Mercedes L. Kuroski de Bold

The amino acid sequence of an atrial peptide with potent diuretic and natriuretic properties

A 28 amino acid peptide with diuretic and natriuretic activity has been purified from rat atrial muscle. The primary structure of this atrial peptide is H-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly- (sequence in text) Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-(Arg)-Tyr-OH. The biological activity of this peptide is identical to that of atrial natriuretic factor and cardionatrin I isolated from rat atria.

The endocrine function of the heart

Atrial cardiocytes in the heart of mammals produce in a regulated manner the polypeptide hormones atrial natriuretic factor (ANF, ANP) and brain natriuretic peptide (BNP). The biological actions of ANF and BNP are similar; they include the modulation of systems that tend to increase extracellular fluid volume and blood pressure, such as the renin-angiotensin system and the sympathetic nervous system. Additionally, both hormones have potent growth-regulating properties. ANF and BNP signal by activating membrane-bound guanylyl cyclase receptors, leading to an increase in intracellular cGMP and thus affecting the activity of cGMP-regulated enzymes and ion channels. Under chronic hemodynamic overload, cardiac ANF and BNP synthesis and secretion are increased. This increase is viewed as a cardioprotective mechanism, given the beneficial effects of ANF and BNP on cardiac preload, afterload and cardiovascular growth. As discussed in this review, some basic facts regarding the synthesis and secretion of ANF and BNP and their peripheral effects remain to be clarified. Nevertheless, at the clinical level, the elevation of circulating ANF and BNP in heart failure or following acute coronary syndromes has been shown to have diagnostic and prognostic implications. Moreover, these peptides themselves hold promise as therapeutic agents in the treatment of heart failure. Additional pharmaceutical applications might be gleaned from current preclinical and clinical studies showing beneficial effects of ANF or BNP in the treatment of hypertension, bronchospasm and in tissue remodeling following acute myocardial infarction.

Evidence for Load-Dependent and Load-Independent Determinants of Cardiac Natriuretic Peptide Production

BACKGROUND In hypertension with cardiac hypertrophy, the specific contributions to increased production of the cardiac natriuretic peptides (NP) atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) by load and the hypertrophic process are not known. In the present work we determine ANF and BNP synthesis and secretion in the aortic-banded rat treated with dosage schedules of the ACE inhibitor ramipril that result in the prevention or regression of both hypertension and hypertrophy (high dosage) or in the prevention or regression of hypertrophy alone with persistent hypertension (low dosage). Myosin heavy chain (MHC) isoform switch was studied as an indicator of ventricular cardiocyte hypertrophy as well as the levels of collagen III mRNA as a measure of changes in extracellular matrix. METHODS AND RESULTS Ramipril was administered for 6 weeks just after suprarenal aortic banding, or rats were banded for 6 weeks, after which ramipril was administered during the following 6 weeks. Banding caused an increase in blood pressure, left ventricular weight-to-body weight ratio, plasma and ventricular NP, ventricular NP mRNA, collagen III, and beta-MHC mRNA. Ramipril at 1 mg/kg normalized all these parameters while ramipril at 10 micrograms/kg normalized left ventricular weight-to-body weight ratio but not blood pressure. Plasma and ventricular NP content and mRNA levels were partially normalized by ramipril (10 micrograms/kg). Ramipril (10 micrograms/kg) prevented increased collagen III mRNA levels but did not affect beta-MHC mRNA levels. CONCLUSIONS (1) NP production and secretion in aortic-banded rats are independently related to increased blood pressure and hypertrophy. (2) A load-dependent component is more important than a load-independent component in regulating left ventricular NP production. (3) ANF production is more sensitive than BNP production to the load-independent component. (4) Low-dose ramipril treatment reverses hypertrophy and the increased collagen III expression but does not reverse the increased beta-MHC isoform expression, suggesting that these are independently regulated processes. (5) Aortic banding and ACE inhibition do not affect atrial NP production and content.

Natriuretic Peptide System Gene Expression in Human Coronary Arteries

The natriuretic peptides (NPs) ANF, BNP, and CNP have potent anti-proliferative and anti-migratory effects on vascular smooth muscle cells (SMCs). These properties make NPs relevant to the study of human coronary atherosclerosis because vascular cell proliferation and migration are central to the pathophysiology of atherosclerosis. However, the existence and cytological distribution of NPs and their receptors in human coronary arteries remain undetermined. This has hampered the development of hypotheses regarding the possible role of NPs in human coronary disease. We determined the pattern of expression of NPs and their receptors (NPRs) in human coronary arteries with atherosclerotic lesions classified by standard histopathological criteria as fatty streak/early atherosclerotic lesions, intermediate plaques, or advanced lesions. The investigation was carried out using a combination of immunocytochemistry (ICC), in situ hybridization (ISH), and semiquantitative polymerase chain reaction (PCR). Both by ICC and ISH, ANF was found in the intimal and medial layers of all lesions. BNP was highly expressed in advanced lesions where it was particularly evident by a strong ISH signal but weak ICC staining. CNP was demonstrable in all types of lesions, giving a strong signal by ISH and ICC. This peptide was particularly demonstrable in the endothelium, as well as in the SMCs of the intima, media, and vasa vasorum of the adventitia and in macrophages. By ISH, NPR-A was not detectable in any of the lesions but both NPR-B and NPR-C were found in the intimal and the inner medial layers. By RT-PCR, mRNA levels of all NPs tended to be increased in macroscopically diseased arteries, but only the values for BNP were significantly so. No significant changes in NPR mRNA levels were detected by PCR. In general, the signal intensity given by the NPs and their receptors by ICC or ISH appeared dependent on the type of lesion, being strongest in intermediate plaques and decreasing with increasing severity of the lesion. This study constitutes the first demonstration of NPs and NPR mRNAs in human coronary arteries and supports the existence of an autocrine/paracrine NP system that is actively modulated during the progression of atherosclerotic coronary disease. This suggests that the coronary NP system is involved in the pathobiology of intimal plaque formation in humans and may be involved in vascular remodeling.

Estrogen, natriuretic peptides and the renin–angiotensin system

There are significant gender-specific differences in the incidence of hypertension and the clinical outcome of cardiovascular disease between premenopausal women and age-matched men, suggesting that sex hormones such as estrogen (E) might be responsible for the observed cardioprotective effects. This cardioprotective action of E is thought to involve lipoproteins. However, the effect of E on the lipid profile accounts for about 50% of the reduction in cardiovascular disease, indicating that there might be other mechanisms by which E exerts its cardioprotective effects. At present, the underlying mechanism of E action is poorly understood. In this review, the interplay between E, the natriuretic peptides (NP) and the renin-angiotensin system (RAS) is examined. It is hypothesized that E might, through endocrine and/or paracrine action, modulate cardiac NP in females by affecting the RAS either directly or indirectly.

Dissociation of cardiac hypertrophy, myosin heavy chain isoform expression, and natriuretic peptide production in DOCA-salt rats.

We examined the relationship between cardiac hypertrophy, myosin heavy chain (MHC) isoform expression, and production of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) before and after the development of DOCA-salt hypertension. DOCA-salt rats exhibited significant left ventricular hypertrophy at the prehypertensive stage (1 week of treatment), without MHC isoform switch or change in natriuretic peptide gene expression. In the hypertensive stage (5 weeks of treatment), pronounced left ventricular hypertrophy was observed, and this was characterized by an increase in beta-MHC protein, resulting in a switch from 90% alpha-MHC to 51% alpha-MHC and 49% beta-MHC. ANF and BNP mRNA levels and peptide content were significantly increased at this stage. Unexpectedly, the MHC isoform switch was evident in the non-hypertrophied right ventricle to the same degree as in the left ventricle. Natriuretic peptide production was also increased in the right ventricle at 5 weeks of treatment, but to a lesser degree than in the left ventricle. In contrast, in the hypertrophied left atrium there was no MHC isoform switch, while ANF and BNP mRNA levels were augmented. Plasma ANF was significantly increased in the prehypertensive stage; this was accompanied by a partial depletion of atrial ANF stores. Plasma BNP was increased only in the hypertensive stage, reflecting an increase in ventricular BNP synthesis and secretion. These results suggest that 1) cardiac hypertrophy, MHC isoform expression, and stimulation of natriuretic peptide production are processes that may be dissociated from each other; 2) increases in plasma ANF without a concomitant increase in plasma BNP reflect atrial hemodynamic overload, while increases in both ANF and BNP in plasma are associated with ventricular hypertrophy; and 3) there exist differences in the storage, secretion, and processing patterns of ANF and BNP in the atria.

Optimization of atrial natriuretic factor radioimmunoassay.

The development and long-term performance of a radioimmunoassay method for cardionatrin (C I = ANF 99-126) is described. The method was evaluated for accuracy, specificity and precision using different protocols and in various species. The antiserum raised against C I cross-reacts 100% with both human and rat C I and 122% with cardionatrin IV (C IV = ANF 1-126). Dextran-coated charcoal (DCC) and double antibody (DA) disequilibrium protocols were used for the separation of free from antibody-bound radioactivity. The sensitivity, coefficient of variation within assay and between assay for DCC was 4.6 pg/tube, 3.1% and 13.8% respectively, and 1.5 pg/tube, 3.8% and 9.1% for the DA. The mean normal plasma C I levels in human, rat and dog after plasma acidification and extraction using the DA method was 49.7 +/- 4.0; 253.6 +/- 19.6 and 59.9 +/- 3.4 pg/mL respectively.

Relationship between natriuretic peptides and inflammation: proteomic evidence obtained during acute cellular cardiac allograft rejection in humans.

BACKGROUND Cardiac natriuretic peptides (NPs) atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) are polypeptide hormones secreted by the heart. Previously, we found that BNP, but not ANF, plasma levels may increase during an acute cellular cardiac allograft rejection episode. In vitro, the pro-inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) produced a selective increase of BNP gene expression and secretion. Other pro-inflammatory cytokines had no such effects. METHODS We identified cytokines associated with the selective upregulation of BNP during cardiac allograft rejection using a proteomics approach to measure 120 cytokines and related substances in the plasma of 16 transplant patients before, during and after an acute rejection episode. The values obtained were correlated with BNP plasma levels. Cytokines identified as being significantly related to BNP plasma levels were tested in neonatal rat ventricular cardiocytes in culture for their ability to selectively promote BNP secretion. The signaling pathway related to this phenomenon was pharmacologically characterized. RESULTS Regulated-on-activation, normal T-expressed and secreted (RANTES), neutrophil-activating protein-2 (NAP-2) and insulin growth factor binding protein-1 (IGFBP-1) had significant correlations with BNP plasma levels during Grade 3A (Grade 2 revised [2R]) or above rejection as diagnosed by endomyocardial biopsy score according to the International Society for Heart and Lung Transplantation (ISHLT) grading system. In rat neonatal ventricular cardiocyte cultures, IGFBP-1 and RANTES were capable of promoting BNP, but not ANF secretion, as observed in rejecting patients. The BNP-promoting secretion activity of the identified cytokines was abolished by SB203580, a specific p38 MAP kinase inhibitor. CONCLUSIONS This work shows that cytokines other than pro-inflammatory cytokines correlate with BNP plasma levels observed during acute cardiac allograft rejection, and that the substances identified have in common p38 signaling. This finding provides a unifying mechanistic explanation regarding the relationship between inflammation and cardiac hormone production in acute cardiac allograft rejection.

Atrial Natriuretic Factor and Brain Natriuretic Peptide Gene Expression in the Spontaneous Hypertensive Rat During Postnatal Development

Abstract The increase in natriuretic peptides (NP), atrial natriuretic factor (ANF), and brain natriuretic peptide (BNP) production and release by cardiocytes that occurs in hypertension has been considered to be a compensatory mechanism against ventricular overload. Studies on NP production in the spontaneously hypertensive rat (SHR), an experimental model of human hypertension, have produced controversial results and were carried out when hypertension was already established (> 17 weeks). At this time, age-related physiologic and molecular changes in cardiac muscle are difficult to separate from those related to hypertension, ie, increased ANF production and plasma levels. In addition, most of the studies used male rats because the rate of increase in arterial blood pressure—as well as the level to which it rises—is greater in males than in females. Studies of a similar nature using female SHR are not available. The aim of this work was to determine 1) whether ANF and BNP production and secretion increase with the development of hypertension in genetically hypertensive rats; 2) whether a sexual dimorphism in ANF and BNP production and secretion is present in the genetically hypertensive rat during the development of hypertension; and 3) whether the demand for ANF and BNP is the same from each chamber of the heart under these experimental conditions. Age-matched male and female SHR, Wistar-Kyoto (WKY), and Sprague Dawley (SD) rats at 2, 4, and 8 weeks of age were used. The normotensive SD were included to provide a wider basis for baseline findings, as WKY rats are not always a suitable control for SHR due to genetic variations. Natriuretic peptide plasma levels and tissue content were measured by radioimmunoassay. ANF, BNP, as well as α- and β-myosin heavy chain (MHC) mRNA were estimated by Northern blot analysis. Blood pressure (BP) of more than 150 mm Hg was found only in 8-week-old male SHR. Plasma immunoreactive (ir)ANF and irBNP increased significantly at puberty (8 weeks) in both male and female SHR. The earliest molecular change encountered during the development of hypertension was a significant increase in BNP mRNA in the right and left atria from both male and female 8-week-old SHR. In the ventricles from both male and female SHR, there was no increase in the ratio of left ventricular wet weight/body weight, no increase in ventricular ANF mRNA transcripts, and no myosin heavy chain isoform switch (a protein marker of hypertrophy). irBNP ventricular concentration, however, increased significantly in both male and female SHR, but only in female SHR was there a concomitant increase in BNP mRNA. These results suggest that 1) ANF and BNP production are not coordinated in all cardiac compartments during the development of hypertension; 2) upregulation of BNP in the atria from male and female SHR is the earliest event detected at 8 weeks; 3) the prehypertensive stage, in the genetically hypertensive female rats, is associated with an increase in ventricular irBNP concentration and BNP mRNA; 4) there is a dissociation between BP and plasma levels of NP; and 5) as well, there is a dissociation between NP gene expression and MHC isoform switch. The regulation of NP is not coordinated in either gender during the development of hypertension. The activation of the BNP gene in female SHR suggests that BNP might play an important role at the onset of hypertension.

Angiotensin II receptor antagonism reverts the selective cardiac BNP upregulation and secretion observed in myocarditis.

The cardiac natriuretic peptides atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) are discoordinately regulated in myocardial inflammation associated with acute allograft rejection in humans and during in vitro exposure of cardiocyte cultures to some proinflammatory cytokines. We used experimental autoimmune myocarditis (EAM) to determine whether the discoordinate regulation of ANF and BNP was specific to the situations above or was generally associated with other types of myocardial inflammation. The dependency of this process to angiotensin signaling was also determined, given that previous work demonstrated beneficial effects of the angiotensin receptor blocker olmesartan in myocarditis. Histopathological changes, plasma and cardiac ANF, BNP, and selected cytokines gene expression as well as plasma cytokine levels using a cytokine array were determined in EAM, angiotensin receptor blocker-treated, and control rats. It was found that EAM specifically increases BNP but not ANF circulating levels, thus mimicking the findings in acute cardiac allograft rejection and the effect of some proinflammatory cytokines on cardiocyte cultures in vitro. Plasma cytokine array and real-time PCR revealed that lipopolysaccharide-induced CXC chemokine, monocyte chemotactic protein-1, and tissue inhibitor of metalloproteinase-1 were increased in plasma and in the myocardium of EAM rats. Olmesartan treatment reversed virtually all neuroendocrine and histopathological cardiac changes induced by EAM, thus providing a mechanistic insight into this phenomenon. It is concluded that the inflammatory process contributes specific cytokines, leading to the disregulation of cardiac ANF and BNP production observed during myocardial inflammation, and that this process is angiotensin receptor 1 dependent.