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Am J Physiol Heart Circ Physiol 275: H1200-H1208, 1998;
0363-6135/98 $5.00
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Vol. 275, Issue 4, H1200-H1208, October 1998

Amino-terminal proBNP in ovine plasma: evidence for enhanced secretion in response to cardiac overload

C. J. Pemberton, T. G. Yandle, M. T. Rademaker, C. J. Charles, G. D. Aitken, and E. A. Espiner

Department of Endocrinology, Christchurch School of Medicine, Christchurch 1, New Zealand

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

We have recently identified a novel amino-terminal fragment of pro-brain natriuretic peptide (NT-proBNP) in the circulation of humans, the concentration of which increases progressively as the left ventricle fails. To clarify the origins of NT-proBNP in experimental animals, we have developed an RIA for NT-proBNP based on residues 52-71 of ovine proBNP-(1---103) and used it to study cardiac processing, secretion, and metabolism of BNP in sheep with cardiac overload induced by coronary artery ligation (CAL) or rapid left ventricular pacing (rLVP). The concentration of NT-proBNP in left atrial plasma extracts drawn from normal control sheep was threefold that of mature BNP. Size-exclusion and reverse-phase HPLC analyses of plasma extracts coupled to RIA revealed a single peak of immunoreactive (ir) NT-proBNP [~8,000 relative molecular weight (Mr)], quite distinct from a single peak of ir-mature BNP (~3,000 Mr). In contrast, ovine cardiac tissue contained only a single immunoreactive peak of high-molecular-weight BNP (~11,000 Mr), consistent in size with proBNP-(1---103). Sampling from the cardiac coronary sinus in normal control sheep (n = 5) and sheep with CAL (n = 5) revealed that the molar ratio of NT-proBNP to mature BNP was similar. There was a significant gradient of both mature and NT-proBNP across the heart in normal sheep, whereas after CAL the gradient was significant for mature BNP only. In both forms of cardiac overload (CAL and rLVP), left atrial plasma levels of NT-proBNP were significantly increased above normal levels, in contrast with mature BNP levels, which were raised only in the rLVP group of animals. Blockade of natriuretic peptide metabolism in sheep with heart failure (induced by rLVP) raised mature BNP levels threefold but did not affect levels of NT-proBNP. In conclusion, these studies show that NT-proBNP is formed from proBNP stores during secretion and, compared with mature BNP, accumulates in plasma because metabolism of NT-proBNP appears to differ from that of mature BNP. Although its function, if any, remains unclear, plasma NT-proBNP may prove to be a sensitive marker of cardiac overload and/or decompensation.

natriuretic peptide; high-performance liquid chromatography; neutral endopeptidase; cardiac processing

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

ATRIAL NATRIURETIC PEPTIDE (ANP), secreted by the atrium, and brain natriuretic peptide (BNP), secreted largely by the cardiac ventricle, are circulating hormones responsive to increases in intracardiac pressure (29). Acting via specific receptors (6, 8) located within the kidney, adrenal cortex, and the vascular system (19), both hormones serve to maintain natriuresis, inhibit aldosterone, and reduce intravascular volume and pressures, the combined actions of which help to control arterial pressure and cardiac filling pressures and output (10).

Although regulated by distinct genes, both ANP (33) and BNP (23, 18) are synthesized within cardiac myocytes as high-molecular-weight precursors. In all mammals studied to date, ANP is stored in atrial granules as a 126-amino acid precursor, proANP (33), which is cleaved during secretion to release the mature (bioactive) low-molecular-weight form, ANP-(99---126) (34, 38, 29), and an amino-terminal form, ANP-(1---98) (35), also known as NT-proANP (32, 17). Much less is known of BNP processing and secretion, which, in contrast with the regulated secretion of ANP from atrial stores, appears to be largely constitutive (18, 23, 30). We have recently identified an 8,600 relative molecular weight (Mr) amino-terminal form of proBNP-(1---108) (NT-proBNP) in human cardiac tissue (40) and plasma extracts (16) that is consistent in size with proBNP-(1---76). Several recent observations from our laboratory suggest that NT-proBNP is actively secreted by the human heart. First, NT-proBNP [distinct from proBNP-(1---108) and mature BNP-32] is found in cardiac tissue extracts (40). Second, plasma levels of NT-proBNP increase progressively as left ventricular function deteriorates (15). Third, although high-molecular-weight proBNP-like immunoreactivity circulates in human plasma (39), minimal processing of proBNP-like material occurs in blood or plasma (14). These findings strongly suggest that the amino-terminal form of proBNP is actively secreted by the heart and call for more detailed studies of cardiac processing, secretion, and metabolism of NT-proBNP in experimental animals.

Our recent cloning of the ovine BNP gene (1), yielding knowledge of the amino acid sequence of pro-ovine BNP [proBNP-(1---103)] and the likely processing sites (26) within the ovine proBNP sequence, led us to generate site-specific antisera and, subsequently, an RIA for ovine NT-proBNP. We now report the first evidence for the presence of NT-proBNP in a species other than human and its enhanced secretion in ovine models of cardiac overload, together with comparative studies of the metabolism of mature and amino-terminal BNP forms in sheep with experimental heart failure.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

RIA for Ovine NT-proBNP

Peptides. Several lines of evidence suggest that the mature (carboxy-terminal proBNP) forms in sheep comprise 26- to 29-amino acid peptides (26), consistent with putative furin/PC3 processing sites (Arg-4-X-X-Arg-1) (30, 31). These sequences occur at Lys74-Met75-Met76-Arg77 and Arg71-Gly72-Pro73-Lys74 in ovine proBNP-(1---103) (1). The former processing site would yield ovine NT-proBNP-(1---77), whereas the latter would yield NT-proBNP-(1---74). Accordingly, two peptides were synthesized by Chiron Mimotopes (Melbourne, Australia). The first was authentic ovine proBNP-(52---71) (Pro52-Ala-Ala-Ala-Pro-Ala-Gly-Phe-Leu-Gly-Pro-His-His-Ser-Leu-Leu-Gln-Ala-Leu-Arg71) to be used as immunogen, whereas, in the second peptide sequence, Tyr replaced residue Pro52 in the otherwise identical peptide. The latter peptide was synthesized for the preparation of radiolabeled tracer. The purity of these peptides as assessed by mass spectrometry was >47 and 57%, respectively.

Conjugation and immunization. Synthetic ovine proBNP Pro52-Arg71 (2 mg) was conjugated to BSA (2 mg; Sigma-Aldrich, Penrose, Auckland, NZ) in distilled water over 12-16 h at room temperature using 200 mg of carbodiimide at pH 6.0-6.2. The conjugate was dialyzed against three changes of 0.9% saline over 24 h at 4°C, emulsified at room temperature with an equal volume of complete Freund's adjuvant (Sigma-Aldrich), and injected intradermally into four New Zealand White rabbits over five or six sites. Rabbits were bled 10-12 days after injection, and the procedure was repeated over 4- to 6-wk intervals until an adequate titer was obtained.

Preparation of ovine proBNP 125I-labeled Tyr0-proBNP-(53---71). Ovine Tyr0-proBNP-(53---71) (5 µg) was iodinated using 0.5 mCi Na125I in the presence of 10 µg chloramine T in 5 µl of 0.5 M phosphate buffer, pH 7.3, for 20-25 s. The reaction was stopped by the addition of 50 µg cysteine HCl in 5 µl phosphate buffer. The resulting mixture was loaded onto a 10-cm RP300 Brownlee HPLC column (Applied Biosystems, San Jose, CA) and eluted with a gradient from 0 to 60% acetonitrile in 49 mM phosphate buffer (pH 2.9) over 30 min at 1 ml/min. Fractions (0.5 ml) were collected. One major peak of ovine 125I-labeled Tyr0-proBNP-(53---71) eluted at a concentration of 34% acetonitrile and contained >65% of the total radioactivity.

NT-proBNP RIA procedure. Antiserum 543-5 (drawn from rabbit 543, 5th bleed) was used in the RIA at a final dilution of 1:3,000. The cross-reactivities of this antiserum with the synthetic peptides human (h) BNP-32, hproBNP-(62---76), porcine (p) BNP-26, pBNP-32, hANP-(99---126), C-type natriuretic peptide 22, and endothelin-1 were all <0.01%; cross-reactivity with human adrenomedullin was <0.05%, angiotensin II <0.03%, hproANP-(1---30) <0.02%, and hproBNP-(1---21) <0.25%. Because complete synthesis of the entire putative NT-proBNP peptide [NT-proBNP-(1---77)] was impractical, standards were prepared from synthetic ovine proBNP-(52---71) (Chiron Mimotopes) with the purity data supplied taken into account. All sample extracts, radioactive tracer, standard, and antiserum solutions were diluted in assay buffer, pH 7.4 (26). The assay incubate consisted of 100 µl of extracted sample or standard (0-640 fmol/tube) combined with 100 µl of antiserum and 100 µl (10,000 counts/min) of the ovine proBNP 125I-labeled Tyr0-proBNP-(53---71). The tubes were vortexed and incubated at 4°C for 24 h. Free and bound NT-proBNP were separated by a solid-phase second-antibody method (donkey anti-rabbit Sac-Cel, Immunodiagnostic Systems, Boldon, UK). Sac-Cel (1 ml) diluted in 5% dextran solution (final Sac-Cel concentration 5%) was added to each tube, and the solution was vortexed and incubated at room temperature for 30 min. The tubes were centrifuged for 15 min at 2,500 g at 20°C and the supernatant decanted. The resulting pellet was counted in a Gammamaster (LKB, Uppsala, Sweden).

Mature BNP RIA procedure. Mature BNP [representing ovine proBNP-(74/77---103)] was extracted on Vycor glass beads and assayed as previously described (26).

HPLC of Immunoreactive Ovine NT-proBNP and Mature BNP

Before HPLC, plasma samples were extracted with the use of Vycor glass or SepPak C18 cartridge methods. Cardiac tissue samples were processed as previously reported (26). For all HPLC procedures an LC-10AD pump (Shimadzu, Kyoto, Japan) was used. Size-exclusion HPLC (SEHPLC) was performed on a TSK-GEL G2000SW (Toyo Soda, Tokyo, Japan) column (7.5 × 600 mm) equilibrated with 20% acetonitrile in 0.1% trifluoracetic acid (TFA) at 0.5 ml/min with 0.5-ml fractions collected. The column was calibrated with the following Mr standards: bovine thyroglobulin, 90,000 Mr; cytochrome c, 12,384 Mr; aprotinin, 6,500 Mr; and synthetic porcine (ovine) BNP-26, 2,867 Mr. BNP immunoreactivity identified by SEHPLC was further resolved by reverse-phase HPLC (RPHPLC) with the use of a 22-cm RP300 Brownlee cartridge column (Applied Biosystems) with a gradient from 0 to 60% acetonitrile in 0.1% TFA (at 40°C) over 60 min at 1 ml/min, and 0.5-ml fractions were collected. Fractions were dried under a stream of air at 37°C after the addition of 10 µl of 1% Triton X-100 (Sigma, St. Louis, MO) and reconstituted in 0.5 ml of buffer.

Extraction of BNP Immunoreactivity From Ovine Plasma and Cardiac Tissue

Plasma. Two separate methods were assessed for the extraction and isolation of BNP forms from ovine plasma, solid-phase SepPak C18 cartridges (26) and Vycor glass (Corning, NY). With the latter method, a ratio of 4 ml of plasma to 1 ml of 25 mg/ml Vycor glass (in distilled water) was mixed for 30 min at 4°C. After centrifugation (2,500 g at 4°C for 15 min) and aspiration of the supernatant, the pellet of glass-bound peptide was washed twice with distilled water and eluted by continuous mixing with 2 ml of 60% acetone-0.05 M HCl (redistilled) for 30 min at 4°C. The supernatant obtained after centrifugation (2,500 g at 4°C for 15 min) was transferred to glass tubes containing 10 µl of Triton X-100 (Sigma) and dried under a stream of air at 37°C. The dried extract was reconstituted in RIA buffer and stored at -20°C until RIA. The mean recoveries of synthetic mature ovine BNP added to plasma were 90 ± 1% (n = 3) and 85 ± 3% (n = 5) for SepPak and Vycor extraction methods, respectively. Because the molecular nature of endogenous ovine NT-proBNP was unknown (hence, it is unavailable in a purified form), calculation of its recovery from plasma with the use of either extraction method was not determined.

Cardiac tissue. proBNP-(1---103) and other possible processed forms were extracted from the atrium and ventricle of one normal control sheep heart, as previously described (26).

Physiological Studies

Plasma levels of NT-proBNP and mature BNP were measured in normal control sheep and in two separate models of cardiac overload: coronary artery ligation (CAL) and experimental heart failure induced by rapid left ventricular pacing (rLVP). An additional study examined the effect on plasma NT-proBNP and mature BNP hormone levels of inhibition of the two main degradation pathways of the natriuretic peptides, i.e., natriuretic peptide clearance receptor (NPR-C) and neutral endopeptidase (NEP) activity. All experimentation was undertaken after approval by the Christchurch School of Medicine Animal Ethics Committee (Christchurch, New Zealand).

Coronary artery ligation. Five animals were instrumented via a left lateral thoracotomy performed under general anesthesia induced by thiopental (17 mg/kg) and maintained with halothane-nitrous oxide. Polyvinyl chloride (PVC) catheters were inserted into the coronary sinus (via the azygous vein) and left atrium for blood sampling. Two surgical sutures were positioned as follows: the first was placed around the left anterior descending (LAD) coronary artery (~40% anterior to the apex of the heart), and the second suture embraced the second diagonal of the LAD coronary artery at a position in line with the first. At time t = 0 min, both sutures were closed off, thus ligating both branches of the LAD coronary artery. Incisions were sutured, and the animals were given intramuscular pethidine (50 mg) and returned to metabolic cages with free access to food and water. CAL in all animals resulted in moderate to severe myocardial infarctions as confirmed by 1) a marked S-T segment elevation determined by acute electrocardiogram measurements, 2) a 17-fold increase in creatinine kinase and a 400-fold increase in troponin T cardiac enzyme concentrations, 3) a marked decrease in left ventricular ejection fraction from 56 ± 2% to 34 ± 5%, and 4) visual confirmation of ventricular infarct postmortem (unpublished observations).

Blood was drawn from the coronary sinus and left atrial catheters at 30 min preligation (normal control) and at 3 h postligation (CAL). It should be noted that 30-min-preligation samples were taken from anesthetized animals, whereas 3-h-postligation samples were drawn from conscious animals after completion of surgery and receipt of 50 mg intramuscular pethidine. Blood was taken into chilled 10-ml collection tubes containing 100 µl of 15 mg/ml Na3-EDTA, inverted for 30 s, and immediately centrifuged at 2,500 g at 4°C for 15 min to prepare plasma. Plasma was then stored at -80°C before extraction.

rLVP-induced heart failure. Five animals were prepared as previously described (11, 27). Briefly, a left lateral thoracotomy was performed, and two PVC catheters were inserted into the left atrium for blood sampling and left atrial pressure (LAP) determination, respectively; a Königsberg (P 6.0) high-fidelity pressure-tip transducer was inserted into the aorta for measurement of mean arterial pressure (MAP); an electromagnetic flow probe was placed around the ascending aorta to measure cardiac output; a 7-Fr Swan-Ganz catheter was inserted in the pulmonary artery for infusions; and a 7-Fr His-bundle electrode was stitched subepicardially to the wall of the left ventricle for subsequent LV pacing. Animals received 50 mg intramuscular pethidine postoperatively and were allowed to recover in metabolic cages for 14 days before the initiation of pacing at 225 beats/min. Animals had free access to food and water at all times. Heart failure was induced by rLVP at 225 beats/min for 7 days, as previously reported (11, 27). Blood samples were drawn from the left atrial catheter before the initiation of pacing (normal control) and after 7 days of continuous pacing (rLVP). Plasma was prepared and stored as for the CAL study. Hemodynamic measurements were not recorded.

Inhibition of natriuretic peptide degradation pathways during rLVP-induced heart failure. Five sheep with heart failure induced by rLVP received in balanced random order either 1) vehicle or 2) combined administration of an NEP inhibitor, SCH-32615 (Schering-Plough, NJ), and the NPR-C ligand C-ANP-(4---23) (Scios Nova, Palo Alto, CA). SCH-32615 was given as intravenous boli in three incremental doses (1, 5, and 25 mg/kg) at 90-min intervals, overlaying a continuous intravenous infusion of C-ANP-(4---23) (also given as 3 incremental doses of 20, 100 and 500 pmol · kg-1 · min-1 each) for 90 min. Vehicle was 10 ml of 0.9% saline for intravenous boli and 60 ml of Haemaccel (Behring, Frankfurt, Germany) for infusions. All treatments were administered by pulmonary artery catheter. All blood samples were taken from the left atrium with the sheep standing quietly in the metabolic crate. Concomitant hemodynamic and blood samples were obtained immediately pretreatment (t = 0) and t = 90, 180, and 270 min posttreatment (i.e., at the end of each incremental dose but before the next increment had started). Plasma samples were stored at -80°C until extraction.

Data Presentation and Statistical Analysis

When appropriate, data are presented as means ± SE. Statistical analysis was performed on two means using the two-tailed Student's t-test with the computer program SigmaStat (Jandel Scientific, Corte Madera, CA). For treatment- and time-related analysis, a two-way repeated-measures ANOVA was performed using the statistical computer program BMDP 2V (BMDP Statistical Software, San Francisco, CA). When significance was obtained by two-way ANOVA, a priori Fishers protected least significant difference tests were applied to identify individual points of significance. In all statistical analysis, significance was defined as P < 0.05.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Radioimmunoassay for Ovine NT-proBNP

Three of the four rabbits produced antisera, and among these, antiserum 543-5 gave the best binding of radiolabeled tracer and the most sensitive standard curves for assay development. With the use of antiserum 543-5 and proBNP-(52---71) as standard, the assay had a mean zero binding of 20.0 ± 0.4%, a detection limit of 3.3 ± 0.3 fmol/tube (11 pmol/l), and an IC50 (concentration displacing 50% of tracer) of 82.3 ± 1.9 fmol/tube (247 pmol/l) based on data obtained from eight consecutive assays. Nonspecific binding, with the use of assay buffer (2.7 ± 0.1%) or distilled water (0%), was low. As shown in Fig. 1, the dilutions of SepPak and Vycor extracts of plasma (drawn from sheep 3 h post-CAL) and normal control cardiac tissue extracts displaced in parallel with the standard curve.


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  		Fig. 1.   Dilution curves for sample extracts from plasma (Vycor and SepPak extracts) and cardiac tissue compared with standard curve for assay of ovine amino-terminal pro-brain natriuretic peptide (NT-proBNP) based on synthetic ovine NT-proBNP-(52---71) standards and ovine NT-proBNP 125I-labeled Tyr0-proBNP-(53---71) tracer (125I-Tyr0-labeled Ala53-Arg71). Numbers refer to dilution factors for plasma (horizontal bar, range 1- to 32-fold) and cardiac tissue extracts (5- to 160-fold).

Molecular Forms of NT-proBNP and Mature BNP in Ovine Plasma

Having established that immunoreactive (ir) NT-proBNP was present in ovine plasma extracts, ir-NT-proBNP was further characterized with SEHPLC and RPHPLC. The same fractions were also assayed for ir-mature BNP. As shown in Fig. 2, Vycor extracts of plasma drawn from normal animals (normal control, n = 2) and sheep with cardiac overload (CAL, n = 2; rLVP, n = 2) show a single peak of ovine ir-NT-proBNP using SEHPLC. In every case this material eluted with a molecular weight approximating 8,000 (Fig. 2). The molecular form(s) of ir-mature BNP in these same plasma extracts were quite distinct from ir-NT-proBNP form(s), eluting later on SEHPLC (Fig. 2) and in keeping with synthetic ovine BNP-26 standard. As shown in Fig. 3, further discrimination of ir-NT-proBNP and ir-mature BNP molecular forms was obtained using RPHPLC. Thus, using RPHPLC, both NT-proBNP and mature BNP forms were consistently separated into two distinct peaks of immunoreactivity. Identical SEHPLC and RPHPLC profiles were obtained from SepPak C18 extracts of plasma (data not shown). For both NT-proBNP and mature BNP forms, >85% of the immunoreactivity measured in individual extracts was recovered after separation on HPLC. In plasma drawn from the coronary sinus of normal control animals (Fig. 2A) and in coronary sinus plasma drawn from CAL animals (Fig. 2B), the ratio of NT-proBNP to mature BNP was ~1:1. However, in plasma drawn from the left atrium of the rLVP group of animals, the ratio of NT-proBNP to mature BNP was ~6:1 (Fig. 2C).


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  		Fig. 2.   G2000 size-exclusion HPLC (SEHPLC) analysis of immunoreactive (ir-) NT-proBNP (filled circles) and ir-mature BNP (open circles) in Vycor extracts of ovine plasma from 3 groups [normal control (A), coronary artery ligation (CAL; B), and rapid left ventricular pacing (rLVP; C)] of experimental sheep. In all cases, a similar peak of ir-NT-proBNP was observed with a relative molecular weight (Mr) approximating 8,000, quite distinct from a lower molecular weight peak of ir-mature BNP, which eluted consistent with synthetic porcine BNP-26 standard. Identical elution profiles were observed in SepPak C18 extracts of the same plasma samples. Data for A and B were obtained from coronary sinus plasma, whereas data for C were obtained from left atrial plasma. Arrows indicate elution positions: 1, column void volume; 2, cytochrome c (12,384 Mr); 3, aprotinin (6,500 Mr); and 4, synthetic porcine BNP-26 (= ovine BNP-26; 2,867 Mr).


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  		Fig. 3.   Reverse-phase HPLC (RPHPLC) analysis of ir-NT-proBNP (filled circles) and ir-mature BNP (open circles) in Vycor extracts of normal (A) and CAL (B) coronary sinus plasma. SepPak C18 extracts gave identical profiles on SEHPLC. Arrow (4) indicates elution time of synthetic porcine BNP-26 standard (2,867 Mr).

Immunoreactive Levels and Molecular Forms of BNP in Cardiac Tissue Extracts

BNP immunoreactivity in the atrial tissue extract was 62 pmol/g wet weight as detected by the NT-proBNP RIA and 110 pmol/g wet weight by the mature BNP RIA. BNP immunoreactivity in the ventricular extract was ~9 pmol/g wet weight by NT-proBNP RIA and 7 pmol/g wet weight by mature BNP RIA. With the use of two separate HPLC systems (SEHPLC/RPHPLC), NT-proBNP and mature BNP immunoreactivity in atrial tissue extracts (Fig. 4, A and B) and ventricular tissue extracts (Fig. 4, C and D) coeluted as single peaks corresponding to a molecular weight approximating 11,000. In agreement with our previous report (26), there was no evidence for processed NT-proBNP or mature BNP in cardiac extracts. Thus the ovine heart contains only high-molecular-weight immunoreactive BNP, i.e., proBNP-(1---103).


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  		Fig. 4.   G2000 SEHPLC (A and C) and RPHPLC (B and D) profiles of atrial tissue (A and B) and ventricular tissue (C and D) extracts assayed for NT-proBNP (filled circles) and mature BNP (open circles) immunoreactivity. Note that tissue profiles presented here were generated on a different G2000 SEHPLC column from that used for Fig. 2. Both columns, however, were calibrated using the same molecular weight standards; thus arrows represent the same markers as in Fig. 2.

Immunoreactive Levels of NT-proBNP and Mature BNP in Ovine Plasma: Effect of Cardiac Overload

As shown in Fig. 5, mean levels of NT-proBNP and mature BNP obtained with Vycor extraction were similar to those in plasma drawn from the coronary sinus of normal control sheep (n = 5). Compared with simultaneous plasma samples drawn from the left atrium, significant step-ups in the concentrations of both NT-proBNP (2-fold, P = 0.028) and mature BNP (7-fold, P < 0.001) forms occurred across the heart. A similar pattern is evident in the CAL group [step-up across the heart 1.5-fold (not significant) and 13-fold (P < 0.001) for NT-proBNP and mature BNP forms, respectively]. Thus gradients across the heart were greater for mature BNP than for NT-proBNP.


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  		Fig. 5.   Comparison of immunoreactive levels of NT-proBNP (filled bars) and mature BNP (shaded bars) in coronary sinus (CS) and left atrial (LA) plasma extracts drawn from 3 groups of experimental sheep: normal control (n = 5), CAL (n = 5), and rLVP (n = 5). CAL samples were drawn at 3 h postligation, whereas rLVP samples were drawn after 7 days of pacing. Levels are means ± SE. ** P < 0.05 vs. normal control level of same fragment in same source. Psi  P < 0.05 for NT-proBNP level vs. mature BNP level in same sample.

Compared with left atrial plasma levels in the normal control group, NT-proBNP levels were significantly raised in CAL (P = 0.036) and rLVP (P < 0.001) groups of animals, whereas levels of mature BNP were significantly raised (P < 0.001) only in the rLVP group. However, the mean NT-proBNP concentration in left atrial plasma was significantly higher than simultaneously drawn plasma levels of mature BNP in all three experimental groups (normal control = 3-fold, P < 0.001; CAL = 7-fold, P = 0.016; rLVP = 5-fold, P < 0.01).

Inhibition of Natriuretic Peptide Degradative Pathways During rLVP-Induced Heart Failure

Before the experimental intervention, the mean left atrial plasma level of NT-proBNP (~270 pmol/l) was more than fourfold that of the mature BNP level (~60 pmol/l) in sheep paced at 225 beats/min for 7 days (P < 0.01, n = 5). As shown in Table 1, there was a greater than threefold rise (P < 0.001 compared with normal control, n = 5) in plasma mature BNP levels when degradative pathways were blocked by the combined administration of SCH-32615 and C-ANP-(4---23), whereas levels remained unchanged when vehicle alone was given. In contrast, plasma levels of NT-proBNP were unchanged by inhibition of degradative pathways.

                              
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  		Table 1.   Effect of combined NPR-C/NEP inhibition on hormonal and hemodynamic parameters in five sheep with pacing-induced heart failure

The effect of combined NEP/NPR-C inhibition on hemodynamic indexes in ovine heart failure has been previously reported (28). In accordance with this previous report, there was an immediate and significant fall in MAP during the first dose of combined NEP/NPR-C inhibition (P < 0.001 vs. vehicle; Table 1). MAP continued to fall in a dose-dependent manner throughout the study, with the greatest fall (8 mmHg, P < 0.001 vs. vehicle) obtained at the end of the highest dose period. Cardiac output increased progressively and was significantly higher (P < 0.001 vs. vehicle) at t = 270 min (Table 1). Thus, in accordance with increases in mature BNP levels in left atrial plasma during combined NEP/NPR-C inhibition, there were significant improvements in arterial and cardiac hemodynamics in these sheep with established heart failure.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

These results establish the presence of NT-proBNP as a circulating hormone in sheep. To the best of our knowledge, this is the first report of the amino-terminal form of proBNP in the circulation of experimental animals. These findings, together with our previous findings in humans (15, 16), strongly suggest that the production of NT-proBNP forms is intrinsic to the process of BNP secretion in mammals. HPLC analysis confirmed that NT-proBNP, extracted from ovine plasma, was high-molecular-weight material (~8,000 Mr), consistent with its production by cleavage between Lys74-Met75 and/or Arg77-Asp78 of the ovine proBNP sequence (1). In showing a significant gradient across the heart, the present work supports the view that NT-proBNP is secreted (in concert with mature BNP) by the heart into the systemic circulation, where its metabolism (uptake by NPR-C and hydrolysis by NEP) appears to differ markedly from that of mature BNP-26/29 (7, 28).

The establishment of a specific RIA for NT-proBNP was made feasible by the synthesis of the immunogen proBNP-(52---71) and the subsequent generation of antiserum directed against the carboxy terminus of the putative proBNP-(1---74/77) fragment. This antiserum showed no cross-reactivity with mature BNP (ovine or human forms) or with the relevant human NT-proBNP sequence. Ovine NT-proBNP proved to be equally extractable from plasma using Vycor glass or SepPak C18 methods, in contrast to our experience with human plasma (15, 16) in which the NT-proBNP fragment was poorly extracted on Vycor glass. This contrast may result from the differing polarities of the two peptides. Amino acid residue analysis reveals human NT-proBNP (19, 24) to possess an overall neutral charge, whereas ovine NT-proBNP (1) has a moderately negative charge that may affect the relative binding of the peptide to charged surfaces such as glass. A comparison of extraction procedures in the present study showed SepPak and Vycor methods to be equally efficient at retaining mature ovine BNP. However, in the absence of purified NT-proBNP, suitable for addition to ovine plasma, the true recovery of this peptide using either extraction method cannot be determined.

HPLC profiles of ovine cardiac tissue extracts revealed only one BNP form (proBNP-like immunoreactivity), with no evidence of processed NT-proBNP or mature BNP forms. These findings contrast with those from humans in which at least three forms (NT-proBNP, intact proBNP, and mature BNP) are present in cardiac tissue extracts (37, 40). With respect to ANP, at least in the human and rat, cardiac stores contain high-molecular-weight proANP (22, 33), whereas coronary sinus plasma extracts contain both mature ANP and NT-proANP in equimolar ratios (5, 12, 17, 32). Thus the current study provides further evidence that storage and secretion of BNP forms appears to be a species-specific process, in contrast with the synthesis and secretion of ANP, which appear to be less variable across species. Differences between humans and sheep extend further to circulating BNP forms. SEHPLC and RPHPLC revealed ir-NT-proBNP in ovine plasma to be ~8,000 Mr and quite distinct from ir-mature BNP. Furthermore, the molecular form of the peptide did not alter in the settings of CAL or rLVP. These data are in agreement with studies on human NT-proBNP in plasma (15, 16), which was also shown to comprise one immunoreactive peak under similar HPLC conditions. In sheep plasma there appear to be only two major forms representing the cleaved products of ovine proBNP-(1---103), i.e., NT-proBNP [presumed to be proBNP-(1---74) and/or NT-proBNP-(1---77)] and mature BNP forms (26). However, in humans, as well as the presence of NT-proBNP and mature BNP (BNP-32), plasma also contains a high-molecular-weight (~10,000 Mr) form of BNP (39), which may be a form of proBNP-(1---108) with several amino acids deleted from the amino terminus (14, 16). These findings point to more complete processing of BNP in sheep during secretion than appears to occur in humans. However, in making these comparisons it must be noted that the relevant antibodies (to NT-proBNP forms) differ, with the human antibody directed toward proBNP-(1---14) (15, 16), whereas the present sheep antibody is directed toward proBNP-(52---71). In this context it is relevant to note that quantification of the stored form [higher molecular weight, presumably proBNP-(1---103)] in ovine cardiac tissue extracts gave lower RIA levels using the NT-proBNP antiserum than were found using antiserum directed against the mature BNP (carboxy terminus) region of the hormone. We have made similar observations using antisera directed to mid-molecule segments of intact polypeptides, suggesting that the terminus of the polypeptide is preferentially recognized, compared with internal sites of the molecule that may be protected by folding or other conformational changes within the protein molecule.

Immunoreactive levels of NT-proBNP in coronary sinus plasma were similar to mature BNP levels in both normal control and CAL groups of animals. However, compared with mature BNP levels, left atrial plasma levels of NT-proBNP were approximately threefold higher in normal control, sevenfold higher in CAL, and fivefold higher in rLVP groups of animals, respectively. In contrast, the step up in concentration across the heart was less for NT-proBNP than for mature BNP. These data are consistent with the view that the halflife of circulating plasma NT-proBNP could be longer than that of mature BNP. It should be noted that the half-life of circulating NT-proANP in rat plasma is eightfold longer than that of mature ANP (35). Although the half-life of NT-proBNP in humans (15) and sheep is unknown, studies in both species show an increased ratio of NT-proBNP to mature BNP in states of cardiac impairment. Thus, in the human, the NT-proBNP-to-mature BNP ratio in normal peripheral plasma is 1:1, whereas this ratio is ~4:1 in New York Heart Association (NYHA) class I patients and 5:1 in NYHA class III patients (15). Taken together, these data strongly suggest that the plasma half-life of NT-proBNP in normal health is longer than that of mature BNP and that this ratio may increase in experimental heart failure. However, because of the lack of purified or synthetic ovine NT-proBNP for infusion/bolus half-life studies, we are currently investigating novel and alternative methods of determining the half-life of the peptide in sheep. Indeed, preliminary results from our laboratory suggest that the in vivo half-life of NT-proBNP is approximately three- to fourfold longer than that of mature BNP (Pemberton, C. J., J. H. Livesey, T. G. Yandle, and E.A. Espiner, unpublished observations), but much more work is required to verify this.

Possible sites of NT-proBNP clearance from plasma have not been studied, but it is known that NT-proBNP and mature BNP appear to be handled similarly across the kidney and tissues of the lower limb in humans (15). Studies comparing loss of mature BNP and NT-proBNP across tissues in sheep need to be undertaken to further assess sites of degradation/clearance. To date, two major degradative pathways of natriuretic peptide metabolism have been identified: uptake by NPR-C (19, 21) and hydrolysis by NEP (3, 10, 29). Combined NPR-C and NEP inhibition in normal sheep (7) and in sheep with pacing-induced heart failure (28) significantly elevates mature ANP/BNP levels in plasma, significantly reduces arterial pressures, and promotes diuresis/natriuresis. Furthermore, in sheep both pathways appear to have an equal role in the removal of mature ANP/BNP from plasma (7, 28). In the present study, combined NEP/NPR-C inhibition significantly increased left atrial plasma levels of mature BNP, whereas NT-proBNP levels were unaffected. During the treatment period, MAP and intracardiac pressures fell significantly and cardiac output increased, consistent with the known effects of increasing plasma levels of mature ANP and BNP. These responses to combined NEP/NPR-C inhibition suggest that NT-proBNP is not subject to degradation via NPR-C uptake (21) or hydrolysis by NEP (3). Furthermore, the lack of fall in plasma NT-proBNP suggests that cardiac secretion of BNP was not reduced, despite the significant drop in MAP and evidence of cardiac unloading. However, because coronary sinus plasma levels of NT-proBNP and mature BNP were not measured, there is a possibility that plasma levels of NT-proBNP remained stable because a (small) reduction in secretion was balanced by a drop in metabolic clearance. Full assessment of NT-proBNP as a marker of cardiac secretion will therefore require measurement of coronary sinus plasma levels (and gradients across the heart) coupled with rigorous and precise calculation of its half-life in plasma.

The confirmation of NT-proBNP as a circulating peptide now calls for studies addressing its possible bioactivity. Such studies, along with possible interactions with other BNP forms, must await purification and amino acid sequencing of the hormone. Despite the lack of knowledge concerning bioactivity of NT-proBNP (and NT-proANP), there has been increasing interest in the value of plasma natriuretic peptide levels as markers of cardiac function (9, 13, 25, 36). Several studies indicate that NT-proANP has potential as a marker of symptomless left ventricular dysfunction (20), and plasma levels of the peptide have been reported to be a good neurohumoral indicator of cardiac and renal function (2) and cardiovascular mortality after myocardial infarction (13). In accordance with these proposals, plasma levels of NT-proANP are raised in patients with essential hypertension, congestive heart failure, and chronic renal failure (4, 5). Recent studies have suggested that NT-proBNP levels in human plasma could be an even more sensitive marker of cardiac (ventricular) impairment (15) and that NT-proBNP may be a more predictive indicator of mortality after myocardial infarction (Richards, A. M., personal communication). The present data in sheep suggest that NT-proBNP may be a sensitive marker of cardiac decompensation, particularly because peripheral venous levels increased some 500%, whereas mature BNP levels increased only 50%, after coronary ligation. It remains to be seen whether lesser degrees of left ventricular dysfunction are detected by rises in NT-proBNP and how such changes relate to changes in the levels of other natriuretic peptides.

    ACKNOWLEDGEMENTS

This work was supported by the Health Research Council, National Heart Foundation, and Lotteries Foundation of New Zealand. C. J. Pemberton is the recipient of a Postgraduate Fellowship from the National Heart Foundation of New Zealand.

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: C. J. Pemberton, Dept. of Endocrinology, Christchurch Hospital, Private Bag 4710, Christchurch, New Zealand.

Received 10 March 1998; accepted in final form 23 June 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Heart Circ Physiol 275(4):H1200-H1208
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society


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