial natriuretic factor (ANF) prohormone in modulating renal sodium and water handling has not been defined. Since water immersion to the neck (NI) provides an acute central volume expansion identical to that produced by 2 liters of saline but without plasma compositional change, immersion to the neck was used to assess the N-terminal and C-terminal portions of the ANF prohormone response to acute central blood volume expansion in seven seated sodium-replete normal subjects. Both the C terminus, which contains amino acids 99-126 and is identical to ANF, and the whole N terminus (i.e., amino acids 1-98) increased promptly with NI and peaked after 1 hr of immersion. A Mr 3900 peptide from the midportion of the N terminus consistent with amino acids 31-67 (i.e., pro-ANF-31-67) also increased with NI and followed a pattern of increasing circulating concentration nearly identical to that of the whole N terminus of the prohormone, except that its maximal concentration was at the second hour of the 3 hr of NI. With cessation of immersion, ANF decreased to preimmersion levels within 1 hr whereas the N terminus and pro-ANF-31-67, although their circulating concentrations were decreasing, were still significantly elevated at 1 hr. These findings suggest that the increase in plasma ANF, the N terminus of the ANF prohormone, and pro-ANF-31-67 from the midportion of the N terminus, with natriuretic properties similar to ANF, contribute to the natriuretic response to NI, implying a physiologic role for these atrial peptides in modulating volume homeostasis in humans.     

Degradation of human atrial natriuretic peptide by human brain membranes

Human atrial natriuretic peptide (Ser 99-Tyr 126) was rapidly degraded by both choroid plexus and hypothalamic membranes with a complex pattern of cleavage. The use of protease inhibitors allowed a preliminary characterization of the enzymes involved in the hydrolysis of the Ser-Phe and Phe-Arg bonds of iodine-labelled atrial natriuretic peptide.The C-terminal tripeptide was generated by three different enzymatic activities acting on the Ser-Phe bond: endopeptidase 24.11, a phosphoramidon-insensitive metallopeptidase and a thiol protease. Peptides like substance P, neurotensin, bradykinin inhibited the cleavage of the Ser-Phe bond of atrial natriuretic peptide. The C-terminal tripeptide was further degraded by aminopeptidases. Cleavage of the C-terminal dipeptide was inhibited by aprotinin, suggesting the contribution of brain kallikrein in the formation of this metabolite.These results show that many different proteases were involved in the hydrolysis of the C-terminal sequence of atrial natriuretic peptide, at least in vitro and underline the complexity of neuropeptide catabolism by brain preparations.     

Cloning of a cDNA encoding porcine brain natriuretic peptide

Complimentary DNA (cDNA) clones encoding porcine brain natriuretic peptide (BNP) were isolated from a porcine atrial cDNA library. The longest of the cDNA clones (1507 nucleotides) apparently originated from an unprocessed messenger RNA, since the nucleotide sequence encoding BNP-26 was interrupted by an intron of 554 nucleotides. A partial cDNA clone representing processed BNP mRNA was prepared by polymerase chain reaction. A comparison of the sequence of these two cDNAs reveals the presence of an additional intron within the sequence encoding the BNP precursor. The identification of these introns suggests that the BNP gene structure differs from the atrial natriuretic peptide gene in the location of intron 2. BNP mRNA encodes a propeptide of 131 amino acids, including a signal peptide domain (25 amino acids) and a prohormone domain (106 amino acids). Like atrial natriuretic peptide, the bioactive BNP sequence is localized at the carboxyl terminus of the prohormone. Although the carboxyl-terminal peptide sequences of porcine atrial natriuretic peptide and BNP are well conserved, there is relatively little homology within their propeptide regions.     

Atrial natriuretic factor prohormone peptides are present in a variety of tissues.

Utilizing two sensitive and specific radioimmunoassays which immunologically recognize 1) the 98 amino acid (a.a.) N-terminus and 2) the 28 a.a. C-terminus (i.e., a.a. 99-126) of the 126 a.a. atrial natriuretic (ANF) prohormone, various tissues including aorta, kidney, small intestine, colon, liver, spleen, lung, and testis were investigated to determine if the ANF prohormone was present in any of these tissues in addition to its previously demonstrated presence in heart and brain. Aorta with 62.3 +/- 3 ng of the N-terminus/g of tissue and 51.6 +/- 1.8 ng of the C-terminus of the ANF prohormone/g of tissue had the highest concentration of the ANF prohormone of the previously undescribed ANF prohormone-containing tissues. The next highest concentration of the ANF prohormone was in the intestine, followed by lung and spleen. Pancreas, liver and kidney had similar levels of immunologically recognized ANF prohormone (approximately 1/50 of the aorta), while the testis and cerebrum had low levels. These results suggest that a much larger variety of tissues synthesize and/or store the ANF prohormone than is presently thought.     

Identification of an endogenous protease that processes atrial natriuretic peptide at its amino terminus

We have partially purified a thiol-dependent protease from bovine atrial tissue that cleaves the Arg98-Ser99 bond of rat natriuretic peptide (Gly96-Tyr126) to produce the natriuretic Ser99-Tyr126 peptide (cardionatrin I). This was the only hydrolytic product we detected. The existence of the atrial natriuretic peptide system implicates the mammalian heart as an endocrine organ which participates in the hormonal regulation of extracellular fluid volume, electrolyte balance and vascular tone. This enzyme appears to be part of that system. The atrial protease also hydrolyzes the Arg-2-Napthylamide bond of natriuretic peptide stand-in substrates; on the basis of relative Vmax/Km as a measure of substrate specificity, Bz-Leu-Arg-Arg-2-Napthylamide (NA) greater than Bz-Leu-Arg-2-NA greater than Arg-2-NA. There is little or no cleavage between the Arg-Arg pair of the first substrate. Since in the Gly96-Tyr126 peptide the Arg-Arg pair is not the principle cleavage site for this enzyme, it is very unlikely that it is a principle cleavage site for this enzyme in pro-atrial natriuretic factor. It is possible that it is a cleavage site for a different enzyme or the pair may serve as a signal for cleavage at Arg98.