Discovery of New Cardiovascular Hormones for the Treatment of

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Cardiovascular & Haematological Disorders-Drug Targets, 2007, 7, 47-62 47 1871-529X/07 $50.00+.00 © 2007 Bentham Science Publishers Ltd. Discovery of New Cardiovascular Hormones for the Treatment of Conges- tive Heart Failure David L. Vesely * Departments of Internal Medicine, Molecular Pharmacology and Physiology, University of South Florida Cardiac Hormone and Health Sciences Center and James A.

Haley Veterans Medical Center, Tampa, Florida, USA Abstract: In 1628, Harvey first correctly described the heart as a pump. It was another 350 years before the heart was es- tablished as an endocrine gland that synthesized a family of peptide hormones that regulate blood volume and blood pres- sure. There are now five peptide hormones made in the heart which have been demonstrated to have beneficial effects in persons with congestive heart failure.

One of these peptide hormones i.e. brain natriuretic peptide (BNP) is commercially available and has been widely used in the United States for the treatment of acute congestive heart failure under the name Nesiritide/Natrecor ® . Nesiritide has one serious side effect, i.e.

it may worsen renal function in persons with acute decom- pensated cardiac failure. The best of these peptide hormones for the treatment of chronic heart failure is a cardiac hormone named vessel ... more. less.

dilator which enhances sodium and water excretion 4- to 5-fold in persons with congestive heart failure but vessel dilator 9s biologic effects lasts six hours compared to less than 30 minutes for BNP, without the deleterious effects of BNP on renal function. This review will focus on six cardiac hormones 9 discovery, identification and comparison of their beneficial effects and side effects in humans with congestive heart failure. Key Words: Cardiac hormones, congestive heart failure, treatment, natriuretic peptides, diuretics, sodium excretion. I. INTRODUCTION In 1628, Harvey [1], correctly described the heart as a pump 3 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 properties being control of blood volume and blood pressure [2-5]. The history of the experimentation leading to discovery of the cardiac peptides 9 hormonal sys- tem has followed two pathways: physiological and anatomi- cal. A. History: Physiological Studies 1. Association of the Heart and Renal Function In 1847, Harthshorne suggested that the heart possessed volume receptors capable of sensing the cfullness of blood- stream d induced by whole body immersion, which he clearly recognized had a diuretic effect [6]. This observation re- ceived little further notice until 1935, when John Peters made the same observation that cthe fullness of the blood- stream may provoke the diuretic response on the part of the kidney d [7]. This concept then received experimental verifi- cation when it was shown that expansion of blood volume increased urine flow [8-10]. Peters also suggested that the diuretic response was secondary to the ability of the heart or something very near the heart to csense the fullness of the bloodstream d [7]. *Address correspondence to this author at the Molecular Pharmacology and Physiology, Director, USF Cardiac Hormone Center, J. A. Haley Veterans Medical Center-151, 13000 Bruce B. Downs Blvd., Tampa, Florida 33612, USA; Tel: (813) 972-7624; Fax: (813) 972-7623; E-mail: david.vesely@med.va.gov 2. Balloon Distention of Atria Experimental evidence of an association between cardiac atria and renal function was provided in 1956 by Henry, Gauer, and Reeves, who observed that balloon distention of the left atrium in anesthetized dogs was associated with an increase in urine flow [11]. 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 his colleagues concluded that stretch receptors in the left atrium must be present [11,12]. Henry et al . noted the diuresis but did not investigate whether it was accompanied with an in- crease in salt excretion (natriuresis) [11,12]. It is well estab- lished now, however, that balloon distention of the cardiac atria does cause a natriuresis as well as a diuresis [13-15]. That animals with denervated hearts and/or denervated kid- neys respond to an increase in atrial pressure to produce a diuresis [16] suggests a hormonal pathway between the heart and the kidney. Part of this hormonal pathway involves car- diac peptide hormones. B. History of Cardiac Hormones: Anatomical Studies 1. Atrial Granule Structure Shortly before Henry and his colleagues reported their observation that balloon distention of atria caused a diuresis [11], Kisch, in 1955 utilizing electron microscopy, described dense granules that were located in the atria but not in the ventricles of mammals [17]. 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 [18,19]. Jamieson and Palade [20] demonstrated that such granules are present in cardio- cytes of the atria of all mammals, including humans. These authors suggested that these granules resemble other gran- ules that release polypeptide hormones [20]. Ultrastructural 48 Cardiovascular & Haematological Disorders-Drug Targets, 2007 , Vol. 7, No. 1 David L. Vesely cytochemistry has shown that these granules consist of pro- teins [21]. 2. Effect of Salt Intake on Atrial Granules of the Heart In 1958, Poche demonstrated that the number of granules present in the heart was influenced by changes in food and water intake [22]. There was then an 18-year hiatus before the next investigation of the effect of salt and water metabo- lism on the number of atrial granules at which time Marie, Guillemont, and Hatt demonstrated that the number of gran- ules in the atrial cardiocytes increased when the amount of sodium in an animal 9s diet was reduced [23]. deBold then performed the corollary experiment of documenting that decreased granularity resulted from sodium loading [24]. Thus, atrial granularity varies inversely with the amount of serum sodium present, which has the implication that the granules must store some substance that is interrelated with sodium balance. These studies led deBold et al . to perform the classic endocrine experiment of infusing minced rat heart extracts into other rats while monitoring the effect of these extracts on renal function [25]. 3. Atrial Extracts and Natriuresis In 1922, Banting and Best utilized a classic endocri- nological technique in their discovery of insulin [26]. They pulverized pancreas, filtered the crude tissue extract, and found that it produced hypoglycemia in an experimental dog [26]. deBold and his colleagues, in 1981, 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 a diuresis and natriuresis, with urine flow increasing 10-fold and sodium and chloride excretion increasing 30- fold [25]. This simple but elegant experiment led to the dis- covery of the cardiac hormones that have the most potent endogenous natriuretic activity of any substance yet de- scribed [27]. Atrial natriuretic peptide (ANP) one of the pep- tide hormones 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 to- day [27]. Other investigators quickly confirmed this natri- uretic action [28], as well as the ability of atrial extracts to cause vasodilation [29,30]. It was found that these effects were at least partially due to a peptide(s) that is present in all mammalian atria [25,27]. It was then demonstrated that the crude atrial extracts had significantly more natriuretic and diuretic properties than pure synthetic ANP suggesting that another or other peptide hormone(s) with natriuretic proper- ties was/were present in the atrial extracts [15,31,32]. 4. Discovery that the Atrial Natriuretic Peptide Gene Syn- thesizes Four Peptide Hormones Rather than Just one Pep- tide The reason that the atrial extracts have significantly more natriuretic properties than pure ANP itself was clarified when Vesely, et al . demonstrated that there were actually four peptide hormones made by the atrial natriuretic peptide (ANP) gene [33]. The three other peptide hormones synthe- sized by this gene vasodilate blood vessels [33] and cause a natriuresis and diuresis in animals [31-44] and humans [45- 50]. Twenty years ago (in 1987), Vesely, et al . elucidated that part of these four peptide hormones mechanism(s) of action was mediated via cyclic GMP [51]. These peptide hormones, numbered by their amino acid (a.a.) sequences beginning at the N-terminal end of the ANP prohormone consist of the first 30 a.a. of the prohormone (i.e., proANP 1- 30; long acting natriuretic peptide, LANP), a.a. 31-67; (i.e. proANP 31-67; vessel dilator), a.a. 79-98 (proANP 79-98, kaliuretic peptide) and a.a. 99-126 (atrial natriuretic peptide, ANP), Fig. ( 1 ). A number of laboratories have since con- firmed that these four cardiac hormones have biologic effects [39,40,52-54]. Vesely, et al . was also the first to demonstrate that all four of these peptides circulate in animals [38,51,55] and humans [56-70]. These discoveries demonstrated that these peptides were actually hormones since they circulate in the blood and have biological effects at distant target tissues. The discovery that these cardiac hormones circulate has been confirmed by a number of laboratories [71-74]. After these four cardiac hormones were discovered, a search of porcine brain cDNA library in 1988 revealed a fifth cardiac homone named brain natriuretic peptide (BNP) [75]. This peptide is now considered to have been misnamed because there is 10- fold more of BNP in the heart than in the brain [75,76]. Be- cause there is 10-fold more BNP in the heart than the brain [75,76] it has been suggested that this peptide be called B- type natriuretic peptide rather than brain natriuretic peptide. Naming this peptide B-type natriuretic peptide, however, implies that it was the second cardiac peptide discovered which, as above, is incorrect as it was the fifth cardiac pep- tide hormone discovered. Thus, the majority of investigators have continued to use brain natriuretic peptide, i.e. the origi- nal name for this peptide. A sixth member of the cardiac peptide family was also found first in the brain in 1990 and was named C-type natriuretic peptide [77] II. CARDIAC HORMONE FAMILY: SYNTHESIS OF THREE PROHORMONES This family of peptide hormones are synthesized by three different genes in the heart [78-84] and then stored as three different prohormones (i.e., 126 amino acid [a.a.] atrial na- triuretic peptide (ANP), 108 a.a. brain natriuretic peptide (BNP), and 103 a.a. C- type natriuretic peptide (CNP) pro- hormones) [84,85]. In healthy adults, the ANP prohormone's main site of synthesis is the atrial myocyte with its mRNA being 30 to 50 fold higher than that observed in the ventricle [86] but it is also synthesized in a variety of other tissues as well [86,87]. III. PEPTIDE HORMONES ORIGINATING FROM THE ANP, BNP AND CNP PROHORMONES Within the 126 a.a. ANP prohormone encoded by a sin- gle gene are four peptide hormones, Fig. ( 1 ) with blood pres- sure lowering, natriuretic, diuretic, and/or kaliuretic proper- ties as outlined above. The BNP and CNP genes, on the Discovery of New Cardiovascular Hormones Cardiovascular & Haematological Disorders-Drug Targets, 2007 , Vol. 7, No. 1 49 other hand, appear to each synthesize only one peptide hor- mone within their respective prohormones, i.e., BNP and CNP [82,83,88,89]. IV. MOLECULAR BIOLOGY OF THE CARDIAC HORMONAL SYSTEM A. ProANP Gene The gene encoding the synthesis of atrial natriuretic pep- tide prohormone (proANP), with which processing forms four cardiac hormones, consists of three exon (coding) se- quences separated by two intron (intervening) sequences which encode for a mature mRNA transcript approximately 900 bases long [90-95]. Translation of human ANP prohor- mone mRNA results in a 151 amino acid (a.a.) preprohor- mone [90,93-95]. Exon 1 encodes the 5'-untranslated region, the hydrophobic signal peptide (i.e., leader segment), and the first 16 a.a. of the ANP prohormone (i.e., the first 16 a.a. of long acting natriuretic peptide) [90,93-96]. The signal pep- tide, which is important for the translocation of this precur- sor peptide from the ribosome into the rough endoplasmic reticulum [93], is cleaved from the preprohormone (151 a.a.) in the endoplasmic reticulum. The resulting prohormone of 126 a.a. is the storage form for the four cardiac hormones synthesized by the proANP gene within tissues and is the major constituent of the atrial granules [90,93,95,97]. 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), Fig. ( 1 ). Exon 3 encodes for the terminal tyrosine (i.e., a.a. number 126 of the ANP prohormone) in humans and terminal 3 a.a. (Try-Arg-Arg) in rat, rabbit, cow and mice [90,93-96]. Dele- tion of this terminal tyrosine residue encoded by exon 3 does affect 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 [3]. Exon 2 encodes for the rest of the prohormone (i.e., a.a. 17-125 in humans) [90,93-95]. There is considerable homology in the proANP gene among species, particularly within the encoding and 5' flank- ing sequences [95,98]. The homology decreases in the in- trons of the proANP gene [95]. The sequences encoding the atrial natriuretic peptide prohormone are more highly con- served between the human and rodent gene than the interven- ing sequences or the 5' and 3' untranslated sequences [95,98]. Comparison of human, rat, and mouse genomic sequences and dog and rabbit cDNAs demonstrates maximum homol- ogy in the regions encoding proANP (93 of the 126 a.a. resi- dues are identical in all five species), whereas in the hydro- phobic leader sequence only 5 of the 26 residues are identi- cal [82,95]. The proANP gene has many features common to all eukaryotic genes [99], including a TATTA box (T = thymine, A = adenine), intervening sequences bounded by GT-AG splicing signals (G = guanine), and a consensus se- quence found in promoted regions. Also, AATAA poly- adenylation addition signals at the three transcription start sites for human [95,100] and rat [101] proANP mRNAs are located at ~30 base pairs 3' to the respective TATAA boxes- that is, about 95 base pairs before the initiation codon. The overall size of the proANP transcripts is 950-1050 nucleo- tides [86,97,102]. There is a "cap" sequence at the 5' end of proANP mRNA and a terminal codon (human mRNA only), as in the case with specific mRNA from most eukaryotic cells [103]. An interesting feature of the human proANP gene is a consensus sequence for a putative glucocorticoid hormone regulatory element within the second intron [94, 102,104]. V. STRUCTURE OF THE CARDIAC HORMONES a. Atrial Natriuretic Peptide: Comparison of Amino Acid Sequences Among Species The amino acid (a.a.) sequence of the whole ANP pro- hormone synthesized by the above gene is strikingly ho- mologous among many species with differences clustered at the extreme carboxy terminal end of the prohormone i.e. Fig. (1). The atrial natriuretic peptide prohormone gene in the heart synthesizes a 126 amino acid (a.a.) prohormone with which proteoly tic processing results in the formation of four cardiac hormones. These four cardiac hormones are 1) long acting natriuretic peptid e (LANP) consists of the first 30 amino acids of the 126 a.a. prohormone, 2) vessel dilator (VDL), a.a. 31-67 of the prohormone, 3) kali uretic peptide (KP), a.a. 79-98 of this prohormone and 4) atrial natriuretic peptide (ANP), consisting of a.a. 99-126 of the 126 a.a. prohormo ne. Reprinted with permission from Sun Y, et al . Anticancer Research 26: 3217-3222, 2006. 50 Cardiovascular & Haematological Disorders-Drug Targets, 2007 , Vol. 7, No. 1 David L. Vesely ANP as determined by a number of investigators [79,93, 95,98,100,101,105-112]. The 28 a.a. carboxy terminal end of the ANP prohormone, i.e., ANP, has 93% homology in five species, i.e., humans, rats, mice, rabbits, and dogs [91,92, 96]. In each species, the C-terminus is distinguished from the rest of the prohormone by forming a 17 a.a. ring structure via joining by a disulfide bond between the two cysteine resi- dues (105 and 121 of the prohormone). The ring structure originally was believed to be absolutely necessary for bio- logical activity [113,114]), but linear forms (same amino acids in linear form) without a ring structure have since been shown also to have biological activity [115]. For full natri- uretic and vasorelaxant acivity, the Phe-Arg-Tyr (i.e., a.a. 124-126) at the COOH terminus [113] and a.a. 99-104 of the NH 2 terminus of ANP are necessary [2]. A single arginine, as found at a.a. 98 of the prohormone, appears to be the pro- teolytic processing signal in the ANP prohormone and in other peptide prohormones also [3,116]. This processing signal helps to ensure that ANP is formed during proteolytic processing, cleaving it away from its adjacent hormone kali- uretic peptide which contains this terminal arginine. Except for the addition of the C-terminal Arg-Arg in ro- dents during synthesis, there is only a single amino acid dif- ference in the mammalian forms of ANP, which is at posi- tion 110 of the 126 a.a. prohormone. In rodents (rats, mice, and rabbits), this amino acid is isoleucine, whereas in higher mammals (humans, dogs, pigs, and cows) the amino acid at this position is methionine [82,94,95,98,105,116-118]. b. Long Acting Natriuretic Peptide: Comparison of Amino Acid Sequences Among Different Species Long acting natriuretic peptide has 20 of its 30 amino acids exactly the same in these five species, and another six of the remaining ten amino acids are exactly the same in four out of the five species [82,94,105,116,117]. Of the remaining four amino acids, two are exactly the same in three out of the five species. Thus, only two amino acids (5 and 28 of the prohormone) are not the same in the majority of the species, and these two positions have the same amino acids in at least two of the five species [82,94,105,116,117]. c. Vessel Dilator: Comparison of Amino Acid Sequences Among Species Vessel dilator, has 25 of its 37 amino acids being identi- cal the same five species [94,95,98,117,118]. Six of the re- maining twelve a.a. are the same in four of the five species, and three of the remaining six a.a. are the same in three out of the five species. Thus, only three amino acids (33,42, and 43 of the prohormone) are not the same in the majority of the species [94,95,98,117,118]. d. Kaliuretic Peptide: Comparison of Amino Acid Se- quences Among Species Kaliuretic peptide has highly conserved sequence among the above five species with 16 of its 20 amino acids being the same in all five [82,94,105,116-118]. One of the remain- ing four amino acids is the same in four species, whereas two of the other three amino acids are the same in three of the five species. With only one amino acid not the same in kali- uretic peptide in the majority of the five species tested, this peptide is also remarkably conserved among the species. This extraordinary conservation among species of LANP, vessel dilator, kaliuretic peptide is not observed in the BNP prohormone where there is a marked difference in amino acid sequence homology among species with respect to spe- cies 9 amino acid homology [75,80,82,89,119]. VI. MECHANISM(S) OF ACTION OF THE FOUR CARDIAC HORMONES THAT ARE GENE PROD- UCTS OF ProANP GENE Part of the intracellular mechanism of action of the four cardiac hormones encoded by the proANP gene is that after they bind to their specific receptors they enhance membrane bound guanylate cyclase [120] to cause an increase in the intracellular messenger cyclic GMP, Fig. ( 2 ), [121]. Cyclic GMP then stimulates a cyclic GMP-dependent protein kinase which phosphorylates protein(s) within the cell producing physiologic effects, Fig. ( 2 ). The receptors for ANP which mediate ANP's biologic effects (e.g., natriuretic peptide [NPR]-A and B-receptors) were the first receptors demon- strated to contain guanylate cyclase and a protein kinase within the receptors themselves [85,122,123], Fig. ( 2 ). The NPR-A receptor has a 441 a.a. extracellular portion that binds ANP, which, in turn, activates the catalytic portion of guanylate cyclase within the cell, Fig. ( 2 ). The protein kinase within this receptor has an inhibitory influence on guanylate cyclase until this receptor is activated by ANP or BNP [85,123]. This receptor is attached to the membrane via a 21 a.a. portion of the receptor, Fig. ( 2 ). VII. REGULATION OF THE ProANP GENE A. Enhancement of ProANP Gene Expression 1. Stretch Mechanical stretch, or more specifically tension deliv- ered across the atrial wall is a potent activator of proANP gene expression and/or secretion [54,124,125]. In animals, any increase in volume secondary to an increase in sodium intake results in an increased release of the ANP prohormone peptides [35,36,45]. 2. Thyroid Hormones Thyroid hormones i.e., thyroxine (T 4 ) and triiodothy- ronine (T 3 ) also help to regulate proANP gene expression [126,127]. T 3 , at concentrations of 10 -10 M to 10 -9 M, in- creases proANP mRNA levels two-fold in atria and four-fold in ventricular cardiac myocytes in culture [126]. The effect of thyroid hormones on proANP mRNA is paralleled by the increase in the circulating concentrations of the gene prod- ucts of this synthesis, i.e., vessel dilator, LANP, and ANP in persons with hyperthyroidism [128]. The effect of thyroid hormones on ANP mRNA levels has also been evaluated by comparing hypothyroid, euthy- roid, and hyperthyroid rat proANP mRNA in both atria and ventricles [127]. Right atrial proANP mRNA content in hy- Discovery of New Cardiovascular Hormones Cardiovascular & Haematological Disorders-Drug Targets, 2007 , Vol. 7, No. 1 51 pothyroidism and hyperthyroidism was 41 percent and 176 percent, respectively, of euthyroidism [127]. Ventricular proANP mRNA content in hypothyroidism and hyperthy- roidism was 31 percent and 178 percent, respectively, of that in euthyroidism [127]. These changes in proANP mRNA parallel very closely the circulating concentrations of ANP, vessel dilator, and LANP in humans, which are decreased in hypothyroidism and increased in hyperthyroidism [128,129]. In hyperthyroidism, vessel dilator, LANP and ANP increase 4-fold, 3-fold, and 2-fold, respectively, in the circulation compared to 54 healthy adults [128]. When the hyperthyroid subjects were treated with the anti-thyroid drug pro- pylthiouracil (PTU) the circulating concentrations of LANP, vessel dilator and ANP decreased 50% after one week of treatment with a simultaneous 50% decrease in serum triio- dothyronine (T 3 ) levels [128]. In hypothyroidism, the circu- lating concentrations of LANP and vessel dilator are 50% less and ANP 1/3 less than that of healthy adults [128]. The circulating concentrations of these natriuretic hormones re- turn to normal in persons with hypothyroidism with thyroid hormone treatment [128]. 3. Glucocorticoids Dexamethasone,at a dose of 1 mg/day, increases proANP mRNA levels in both atria and ventricles of the rat approxi- mately two-fold [102]. There is negative feedback between cortisol and the gene products of proANP gene expression in that vessel dilator, LANP, kaliuretic hormone, and ANP de- crease the circulating concentration of cortisol [130]. This decrease in cortisol is due, at least partially, 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 [130]. VIII. TRANSGENIC KNOCKOUT AND/OR MICE OVEREXPRESSING ProANP GENE Transgenic mice with an 11 base pair deletion in exon 2 of the proANP gene have increased blood pressure in ho- mologous (-/-) mice of 8 to 23 mm Hg compared to wild type (+/+) mice [131]. Exon 2 of the proANP gene encodes for vessel dilator, kaliuretic peptide and ANP [5,85]. Exon 2 homozygote mutants have no circulating ANP and they be- come hypertensive when fed a standard diet [131]. Het- erozygotes (+/-) with this base pair deletion in exon 2 are salt-sensitive and become hypertensive (systolic blood pres- sure increases 27 mm Hg) on a high salt (8%) diet [131]. Mice which overexpress the proANP gene, on the other hand, become hypotensive [132]. IX. HUMAN DISEASES WITH UPREGULATION OF ProANP GENE A. Cerebrovascular Disease i.e., Stroke - Hypertension A genetic linkage study (Physicians Health Study) of 22,071 male physicians, all of which had no history of Fig. (2). Structure of natriuretic peptide (NPR)-A (active) receptor. The extracellular portion of the receptor binds ANP and/or BNP, wh ich activates the catalytic portion of guanylate cyclase within the cell, which catalyzes the conversion of guanosine triphosphate (GTP) to the intracellular messenger cyclic GMP. The structure illustrated was drawn utilizing the amino acid sequences determined for the N PR-A recep- tor [120]. 52 Cardiovascular & Haematological Disorders-Drug Targets, 2007 , Vol. 7, No. 1 David L. Vesely stroke, were investigated from 1982 to 1999 [133]. DNA extracted from peripheral white blood cells of those indi- viduals 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 2-fold (P<0.01) increased risk of stroke [133]. The individuals who had cerebrovascular accident (i.e., stroke) had significantly (P<0.001) higher systolic and diastolic blood pressures than the persons who did not have a stroke [133]. The molecular variant of the proANP gene was found to be an independent risk factor (in addition to hyper- tension) for a cerebrovascular accident [133]. This molecular variant was found to be responsible for a valine-to-methio- nine transposition in the peptide hormone synthesized by exon 1, i.e., long acting natriuretic peptide (LANP) [3]. (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 posi- tion 7 of LANP [3]. Amino acid #7 (i.e. valine) of the ANP prohormone is highly conserved among different species [82,94,105,116,117]. In this human study (Physician's Health Study) of cerebrovascular accidents there was not any defect in the structure or expression of the proBNP gene [133]. Thus, this large prospective, case-controlled study revealed that carrier status for a mutation in the exon encoding for LANP is associated with a significantly increased (2-fold) risk of stroke [133]. It is important to note that in humans, blood pressure and the cardiac hormones correlate through- out the 24-hour period in a circadian relationship [68,69]. Long acting natriuretic peptide reflects salt sensitivity in hypertension-prone individuals even before they develop hypertension [134]. B. Long Acting Natriuretic Peptide and Stroke Long acting natriuretic peptide (LANP) has potent vaso- dilatory properties in both animals [33]) and humans [45,46]. Antisera to LANP (i.e., to block the biologic activity of this peptide hormone) results in a significant increase in mean arterial pressure from 112±12 mm Hg to 131±9 mm Hg in normotensive animals and exacerbates hypertension in spon- taneously hypertensive rats (SHR) from 140±10 mm Hg to 159±9 mm Hg [44]. This antisera data suggest an important physiological role for long acting natriuretic peptide in the regulation of arterial pressure [44]. In the brain of stroke-prone rats, the expression ANP prohormone gene (which synthesizes LANP) is dramatically reduced [135]. Through genotype/phenotype cosegregation analysis of F 2 intercross, from the crossbreeding of stroke- prone and stroke-resistant spontaneously hypertensive rats (SHRs) it was found that a different point mutation in the ANP gene confers a stroke delaying effect [136]. There are no mutations in the BNP gene and no differences in BNP gene expression between stroke-prone and stroke-resistant animals [135,136]. Thus, these studies in stroke-prone ani- mals suggest that the gene synthesizing the ANP prohor- mone may be protective against cerebrovascular accidents and this data is consistent with the human data that mutations in one or more portions of this gene [133] can lead to an in- creased incidence of cerebrovascular accidents. Further evidence of the importance of the peptide hor- mones synthesized by the ANP prohormone gene is from gene knockout studies in mice, which all develop salt- sensitive hypertension within one week leading to stroke when this gene is knocked out [131]. The BNP gene does not upregulate to prevent hypertension and/or stroke when there is proANP gene knockout [131]. Downregulation of the proANP gene within the brain in the stroke-prone rats (SHRs) has further been found to co-segregate with the oc- currence of early strokes in their F 2 descendents [135]. C. Natriuretic Peptide Hormones and Hypertension Hypertension is an independent risk factor in addition to mutation in the ANP prohormone gene [133] for a cere- brovascular accident. The original hypothesis for hyperten- sion was that there was a defect in the production of the blood pressure lowering cardiac hormones [137,138]. Ex- perimental evidence revealed that rather than being de- creased these blood pressure lowering peptides are elevated in the circulation in an apparent attempt to overcome the elevated blood pressure [60,137-139]. ANP increases in es- sential hypertension [137,138] and in persons with pheo- chromocytomas [139]. The hypertension associated with pheochromocytomas is characterized by increased circulat- ing concentrations of vessel dilator, long acting natriuretic peptide (LANP) and ANP [139,140]. Each of these blood pressure lowering hormones increase further with surgical manipulation-induced increases in blood pressure, and then these cardiac hormones return to normal after surgical re- moval of the pheochromocytomas and lowering of blood pressure [139,140]. The hypertension of obesity also is asso- ciated with increased circulating concentrations of ANP [60] which decreases into the normal range with weight-reduc- tion-induced decrease of the high blood pressure. In a normal pregnancy, the cardiac hormones increase in each trimester with the plasma volume expansion which accompanies a normal pregnancy [141]. ANP, vessel dilator and LANP in- crease dramatically with the hypertension of pre-eclampsia compared to their circulating concentrations in healthy preg- nant women [62]. If one knocks out the ANP prohormone gene which syn- thesizes the four atrial natriuretic peptides (Fig. 1 ), within one week the animals develop salt sensitive hypertension [131] while, on the other hand, transgenic mice overexpress- ing the ANP prohormone gene, develop hypotension [132]. In addition to directly vasodilating vasculature, kaliuretic peptide and ANP inhibit the release of the potent vasocon- trictive peptide endothelin which is produced by the vascular endothelium [46]. The cardiac hormones, thus, appear very important in blood pressure control in both health and in pathological diseases. D. Congestive Heart Failure In congestive heart failure (CHF) there is upregulation of proANP gene expression [142-144]. The increase in proANP gene expression is, however, not in the atria of the heart, but rather the increase in the proANP gene expression is in the ventricle of the heart [142-144]. Steady state proANP Discovery of New Cardiovascular Hormones Cardiovascular & Haematological Disorders-Drug Targets, 2007 , Vol. 7, No. 1 53 mRNA increases 4.3 ± 0.06-fold in the ventricle of CHF animals compared to sham animals [144]). In persons with congestive heart failure there is no defect in the production of these peptides from the ANP prohormone but rather each are increased within the circulation in the attempt by the heart to overcome the abnormal sodium and water retention by releasing more of these peptides which cause sodium and water excretion [47,48,57]. As part of the pathophysiology of CHF, for example, vessel dilator and LANP increase in direct proportion to the severity of CHF as classified by the symptomatic New York Heart Association (NYHA) [57]. The ANP clearance receptor pathway has been found not to be linked to the renal ANP resistance and/or avid sodium retention observed in CHF [145]. X. SECRETION OF NATRIURETIC PEPTIDES The main physiological stimulus to secretion of these peptide hormones to control blood volume appears to be an increase in pressure within the atria [54,124,125,146-148]. An increase of 4 to 6 mm Hg of pressure within the atria releases the four cardiac hormones from the ANP prohor- mone [55]. These peptide hormones, in turn, decrease the volume returning to the heart secondary to their causing a diuresis and natriuresis [34,40,55]. Cardiac pacing with rapid heart rates at 125 beats/minute and above release the cardiac hormones into the circulation [58]. Both atrial and ventricu- lar arrhythmias with heart rates of 125 beats/min and above release these cardiac hormones and increase their circulating concentratons in humans [61]. Hypoxia and a variety of humoral factors (endothelin, glucocortiods, acetylcholine, adrenergic agonists) have been suggested as contributing to the release of these cardiac hormones but the majority of these humoral factors 9 effects are to increase ANP prohor- mone gene synthesis as outlined above rather than release per se [147,148]. With respect to hyperosmolarity, the threshold for ANP release is as low as 10 mosmol/kg H 2 O and this is regulated by a cross-talk between sarcolemmal-L type Ca 2+ channel and the sarcoplasmic Ca 2+ release [149]. ANP, vessel dilator, LANP, and kaliuretic peptide have a negative feed back mechanism whereby their inhibit their own and each others 9 release [46]. CNP also inhibits ANP 9s release [150]. XI. BIOLOGIC EFFECTS OF THE NATRIURETIC HORMONES AND THEIR MECHANISM(S) OF AC- TION A. ANP 1. Vasodilation deBold et al 9s original report that crude atrial extracts cause diuresis and natriuresis also indicated that these ex- tracts could decrease mean arterial pressure [25]. Crude atrial extracts were then shown to possess vasorelaxant activity in isolated aortic segments [29,151,152]. Synthetic ANP can also cause vasodilation in vitro [153-156]. Both crude [151] and synthetic [157] ANP decrease total peripheral resistance. There is a diversity in the ability of ANP to cause vasodila- tion of preconstricted vessels [152,158]. There is variability in relaxing different isolated vasculature preparations by ANP. Large central arteries (i.e., aorta and renal) relax, whereas more distal (i.e., ear and basilar) arteries are refrac- tory to nanomalor concentrations of ANP [3,159]. Pulmo- nary, femoral, and iliac arteries are intermediate in their re- sponse to ANP [3]. One exception to small arteries not re- sponding well upon the addition of ANP are the carotid ar- teries, which relax [159]. In general, veins do not appear to vasodilate with the addition of ANP as well as arteries do, but ANP has been shown to relax peripheral veins in addi- tion to aortic rings [154]. ANP produces a dose-dependent reduction in systemic blood pressure in humans [160]. a. Mechanism of ANP-Induced Vasodilation The vasodilation observed with ANP is endothelium- independent [51,155,161]. It is mediated by cyclic GMP, which is increased after enhancement of membrane-bound guanylate cyclase by ANP [51,155,162]. Cyclic GMP itself has been demonstrated to cause vasodilation [155,161,163]. The vasorelaxation with ANP appears independent of cal- cium [51,164]. There is no change in cyclic AMP occurring during the same period of time that cyclic GMP increases [51,155]. With respect to calcium, ANP can both increase cyclic GMP and cause vasodilation without any calcium in the incubation media [51,121,155,162]. 2. Natriuresis In the classic experiment by deBold et al . (25) in 1981, crude atrial extracts caused a marked diuresis and natriuresis. Since that time, purified synthetic ANP has also been found to cause a natriurersis and diuresis [165-168]. This natriure- sis is thought to involve guanylate cyclase which is present in the proximal tubule of the kidney and cyclic GMP pro- duced by this enzyme increases amiloride-sensitive 22 Na uptake in the phosphorylated brush border membranes of the proximal tubule suggesting that ANP 9s intracellular mediator cyclic GMP directly stimulates the Na + -H + antiporter in the proximal tubule [169]. B. Long Acting Natriuretic Peptide, Vessel Dilator, and Kaliuretic Peptide 1. Vasodilaton LANP, vessel dilator, and kaliuretic peptide each cause an endothelium-independent vasodilation of vasculature [51] similar to ANP 9s endothelium-independent vasodilation of vasculature [55,156,162]. The amount of vasodilation in vi- tro with these peptide hormones is similar to that observed with addition of ANP [51]. These same peptide hormones have significant blood pressure-lowering effects in vivo [34]. When infused over two hours at the same 100 ng·kg body weight·min concentration in experimental rats vessel dilator and ANP were found to decrease blood pressure from an average of 145 to 124 mm Hg (P<0.05) and from 143 to 123 mm Hg (P<0.05), respectively [34]. Long acting natriuretic peptide lowered blood pressure from a mean of 138 to 122 mm Hg (P<0.05), whereas kaliuretic peptide decreased blood pressure from a mean of 155 to 138 mm Hg (P<0.05) [34]. Bloodpressuredidnot change in the control animals through- 54 Cardiovascular & Haematological Disorders-Drug Targets, 2007 , Vol. 7, No. 1 David L. Vesely out the 120-minute pre-experimental period or during the 120-minute experimental period [34]. Similar to ANP, these peptide hormones mechanism of vasodilating vasculature is mediated by cyclic GMP [51], i.e., each of these peptide hormones 9 mechanism of action involves enhancement of the enzyme guanylate cyclase with resultant increase in the intracellular messenger cyclic GMP [51]. The enhancement of guanylate cyclase activity by these atrial peptides is cal- cium-independent in vasculature [51]. 2. Long Acting Natriuretic Hormone, Vessel Dilator and Kaliuretic Peptide: Natriuresis-Mechanism of Action Comparison of the relative natriuretic and diuretic poten- cies the same 100-ng/kg body wt/min dose of vessel dilator, LANP and ANP revealed that they have significant natri- uretic properties in healthy humans, but kaliuretic peptide had no significant natriuretic effects [45]. The natriuretic properties of vessel dilator are especially impressive in light of the fact that ANP has been found to be a more potent na- triuretic and diuretic agent than furosemide [27] and that vessel dilator and LANP circulate normally at a 17- to 22- fold higher concentration than ANP and 33- to 48-fold higher than BNP and 124- to 177-fold higher than CNP [57,59,68,71-74] while at the same low concentrations as ANP and BNP experimentally vessel dilator has much stronger natriuretic effects [45]. In persons with congestive heart failure, vessel dilator has 4-fold more potent natriuretic and diuretic effects than ANP or BNP [47]. This 17- to 22- fold higher circulating concentration of vessel dilator and LANP compared to ANP is found both during basal condi- tions and with release secondary to physiological stimuli such as head-out water immersion where the atria are stretched releasing these peptides [59,63]. It is therefore pos- sible that vessel dilator and LANP with higher concentra- tions in the circulation may contribute more to natriuresis and diuresis that is observed secondary to normal physio- logical stimuli than does ANP since vessel dilator and LANP are seventeen-fold higher in circulation and have equally potent biologic effects when utilized at the lower circulating concentration of ANP [45,47,48]. Vessel dilator 9s biologic effects last significantly longer than ANP (i.e., six hours ver- sus 30 minutes) [45,47,48]. Vessel dilator and ANP are markedly different, however, with respect to potassium ex- cretion [45]. Vessel dilator is the only one of the four cardiac hormones from the ANP prohormone that does not signifi- cantly enhance potassium excretion [34,45]. This potassium- sparing effect of vessel dilator makes it distinctly different from ANP, LANP, and kaliuretic peptide. LANP has similar but more modest diuretic, natriuretic and kaliuretic proper- ties when compared to ANP at the same concentration [45]. Kaliuretic peptide does not significantly enhance the frac- tional excretion of sodium (FE Na ), but it is the only cardiac hormone that significantly enhances the fractional excretion of potassium in healthy humans (FE k ). (Fractional excretion of sodium or potassium is the percentage of glomerular- filtered sodium or potassium that is excreted into the urine) [45]. Physiological maneuvers such as head-out-of-water immersionresulting in a central hypervolemia release LANP, vessel dilator, kaliuretic peptide and ANP and produce natri- uresis and diuresis [59,63,64]. The natriuretic effects of long acting natriuretic peptide, kaliuretic peptide and vessel dilator have distinctly different mechanism(s) of action from ANP in that they inhibit renal Na + -K + -ATPase secondary to their ability to enhance the synthesis of prostaglandin E 2 [37,51] which ANP does not do [37,51,170]. ANP, BNP, and CNP 9s effects in the kidney are thought to be mediated by cyclic GMP [2,4,31,147]. D. Brain Natriuretic Peptide and C-Type Natriuretic Peptide 1. BNP: Biologic Effects Brain natriuretic peptide (BNP) is a 32 a.a. peptide in humans [45 a.a. in rat] with similar diuretic and natriuretic effects and short half-life as ANP [75]. BNP has remarkable sequence homology to ANP with only 4 a.a. being different in the 17 a.a. ring structure formed by a disulfide bond common to both peptides [75,80,89,147]. Although BNP was named [75] for where it was first isolated (i.e., porcine brain cDNA), the main source of its synthesis and secretion is the heart (10-fold greater than brain) [31,76,84,89, 147,171]. As with ANP, the highest levels of BNP are found in the atria of the heart [31,76]. BNP levels in the atria, how- ever, are less than 1% of ANP levels [76]. The immunoreac- tive level of BNP within the ventricles is only 1% of BNP 9s concentration within the atria i.e., 99% of BNP is found in the atria [76]. BNP, however, has been termed a cventricu- lar d peptide based upon ventricular BNP mRNA levels being similar to those in the atria in one study and the ventricles are much larger than the atria [81]. 2. CNP: Circulating Concentrations and Biologic Effects C-type natriuretic peptide (CNP) is a 22 a.a. peptide with remarkable similarity to ANP and BNP in its amino acid sequence but lacks the carboxy terminal tail of ANP and BNP [77,88,172]. CNP was found originally in the brain but more recent studies suggest that it is also present within the heart [173]. The amount of CNP within the heart, however, isverylow and only small amounts are present within plasma [173]. Two CNP forms, 22 and 53 a.a. in length, have been identified within plasma [88,174]. Each is derived from a single CNP prohormone, with the 22 a.a. form contained within the carboxy terminal portion of the 53 a.a. form. The 22 a.a. form predoiminates in plasma and is more potent than the 53 a.a. form in humans [77,88,172,174]. The plasma concentration of CNP is very low with some authors report- ing that CNP is not normally detectable but becomes detect- able only in renal failure [175] and congestive heart failure [176]. CNP has been found to have little effect on renal vasoconstriction [177]. Although CNP has been reported to have natriuretic effects in some animals, when infused in humans at physiological concentrations and in concentra- tions that reached 4- to 10-fold above those observed in dis- ease states, CNP did not affect renal function [172]. Thus, in healthy humans CNP had no effect on renal hemodynamics, systemic hemodynamics, intrarenal sodium handling, sodium Discovery of New Cardiovascular Hormones Cardiovascular & Haematological Disorders-Drug Targets, 2007 , Vol. 7, No. 1 55 excretion or on plasma levels of renin and aldosterone [172]. In another study of infusion of CNP in healthy humans, CNP was increased 60-fold in plasma and there was no significant hemodynamic or natriuretic effects [178]. The authors of this study concluded that it is unlikely that CNP has any role in endocrine physiology [178]. There is one study in humans where infusion of CNP to increase CNP plasma levels 550- fold above normal levels caused a 1 -fold increase in urine volume and sodium excretion [179]. With this very high plasma concentration of CNP, both ANP and BNP also in- creased 2.4-fold [179], which may have been the cause of the natriuresis and diuresis observed. Each of these studies sug- gest that CNP does not contribute physiologically to any natriuresis or diuresis in healthy humans [172,178,179]. CNP 9s main site of synthesis is vascular endothelium [179, 180]. CNP acts as a paracrine endothelium-derived hyperpo- larizing factor (EDHF) via activation of NPR-C receptor and the opening of a G-protein-gated inwardly rectifying K+ channel (GIRK) in mesenteric resistance arteries to mediate vasodilation [180]. In conduit vessels, on the other hand, CNP induces relaxation via a cyclic GMP-dependent mecha- nism [180]. XII. CONGESTIVE HEART FAILURE The prevalence of congestive heart failure is markedly increasing to epidemic proportions while other heart disease such as acute myocardial infarction the incidence is declin- ing [47]. A. Nesiritide (BNP) Currently, only one of the cardiac hormones is commer- cially available in the United States, i.e., BNP (Nesiri- tide/Natrecor, SCIOS, Sunnyvale, CA). Nesiritide (BNP) is currently used via an 3 to 8-hour infusion ( 0.03 g·kg - 1 ·min -1 ) for persons with acute, decompensated heart failure. Nesiritide in a double-blind, placebo controlled randomized trial of 20 CHF patients caused a small increase in urine vol- ume (90 ± 38 mL/hr vs . 67 ± 27 mL/hr) from baseline values [181]. Further evaluation has revealed that there is no sig- nificant natriuresis [182] or diuresis [183,184] with nesiritide (BNP) infusion in humans with CHF. The natriuretic and diuretic effects of BNP in persons with CHF are markedly blunted compared to healthy individuals [181-184]. In CHF animals, BNP also had no significant natriuretic or diuretic effects [89,185]. BNP has hemodynamic effects in CHF in- dividuals with lowering of pulmonary capillary wedge pres- sure (PCWP) [182,184]. BNP can cause significant hypoten- sion in CHF subjects [182,184] with an incidence of 27% reported with 0.06 g/kg/min [184]. In some individuals the hypotension secondary to nesiritide was severe enough to cause seizures. The high incidence of hypotension at this dose is the reason this dose was not approved for use in hu- mans by the United States Food and Drug Administration (FDA). Nesiritide made it to market with a superb marketing campaign i.e., although it causes no significant natriuresis or diuresis in the doses allowed by the FDA for persons with CHF, it became commercially available since the CHF indi- viduals cfelt better d i.e., their subjective symptoms improved and because it lowered pulmonary capillary wedge pressure. It was approved by the FDA on csymptoms improvement d and hemodynamic changes (decreased PCWP) only with no demonstrated effects of enhancing a natriuresis or causing a diuresis that cause improvement in CHF. B. Risk of Worsening Renal Function with Nesiritide (BNP) in Patients with Acutely Decompensated Heart Failure In addition to the hypotension caused by nesiritide (i.e., BNP) in persons with CHF, there is a suggestion that nesiri- tide significantly increases the risk of worsening renal func- tion in persons with acute decompensated congestive heart failure [186]. This study of randomized clinical trials com- paring nesiritide with either placebo or other current treat- ments of CHF were identified by electronic and manual searches and thorough review of FDA files available via the FDA website [186]. This study included a systematic review of FDA documents released by the Cardiovascular and Renal Drug Advisory committee for meetings in 1997 and 2001 which included the New Drug Application submission pre- pared by SCIOS, Inc. [187-190] and data from SCIOS, Inc. Medical Affairs Dept [191-193]. In addition, the authors of the cBNP worsens renal function d studies [186] did a manual search of annual meetings of the American Heart Associa- tion, American College of Cardiology and Heart Failure So- ciety of America. From these sources, seven unique random- ized multi-center trials were identified [186]. Trials were selected for this meta-analysis when they fulfilled each of the following characteristics: randomized, double-blind, par- allel-group study on patients with acutely decompensated chronic heart failure with effect on serum creatinine re- ported. Worsening renal function was defined as an increase in serum creatinine >0.5 mg/dL. Relative risk across all stud- ies was determined by meta-analysis with Mantel-Haenszel fixed-effects model (RR MH ). Risk of dialysis and medical intervention for worsening renal function were compared between therapies. Frequency of worsening renal function was determined from five randomized studies that included 1269 patients [186]. Use of FDA-approved doses of nesiritide ( 0.03 g·kg - 1 ·min -1 ) [192] significantly increased the risk of worsening renal function compared with non-inotrope-based treatments of CHF (RR MH , 1.52; 95% CI, 1.16 to 2.00; P=0.003) and compared to all other therapies of CHF, including non- inotrope- and inotrope-based therapies (RR MH , 1.54; 95% CI, 1.19 to 1.98; P=0.001) [186]. Of the persons receiving nesiri- tide (BNP) 22% (range 18 to 27%) had a worsening of renal function with creatinine increasing greater than 0.5 mg/dL [186]. Even with low-dose nesiritide ( 0.015 g· kg -1 ·min -1 ) in 906 patients with CHF there was a 22% incidence of worsening of renal function [186]. Thus, low-dose nesiritide ( 0.015 g·kg -1 ·min -1 ) significantly increased risk (P=0.012 and P=0.006 compared with non-inotrope- and inotrope- based treatments of CHF, respectively), as did BNP adminis- tered at any dose up to 0.06 g· kg -1 ·min -1 (P=0.002 and P=0.001, respectively) [186]. 56 Cardiovascular & Haematological Disorders-Drug Targets, 2007 , Vol. 7, No. 1 David L. Vesely Compared with any other therapy of congestive heart failure, BNP (i.e., nesiritide) administered in FDA-approved doses, or at doses lower than the approved doses, increased the risk of worsening renal function [186]. The frequency of worsening renal function requiring medical intervention was increased (almost 3-fold) in the nesiritide -treated patients [186]. Because increases in creatinine 0.5 mg/dL in patients with CHF indicate a worse prognosis, with a significantly increased risk of death [194] (hazard ratio 2.86; 95% CI, 1.55 to 5.26) that can be predicted with high specificity (82%; 195), this meta-analysis of the company 9s data and clinical trials raised serious questions regarding the contin- ued use of BNP to treat CHF [186]. The authors of this study concluded that the short-term effects of BNP (nesiritide) of improving symptoms in persons with CHF may not be suffi- cient to ensure long-term safely and giving BNP should be balanced against the possibility of worsening renal function in light of increased mortality associated with increasing the creatinine above 0.5 mg/dL [186]. In addition to the above studies, several other studies [196,197] indicating a strong association between elevations in serum creatinine levels and the possibility of death suggest that BNP could be associated with clinically relevant risk [186]. With respect to increased death with giving Nestiride ® , in a follow-up to the above study [186] a pooled analysis of randomized control trials with nesiritide through December 2004 from the FDA, Scios, Inc. and the scientific literature where 485 CHF patients treated with nesiritide were com- pared to 377 control CHF patients suggested that there was an increased risk of death (i.e., more than double, P=0.04) within the first 30 days of receiving nesiritide for acutely decompensated heart failure [198]. This increased risk with nesiritide was observed at 30 days (end of study) after giving nesiritide [198]. The authors of the above article have sug- gested that controlled trial(s) be done before any further use of nesiritide for heart failure because of the possibility of an increased risk of death with nesiritide [198]. C. Atrial Natriuretic Peptide Atrial natriuretic peptide (ANP) is commercially avail- able in Japan under the brand name Carperitide (Astella Pharma, Osaka, Japan). Although ANP causes a natriuresis and diuresis in healthy humans, it has markedly attenuated natriuretic response in humans with CHF [199,200] and in animal models of CHF [201,202]. High dose administration of ANP produces little or no diuresis in humans with CHF [199,200]. ANP, like BNP, thus, has significantly blunted natriuretic and diuretic effects in persons with CHF com- pared to healthy subjects [199,200]. ANP, like BNP, has hemodynamic effects in persons with CHF and can cause severe hypotension [47,200]. D. Vessel Dilator Vessel dilator is the cardiac hormone with the most sig- nificant natriuretic and diuretic effects of the natriuretic pep- tide hormones for the treatment of CHF [47]. When given to patients with chronic New York Heart Association (NYHA) class III CHF intravenously for 60 min, vessel dilator in- creases urinary flow 5-fold i.e., 7.55 ± 0.75 compared with 1.56 ± 0.35 mL/min excreted at baseline ( vs a similar 4-fold increase in healthy individuals i.e., 3.4 ± 1.1 vs 0.9 ± 0.2 mL/min excreted at baseline) and sustains this increase for 3 hours after the infusion is stopped (P<0.01) [47]. At the end of the 60-minute infusion, mean urinary flow was 7.55 ± 0.75 mL/min and it was 6.7 ± 0.7 mL/min three hours after the infusion was stopped [47]. These values are 4.8- and 4.3- fold higher, respectively, than the baseline urinary flow of the patients with CHF which was 1.56 ± 0.35 mL/min [47]. One patient of the CHF patients in this investigation who was studied for a longer period exhibited a two-fold increase in urinary volume and flow for six hours after vessel dilator infusion was stopped [47]. Vessel dilator also enhanced sodium excretion 5-fold at the end of its infusion in CHF subjects (12.2 ± 3.6 vs 2.6 ± 1.0 Eq of Na + excreted per minute; P<0.01) versus no na- triuresis with ANP or BNP. Vessel dilator caused a three- fold enhancement of sodium excretion within 20 minutes (10.1 ± 1.9 vs 2.6 ± 1.0 Eq of Na + excreted per minute); and three hours after the vessel dilator infusion was stopped, so- dium excretion was still 3-fold greater (i.e., 7.8 ± 1.8 vs 2.6 ± 1.0 Eq of Na + per minute) than baseline sodium excretion (P<0.01) [47]. Vessel dilator increased fractional excretion of sodium (FE Na ) in patients with CHF up to a maximum of 6-fold (6.7% vs 1% in control) (P<0.001) [47]. Thus, the FE Na doubled (2% vs 1%) 20 minutes after vessel dilator infusion began, and it was 4.5-fold greater than baseline at the end of the infusion (4.5% vs 1% at baseline) [47]. Thus, the natriuretic and diuretic effects of vessel dilator are not blunted in persons with CHF, as those of BNP and ANP are, compared to healthy adults [47]. During the vessel dilator infusion, systemic vascular re- sistance decreased 24% from a mean of 1127 ± 18 to 857 dynes·s·cm -5 . Pulmonary vascular resistance decreased 25% (from 129 ± 6 to 97 ± 4 dynes·s·cm -5 ; Fig. ( 3 ). Pulmonary capillary wedge pressure decreased 33% (from 21 ± 3 to 14 ± 3 mm Hg) and central venous pressure decreased 27% (from 8.25 ± 1.48 to 6.00 ± 1.00 mm Hg) Fig. ( 3 ). Thus, ves- sel dilator very significantly (p<0.001) decreases pulmonary capillary wedge pressure, i.e. the only objective finding with BNP for which it was given FDA approval to treat persons with CHF. Heart rate and mean pulmonary artery pressure did not change significantly. Vessel dilator increased cardiac output 34% (from 5.4 ± 0.9 to 7.9 ± 1.2 L/min) and cardiac index 35% (from 2.66 ± 0.01 to 3.58 ± 0.01 L·min -1 ·m -2 ) [47]. Vessel dilator also increased stroke volume index by 24% (from 0.034 ± .003 to 0.042 ± .002 mL/m 2 ) [47]. Thus, vessel dilator decreases systemic vascular resis- tance and increases the cardiac index, Fig. ( 3 ). These results are beneficial for persons with CHF, whose baseline state is characterized by increased systemic resistance and a reduced cardiac index. The decrease in systemic vascular resistance secondary to arterial vasodilation by vessel dilator also de- creases the back-pressure on the heart, which results in de- Discovery of New Cardiovascular Hormones Cardiovascular & Haematological Disorders-Drug Targets, 2007 , Vol. 7, No. 1 57 creased left atrial pressure. Systemic blood pressure and sys- temic vascular resistance decrease following treatment with vessel dilator, which suggests improvement in both afterload and preload. In patients with CHF, when ventricular function is on the steep portion of the pressure-volume curve, preload reduction may decrease ventricular wall stress, resulting in improved cardiac output. This was found to be the case with vessel dilator administration [47]. Vessel dilator also lowers pulmonary capillary wedge pressure (PCWP) to a greater extent than BNP (nesiritide) which is nesiritide 9s only known beneficial effect in CHF [47]. There were no side effects with the infusion of vessel dilator in persons with CHF [47]. Vessel dilator does not worsen renal function as does BNP. On the contrary, vessel dilator markedly improves renal function when acute renal failure (ARF) occurs [5]. E. Long Acting Natriuretic Peptide Long acting natriuretic peptide (LANP) enhances urine flow 4.3-fold (3.8 ± 1.8 vs 0.9 ± 0.2 mls/min excreted at baseline) while increasing sodium excretion 2.5-fold in healthy humans (i.e., 8.3 ± 3.2 vs 3.5 ± 1.4 Eq Na + excreted per minute at baseline) [45]. In persons with congestive heart failure (CHF), LANP at 0.1 g/kg body weight/min for 1 hour increases urine flow 2-fold (2.75 ± 0.65 vs 1.43 ± 0.33 mls of urine per minute) and causes a 1.3-fold increase in natriuresis (i.e., 3.8 ± 1.1 vs 2.9 ± 1 Eq Na + excreted/min at baseline [48]). Thus, urine flow secondary to LANP was blunted compared to its effects in healthy individuals [48]. The FE Na increased 3-fold (1.4% vs 0.4% at baseline) secon- dary to LANP in patients with CHF [48]. F. Kaliuretic Peptide Kaliuretic peptide enhances urine flow 2-fold i.e., 2.8 ± 0.8 vs 1.2 ± 0.3 mls/min in healthy humans [45]. Kaliuretic peptide (100 ng/kg body weight/minute) given intravenously for 60 minutes to NYHA class III CHF subjects increases urine flow 4-fold i.e., 2.7 ± 1.2 vs 0.7 ± 0.2 mls/min in the first 20 min; P<0.001), which was still 4-fold increased (2.87 ± 0.7 vs 0.7 ± 0.2 ml/min baseline 2.5 hours after its infusion was stopped [50]. Kaliuretic peptide enhance sodium excre- tion 4-fold (4.3 ± 1.8 vs 1.0 ± 0.1 Eq of Na + /min) in the first 20 minutes of its infusion in subjects with CHF [50]. Sodium excretion was increased 2-fold (2.3 ± 0.6 vs 1.0 ± 1 Eq of Na + /min) 2.5 hours post-infusion [50]. The FE Na doubled (1.4% vs 0.7%) 20 minutes after beginning the kali- uretic peptide infusion and was 3-fold (1.7% vs 0.7%) in- creased 2.5 hours postinfusion. Kaliuretic peptide increased the urinary excretion rate of K + and the fractional excretion of K + (FE K+ ) 4- (i.e., 4.6 ± 0.6 vs 1.1 ± 0.2 Eq/min baseline) and 2-fold (3.74% vs 2.29% at baseline; P<0.05), respec- tively [50]. Kaliuretic peptide 9s ability to enhance sodium excretion is not blunted but rather enhanced in persons with CHF compared to healthy individuals [50]. Vessel dilator, however, enhanced sodium and FE Na more than kaliuretic peptide and more than ANP, BNP and LANP in persons with chronic NYHA Class III CHF. Vessel dilator, LANP, and kaliuretic peptide, as opposed to ANP and BNP have never caused a hypotensive episode when given to either healthy animals or healthy humans [34,45,46] or when given to hu- mans with sodium and water retention [38,47,50]. Fig. (3). Vessel dilator decreases systemic vascular resistance (SVR), pulmonary vascular resistance (PVR), pulmonary capillary wedge pressure (PCWP) and central venous pressure (CVP in persons with congestive heart failure. The decrease in every measurement wa s sig- nificant (p<0.05) when evaluated by repeated analysis of variance ANOVA. No significant changes were found in heart rate (HR) o r pulmo- nary artery pressure (PAP). Cardiac output (CO), cardiac index (CI) and stroke volume index (SVI) were significantly (p<0.05) i ncreased when evaluated by repeated measures of ANOVA. There was no change in any of these parameters in patients with congestive heart failure who received saline vehicle only. Reprinted from reference 47 with permission of the American Heart Association. 58 Cardiovascular & Haematological Disorders-Drug Targets, 2007 , Vol. 7, No. 1 David L. Vesely ACKNOWLEDGEMENTS This work was supported in part by a Merit Award from the United States Department of Veterans 9 Affairs, a grant from the Darren Malenski Foundation, and grants from the American Heart Association, Florida/Peurto Rico affiliate. REFERENCES [1] Harvey, W. Exercitatio Anatomica de motu cordis et sanguinis animalibus . Francofurti Guilielem Fitzeri, 1628. C D Leake (trans). Charles C. Thomas, Springfield, IL, 1928 . [2] Brenner, B.M.; Ballermann, B.J.; Gunning, M.E.; Zeidel, M.L. Diverse biological actions of atrial natriuretic peptide. Physiol. Rev., 1990 , 70 , 665-699. [3] Vesely, D.L. Atrial Natriuretic Hormones . Englewood Cliffs, N.J.: Prentice Hall, 1992 , pp. 1-256. [4] Vesely, D.L. Atrial natriuretic peptides in pathophysiological dis- eases. Cardiovasc. Res., 2001 , 51 , 647-658. [5] Vesely, D.L. Natriuretic peptides and acute renal failure. Am. J. Physiol., 2003 , 285 , F167-F177. [6] Harthshorne, H. Water versus hydrotherapy .; Lloyd P Smith Press, Philadelphia, 1847 , pp. 2 8. [7] Peters, J.P. Body water: the exchange of fluids in man, Charles C Thomas, Springfield, IL, 1935 , pp. 287. [8] Borst, J.G.G. The maintenance of an adequate cardiac output by the regulation of the urinary excretion of water and sodium chloride; an essential factor in the genesis of edema. Acta. Med. Scand., 1948 , 130 (Suppl., 207), 1-71. [9] Zuidema, G.D.; Clarke, N.P.; Reeves, J.L.; Gauer, O.H.; Henry, J.P. Influence of moderate changes in blood volume on urine flow. Am. J. Physiol., 1956 , 186 , 89-92. [10] Gauer, O.H.; Henry, J.P. Criculatory basis of fluid volume control. Physiol. Rev., 1963 , 43 , 423-481. [11] Henry, J.P.; Gauer, O.H.; Reeves, J.L. Evidence of the atrial loca- tion of receptors influencing urine flow. Circ. Res., 1956 , 4 , 85-90.

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