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Atrial Natriuretic Factor and Family of Natriuretic Peptides
Atrial Natriuretic Factor and Family of Natriuretic Peptides Kenneth K.-Y. Ma and Adolfo J. de Bold University of Ottawa, Ottawa, Ontario, Canada
Glossary cis-acting elements Short DNA consensus sequences located upstream (50 -flanking region) of the transcription start point. They can be either enhancers, which increase transcription, or silencers, which decrease transcription. diuresis The increased excretion of urine, especially in greater than normal amounts. exon Segment of a gene that corresponds to a sequence in the final processed RNA transcript of that gene. heart failure Defective cardiac filling and/or impaired contraction and emptying, resulting in a heart’s inability to pump a sufficient amount of blood to meet the needs of the body tissues or to be able to do so only with an elevated filling pressure. hypertrophy An increase in the size of a tissue or an organ due to an increase in the size of its component cells. Contrast with hyperplasia. immediate-early gene (IEG) Class of genes whose expression is low in quiescent cells, but whose transcription is activated within minutes of stimulation. Many IEGs encode trans-acting factors. intron A noncoding sequence of DNA within a gene that is transcribed into nuclear RNA, but is subsequently removed by RNA splicing in the nucleus and rapidly degraded. natriuresis The excretion of sodium in the urine, especially in greater than normal amounts. g0045
TATA-box A consensus segment, 50 -TATAAA-30 , that is 19–27 bp upstream from the start point of eukaryotic structural genes to which RNA polymerase II binds. Also known as a Goldberg-Hogness box. trans-acting factors DNA-binding proteins that recognize cis-acting elements on the 50 -flanking region of the regulated gene.
T
he atrial natriuretic factor family of natriuretic peptides (NPs) consists of three members: atrial natriuretic factor (ANF), brain natriuretic peptide or B-type natriuretic peptide
Encyclopedia of Endocrine Diseases, Volume 1. ß 2004 Elsevier Inc. All rights reserved.
(BNP), and C-type natriuretic peptide (CNP). These polypeptide hormones play central roles in maintaining fluid and electrolyte balance and circulatory homeostasis. In mammals, the bulk of ANF and BNP is produced and secreted by the cardiac muscle cell (cardiocyte), whereas CNP is produced mainly by the vascular endothelium and brain.
PHYSIOLOGY, STRUCTURE, BIOSYNTHESIS, AND REGULATION OF ANF Physiology, Structure, and Biosynthesis of ANF Physiological Actions of ANF In 1981 it was demonstrated that injections of heart atrial extracts into rats gave rise to pronounced diuresis, natriuresis, and lowered blood pressure. Within a short time, a peptide with the same biological properties as the crude atrial extracts was isolated and its amino acid sequence was identified. This peptide, now known as atrial natriuretic factor (also known as atrial natriuretic peptide), firmly established the heart as an endocrine organ and heralded a new era of research on the control and maintenance of blood pressure, blood volume, and vascular tone. ANF is a potent natriuretic and diuretic agent, owing these properties to both its renal hemodynamic actions and its direct tubular actions. By simultaneous dilation of afferent arterioles and constriction of efferent arterioles, ANF increases the glomerular filtration rate and filtration fraction. ANF will directly inhibit water reabsorption by the renal cortical collecting duct and contributes to inhibition of Na+ reabsorption by the renal inner medullary collecting duct. ANF also has profound effects on the cardiovascular system. Acute administration of ANF causes a fall in arterial pressure due to a reduction in cardiac output mediated by a decreased preload and vascular resistance. The decreased preload is believed to be a consequence of venodilation and reduction of the intravascular volume. Furthermore, ANF also reduces
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sympathetic tone by inhibiting arterial baroreceptor response and suppression of catecholamine release from autonomic nerve endings. The renin–angiotensin–aldosterone system (RAAS) is also a target for ANF. ANF will directly reduce renin secretion, which has the cascade effect of lowering circulating levels of angiotensin II (Ang II), a potent vasoconstrictor and stimulator of aldosterone. ANF can also directly inhibit aldosterone synthesis and secretion from the glomerulosa cells of the adrenal cortex. The central actions of ANF include inhibition of secretion of vasopressin and of salt and water intake. ANF also possesses important anti-mitotic actions on vascular endothelial cells, smooth muscle cells, and cardiac fibroblasts. Structure and Biosynthesis of ANF The sequencing of ANF permitted the generation of oligonucleotide probes that were used to isolate cDNA clones for ANF-coding sequences. The mRNA is approximately 1 kb long and encodes a preproANF that contains between 149 and 153 amino acids depending on the species (Fig. 1). The human amino acid sequence shares strong homology with peptides from other species including rat, mouse, and pig. In the human atria, ANF is stored mainly within organelles referred to as specific atrial granules as a prohormone called proANF1–126. Subsequent processing releases into the bloodstream the biologically active hormone, ANF99–126. Normal human plasma concentrations of ANF are approximately
Figure 1 Structure of the gene and biosynthetic pathway of human ANF. Solid black sections are those that code for the mature ANF99– 126. Gray sections code for NH2-terminal fragments and the striped section codes for the signal peptide. Numbers indicate the amino acid position relative to the sequence encoded in proANF. T indicates the location of the TATAAA-box. UTR, untranslated region.
Atrial Natriuretic Factor and Family of Natriuretic Peptides
6 fmol/ml. The biological half-life of ANF99–126 is very short (0.5–2 min). All members of the ANF family of natriuretic peptides [ANF, B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP)] share a common central ring structure formed by a disulfide bridge (positions 105 and 121 for ANF). Disruption of this 17-member ring leads to a loss of biological activity (Fig. 2).
ANF Gene Regulation The ANF gene is mapped to chromosome 1 in humans, chromosome 4 in mouse, and chromosome 5 in rats. Structurally, the ANF gene is similar in all mammals and consists of three exons and two introns (Fig. 1). Tissue-Specific and Developmental Regulation of ANF Gene Expression Atrial expression of ANF rises continuously during fetal and postnatal development to comparatively high levels (1–3% of total atrial mRNA), whereas ventricular expression of ANF, although quite high during fetal development, falls quickly after birth (1% of atrial levels). Extracardiac (aortic arch, hypothalamus, pituitary, and kidney) sites of synthesis have also been documented, but the major site of ANF production remains the atria. In adulthood, ANF expression by the heart can be markedly increased by mechanical stretch of the cardiocyte and/or by neuroendocrine hormones. Cis-acting elements responsible for tissue- or stagespecific expression of ANF have been identified on the 50 -flanking promoter of the ANF gene. The characteristic TATAA-box motif for binding the ubiquitous transcription initiation factor IID (TFIID) transcription factor is 30 bp upstream of the transcription initiation site (CAP site). Within the 700 bp ANF promoter, certain regions that confer chamber and temporal-specific activity have been identified. A CArG motif at 405 bp plays a critical role in the ventricular and stage-specific activity of ANF expression. Furthermore, located between 380 and 300 is cardiac regulatory element (CARE). CARE and the trans-acting factor Catf-1 are active in the embryonic atria and ventricle but not in the postnatal ventricular cardiocyte. Finally, there are a few elements that are important in maintaining basal expression of ANF. These are located in the proximal promoter from 135 to 1 and include GATA-, Nkx2.5 response element (NKE), and T-box 5 (Tbx5)-binding elements.
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Atrial Natriuretic Factor and Family of Natriuretic Peptides
Figure 2 The biologically active forms of the natriuretic peptides. Amino acids that are shared by all members of the natriuretic peptide family are identified by filled circles. A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine. Mechanical and Neuroendocrine Regulation of ANF Gene Expression Acute Mechanical Myocardial Stretch ANF is continuously released from the atria of the heart, but appropriate mechanical stretch can increase the rate of ANF secretion. In acute experiments with isolated atrial preparations, increases in atrial wall stretch are met with corresponding increases in ANF secretion with no change in ANF gene expression. Despite continued mechanical stimulation, the acute increase in ANF release eventually decays to baseline values within minutes. The precise mechanism underlying mechanotransduction of stretch-induced ANF release remains to be elucidated. Neuroendocrine Hormones or Agents Numerous neuroendocrine agents can also increase ANF gene expression and release. a1-Adrenergic agonists (e.g., phenylephrine) and vasoactive peptides, such as endothelin-1 (ET-1) and Ang II, are potent stimulators of ANF gene expression and release. The vasoconstrictor ET-1 is synthesized and released by the endothelial and mesothelial cells. It is believed that ET-1 can act in a paracrine fashion in vivo to modulate ANF gene expression and release. Ang II, an effector molecule of RAAS, is a potent vasoconstrictor as well as a growth factor for cardiocytes. Ang II, ET-1, and catecholamines bind to the heterotrimeric G protein heptahelical transmembrane family of receptors. In general, activation of the receptor leads to the recruitment of secondary effector molecules that activate cytosolic and/or nuclear substrates. In particular, Ang II, ET-1, and phenylephrine will rapidly stimulate protein kinase C and the mitogen-activated protein kinase, p44/42 (ERK), which in
turn will activate trans-acting factors responsible for the increase in ANF expression. These include the immediate-early genes (IEGs), such as the c-fos and c-jun heterodimer, that can bind to activator protein-1 (AP-1) consensus elements on the 50 -flanking region of the ANF promoter. Hormonal ligands for nuclear receptors, such as glucocorticoids, have also been shown to up-regulate ANF gene expression and release. A cytokine produced by the cardiocyte, called cardiotrophin I, has been shown to up-regulate ANF gene expression. Chronic Hemodynamic Overload and Changes in ANF Expression Under chronic hemodynamic overload, mature terminally differentiated cardiocytes will respond with cellular hypertrophy to normalize wall stress. Importantly, ANF gene expression and release are markedly increased not only from the atrial cardiocytes, but also from the ventricular cardiocytes by the hypertrophic stimuli. This can translate into a 100fold increase in ANF plasma concentrations in certain pathophysiological conditions such as chronic congestive heart failure. Hypertrophic stimuli, such as prolonged myocardial stretch or humoral growth factors, will activate the IEGs, such as c-myc, c-fos, and/or c-jun. This is followed by activation of the characteristic embryonic gene program seen during cardiac development. Molecularly, this gene program accounts for the re-expression of ANF, BNP, bmyosin heavy chain, and skeletal a-actin in the hypertrophic adult ventricle. The precise mechanisms responsible for the chronically increased ventricular ANF gene expression and secretion have not been fully elucidated. In vivo
298
Atrial Natriuretic Factor and Family of Natriuretic Peptides
experiments have demonstrated that there may be two components responsible for these changes in natriuretic peptide expression: one that is dependent on hemodynamic load (i.e., mechanical stretch) and one that is load independent (i.e., direct effects of humoral growth factors, such as ET-1 and Ang II, on cardiocytes).
OTHER MEMBERS OF THE ANF FAMILY OF NATRIURETIC PEPTIDES Physiology, Structure, Biosynthesis, and Regulation of BNP Physiological Actions of BNP Brain natriuretic peptide was originally isolated from porcine brain. However, it was later discovered that the highest concentrations were found in the heart. BNP, like ANF, is a potent natriuretic, diuretic, vasorelaxant, anti-mitotic factor as well as an antagonist of the renin–angiotensin–aldosterone axis. Of interest, however, BNP may have functions not associated with ANF. BNP has been shown to have anti-fibrotic properties. This effect may be particularly important in cardiovascular diseases in which cardiac fibrosis contributes to the progression to heart failure. Structure and Biosynthesis of BNP BNP is synthesized as a 121- to 134-amino-acid preprohormone (Fig. 3). In humans, subsequent processing releases a mature biologically active 32amino-acid carboxy-terminal fragment. Unlike ANF or CNP, the amino acid sequence of BNP is not as highly conserved and may differ by as much as 50% between species. The plasma half-life of BNP is approximately 22 min, which is approximately six times longer than that of ANF. The normal circulating BNP level in humans is approximately 0.9 fmol/ml. Regulation of BNP Gene Expression The BNP gene is mapped to chromosome 1 in human and to chromosome 4 in mouse. Structurally the BNP gene is similar to the ANF gene in that it is composed of three exons and two introns. However, there are important differences between the regulation of BNP and ANF at the transcriptional and posttranscriptional levels. Hemodynamic overload increases both atrial and ventricular expression of BNP (and ANF) dramatically. In pathological states such as chronic congestive heart failure, the plasma BNP level can increase 1000-fold.
Figure 3 Structure of the gene and biosynthetic pathway of human BNP. Solid black sections are those that code for the mature BNP77–108. Gray section codes for the NH2-terminal fragment and the striped section codes for the signal peptide. Numbers describe the amino acid position relative to the sequence encoded in proBNP. G’s indicate the location of the GATAAA sequences (TATAAA-box). UTR, untranslated region.
The 50 -flanking region of the BNP gene resembles more of an erythroid- than of a muscle-specific promoter. Cis-acting elements within 114 bp upstream of the CAP site appear to be responsible for conferring not only tissue-specific expression but also hemodynamic stress responsiveness. This region of the promoter contains conserved AP-1 and GATA recognition sites. GATA transcription factors are believed to be important in both basal and stimulated expression of BNP. In response to certain hypertrophic stimuli, BNP mRNA is rapidly up-regulated in a protein synthesisindependent manner with quick turnover. These features are characteristic of an IEG. In support of this, the BNP 30 -untranslated region contains many AUrich elements that may confer instability to the BNP transcript (mRNA t1/2 60 min) not found in ANF mRNA, which is quite stable. Interestingly, BNP mRNA stability can be enhanced after treatment with phenylephrine or ET-1. In addition to neurohumors such as ET-1 and phenylephrine, BNP gene expression and secretion can also be up-regulated by pro-inflammatory cytokines, such as interleukin-1b and tumor necrosis factor a (TNFa). In summary, the significant increase in BNP expression during overload may be due a combination of specific transcriptional and posttranscriptional mechanisms.
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Physiology, Structure, Biosynthesis, and Regulation of CNP Physiological Actions of CNP CNP is found in the cerebellum, hypothalamus, anterior pituitary, kidney, and the vascular endothelial cells but not in the heart. The central actions of CNP include antagonizing angiotensin II-mediated increases in vasopressin, which in turn decreases salt and water intake. The natriuretic activity of CNP is only approximately 1% of that of ANF. CNP has potent antigrowth properties on vascular smooth muscle cells, suggesting that CNP may be an important regulator of blood vessels. Structure and Biosynthesis of CNP CNP is the most conserved of all natriuretic peptides, with 90% homology observed among human, mouse, pig, and rat. Produced from a 126-amino-acid preprohormone, the bioactive hormone, unlike ANF or BNP, is only 22 amino acids long, because CNP has no carboxy-terminal tail (Fig. 2). Regulation of CNP Gene Expression The gene for CNP is located on chromosome 2 in humans, in contrast to the ANF and BNP genes, which are located on chromosome 1 in humans. The
mouse CNP gene consists of least two exons and one intron. The 50 -flanking region of CNP contains numerous cis-acting regulatory elements including dinucleotide CA repeats, cyclic AMP-response element-like, nuclear factor kB, and shear stress recognition sites. Cytokines, such as transforming growth factor-b and TNFa, can up-regulate CNP.
NATRIURETIC PEPTIDE RECEPTORS Many of the biological actions of the natriuretic peptides are mediated through association with specific high-affinity receptors on the surface of target cells and the generation of cyclic guanosine monophosphate (cGMP) (Fig. 4). There are three natriuretic peptide receptors (NPR-A, NPR-B, and NPR-C). NPR-A and NPR-B are linked to guanylate cyclase and, on activation of the receptor, cGMP is formed. cGMP targets may include cGMP-dependent protein kinases and cGMP-gated ion channels. NPR-A binds both ANF and BNP, with preference for ANF. NPR-B binds CNP with far less preference for either ANF or BNP. NPR-C is the clearance receptor and binds CNP with slightly greater affinity than ANF or BNP. Circulating natriuretic peptides are also inactivated by neutral endopeptidases present within renal tubular and vascular cells.
Figure 4 The natriuretic peptide receptors. Binding of ANF and BNP to NPR-A and CNP to NPR-B stimulates intrinsic guanylyl cyclase activity of the receptor. The transduction of ligand binding to cGMP generation is ATP dependent and requires participation from the KHD portion of the receptor. NPR-A, natriuretic peptide receptor-A; NPR-B, natriuretic peptide receptor-B; NPR-C, natriuretic peptide receptor-C; KHD, kinase-like homology domain; NEP, neutral endopeptidase 24.11.
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NATRIURETIC PEPTIDES AND PATHOPHYSIOLOGY A hallmark of chronic congestive heart failure (CHF) is enhanced atrial and ventricular synthesis and release of ANF and BNP secondary to increased hemodynamic load and humoral stimulation. The severity of heart failure correlates well with plasma levels of ANF and BNP such that these peptides are reliable diagnostic and prognostic markers. In severe cases, the plasma level of BNP may surpass that of ANF. It is very likely that the increases in ANF and BNP serve to limit or delay the progression of CHF, in part by its renal, vascular, and endocrine actions.
Atrial Natriuretic Factor and Family of Natriuretic Peptides
heart failure are based on the application of the knowledge base developed about the natriuretic peptides. These include systemic administration of natriuretic peptides and pharmacological approaches, such as the dual angiotensin-converting enzyme and neutral endopeptidase inhibitors. The beneficial outcomes of these agents only highlight the importance of further study of the endocrine heart and the ANF family of natriuretic peptides.
See Also the Following Articles Aldosterone in Congestive Heart Failure Peptides . Renin
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Natriuretic
SUMMARY
Further Reading
The atrial natriuretic factor family of natriuretic peptides plays an important role in regulating blood pressure, extracellular fluid homeostasis, and cardiovascular growth under normal physiological conditions and in diseased states. Natriuretic peptides defend against excess salt and water retention, promote vascular relaxation, inhibit RAAS, inhibit sympathetic outflow, and have potent anti-mitotic properties. For many pathophysiological cardiovascular conditions, ANF and particularly BNP have become important diagnostic and prognostic tools. At present, many clinical treatment strategies for
de Bold, A. J., and Bruneau, B. G. (2000). Natriuretic peptides. In “Handbook of Physiology, Section 7, The Endocrine System, Volume III” ( J. C. S. Fray, ed.), pp. 377–409. Oxford University Press, London. de Bold, A. J., Bruneau, B. G., and Kuroski de Bold, M. L. (1996). Mechanical and neuroendocrine regulation of the endocrine heart. Cardiovasc. Res. 31, 7–18. Durocher, D., Gre´ pin, C., and Nemer, M. (1998). Regulation of gene expression in the endocrine heart. Rec. Prog. Horm. Res. 53, 7–23. Gardner, D. G., Wu, J., and Kovacic-Milivojevic, B. (1998). Cellular and molecular aspects of the A-type natriuretic peptide. In “Natriuretic Peptides in Health and Disease” (W. K. Samson and E. R. Levin, eds.), pp. 71–84. Humana Press, Totowa, NJ.
Glossary cis-acting elements Short DNA consensus sequences located upstream (50 -flanking region) of the transcription start point. They can be either enhancers, which increase transcription, or silencers, which decrease transcription. diuresis The increased excretion of urine, especially in greater than normal amounts. exon Segment of a gene that corresponds to a sequence in the final processed RNA transcript of that gene. heart failure Defective cardiac filling and/or impaired contraction and emptying, resulting in a heart’s inability to pump a sufficient amount of blood to meet the needs of the body tissues or to be able to do so only with an elevated filling pressure. hypertrophy An increase in the size of a tissue or an organ due to an increase in the size of its component cells. Contrast with hyperplasia. immediate-early gene (IEG) Class of genes whose expression is low in quiescent cells, but whose transcription is activated within minutes of stimulation. Many IEGs encode trans-acting factors. intron A noncoding sequence of DNA within a gene that is transcribed into nuclear RNA, but is subsequently removed by RNA splicing in the nucleus and rapidly degraded. natriuresis The excretion of sodium in the urine, especially in greater than normal amounts. g0045
TATA-box A consensus segment, 50 -TATAAA-30 , that is 19–27 bp upstream from the start point of eukaryotic structural genes to which RNA polymerase II binds. Also known as a Goldberg-Hogness box. trans-acting factors DNA-binding proteins that recognize cis-acting elements on the 50 -flanking region of the regulated gene.
T
he atrial natriuretic factor family of natriuretic peptides (NPs) consists of three members: atrial natriuretic factor (ANF), brain natriuretic peptide or B-type natriuretic peptide
Encyclopedia of Endocrine Diseases, Volume 1. ß 2004 Elsevier Inc. All rights reserved.
(BNP), and C-type natriuretic peptide (CNP). These polypeptide hormones play central roles in maintaining fluid and electrolyte balance and circulatory homeostasis. In mammals, the bulk of ANF and BNP is produced and secreted by the cardiac muscle cell (cardiocyte), whereas CNP is produced mainly by the vascular endothelium and brain.
PHYSIOLOGY, STRUCTURE, BIOSYNTHESIS, AND REGULATION OF ANF Physiology, Structure, and Biosynthesis of ANF Physiological Actions of ANF In 1981 it was demonstrated that injections of heart atrial extracts into rats gave rise to pronounced diuresis, natriuresis, and lowered blood pressure. Within a short time, a peptide with the same biological properties as the crude atrial extracts was isolated and its amino acid sequence was identified. This peptide, now known as atrial natriuretic factor (also known as atrial natriuretic peptide), firmly established the heart as an endocrine organ and heralded a new era of research on the control and maintenance of blood pressure, blood volume, and vascular tone. ANF is a potent natriuretic and diuretic agent, owing these properties to both its renal hemodynamic actions and its direct tubular actions. By simultaneous dilation of afferent arterioles and constriction of efferent arterioles, ANF increases the glomerular filtration rate and filtration fraction. ANF will directly inhibit water reabsorption by the renal cortical collecting duct and contributes to inhibition of Na+ reabsorption by the renal inner medullary collecting duct. ANF also has profound effects on the cardiovascular system. Acute administration of ANF causes a fall in arterial pressure due to a reduction in cardiac output mediated by a decreased preload and vascular resistance. The decreased preload is believed to be a consequence of venodilation and reduction of the intravascular volume. Furthermore, ANF also reduces
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sympathetic tone by inhibiting arterial baroreceptor response and suppression of catecholamine release from autonomic nerve endings. The renin–angiotensin–aldosterone system (RAAS) is also a target for ANF. ANF will directly reduce renin secretion, which has the cascade effect of lowering circulating levels of angiotensin II (Ang II), a potent vasoconstrictor and stimulator of aldosterone. ANF can also directly inhibit aldosterone synthesis and secretion from the glomerulosa cells of the adrenal cortex. The central actions of ANF include inhibition of secretion of vasopressin and of salt and water intake. ANF also possesses important anti-mitotic actions on vascular endothelial cells, smooth muscle cells, and cardiac fibroblasts. Structure and Biosynthesis of ANF The sequencing of ANF permitted the generation of oligonucleotide probes that were used to isolate cDNA clones for ANF-coding sequences. The mRNA is approximately 1 kb long and encodes a preproANF that contains between 149 and 153 amino acids depending on the species (Fig. 1). The human amino acid sequence shares strong homology with peptides from other species including rat, mouse, and pig. In the human atria, ANF is stored mainly within organelles referred to as specific atrial granules as a prohormone called proANF1–126. Subsequent processing releases into the bloodstream the biologically active hormone, ANF99–126. Normal human plasma concentrations of ANF are approximately
Figure 1 Structure of the gene and biosynthetic pathway of human ANF. Solid black sections are those that code for the mature ANF99– 126. Gray sections code for NH2-terminal fragments and the striped section codes for the signal peptide. Numbers indicate the amino acid position relative to the sequence encoded in proANF. T indicates the location of the TATAAA-box. UTR, untranslated region.
Atrial Natriuretic Factor and Family of Natriuretic Peptides
6 fmol/ml. The biological half-life of ANF99–126 is very short (0.5–2 min). All members of the ANF family of natriuretic peptides [ANF, B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP)] share a common central ring structure formed by a disulfide bridge (positions 105 and 121 for ANF). Disruption of this 17-member ring leads to a loss of biological activity (Fig. 2).
ANF Gene Regulation The ANF gene is mapped to chromosome 1 in humans, chromosome 4 in mouse, and chromosome 5 in rats. Structurally, the ANF gene is similar in all mammals and consists of three exons and two introns (Fig. 1). Tissue-Specific and Developmental Regulation of ANF Gene Expression Atrial expression of ANF rises continuously during fetal and postnatal development to comparatively high levels (1–3% of total atrial mRNA), whereas ventricular expression of ANF, although quite high during fetal development, falls quickly after birth (1% of atrial levels). Extracardiac (aortic arch, hypothalamus, pituitary, and kidney) sites of synthesis have also been documented, but the major site of ANF production remains the atria. In adulthood, ANF expression by the heart can be markedly increased by mechanical stretch of the cardiocyte and/or by neuroendocrine hormones. Cis-acting elements responsible for tissue- or stagespecific expression of ANF have been identified on the 50 -flanking promoter of the ANF gene. The characteristic TATAA-box motif for binding the ubiquitous transcription initiation factor IID (TFIID) transcription factor is 30 bp upstream of the transcription initiation site (CAP site). Within the 700 bp ANF promoter, certain regions that confer chamber and temporal-specific activity have been identified. A CArG motif at 405 bp plays a critical role in the ventricular and stage-specific activity of ANF expression. Furthermore, located between 380 and 300 is cardiac regulatory element (CARE). CARE and the trans-acting factor Catf-1 are active in the embryonic atria and ventricle but not in the postnatal ventricular cardiocyte. Finally, there are a few elements that are important in maintaining basal expression of ANF. These are located in the proximal promoter from 135 to 1 and include GATA-, Nkx2.5 response element (NKE), and T-box 5 (Tbx5)-binding elements.
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Atrial Natriuretic Factor and Family of Natriuretic Peptides
Figure 2 The biologically active forms of the natriuretic peptides. Amino acids that are shared by all members of the natriuretic peptide family are identified by filled circles. A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine. Mechanical and Neuroendocrine Regulation of ANF Gene Expression Acute Mechanical Myocardial Stretch ANF is continuously released from the atria of the heart, but appropriate mechanical stretch can increase the rate of ANF secretion. In acute experiments with isolated atrial preparations, increases in atrial wall stretch are met with corresponding increases in ANF secretion with no change in ANF gene expression. Despite continued mechanical stimulation, the acute increase in ANF release eventually decays to baseline values within minutes. The precise mechanism underlying mechanotransduction of stretch-induced ANF release remains to be elucidated. Neuroendocrine Hormones or Agents Numerous neuroendocrine agents can also increase ANF gene expression and release. a1-Adrenergic agonists (e.g., phenylephrine) and vasoactive peptides, such as endothelin-1 (ET-1) and Ang II, are potent stimulators of ANF gene expression and release. The vasoconstrictor ET-1 is synthesized and released by the endothelial and mesothelial cells. It is believed that ET-1 can act in a paracrine fashion in vivo to modulate ANF gene expression and release. Ang II, an effector molecule of RAAS, is a potent vasoconstrictor as well as a growth factor for cardiocytes. Ang II, ET-1, and catecholamines bind to the heterotrimeric G protein heptahelical transmembrane family of receptors. In general, activation of the receptor leads to the recruitment of secondary effector molecules that activate cytosolic and/or nuclear substrates. In particular, Ang II, ET-1, and phenylephrine will rapidly stimulate protein kinase C and the mitogen-activated protein kinase, p44/42 (ERK), which in
turn will activate trans-acting factors responsible for the increase in ANF expression. These include the immediate-early genes (IEGs), such as the c-fos and c-jun heterodimer, that can bind to activator protein-1 (AP-1) consensus elements on the 50 -flanking region of the ANF promoter. Hormonal ligands for nuclear receptors, such as glucocorticoids, have also been shown to up-regulate ANF gene expression and release. A cytokine produced by the cardiocyte, called cardiotrophin I, has been shown to up-regulate ANF gene expression. Chronic Hemodynamic Overload and Changes in ANF Expression Under chronic hemodynamic overload, mature terminally differentiated cardiocytes will respond with cellular hypertrophy to normalize wall stress. Importantly, ANF gene expression and release are markedly increased not only from the atrial cardiocytes, but also from the ventricular cardiocytes by the hypertrophic stimuli. This can translate into a 100fold increase in ANF plasma concentrations in certain pathophysiological conditions such as chronic congestive heart failure. Hypertrophic stimuli, such as prolonged myocardial stretch or humoral growth factors, will activate the IEGs, such as c-myc, c-fos, and/or c-jun. This is followed by activation of the characteristic embryonic gene program seen during cardiac development. Molecularly, this gene program accounts for the re-expression of ANF, BNP, bmyosin heavy chain, and skeletal a-actin in the hypertrophic adult ventricle. The precise mechanisms responsible for the chronically increased ventricular ANF gene expression and secretion have not been fully elucidated. In vivo
298
Atrial Natriuretic Factor and Family of Natriuretic Peptides
experiments have demonstrated that there may be two components responsible for these changes in natriuretic peptide expression: one that is dependent on hemodynamic load (i.e., mechanical stretch) and one that is load independent (i.e., direct effects of humoral growth factors, such as ET-1 and Ang II, on cardiocytes).
OTHER MEMBERS OF THE ANF FAMILY OF NATRIURETIC PEPTIDES Physiology, Structure, Biosynthesis, and Regulation of BNP Physiological Actions of BNP Brain natriuretic peptide was originally isolated from porcine brain. However, it was later discovered that the highest concentrations were found in the heart. BNP, like ANF, is a potent natriuretic, diuretic, vasorelaxant, anti-mitotic factor as well as an antagonist of the renin–angiotensin–aldosterone axis. Of interest, however, BNP may have functions not associated with ANF. BNP has been shown to have anti-fibrotic properties. This effect may be particularly important in cardiovascular diseases in which cardiac fibrosis contributes to the progression to heart failure. Structure and Biosynthesis of BNP BNP is synthesized as a 121- to 134-amino-acid preprohormone (Fig. 3). In humans, subsequent processing releases a mature biologically active 32amino-acid carboxy-terminal fragment. Unlike ANF or CNP, the amino acid sequence of BNP is not as highly conserved and may differ by as much as 50% between species. The plasma half-life of BNP is approximately 22 min, which is approximately six times longer than that of ANF. The normal circulating BNP level in humans is approximately 0.9 fmol/ml. Regulation of BNP Gene Expression The BNP gene is mapped to chromosome 1 in human and to chromosome 4 in mouse. Structurally the BNP gene is similar to the ANF gene in that it is composed of three exons and two introns. However, there are important differences between the regulation of BNP and ANF at the transcriptional and posttranscriptional levels. Hemodynamic overload increases both atrial and ventricular expression of BNP (and ANF) dramatically. In pathological states such as chronic congestive heart failure, the plasma BNP level can increase 1000-fold.
Figure 3 Structure of the gene and biosynthetic pathway of human BNP. Solid black sections are those that code for the mature BNP77–108. Gray section codes for the NH2-terminal fragment and the striped section codes for the signal peptide. Numbers describe the amino acid position relative to the sequence encoded in proBNP. G’s indicate the location of the GATAAA sequences (TATAAA-box). UTR, untranslated region.
The 50 -flanking region of the BNP gene resembles more of an erythroid- than of a muscle-specific promoter. Cis-acting elements within 114 bp upstream of the CAP site appear to be responsible for conferring not only tissue-specific expression but also hemodynamic stress responsiveness. This region of the promoter contains conserved AP-1 and GATA recognition sites. GATA transcription factors are believed to be important in both basal and stimulated expression of BNP. In response to certain hypertrophic stimuli, BNP mRNA is rapidly up-regulated in a protein synthesisindependent manner with quick turnover. These features are characteristic of an IEG. In support of this, the BNP 30 -untranslated region contains many AUrich elements that may confer instability to the BNP transcript (mRNA t1/2 60 min) not found in ANF mRNA, which is quite stable. Interestingly, BNP mRNA stability can be enhanced after treatment with phenylephrine or ET-1. In addition to neurohumors such as ET-1 and phenylephrine, BNP gene expression and secretion can also be up-regulated by pro-inflammatory cytokines, such as interleukin-1b and tumor necrosis factor a (TNFa). In summary, the significant increase in BNP expression during overload may be due a combination of specific transcriptional and posttranscriptional mechanisms.
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Atrial Natriuretic Factor and Family of Natriuretic Peptides
Physiology, Structure, Biosynthesis, and Regulation of CNP Physiological Actions of CNP CNP is found in the cerebellum, hypothalamus, anterior pituitary, kidney, and the vascular endothelial cells but not in the heart. The central actions of CNP include antagonizing angiotensin II-mediated increases in vasopressin, which in turn decreases salt and water intake. The natriuretic activity of CNP is only approximately 1% of that of ANF. CNP has potent antigrowth properties on vascular smooth muscle cells, suggesting that CNP may be an important regulator of blood vessels. Structure and Biosynthesis of CNP CNP is the most conserved of all natriuretic peptides, with 90% homology observed among human, mouse, pig, and rat. Produced from a 126-amino-acid preprohormone, the bioactive hormone, unlike ANF or BNP, is only 22 amino acids long, because CNP has no carboxy-terminal tail (Fig. 2). Regulation of CNP Gene Expression The gene for CNP is located on chromosome 2 in humans, in contrast to the ANF and BNP genes, which are located on chromosome 1 in humans. The
mouse CNP gene consists of least two exons and one intron. The 50 -flanking region of CNP contains numerous cis-acting regulatory elements including dinucleotide CA repeats, cyclic AMP-response element-like, nuclear factor kB, and shear stress recognition sites. Cytokines, such as transforming growth factor-b and TNFa, can up-regulate CNP.
NATRIURETIC PEPTIDE RECEPTORS Many of the biological actions of the natriuretic peptides are mediated through association with specific high-affinity receptors on the surface of target cells and the generation of cyclic guanosine monophosphate (cGMP) (Fig. 4). There are three natriuretic peptide receptors (NPR-A, NPR-B, and NPR-C). NPR-A and NPR-B are linked to guanylate cyclase and, on activation of the receptor, cGMP is formed. cGMP targets may include cGMP-dependent protein kinases and cGMP-gated ion channels. NPR-A binds both ANF and BNP, with preference for ANF. NPR-B binds CNP with far less preference for either ANF or BNP. NPR-C is the clearance receptor and binds CNP with slightly greater affinity than ANF or BNP. Circulating natriuretic peptides are also inactivated by neutral endopeptidases present within renal tubular and vascular cells.
Figure 4 The natriuretic peptide receptors. Binding of ANF and BNP to NPR-A and CNP to NPR-B stimulates intrinsic guanylyl cyclase activity of the receptor. The transduction of ligand binding to cGMP generation is ATP dependent and requires participation from the KHD portion of the receptor. NPR-A, natriuretic peptide receptor-A; NPR-B, natriuretic peptide receptor-B; NPR-C, natriuretic peptide receptor-C; KHD, kinase-like homology domain; NEP, neutral endopeptidase 24.11.
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NATRIURETIC PEPTIDES AND PATHOPHYSIOLOGY A hallmark of chronic congestive heart failure (CHF) is enhanced atrial and ventricular synthesis and release of ANF and BNP secondary to increased hemodynamic load and humoral stimulation. The severity of heart failure correlates well with plasma levels of ANF and BNP such that these peptides are reliable diagnostic and prognostic markers. In severe cases, the plasma level of BNP may surpass that of ANF. It is very likely that the increases in ANF and BNP serve to limit or delay the progression of CHF, in part by its renal, vascular, and endocrine actions.
Atrial Natriuretic Factor and Family of Natriuretic Peptides
heart failure are based on the application of the knowledge base developed about the natriuretic peptides. These include systemic administration of natriuretic peptides and pharmacological approaches, such as the dual angiotensin-converting enzyme and neutral endopeptidase inhibitors. The beneficial outcomes of these agents only highlight the importance of further study of the endocrine heart and the ANF family of natriuretic peptides.
See Also the Following Articles Aldosterone in Congestive Heart Failure Peptides . Renin
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Natriuretic
SUMMARY
Further Reading
The atrial natriuretic factor family of natriuretic peptides plays an important role in regulating blood pressure, extracellular fluid homeostasis, and cardiovascular growth under normal physiological conditions and in diseased states. Natriuretic peptides defend against excess salt and water retention, promote vascular relaxation, inhibit RAAS, inhibit sympathetic outflow, and have potent anti-mitotic properties. For many pathophysiological cardiovascular conditions, ANF and particularly BNP have become important diagnostic and prognostic tools. At present, many clinical treatment strategies for
de Bold, A. J., and Bruneau, B. G. (2000). Natriuretic peptides. In “Handbook of Physiology, Section 7, The Endocrine System, Volume III” ( J. C. S. Fray, ed.), pp. 377–409. Oxford University Press, London. de Bold, A. J., Bruneau, B. G., and Kuroski de Bold, M. L. (1996). Mechanical and neuroendocrine regulation of the endocrine heart. Cardiovasc. Res. 31, 7–18. Durocher, D., Gre´ pin, C., and Nemer, M. (1998). Regulation of gene expression in the endocrine heart. Rec. Prog. Horm. Res. 53, 7–23. Gardner, D. G., Wu, J., and Kovacic-Milivojevic, B. (1998). Cellular and molecular aspects of the A-type natriuretic peptide. In “Natriuretic Peptides in Health and Disease” (W. K. Samson and E. R. Levin, eds.), pp. 71–84. Humana Press, Totowa, NJ.