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.     

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.     

Studies of ANF processing and secretion using a primary cardiocyte culture model.

In contrast to most other endocrine peptides ANF is stored in the heart as part of a larger prohormone, often called pro-ANF, yet is found in the circulation as a 28 amino acid peptide, called ANF. It has been shown that the conversion of the 126 amino acid pro-ANF to ANF occurs in the heart. This paper summarizes studies from our laboratory that have used a primary neonatal rat heart cell culture system to investigate the location and mechanism of this relatively unusual processing event. We have found that in culture the maintenance of the cells in a glucocorticoid-containing serum-free medium is required to observe processing as occurs in vivo. The cells contain the prohormone while ANF accumulates in the medium. Various experiments with protease inhibitors, pulse-chase biosynthetic labeling, incubation of cells with ANF-related peptides, and enrichment of cultures for myocytes have resulted in our conclusion that the processing of pro-ANF takes place most likely within the cardiac myocyte just prior to, but in concert with secretion. We have expanded on the use of this processing-competent atrial myocyte culture system to investigate mechanisms of stimulated ANF secretion. It has been shown that the activation of several phospholipase C-coupled receptors (e.g., alpha 1-adrenergic and endothelin receptors) produces a robust release of ANF, but only in cultures that have been maintained under appropriate conditions. Further, it is apparent that the phenylephrine- or endothelin-mediated release of ANF depends in part on influx of extracellular calcium (Ca2+o), while the remaining component of stimulated release may depend on mobilization of intracellular calcium. It also appears that these agonists produce an initial phase of stimulated release, occurring within the first 5 min of agonist exposure, independent of Ca2+o, and a sustained phase that persists as long as the agonists remain on the cells, and depends on the presence of Ca2+o and thus calcium influx. Taken together our studies indicate that the hormonal environment may be an important factor directing the development of differentiated endocrine functions by atrial myocytes and may be involved in the regulation of ANF expression, biosynthesis, and secretion.     

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.     

Proteinase activities in bovine atrium and the possible role of mast cell tryptase in the processing of atrial natriuretic factor (ANF).

1. Using low salt, Triton X-100 and high salt extracts of bovine atria, two main proteinases were identified by means of fluorogenic oligopeptide substrates. 2. An acidic proteinase, extracted in low salt and Triton X-100 was identified as cathepsin B, but it caused little hydrolysis of the Z-Gly-Pro-Arg- containing substrate that resembles the cleavage site for activation of pro-ANF. 3. An alkaline proteinase was extracted only with high salt and had characteristics of the serine proteinase tryptase. It cleaved Z-Gly-Pro-Arg- containing substrates more efficiently than others tested and was localized in and around mast cells histochemically. Previously, Imada et al., 1988 (J. biol. Chem. 263, 9515-9519) found an identical enzyme would cleave ANF from pro-ANF. 4. These results suggest therefore that mast cell tryptase may be involved in the activation of ANF from pro-ANF.     

Atrial natriuretic factor (ANF) effects on L-, N-, and P/Q-type voltage-operated calcium channels

1. We have previously reported that atrial natriuretic factor (ANF) decreases neuronal norepinephrine (NE) release. The mechanism that mediates NE release from presynaptic membrane to synaptic cleft is a strongly calcium-dependent process. The modulator effect of ANF may be related to modifications in calcium influx at the presynaptic nerve ending by interaction with voltage-operated calcium channels (VOCCs).2. On this basis we investigated the effects of ANF on K⁺-induced ⁴⁵Ca²⁺ uptake and evoked neuronal NE release in the presence of specific L-, N-, and P/Q-type calcium channel blockers in the rat hypothalamus.3. Results showed that ANF inhibited K⁺-induced ⁴⁵Ca²⁺ uptake in a concentration-dependent fashion. Concentration–response curves to VOCC blockers nifedipine (NFD, L-type channel blocker), -conotoxin GVIA (CTX, N-type channel blocker), and -agatoxin IVA (AGA, P/Q-type channel blocker) showed that all the blockers decreased NE release. Incubation of ANF plus NFD showed an additive effect as compared to NFD or ANF alone. However, when the hypothalamic tissue was incubated in the presence of ANF plus CTX or AGA there were no differences in neuronal NE release as compared to calcium channel blockers or ANF alone.4. These results suggest that ANF decreases NE release by an L-type calcium channel independent mechanism by inhibiting N- and/or P/Q-type calcium channels at the neuronal presynaptic level. Thus, ANF modulates neuronal NE release through different mechanisms involving presynaptic calcium channel inhibition.     

Atrial natriuretic factor: radioimmunoassay and effects on adrenal and pituitary glands

A simple and sensitive radioimmunoassay was developed for measurement of immunoreactive atrial natriuretic factor (IR-ANF) in rat and human plasma and in rat atria. The two atria contain about 20 micrograms ANF per rat. The right atrium contained 2.5 times more ANF than did the left. Ether anesthesia and morphine markedly increased IR-ANF in rat plasma. The concentration of IR-ANF in plasma of clinically normal human subjects was 65.3 +/- 2.5 pg/ml. Paroxysmal tachycardia and rapid atrial pacing significantly increased IR-ANF in human plasma. Two- to seven-fold higher concentrations were found in coronary sinus blood than in the peripheral circulation. In the plasma of rats and humans, circulating ANF is probably a small-molecular-weight peptide. ANF acts on the adrenal and the pituitary. ANF inhibits aldosterone secretion from rat zona glomerulosa and steroid secretion by bovine adrenal zona glomerulosa and fasciculata. ANF stimulated the basal secretion of arginine vasopressin (AVP) in vitro and inhibited KCl-stimulated release of AVP.     

K物质在大鼠心脏内的分布及其对心血管活动的作用

本工作应用特异性K物质放射免疫分析法测定了大鼠心脏的K物质样免疫活性,心房和心室含量分别为19.9±3.5和4.1±0.8pmol/g组织。心脏内的K物质样免疫活性物质主要以单一分子形式存在。用免疫组织化学方法证明K物质免疫活性存在于心脏神经纤维内。大鼠心肌杂交细胞株(CP8401)细胞上含有K物质特异性结合部位,KD为1.61×10~(-10)mol/L,Bmax为8.08×10~(-12)mol/L。用心肌杂交细胞和离体心脏进行体外实验,证明K物质可释放ANF,这种释放可为速激肽受体拮抗剂[D-Pro~2,D-Trp~(7,9)]-SP部份抑制。给麻醉大鼠静脉注射K物质可引起血压降低、心率加快、左心室内压和收缩速度下降。这一作用亦可被[D-Pro~2,D-Trp~(7,9)]-SP所部份抑制。应用大鼠离体灌流心脏模型,发现K物质可升高心脏灌流压、左心室收缩期峰压、左心室内压最大上升速率和加快心率、具有正性变时和变力性作用。 本工作提示,K物质存在于心脏神经纤维内,在心肌细胞上具有K物质的特异性结合位点。K物质可促进心肌细胞释放心钠素,并可调节心脏的功能。     

Expression of atrial natriuretic factor gene in heart ventricular tissue

A novel peptide hormone, atrial natriuretic factor (ANF), was recently isolated and characterized in mammalian atria. This hormone has potent natriuretic, diuretic and vasorelaxant activities. Since ANF bioactivity was initially found in atria but not in ventricles, it was assumed that the ANF gene is specifically expressed in atria. We now report that ANF mRNA is present in ventricular tissue as well as in atria. This is clearly demonstrated by in situ hybridization and by Northern blot analysis. Rat ventricular ANF mRNA concentration is a hundred-fold lower than in atria. As in atria, the 126 amino acids precursor form of ANF is predominant in ventricles and it is present at a thousand-fold lower concentration. The ten-fold discrepancy in the ratio of ANF mRNA to immunoreactivity between atria and ventricles could reflect a higher rate of peptide release in the latter. Thus, ventricular ANF production may be physiologically significant in view of the much larger ventricular mass.     

Basal and stimulated ANF secretion: role of tissue preparation

Our previous results showed that addition of agonists, such as vasopressin and angiotensin, added to incubation medium with freshly excised rat atria caused marked release of atrial natriuretic factor (ANF). This release was in the form of prohormone rather than active peptide. Since others had difficulty reproducing these findings, in the present study we investigated ANF release with and without angiotensin addition in two sets of atrial tissue. In the first, tissue was blotted and carefully cleaned as previously described; in the second, atrial tissue was placed into incubation medium without prior preparation. ANF activity in the medium was measured by radioimmunoassay and receptor assay. Using the immunoassay, basal release of ANF was threefold greater from prepared vs. nonprepared atrial tissue; significant stimulation by angiotensin was seen only in the prepared atria. ANF release measured by radioreceptor assay was 1/5-1/10 of that measured by immunoassay. Taking the difference between the two measurements as an index of prohormone secretion, the results confirm that both basal and stimulated release was primarily in the form of proANF. Scanning electron microscopy revealed that cleaning of the atria had removed the endocardial lining of the tissue. The results thus indicate that an intact endocardium can prevent agonist-induced proANF secretion, suggesting that this tissue may be an important modulator of plasma ANF levels.     

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)     

Capsaicin-sensitive neural pathway mediates atrial natriuretic factor (ANF) release in response to physiological stimuli

Increases in intravascular volume are detected by mechanoreceptors situated at the junctions of the great veins with the atria. We had previously shown that localized distension of the superior vena caval/right atrial junction, simulating increased cardiac preload, elicits release of ANF remotely from the atrial appendage. We proposed that ANF secretion is stimulated via intrinsic neural pathways running from the venoatrial junctions to the appendage. We developed a technique whereby non-adrenergic, non-cholinergic sensory nerves could be selectively destroyed in the heart of adult rats by instilling capsaicin into the pericardial space. Four days later, the animals were killed, and isolated perfused atria were prepared with small balloons positioned so that the superior vena caval/right atrial junction could be discretely stretched. Immunoreactive ANF secretion into the perfusate was measured. Although distension of the venoatrial junction increased ANF secretion from the control atria, there was no such response in the denervated atria. We conclude (A) that local application of capsaicin to the heart of adult rats induces selective functional neural deficits and (B) that information regarding distension of the junction of the great veins and the atria is normally transmitted across the atrium via these nerves to stimulate ANF secretion from peptide stores located in the atrial appendage. We propose that these pathways are crucial to ensure appropriate ANF secretion in response to an increase in circulating blood volume.     

A synthetic oligonucleotide probe encoding for atrial natriuretic peptide detects specific mRNA transcripts in rat heart but not brain using in situ hybridization histochemistry

In situ hybridization histochemical techniques were used in an attempt to demonstrate atrial natriuretic peptide (ANP) messenger RNA (mRNA) in the rat brain. A synthetic oligonucleotide derived from previously reported ANF cDNA sequence was used as a probe. Northern blot analysis of total RNA isolated from rat heart demonstrated that the oligonucleotide recognized a single species of RNA (0.9 kb), a size consistent with previous reports. Rat heart sections revealed dense accumulations of ANF mRNA in the cardiac atria and lesser densities in the ventricles. Rat brain sections hybridized with the same oligonucleotide did not label ANF mRNA accumulations in any neuronal cell bodies. A possible explanation for this latter observation is either sparsely distributed expressing neurons or low expression and high turnover of ANF mRNA in brain.     

Effect of nitric oxide on basal and stretch-induced release of atrial natriuretic factor (ANF) from isolated perfused rat atria

We investigated the effect of the NO donor SNAP (6.7 nM) on basal and stretch-induced ANF release from isolated perfused rat atria. There was no significant difference in basal ANF secretion between the vehicle- and SNAP-infused atria (SNAP: 388+/-63 pg. 100 microl(-1), n = 13 vs. vehicle: 349+/-26 pg. 100 microl(-1), n = 5). Atrial distention caused an increase in ANF secretion in both the buffer- and SNAP-treated groups. SNAP greatly attenuated the stretch-induced increase in ANF (SNAP: 225+/-7 pg. 100 microl(-1), n = 5 vs. vehicle: 448+/-72 pg. 100 microl(-1), n = 13, P < 0.05). The compliance of atria treated with SNAP was lower than that of the vehicle-perfused atria (P < 0.05). Thus, although SNAP appeared to attenuate stretch-induced ANF secretion, there was in fact no significant difference in the ratio of Delta[ANF] to Deltaintraluminal volume (SNAP: 5.8+/-1.3 pg. 100 microl(-1). microl(-1) vs. vehicle: 8.2+/-1.4 pg. 100 microl(-1). microl(-1).). In conclusion, we found no evidence that NO alters the control of basal or stretch-induced ANF secretion. NO can however reduce ANF release by shifting the pressure-volume curve, so that a given increase in atrial pressure is associated with a smaller increase in intraluminal volume and reduced atrial distention.     

吗啡耐受大鼠心房心钠素的释放及其基因转录

本工作应用心钠素放射免疫测定和分子杂交技术首次发现,吗啡耐受大鼠血浆心钠素水平显著降低,心房内心钠素含量明显升高,同时心房内心钠素特异性mRNA水平也相应提高,提示在吗啡耐受时大鼠心房内心钠素的合成和贮存增加,释放减少。     

Homologous IRCM-Serine Protease 1 from pituitary,heart atrium and ventricle: A common pro-hormone maturation enzyme?

IRCM-Serine Protease 1 (IRCM-SP1) has recently been isolated and characterized from porcine pituitary anterior and neurointermediate lobes (Cromlishet al., 1986a,J. Biol. Chem. 261:10850–10858; Cromlishet al., 1986b,J. Biol. Chem. 261:10859–10870). This pituitary serine protease was shown to selectively cleave human proopiomelanocortin (POMC)-derived peptides at both pairs of basic residues and C-terminal to specific Arg residues, all known to be cleavedin vivo. Here, a similar enzyme was isolated from rat heart atria and ventricles. Rat IRCM-SP1 was shown to be highly specific for the same cleavage sites in POMC, as the porcine pituitary homologue. Furthermore, the rat and the porcine enzymes cleave rat pro-Atrial Natriuretic Factor (pro-ANF 1–126) to yield ANF 103–126, 102–126 and 99–126 in that order of preference. This suggests thatin vitro the cleavage sites preferred in pro-ANF resemble those found in brain and hypothalamus. The enzyme is nine times more abundant in atria versus ventricles/mg protein. It is concluded that IRCM-SP1, could well represent a common pro-hormone maturation enzyme for POMC and Pro-ANF and possibly many other pro-hormones.     

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.     

Intra-atrial communication and control of atrial natriuretic factor (ANF) release

Atrial natriuretic factor (ANF) release was studied in isolated perfused atria prepared from rats. When the vein-atrial junction (VAJ) was distended with an inflatable balloon, ANF release into the perfusate was greater in intact atria than in appendectomized atria. It was concluded that distention of the VAJ causes ANF release from the atrial appendage. A cascade experiment was then prepared whereby buffer from one isolated atrium perfused a second atrium. Although the VAJ of the first atrium could be distended by balloon, the atrial appendage was ligated so ANF was not secreted into the perfusate. The second atrium was intact, but no balloon was inserted. Despite the fact that there were no changes in intraluminal pressure, ANF secretion from the second atrium increased when the VAJ of the first atrium was distended. This response was blocked by the endothelin (ET) A receptor antagonist BQ-123. However, no distention-induced changes in ET-1 levels could be found in the perfusate from the first atrium. It is proposed that, in response to changes in distention of the VAJ, ANF is released remotely from the atrial appendage. The mediator does not appear to be ET-1 itself, but rather some factor that stimulates ET-1-induced ANF release within the tissue of the atrial appendage.