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Natriuretic Hormones
Hormones which enhance sodium excretion, i.e., natriuretic peptide hormones, are very important for the maintenance of extracellu/lar fluid volume within a narrow range, despite wide variations in dietary sodium intake. This regulation occurs through a complex interplay of the antinatriuretic renin–angiotensin–aldosterone system and the antinatriuretic renal sympathetic system, which help to conserve sodium when sodium intake is low, and the natriuretic hormones, which enhance sodium excretion whenever sodium excess occurs. Several of the cardiac natriuretic hormones (Figure 37.1) directly inhibit aldosterone secretion and/or indirectly inhibit aldosterone secretion by inhibiting renin release from the kidney to help regulate extracellular fluid volume. This chapter will concentrate on the natriuretic hormones (cardiac, intestinal, renal, and adrenal) in normal renal physiology, their synthesis, secretion, biologic effects, pathophysiological changes with hypertension and renal diseases, and potential for treating diseases such as acute renal failure.
Introduction
Cardiac, Renal, Intestinal, and Adrenal Hormones which Enhance Sodium Excretion
Hormones which enhance sodium excretion, i.e., natriuretic peptide hormones, are very important for the maintenance of extracellu/lar fluid volume within a narrow range, despite wide variations in dietary sodium intake. This regulation occurs through a complex interplay of the antinatriuretic renin–angiotensin–aldosterone system and the antinatriuretic renal sympathetic system, which help to conserve sodium when sodium intake is low, and the natriuretic hormones, which enhance sodium excretion whenever sodium excess occurs. Several of the cardiac natriuretic hormones ( Figure 37.1 ) directly inhibit aldosterone secretion and/or indirectly inhibit aldosterone secretion by inhibiting renin release from the kidney to help regulate extracellular fluid volume. This chapter will concentrate on the natriuretic hormones (cardiac, intestinal, renal, and adrenal) in normal renal physiology, their synthesis, secretion, biologic effects, pathophysiological changes with hypertension and renal diseases, and potential for treating diseases such as acute renal failure.
History of Atrial (Cardiac) Natriuretic Peptide Hormones
In 1628, Harvey first correctly described the heart as a pump or a muscular organ that contracts in rhythm, pushing blood first to the lungs for oxygenation and then through the peripheral vascular system, bringing oxygen and nutrients to every cell in the body. It was another 350 years before the heart was established as an endocrine gland with its main physiologic targets being the kidney and vasculature. The history of experimentation leading to defining the cardiac natriuretic peptide hormonal system (the first of the natriuretic hormonal systems) has followed two pathways: anatomical and physiological.
History of Cardiac Hormones: Anatomical Studies
Atrial Granule Structure
Shortly before Henry and colleagues reported their observation that balloon distention of atria caused a diuresis, in 1955 Kisch, utilizing electron microscopy, described dense granules that were located in the atria, but not in the ventricles of mammals. The presence of these dense granules in the cytoplasm of atrial cardiac myocytes, but not in the ventricles of the heart, was rapidly confirmed by others utilizing electron microscopy. Jamieson and Palade demonstrated that such granules are present in cardiocytes of the atria of all mammals, including humans, and were the first, in 1964, to suggest that these granules resemble other granules that release polypeptide hormones. These granules are usually adjacent to one or occasionally both poles of the nucleus, and are interspersed among the voluminous elements of the Golgi complex and within close proximity to the mitochondria, and are influenced by salt intake reduction.
Ultrastructural cytochemistry has shown that these granules consist of proteins. They incorporate both [ 3 H]-leucine and [ 3 H]-fructose in a pattern identical to other endocrine-secreting cells, with protein synthesis occurring in the Golgi complex. The ultrastructural features of the specific granules of different species are similar, in that they display an amorphous core and a limiting membrane, and generally measure 300–500 nm. The size and number of these granules vary among species, and generally are inversely related to size. Thus, atrial myocytes from large animals such as cows contain fewer and smaller granules than myocytes from small rodents such as rats. In the rat there are up to 600 spherical, electron-opaque granules per cell.
Atrial Extracts and Natriuresis
In 1922, Banting and Best utilized what is now considered a classic endocrinological technique in their discovery of insulin. They pulverized pancreas with buffer, filtered the crude tissue extract, and found that it produced hypoglycemia in an experimental dog. In 1981, deBold and colleagues, utilizing a similar approach, infused the supernatants of extracts of rat cardiac atria and rat ventricles into other rats, and found that the rat atria extracts, but not the extracts from the rat ventricles, caused dramatic diuresis and natriuresis, with urine flow increasing 10-fold, and sodium and chloride excretion increasing 30-fold. This simple but elegant experiment led to the discovery of atrial peptides that have the most potent endogenous natriuretic activity of any substance yet described. Atrial natriuretic peptide(s) isolated from these atrial extracts has been found to be a two-fold stronger natriuretic producing agent than furosemide (Lasix®, which is one of the most potent natriuretic producing drugs utilized in clinical medicine today.) Other investigators quickly confirmed this natriuretic action, as well as the ability of atrial extracts to cause vasodilation. It was rapidly demonstrated that these effects were at least partially due to a peptide(s). Further investigation revealed that the atrial extracts have significantly more natriuresis and diuresis than pure synthetic ANP, suggesting that other peptide hormones with natriuretic properties were in these atrial extracts.
History of Cardiac Hormones: Physiological Studies
Association of Heart and Renal Function
In 1847, Harthshorne suggested that the heart possessed volume receptors capable of sensing the “fullness of bloodstream” induced by whole-body immersion, which he clearly recognized had a diuretic effect. This observation received little further notice until 1935, when John Peters of Yale University made the same proposal that “the fullness of the bloodstream may provoke the diuretic response on the part of the kidney”. This concept then received experimental verification when it was shown that expansion of blood volume increases urine flow. Peters also suggested that the diuretic response was secondary to the ability of the heart, or something very near the heart, to “sense the fullness of the bloodstream”.
Balloon Distention of Atria
Experimental evidence of an association between cardiac atria and renal function was provided in 1956 by Henry et al., who observed that balloon distention of the left atrium in anesthetized dogs was associated with an increase in urine flow. Because the renal response to left atrial distention could not be elicited after the cervical vagi had been cooled to block nerve conduction, Henry and colleagues concluded that stretch receptors in the left atrium must be present. This finding was later extended to the right atrium. In their reports, Henry et al. noted the diuresis, but did not investigate whether it was associated with increased salt excretion (natriuresis). It is well-established now, however, that balloon distention of the cardiac atria causes natriuresis as well as diuresis. Evidence that animals with denervated hearts or denervated kidneys may also respond to an atrial pressure increase to produce diuresis suggests a hormonal pathway between the heart and the kidney. At least part of this hormonal pathway involves hormones made in the heart.
The “Third Factor”
With respect to a possible hormonal agent causing natriuresis and diuresis, de Wardener and colleagues demonstrated in 1961 that saline infusion produced an increase in urine flow and sodium excretion in anesthetized dogs independent of changes in glomerular filtration rate (GFR), which was decreased, and even in the presence of high circulating levels of aldosterone. These experiments gave rise to the popular concept of an unidentified “third factor,” a term coined by Levinsky and Lalone. The other two factors were aldosterone and GFR-affected sodium excretion. The search for this third factor soon focused on a possible hormonal mediator that came to be known as “natriuretic hormone.” Although this mediator (or mediators) from plasma or urine of volume-expanded humans or animals that causes natriuresis when injected into animals was never chemically identified, the evidence points toward this third factor having a peptide structure(s), because acid hydrolysis characteristically inactivated this substance. The “third factor” that was sought for decades now appears to be a family of peptide hormones termed “atrial natriuretic peptides” (ANPs), so named since they are found in their highest concentrations in the atria of the heart, have natriuretic properties, and are peptides. The third factor(s) also has the ability to inhibit Na + ,K + -ATPase in the kidney. Some of the natriuretic peptide hormones synthesized in the heart fill all of the criteria of being the “third factor(s).” Atrial natriuretic peptide (ANP) does not inhibit Na + ,K + -ATPase, so it would not fulfill the criteria of being the “third factor.” Three of the other peptide hormones synthesized by the ANP prohormone gene ( Figure 37.1 ), namely long-acting natriuretic peptide, vessel dilator, and kaliuretic peptide, however, do inhibit renal Na + ,K + -ATPase and fill all of the criteria of being the “third factor(s)” that researchers have sought since the 1960s.
Family of Cardiac Natriuretic Peptide Hormones
At first it was thought that a single peptide was found in atrial extracts, but further investigation revealed a sophisticated endocrine system in the atria (and other tissues including the kidney) in which the atrial natriuretic peptide (ANP) prohormone gene synthesized four peptide hormones ( Figure 37.1 ), and two other genes were present, as reviewed below. The three other peptide hormones synthesized by the ANP prohormone gene – long-acting natriuretic peptide, vessel dilator, and kaliuretic peptide – were first demonstrated to have biologic effects in 1987, and one of their mechanisms of action, via intracellular messenger cyclic GMP, was also elucidated in 1987. Fifth and sixth members of the natriuretic peptide family were identified in 1988, i.e., brain natriuretic peptide (BNP) isolated from a porcine brain cDNA library, and urodilatin, a peptide formed by differential processing of ANP prohormone in the kidney, which was first found in opossum urine. A seventh member of this family was identified in 1990 in brain tissue and termed C-type natriuretic peptide (CNP). A possible eighth member, DNP, was first described in 1992 from the venom of the green mamba snake.
Family of Cardiac Natriuretic Hormones: Synthesis of Three Prohormones
This family of cardiac peptide hormones has been designated atrial natriuretic peptides (ANPs), also known as atrial natriuretic hormones (ANHs). These peptide hormones are synthesized by three different genes, and then stored as three different prohormones (126 amino acid (a.a.) ANP, 108 a.a. BNP, and 103 a.a. CNP prohormones). In healthy adults, the main site of ANP prohormone synthesis is the atrial myocyte with its mRNA being 30–50-fold higher in the atria than that observed in the ventricle, but it is also synthesized in a variety of other tissues including the kidney. The different organs that synthesize the ANPs in the approximate order that they contribute to the synthesis of ANPs are listed in Table 37.1 .
Table 37.1
Site of Synthesis, Molecular Weight, and Hemodynamic and Natriuretic Properties of Natriuretic Peptides
| Molecular Weight (kDa) | Site of Synthesis | MAP | Diuresis | Natriuresis | |
|---|---|---|---|---|---|
| LANP | 3508 | Atria, ventricle, GI, lung, kidney, brain, adrenal | ↓ | ↑ | ↑ |
| Vessel dilator | 3878 | Atria, ventricle, GI, lung, kidney, brain, adrenal | ↓ | ↑ | ↑ |
| Kaliuretic peptide | 2184 | Atria, ventricle, GI, lung, brain, adrenal | ↓ | ↑ | – a |
| ANP | 3078 | Atria, ventricle, GI, lung, kidney, brain, adrenal | ↓ | ↑ | ↑ |
| Urodilatin | 3503 | Kidney | ↓ | ↑ | ↑ |
| BNP | 3462 | Atria, ventricle, brain, adrenal | ↓ | ↑ | ↑ |
| CNP | 2198 | Endothelium, CNS | ↓ | ↑ | – |
| DNP | 4191 | Atria, ventricle | ↓ | ↑ | ↑ |
| Adrenomedullin | 6029 | Adrenal, kidney | ↓ | ↑ | ↑ |
Note: The sites of synthesis are listed in approximate order in which they contribute to synthesis.
ANP: atrial natriuretic peptide; BNP: brain natriuretic peptide; CNP: C-type natriuretic peptide; CNS: central nervous system; DNP: Dendroaspis natriuretic peptide; GI:, gastrointestinal tract; ANP: long-acting natriuretic peptide; MAP: mean arterial pressure.
(With permission, from ref. [ ].)
Cardiac Peptide Hormones Originating from Atrial Natriuretic Peptide Prohormone
Within the 126 a.a. ANP prohormone encoded by a single gene are four peptide hormones ( Figure 37.1 ) with blood pressure lowering, natriuretic, diuretic, and/or kaliuretic (i.e., potassium excreting) properties in both animals and humans. These peptide hormones, numbered by their a.a. sequences beginning at the N-terminal end of the ANP prohormone, consist of the first 30 a.a. of the prohormone (proANP 1–30, long-acting natriuretic peptide [LANP]); a.a. 31–67 (proANP 31–67, vessel dilator); a.a. 79–98 (proANP 79–98, kaliuretic peptide); and a.a. 99–126 (ANP) ( Figure 37.1 ). These peptide hormones which were each discovered before BNP and CNP were named for their most prominent biologic effects rather than the tissue they were first found in, because these peptides are synthesized in many tissues. Brain natriuretic peptide, so named because it was first found in porcine brain cDNA, for example, is actually present in the heart in 10-fold higher concentrations than in the brain. Each of the four peptide hormones from the ANP prohormone circulate in healthy humans, with LANP and vessel dilator concentrations in plasma being 15–20-fold higher than ANP and 100-fold higher than BNP.
BNP and CNP Prohormones
The BNP and CNP genes, on the other hand, appear to synthesize only one peptide hormone each within their respective prohormones, that is, BNP and CNP. The pro BNP gene and its regulation are reviewed in the section on BNP prohormone gene. The biologic effects of BNP and CNP are reviewed in sections on BNP, “Biologic Effects” and CNP, “Circulating Concentrations and Biologic Effects.”
Origination of Peptide Hormones from Prohormones
More than one peptide hormone originating from the same prohormone is common with respect to the synthesis of hormones. Adrenocorticotropin (ACTH), for example, is derived from a prohormone that contains four known peptide hormones. α-MSH, which has natriuretic properties, originates from this same prohormone. ACTH, similar to vessel dilator, originates from the middle of its prohormone. The middle of their respective prohormones is the most common origin of hormones with calcitonin, glucagon, vasoactive intestinal peptide, gastrin, cholecystokinin, and substance P, as well as ACTH and vessel dilator. Several hormones, such as vasopressin (antidiuretic hormone (ADH)), oxytocin, pancreatic polypeptide, angiotensin, and gastrin-releasing peptide, originate from the N-terminus of their respective prohormones, as does long-acting natriuretic peptide (proANF 1–30). The origin of hormones from the C-terminus of their respective prohormones like ANP, BNP, and CNP is less common, with somatostatin, inhibin, and parathyroid hormone (PTH) being the only known C-terminal prohormone-derived peptides. In the case of PTH, 84 of the 90 a.a. in its prohormone are considered to be the C-terminal “active” hormone; thus, it is not a small C-terminal-derived prohormone peptide, but rather nearly the intact PTH prohormone that serves as the actual peptide hormone.
Molecular Biology of the Cardiac Natriuretic Hormonal System
ProANP Gene
The gene encoding the synthesis of atrial natriuretic peptide prohormone (proANP) consists of three exon (coding) sequences separatedby two intron (intervening) sequences which encode for a mature mRNA transcript approximately 900 bases long ( Figure 37.1 ). Translation of human ANP prohormone mRNA results in a 151 a.a. preprohormone. Exon 1 encodes the 5′-untranslated region, the hydrophobic signal peptide (leader segment), and the first 16 a.a. of the ANP prohormone (first 16 a.a. of long-acting natriuretic peptide). The signal peptide, which is important for the translocation of the precursor peptide from the ribosome into the rough endoplasmic reticulum, is cleaved from the preprohormone (151 a.a.) in the endoplasmic reticulum ( Figure 37.1 ). The resulting 126 a.a. prohormone is the storage form for the four atrial natriuretic peptide hormones in tissues and the major constituent of the atrial granules. The first 16 a.a. of this prohormone encoded by exon 1 are, after proteolytic processing of the ANP prohormone, also the first 16 a.a. of long-acting natriuretic peptide (LANP) ( Figure 37.1 ). Exon 3 encodes for the terminal tyrosine (a.a. 126 of the ANP prohormone) in humans, and terminal three a.a. (Try-Arg-Arg) in rat, rabbit, cow, and mouse. Deletion of this terminal tyrosine residue encoded by exon 3 does slightly affect the binding of ANP, but does not appear to contribute to biologic activity, as there is no apparent decrease in biologic activity when this terminal tyrosine is not present. Exon 2 encodes for the rest of the prohormone (a.a. 17–125 in humans).
There is considerable homology in the proANP gene among species, particularly in the encoding and 5′ flanking sequences. The proANP gene has many features common to all eukaryotic genes, including a TATTA box (T=thymine; A=adenine), intervening sequences bounded by GT-AG splicing signals (G=guanine), and a consensus sequence found in promoted regions. An interesting feature of the human proANP gene is a consensus sequence for a putative glucocorticoid hormone regulatory element in the second intron.
The amino acid sequence of the whole ANP prohormone synthesized by the above gene is strikingly homologous among many species with differences clustered at the extreme carboxy terminal end of the prohormone, i.e., where ANP is formed. ,450 In each species, the C-terminus is distinguished from the rest of the prohormone by forming a 17 a.a. ring structure via the joining by a disulfide bond between two cysteine residues (105 and 121 of the prohormone), as schematically shown in Figure 37.2 . The ring structure originally was believed to be absolutely necessary for biologic activity, but linear forms (same amino acids in linear form) without a ring structure have since been shown also to have biologic activity. For full natriuretic and vasorelaxant activity, the Phe-Arg-Tyr (a.a. 124–126) at the COOH-terminus and a.a. 99–104 of the NH 2 -terminus of ANP are necessary. In the dog, it appears that deletion of a.a. 99–102 of prohormone does not affect natriuresis, but deletion of a.a. 103 and 104 decreases natriuretic activity 10-fold. Twenty of 30 a.a. in long-acting natriuretic peptide ( Figure 37.2 ) are exactly the same in the five above species, and another six of the remaining 10 amino acids are exactly the same in four out of the five species. Only three a.a. (33, 42, and 43 of the prohormone) are not the same in vessel dilator ( Figure 37.2 ) in the majority of the five species. Kaliuretic peptide has a highly-conserved sequence among the aforementioned five species, with 16 of its 20 a.a. ( Figure 37.2 ) being the same in all five.
BNP
This extraordinary conservation among species of LANP, vessel dilator, and kaliuretic peptide is not observed in the BNP prohormone, where there is a marked difference in amino acid sequence homology among species.
Tissue-Specific Expression of ProANP Gene
In healthy adult animals and humans, the atrial myocyte is the main site of the ANP prohormone synthesis, but it is also synthesized in a variety of other tissues. ProANP gene expression is 30–50 times higher in the atria of the heart than in extra-atrial tissues. The expression of this gene has been found in kidney, gastrointestinal tract (antrum of stomach, small and large intestine), lung, aorta, central nervous system, anterior pituitary, and hypothalamus. An example of where the proANP gene synthesized peptides localized in the kidney is illustrated in Figure 37.3 .
Mechanisms of Action of Gene Products (i.e., Cardiac Hormones and Urodilatin) of ProANP Gene
Part of the intracellular mechanism of action(s) of the four peptide hormones encoded by the proANP gene is that after they bind to their specific receptors they enhance membrane-bound guanylyl cyclase to cause an increase in the intracellular messenger cyclic GMP ( Figure 37.4 ). Cyclic GMP then stimulates a cyclic GMP-dependent, protein kinase that phosphorylates protein(s) in the cell, producing physiologic effects ( Figure 37.4 ). Cyclic GMP mediates the vasodilation of each of the cardiac hormones. The receptors for ANP that mediate ANPs biologic effects (e.g., ANP-A and -B receptors) are interesting, in that they contain guanylyl cyclase and a protein kinase in the receptors themselves ( Figure 37.5 ). The NPR-A receptor has a 441 a.a. extracellular portion that binds ANP which, in turn, activates the catalytic portion of guanylyl cyclase in the cell ( Figure 37.5 ). The protein kinase in this receptor has an inhibitory influence on guanylyl cyclase until this receptor is activated by ANP or BNP. There is a 21 a.a. portion of this receptor which attaches this receptor to the membrane ( Figure 37.5 ). The natriuresis secondary to the ANP is thought to also be mediated by cyclic GMP. Vessel dilator, LANP, and kaliuretic peptide’s mechanisms of action of producing a natriuresis is via enhancing the synthesis of prostaglandin E 2 , which in turn inhibits Na + K + -ATPase in the kidney, which ANP does not do. Vessel dilator and kaliuretic peptide’s homodynamic effects via vasodilation of blood vessels are, however, mediated by cyclic GNP.
Processing of Atrial Natriuretic Peptide Prohormone in Kidney
ANP prohormone processing is different in the kidney compared to other tissues, resulting in an additional four a.a. added to the N-terminus of ANP (proANP 95–126, urodilatin ( Figure 37.2 )), a peptide first identified in opossum urine. Thus, in the kidney, the identical four a.a. from the C-terminus of kaliuretic peptide are added to ANP to form the peptide urodilatin ( Figure 37.2 ). At first, urodilatin was thought not to circulate, and that it was not a hormone. To be defined as a hormone, a given protein has to be synthesized in a tissue or organ, circulate in the bloodstream, and have biologic effects in another tissue or organ. With a very sensitive radioimmunoassay, it appears that urodilatin does circulate, but in such low concentrations (9–12 pg/ml) that it may not be physiologically relevant. Since urodilatin constitutes less than 1% of the circulating natriuretic hormones, its physiologic importance as a circulating hormone is very limited, with over 99% of the physiologic natriuretic effects being from the other natriuretic hormones. Urodilatin, however, may have paracrine functions, and may mediate the effects of one of the other natriuretic hormones (ANP). Infusion of ANP increases the circulating concentration of urodilatin, suggesting that some ANP effects may be mediated by urodilatin. Infusion of long-acting natriuretic peptide, vessel dilator, and kaliuretic peptide, on the other hand, do not affect the circulating concentration of urodilatin in healthy humans.
Regulation of Atrial Natriuretic Peptide Prohormone Gene
Enhancement of ProANP Gene Expression
Stretch
Mechanical stretch, or more specifically tension, delivered across the atrial wall is a potent activator of proANP gene expression and/or secretion. In animals, an increase of sodium intake results in an increased release of the ANP prohormone peptides.
Thyroid Hormones
Thyroid hormones thyroxine (T 4 ) and triiodothyronine (T 3 ) increase proANP gene expression. The increase in proANP mRNA in hypothyroidism when treated with thyroid hormone is paralleled by the increase in circulating concentrations of the gene products of this synthesis – vessel dilator, LANP, and ANP – in persons with hypothyroidism treated with thyroid hormone.
The changes in proANP mRNA in both hypothyroidism and hyperthyroidism parallel very closely the circulating concentrations of vessel dilator, LANP, and ANP in humans, which are decreased in hypothyroidism and increased in hyperthyroidism. When the hyperthyroid subjects were treated with the antithyroid drug propylthiouracil (PTU) the circulating concentrations of LANP, vessel dilator, and ANP decreased 50% after one week of treatment, with a simultaneous 50% decrease in serum triiodothyronine (T 3 ) levels.
Glucocorticoids
Dexamethasone, at a dose of 1 mg/day, increases proANP mRNA levels in both atria and ventricles of the rat approximately two-fold. There is negative feedback between cortisol and the gene products of proANP gene expression in that the cardiac hormones vessel dilator, LANP, kaliuretic hormone, and ANP decrease the circulating concentration of cortisol. This decrease in cortisol is due, at least in part, to these cardiac hormones decreasing the circulating concentration of the hypothalamic peptide corticotrophin-releasing hormone (CRH), with a resultant decrease in ACTH, which stimulates the production of cortisol.
Mineralocorticoids
Administration of mineralocorticoids to animals causes transient fluid and sodium retention. Despite continued administration of a mineralocorticoid, animals return to sodium balance within a few days, a phenomenon termed “mineralocorticoid escape.” To investigate the role of ANP in mineralocorticoid escape, Ballerman et al. administered DOCA to rats in sodium balance, and found plasma ANP levels and atrial proANP mRNA content increased in rats retaining sodium in response to DOCA. After “escape” from the mineralocorticoid-induced sodium retention, plasma ANP levels returned to baseline and relative atrial proANP mRNA content remained moderately elevated. This increase in proANP mRNA probably resulted from the secondary cardiovascular effects of the steroids (e.g., increased intravascular volume), rather than from a direct effect of the mineralocorticoids on the ANP-secreting cell, as DOCA has no direct effect on proANP mRNA in ANP-expressing neonatal cardiocytes.
Vasoconstrictive Peptides
Several vasoconstrictive peptides, including endothelin, norepinephrine, and angiotensin II, stimulate proANP transcription and secretion. The cardiac hormones, in turn, affect endothelin in a negative-feedback manner.
Calcium
Primary cultures of neonatal rat cardiocytes exposed for 24 hours to 2 mM CaCl 2 in the culture media increase proANP messenger RNA three-fold, and increase secretion of ANP prohormone into the media three-fold. When these cardiocytes were treated with the calcium channel-blocking agents diltiazem, nifedipine or verapamil, both proANP synthesis and secretion decreased to 25–40% of control values.
Transgenic Knockout and/or Mice Overexpressing Atrial Natriuretic Peptide Prohormone Gene
Transgenic mice with an 11 base-pair deletion in exon 2 of the proANP gene ( Figure 37.1 ) have increased blood pressure in homologous ( −/− ) mice of 8–23 mmHg compared to wild-type ( +/+ ) mice. Exon 2 of the proANP gene encodes for vessel dilator, kaliuretic peptide, and ANP ( Figure 37.2 ). Exon 2 homozygote mutants have no circulating ANP, and they become hypertensive when fed a standard diet. Heterozygotes ( +/− ) with this base pair deletion in exon 2 are salt-sensitive and become hypertensive (systolic blood pressure increases 27 mmHg) on a high-salt (8%) diet. Mice that overexpress the proANP gene, on the other hand, become hypotensive.
Human Diseases with Upregulation of Atrial Natriuretic Peptide Prohormone Gene
Cerebrovascular Disease (Stroke and Hypertension)
A genetic linkage study followed 22,071 male physicians, all of whom had no history of stroke, from 1982 to 1999. DNA extracted from peripheral white blood cells of those individuals who had a subsequent stroke revealed that, when compared to those without strokes, these individuals had a molecular variant in exon 1 of the proANP gene that was associated with a two-fold ( p <0.01) increased risk of stroke. The individuals who had cerebrovascular accident (stroke) had significantly ( p <0.001) higher systolic and diastolic blood pressures than the persons who did not have a stroke. This molecular variant of the proANP gene was found to be an independent risk factor (in addition to hypertension) for a cerebrovascular accident. This molecular variant was found to be responsible for a valine-to-methionine transposition in long-acting natriuretic peptide (LANP), i.e., the only peptide hormone synthesized by exon 1. (Exon 1 does not encode for ANP.) In the 16 a.a. of LANP encoded by exon 1 there is only one valine, which is at position 7 of LANP ( Figure 37.2 ). Residue #7 (amino acid #7 of the ANP prohormone) is highly-conserved among different species. In this human study there was no defect in the structure or expression of the proBNP gene. In humans, blood pressure and the cardiac hormones correlate throughout the 24-hour period in a circadian relationship. There is evidence to suggest that long-acting natriuretic peptide reflects salt sensitivity in hypertension-prone individuals, even before they develop hypertension.
LANP and Stroke
Long-acting natriuretic peptide (LANP) has potent vasodilatory properties in both animals and humans. Antisera to LANP (to block the biologic activity of this peptide hormone) results in a significant increase in mean arterial pressure from 112±12 mmHg to 131±9 mmHg in normotensive animals, and exacerbates hypertension in spontaneously hypertensive rats (SHR) from 140±10 mmHg to 159±9 mmHg. These antisera data indicate an important physiological role for long-acting natriuretic peptide in the regulation of arterial pressure. In the brain of stroke-prone rats, the expression ANP prohormone gene (which synthesizes LANP) is significantly reduced. There were no mutations in the BNP gene, and no differences in BNP gene expression between stroke-prone and stroke-resistant animals.
Further evidence of the importance of the peptide hormones synthesized by the ANP prohormone gene derives from studies in mice with the ANP prohormone gene knocked-out: all develop salt-sensitive hypertension within one week leading to stroke. The BNP gene does not upregulate to prevent hypertension and/or stroke when the proANP gene is knocked-out. Downregulation of the proANP gene in the brain in stroke-prone SHRs has further been found to co-segregate with the occurrence of early strokes in their F 2 descendants.
Natriuretic Peptide Hormones and Hypertension
The original hypothesis for hypertension was that there was a defect in the production of the blood pressure-lowering cardiac hormones. Experimental evidence revealed that, rather than being decreased, these blood pressure lowering cardiac hormones are elevated in the circulation in an apparent attempt to overcome the elevated blood pressure. ANP increases in essential hypertension and in persons with pheochromocytomas. The hypertension associated with pheochromocytomas is characterized by increased circulating concentrations of vessel dilator and long-acting natriuretic peptide (LANP), as well as ANP. Each of these blood pressure lowering hormones increase further with surgical manipulation-induced increases in blood pressure, and then these peptides return to normal after surgical removal of the pheochromocytomas and lowering of blood pressure. The hypertension of obesity also is associated with increased circulating concentrations of ANP which decreases into the normal range with weight reduction-induced decrease in high blood pressure.
In pregnancy, cardiac hormones increase in each trimester with the plasma volume expansion which accompanies a normal pregnancy. ANP, vessel dilator, and LANP increase dramatically with the hypertension of pre-eclampsia, compared to their circulating concentrations in healthy pregnant women. If one knocks-out the ANP prohormone gene that synthesizes the four cardiac hormones ( Figure 37.1 ), within one week the animals develop salt-sensitive hypertension while, on the other hand, transgenic mice overexpressing the ANP prohormone gene develop hypotension. In addition to directly vasodilating vasculature, kaliuretic peptide and ANP inhibit the release of the potent vasoconstrictive peptide endothelin which is produced by the vascular endothelium.
Congestive Heart Failure
In congestive heart failure (CHF), proANP gene expression is upregulated. The increase in proANP gene expression is, however, not in the atria of the heart, but rather in the ventricle of the heart. In persons with CHF, there is no defect in the production of the peptides from the ANP prohormone, but rather each are increased in the bloodstream in an attempt by the heart to overcome abnormal sodium and water retention by releasing more of these peptides that cause sodium and water excretion. Vessel dilator and LANP increase in direct proportion to the severity of CHF, as classified by the New York Heart Association (NYHA). The ANP-clearance receptor pathway is not linked to the avid sodium retention and/or to the renal ANP resistance observed in CHF.
Cirrhosis with Ascites
Another salt- and water-retaining state, cirrhosis with ascites, is characterized by increased circulating concentration of the cardiac hormones, and with a 4.1-fold ventricular (but not atrial) increased steady-state proANP messenger RNA. Although the liver expresses proANP messenger RNA, there is no upregulation of proANP gene expression in the liver when ascites develops. Rather, the upregulation of this gene is only in the ventricle of the heart.
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