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Chapter 21 / Cardiovascular Hormones
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21 Cardiovascular Hormones
Willis K. Samson, PhD and Meghan M. Taylor, PhD
CONTENTS
VASOCATIVE HORMONE FAMILIES
NATRIURETIC PEPTIDE FAMILY
ENDDOTHELINS
AM GENE PRODUCTS
CARDIOVASCULAR HORMONES AS DIAGNOSTIC AND THERAPEUTIC TOOLS
1. VASOACTIVE HORMONE FAMILIES
1.1. The Heart as an Endocrine Organ
acterized, their hallmark effects are to unload the vascular tree via a combination of CNS, pituitary, adrenal,
vascular, and renal actions (Fig. 1). This results in
decreased venous return to the pump as a consequence
of increased renal excretion of water and solute,
vasorelaxation in certain vascular beds, increased capillary permeability, and decreased cardiac output. The
third member of this family of hormones, although
exerting many of the same actions as ANP and BNP, is
unique in that it is predominantly produced in the vascular endothelium, not in the heart, and is thought to
act more in a paracrine or an autocrine fashion, regulating primarily vascular tone and growth. Additionally, this hormone, designated C-type natriuretic
peptide (CNP), exerts several CNS actions that oppose
those of ANP and BNP.
For years the central focus of vascular endocrinology was the renin–angiotensin system (see Chapter
23); however, with the discovery of the cardiac hormones and the realization that at least some of their
actions were expressed by a functional antagonism of
the actions of angiotensin, a broader view of the importance of circulating hormones controlling vascular and
renal function took shape. Then this doctrine of endocrine regulation of cardiovascular and renal function
From: Endocrinology: Basic and Clinical Principles, Second Edition
was challenged and expanded by the realization that
(S. Melmed and P. M. Conn, eds.) © Humana Press Inc., Totowa, NJ
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Although the heart had long been considered merely
a muscular pump that performed the physical labor of
the circulation, it has been recognized for more than
six decades that in addition to the contractile ultrastructure, a secretory function was evidenced by densecore granules in the myocytes. Over the past two
decades, the endocrine nature of the heart has been
established, and the physiology and pathophysiology
of the cardiac hormones have been extensively characterized. In both a constitutive and regulated fashion,
myocytes produce two members of a family of hormones designated natriuretic peptides based on their
abilities to stimulate salt and water excretion by direct
renal actions and by actions in other tissues, including
endocrine organs, responsible for the control of fluid
and electrolyte homeostasis. Two members of the natriuretic peptide family, atrial natriuretic peptide (ANP)
and brain natriuretic peptide (BNP, actually a misnomer since very little if any of this peptide is produced
in the central nervous system) are produced in the heart
and released in response to a variety of cues, many
typical of plasma volume overload or hyperosmolality.
Although numerous biologic actions have been char-
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Fig. 1. Summary of biologic actions of ANP and CNP. AVP = vasopressin; ACTH = adrenocorticotropin; GnRH = gonadotropinreleasing hormone; CRH = corticotropin-releasing hormone; CO = cardiac output; UV = urine volume; UNaV = urinary sodium
excretion; NO = nitric oxide; ET = endothelin; T3/T4 = thyroid hormones.
perhaps the largest endocrine organ in the body, by
virtue of its enormous surface area and ubiquitous presence, was the vasculature itself, mainly the endothelium. Not only was one member of the natriuretic
peptide family produced in and released from this tissue, but it became apparent that numerous peptidergic,
as well as nonpeptidergic, factors originating in the
endothelium controlled vascular tone and proliferation.
1.2. Hormones of the Endothelium
The vascular endothelium controls access of bloodborne factors not only to the interstitium, but also to the
contractile and proliferative elements of the vascular
tree, the vascular smooth muscle cells (VSMCs). Additionally, the endothelial cells are positioned optimally
to respond themselves to circulating factors and to transduce those messages to the VSMCs. Many hormonal
messages are in fact delivered to the contractile ele-
ments via factors produced in the endothelium. Much
attention has been focused on the ability of the endothelium to cause vasorelaxation via the generation of a
soluble gas, nitric oxide (NO); however, peptidergic
factors originating in the endothelial cells control VSMC
function as well. Here the role of CNP as a paracrine
factor has been established, and the endothelial cell–
VSMC interface was the setting for the discovery and
characterization of two additional, potent vasoactive
hormones, endothelin (ET) and adrenomedullin (AM).
The ETs are potent hypertensive agents that exert their
effects directly on the VSMCs. AM, on the other hand,
is a potent hypotensive agent, acting in a paracrine or an
autocrine fashion in the vasculature. Thus, both circulating and locally produced vasoactive hormones can
control regional blood flow, and this cellular interface
has provided a model for the paracrine and autocrine
effects of these peptides in other tissues as well. Agonists and antagonists selective for these peptides have
Chapter 21 / Cardiovascular Hormones
been successfully tested in models of cardiovascular
disease and some have even been approved for clinical
use in humans.
2. NATRIURETIC PEPTIDE FAMILY
2.1. Gene Structure and Regulation
The members of the natriuretic peptide family share
structural homology but are products of unique genes.
Expression of separate genes and posttranslational processing of the nascent hormones is very similar. The
genes for ANP and BNP have been localized to the same
chromosome, whereas that for CNP resides on a separate chromosome, further suggesting the similar actions
of ANP and BNP and disparate effects of CNP. Cloning
of the cDNA complementarity to ANP mRNA revealed
the presence of three exons in the ANP gene and the
transcription of a prepro-ANP mRNA that encoded a
151- to 152-amino-acid preprohormone, depending on
species. Removal of the N-terminal signal peptide
results in a prohormone of 126 amino acids, which demonstrates extensive homology across species. This 126amino-acid prohormone is the major form of the peptide
stored in secretory granules in the heart. Stored proANP is processed at secretion to a variety of smaller,
biologically active forms, primarily the mature 28amino-acid, C-terminal fragment. In brain, on the other
hand, the prohormone is further processed to the mature
peptide before packaging in secretory granules. An
additional form of ANP is produced in kidney, this form
being 33 amino acids of the C-terminus by posttranslational processing that includes four more amino acids
on the N-terminus. This isoform, designated urodilatin,
is thought to act as a paracrine regulator of tubular
function. Expression of the BNP gene differs in that the
resultant mRNA is less stable and the final posttranslational product is 32 amino acids long. Finally, CNP
processing is quite similar to that of ANP, with the
exception that the final posttranslational product lacks
the C-terminal extension distal to the shared (within the
natriuretic family) 17-membered disulfide loop, consisting, thus, of only 22 amino acids. In humans, owing
to the presence of an arginine in the prohormone at
position 73, a second form of CNP is present, which is
N-terminally extended, consisting of 53 amino acids.
CNP-22 and CNP-53 exert similar actions in many biologic systems. Note that in all three isoforms, the integrity of the internal disulfide loop is necessary for biologic
activity.
Although little is known about the regulation of CNP
gene transcription, mechanisms for activation of ANP
and BNP gene transcription have been extensively studied. Gene transcription is induced by glucocorticoids,
α-adrenergic agents, growth factors, calcium, and
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physical factors. There is a regional mismatch in the
adult in gene expression of the two peptides, with ANP
expressed primarily in the atria and BNP in the ventricles under basal conditions. Physical factors such as
cardiac overload induce transcription of both genes,
with the appearance of ANP expression in the ventricles
as well. Most striking, however, is the level of induction of the BNP gene in the ventricles, resulting in a
remarkable increase in circulating hormone levels. At
the molecular level, ANP gene transcription is regulated by numerous members of the activating protein1 complex, being induced by c-jun and in most cases
suppressed by c-fos. A close relative of c-fos, fra-1,
exerts biphasic effects, reducing the magnitude of c-jun
activation of ANP gene expression in atriocytes, while
amplifying the induction of expression by c-jun in
ventriculocytes. Thus, the response of the ANP promotor to these early response elements may vary under
unique physiologic conditions, permitting a wider repertoire of control of gene expression.
2.2. Hormone Secretion
2.2.1. PHYSIOLOGIC RELEASE
Plasma levels of ANP and BNP are extremely low
(5–10 and 0.5–1.0 fmol/mL, respectively) and rise in
response to any interventions that increase venous
return and, therefore, atrial pressure and stretch. Pressor
agents can release ANP in vivo and some even act in
isolated tissue in vitro, suggesting direct cellular effects
independent from increased venous return. The natriuretic effects of ANP and BNP are mirrored by the ability of hyperosmolality to stimulate directly, and
indirectly via volume expansion, hormone secretion. In
addition to secretion from the heart, these peptides are
produced in and secreted from or into a variety of other
tissues where distinct biologic actions have been characterized. The absolute contribution of those release
sites to circulating levels of the hormones is in all likelihood minor; however, the potential importance of
paracrine effects of the natriuretic peptides in those other
tissues makes the study of the regulation of release in
noncardiac sites extremely important. Indeed, renal,
CNS, gonadal, and thymic production sites suggest a
diversity of function for the peptides, and the mechanisms responsible for the regulation of secretion first
must be elucidated before the physiologic or pathologic
significance of those production sites is fully understood. Within the CNS, some of the same circulating
factors that can stimulate ANP release from the
myocytes (i.e., vasopressin and ET) similarly stimulate
neuronal production and release of the peptide.
Endothelial cell production of CNP has been clearly
established and the control of peptide secretion partially
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characterized. A variety of cytokines and growth factors (including interleukin-1α [IL-α] and IL-β, tumor
necrosis factor-α [TNF-α], and transforming growth
factor [TGF-β], as well as ANP and BNP) can stimulate
CNP release from endothelial cells. Shear stress and
hypoxia stimulate CNP release in the vasculature, as
they do for ANP and BNP in the heart. Thus, the
endothelial cell can, via CNP secretion, both transduce
the antimitogenic effects of circulating ANP and BNP
and buffer the proliferative effects of circulating cytokines and growth factors.
2.2.2. STATE OF HYPERSECRETION
Elevations in circulating natriuretic peptides have
been reported in a variety of pathophysiologic states.
CNP is remarkably elevated in septic shock, but not in
hypertension or congestive heart failure (CHF). This
again points to the more likely paracrine actions of
CNP within the endothelial cell interface with VSMCs.
CHF, myocardial ischemia, and hypertension all result
in increased ANP and BNP secretion, reflecting possible compensatory mechanisms called into play during
those conditions. Plasma BNP levels in cardiac overload states exceed those of ANP and are used clinically
as diagnostic and prognostic tools to assess the progression and degree of heart failure. Although elevated during those overload states, the bioactivity of ANP and
BNP appears to be reduced owing to a possible combination of effects, including reduced renal perfusion,
receptor downregulation, or the counterregulatory
effects of simultaneous activation of the renin-angiotensin-aldosterone system. Of these three possible
explanations, the best case can be made for the latter.
The increased circulating levels of ANP in critically ill
trauma patients is thought to be a potential cause of
suppressed adrenocorticotropic hormone levels frequently observed, because ANP can act at both the
hypothalamic and pituitary levels to inhibit corticotropin release.
2.3. Sites of Action
Three natriuretic peptide receptor subtypes have
been identified (Fig. 2). Two of these proteins contain
intracellular kinase homology domains (adenosine
triphosphate binding sites) and C-terminal guanylyl
cyclase (GC) domains. Activation of these receptors
results, therefore, in elevated cellular cyclic guanosine
5´-monophosphate (cGMP) levels. Their extracellular
domains share 44% homology, whereas the intracellular domains share 63% homology in the kinase homology domain and 88% homology in the GC domains.
These two receptors have been designated the GC-A
and GC-B receptors and are alternatively called natri-
uretic peptide receptor-A (NPR-A) and NPR-B. A third
receptor subtype, called the clearance receptor or NPRC, shares approx 30% homology with NPR-A and NPRB in the extracellular ligand-binding domain; however,
this receptor lacks the intracellular C-terminal extension (i.e., it is missing the kinase and GC domains). This
receptor was originally thought to have no biologic
activity other than to sequester or clear natriuretic peptides from the extracellular fluid; however, it is now
recognized that NPR-C plays important biologic roles
and signals via a reduction in cyclic adenosine monophosphate (cAMP) levels and possibly a stimulation of
polyinositol phosphate turnover (increased phospholipase C [PLC] activity). This receptor appears to mediate the antimitogenic actions of the natriuretic peptides
in the CNS. A distinct hierarchy of binding affinities
characterizes these receptors with all three forms of
natriuretic peptides binding with equal affinity to NPRC. NPR-A prefers ANP as a ligand (ANP > BNP >>
CNP), whereas NPR-B recognizes more readily CNP
(CNP >> ANP = BNP). Thus, the sites of action of the
natriuretic peptides are determined by the relative distributions of the three receptors, with NPR-B predominating in the brain (e.g., the hypothalamo-hypophyseal
system) and muscular component of the vasculature,
whereas NPR-A is more abundant in the kidney, adrenal
gland, and endothelium. The clearance (NPR-C) receptor is present throughout the body.
2.4. Biologic Actions
Originally CNP was thought to act only in a paracrine
fashion to regulate vascular tone and growth; however,
CNP can also exert cardiovascular, renal, and adrenal
actions when infused intravenously. This may simply
be a reflection of the fact that CNP is produced in a
variety of tissues, and, therefore, multiple paracrine
actions may occur. CNP levels are elevated in chronic
renal failure, and the peptide is produced in kidney,
where it exerts diuretic and natriuretic effects. One can
recognize the sites of action of the natriuretic peptides
by locating receptors, but the assignment of biologic
activity is not as simple. Two reagents that have clarified the receptor subtype responsible for a variety of
natriuretic peptide actions are the clearance receptor
ligand C-ANF4–23, which binds preferentially to NPRC, and the GC antagonist HS-142-1, which blocks the
ability of the natriuretic peptides to signal via activation of GC. By using a combination of methodologic
approaches, it has been realized that although the NPRC controls the antimitogenic effects of the natriuretic
peptides centrally, NPR-B performs a similar function
in the vascular compartment. Within the kidney, multiple receptors are found, explaining the ability of both
Chapter 21 / Cardiovascular Hormones
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Fig. 2. Three members of the natriuretic peptide family, ANP, BNP, and CNP, share 65% homology (indicated by shaded circles)
in the biologically active ring structure formed by the disulfide links and vary in amino acid composition and lengths of their N- and
C-terminal extensions. All three peptides are recognized by the natriuretic peptide C (clearance) receptor (NPR-C); however, the A
receptor (NPR-A) prefers ANP and BNP. The B receptor (NPR-B) recognizes with relative preference CNP. Activation of the three
receptors has been reported to generate the indicated changes in intracellular levels of cGMP or cAMP and/or phosphoinositol (PI)
turnover.
ANP and CNP to act as diuretic and natriuretic agents.
The multiple peripheral effects of the natriuretic peptides are summarized in Fig. 1. Although not all of these
actions seem related to fluid and electrolyte homeostasis, some may instead be related to the antiproliferative
effects of the peptides.
Within the CNS, similar and diverging actions of
ANP and CNP have been described. In most species,
more CNP is produced within the brain than ANP or
BNP and, for the most part, NPR-B and NPR-C predominate within the brain interstitium. It should be recognized that ANP may therefore exert its biologic
actions in the brain by displacing CNP from NPR-C
(clearance receptor). This has been demonstrated to be
the case in the neuroendocrine hypothalamus. There are
other examples where interactive effects via the shared
NPR-C cannot underlie the effects observed. Thus, the
ability of ANP to inhibit the behavioral (water drinking)
and endocrine (prolactin [PRL] secretion) aspects of
fluid and electrolyte homeostasis is opposed by the
stimulatory effects of CNP. Certainly in these cases,
activation of NPR-A must underlie the effects of ANP,
whereas NPR-B must be responsible for the stimulatory
effects of CNP. In the absence of antagonists that can
distinguish between these two GC receptor subtypes,
other methodologies had to be created to make these
distinctions. One such approach is receptor-specific
cytotoxin cell targeting using the plant lectin ricin. With
this approach, evidence for the involvement of the NPRA in the physiologic regulation of salt appetite has been
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obtained, and the importance of NPR-B in the hypothalamic mechanisms controlling neuroendocrine function
has been established.
Which of the pharmacologic actions of the natriuretic peptides are physiologically relevant? A combination of experimental approaches has provided
evidence for the role of the peptides in a variety of
tissues. Use of the selective clearance receptor ligand
C-ANF4–23 established the importance of ligand binding to the NPR-C on astrocyte proliferation (i.e.,
antimitogenic effects). The GC receptor antagonist
HS-142-1 was employed to demonstrate that the natriuretic peptides play important roles in the maintenance
of glomerular filtration and sodium excretion under
basal conditions. Ricin cytotoxin adminstration studies demonstrated the role of endogenous brain-derived
CNP in the neuroendocrine regulation of PRL and
luteinizing hormone secretion, and the importance of
central ANP to the control of sodium homeostasis
(i.e., appetite). Passive immunoneutralization was
employed to demonstrate the physiologic relevance of
the action of ANP to inhibit thirst.
Recently two molecular techniques have provided
additional insight into the physiology of the natriuretic
peptides. Transgenic mouse models of overexpression
of natriuretic peptide have been created. In the case of
the ANP transgene, homozygotes displayed significantly lower blood pressure under basal conditions than
nontransgenic littermates; however, sodium excretion
was not different. Transgene-induced overexpression
of BNP led to increased endochondral ossification and
bony overgrowth. These studies uncovered an action of
BNP that was not revealed in pharmacologic studies,
probably due to the chronic effect of BNP overexpression, something not possible to accomplish in classic
pharmacologic application studies. Transgene-induced
overexpression of CNP or BNP improved postischemic
insult neovascularization by stimulating reendothelialization and suppressing neointimal formation. These
studies strongly suggest a therapeutic option for the
natriuretic peptides in patients with tissue ischemia.
The second molecular approach to the study of the
physiologic relevance of the pharmacologic effects of
the natriuretic peptides is the generation of null mutations (knockouts), which results in the absence of a given
peptide. ANP-null mice are more susceptible to the
hypertensive consequences of high salt ingestion. This
model reveals two important things. First, ANP is not
essential for normal embryonic and postnatal development. Second, endogenous ANP must play some role in
the physiologic mechanisms that protect against the
development of high blood pressure. CNP knockouts
are dwarfs, displaying impaired endochondral ossifica-
Part IV / Hypothalamic–Pituitary
tion, but the condition can be rescued by simultaneous
targeted overexpression of a CNP transgene. These data
complement the results (just discussed) in the CNP
overexpression system alone and further support a significant role of CNP in normal bone development and
turnover. The CNP knockout animals display early
mortality; thus, other important roles of CNP must be
present and are certainly awaiting discovery.
2.5. Potential Therapeutic Uses
Although the action of the natriuretic peptides
appears to be blunted in edematous states, such as CHF,
cirrhosis, and the nephrotic syndrome, therapeutic use
of the peptides in these states may prove at least acutely
advantageous. Certainly, if the mechanism by which
the biologic activity of the peptides has been reduced
in these states can be elucidated, strategies might be
employed that overcome those deficits. In particular, it
has already been demonstrated that in CHF, administration of high doses of ANP and urodilatin can lower
preload and increase diuresis and natriuresis, providing
significant benefit in this life-threatening situation.
Even though plasma ANP levels are elevated in CHF,
further elevation by exogenous administration has provided salutary therapy. Like conventional diuretics,
ANP increases urine volume and urinary sodium excretion, at least in part by inhibiting sodium reabsorption
in the collecting duct by an action on the sodium/chloride transporter. However, unlike current diuretic
agents, ANP inhibits the renin-angiotensin-aldosterone
system by directly inhibiting renin secretion and
thereby lowering aldosterone levels in plasma. In addition, administration of ANP lowers sympathetic tone,
and the combined reduction in plasma renin activity
and sympathetic tone effectively lowers sodium reabsorption in the proximal tubule. ANP also inhibits
tubuloglomerular feedback, and the maintenance of
glomerular filtration even in the face of decreased renal
blood flow may be an important reason for the peptide’s
protective effect on renal function even in low perfusion states such as heart failure. Finally, ANP not only
inhibits arginine vasopressin (AVP) release, but it
blocks AVP’s ability to stimulate water reabsorption in
the collecting duct. ANP therapy in acute heart failure
was approved in Japan almost 10 yr ago, and recently
a synthetic BNP, nesiritide, was approved for use in the
United States.
Because a role of endogenous ANP in the phenomenon has been suggested, perhaps a similar strategy can
be used to induce mineralocorticoid escape. Interest in
the postoperative use of ANP and urodilatin to prevent
acute renal failure has been stimulated by early studies
demonstrating the ability of high pharmacologic doses
Chapter 21 / Cardiovascular Hormones
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Fig. 3. Three members of the mammalian ET peptide family have been identified, each sharing remarkable homology in amino acid
composition. Shaded circles indicate differing amino acids. Three receptor subtypes have been characterized. The ET-A receptor
binds ET with a relative preference indicated by the thickness of the arrows (ET-1 ⱖ ET-2 > ET-3). The ET-B receptor recognizes
equally all three forms of ET. The third receptor, ET-C, found thus far only in nonmammals, prefers ET-3. Sites of receptor expression
are indicated.
of the peptide to reduce the need for hemodialysis/
hemofiltration in these patients.
Most promising in a therapeutic sense is the potential use of the natriuretic peptides as antiproliferative
agents. In a rabbit model of vascular lesions caused by
balloon catheter injury, administration of CNP significantly lowered the resultant intima-to-media ratios,
providing direct evidence for a paracrine action of the
peptide to suppress intimal thickening. Furthermore,
local CNP antagonizes the growth-promoting effects
of angiotensin II (Ang II) and the vascular consequences of cyclosporine A induction of ET release and
subsequent mitogenesis, again predicting a significant
avenue for the prevention of vascular lesions.
3. ENDOTHELINS
3.1. Gene Structure
In addition to the production of endothelial-derived
relaxing factors in the vasculature, it had been known
for some time that the cells lining the blood vessels
produce potent vasoconstrictive substances as well. In
1988, the sequence of a powerful, endogenous vasoconstrictor substance produced by endothelial cells was
identified. This 21-amino-acid peptide was named ET.
It is now recognized that there are at least three forms
of the ETs (ET-1, ET-2, and ET-3), all 21 amino acid
peptides differing by only 2–5 amino acids in the 15membered ring structure formed by two internal disulfide bonds (Fig. 3). Each are products of unique genes
and are first processed similarly into a prohormone
form of 203 amino acids, in the case of ET-1, and then
posttranslationally modified into the 39-amino-acid
prohormone intermediate, big ET. In states of hypersecretion, the prohormone forms appears in plasma;
however, under normal conditions, the mature 21amino-acid form is the major secretory product. The
final cleavage of the prohormone is thought to occur at
secretion and to be catalyzed by a phosphoramidonsensitive metalloproteinase designated ET-converting
enzyme (EC 3.4.24.11). Knowledge of this important
conversion enzyme’s presence has led to potential
therapeutic intervention strategies for interruption of
ET action in states of hypersecretion, because the
prohormone big ET has limited biologic activity.
The human ET-1 gene has five exons and four introns,
with the peptide coded in the second exon. The gene is
transcriptionally regulated via cis elements, including a
GATA-2 protein-binding site and an AP-1 site that is
activated by thrombin, angiotensin II, epidermal growth
factor (EGF), basic fibroblast growth factor (bFGF),
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insulin-like growth factor, and TGF-β. Other transcriptional regulators include vasopressin, the ILs, TNF-α,
and NO, which apparently mediates the ability of heparin to stimulate ET production. Physical factors also
activate transcription, including pressure and anoxia.
Translational regulation is exerted by a variety of factors that also regulate secretion, since little hormone is
stored intracellularly. High-density lipoproteins stimulate production and secretion, whereas insulin not only
stimulates production and secretion, but also augments
ET binding and action. Negative regulation of production and secretion is exerted at the transcriptional level
by NO, and at the translational event by prostaglandins,
prostacyclin, AM, and ANP.
3.2. Hormone Secretion
Fortunately, the majority of the ET produced is
secreted abluminally, away from the vessel lumen, to act
in a paracrine or autocrine fashion. Although levels of
the hormone do rise in certain pathologic conditions, in
general, reflecting tissue damage in most cases, this peptide should be kept out of the circulation because of its
potent vasoconstrictive properties and because in experimental animals, elevation in circulating ET results in
respiratory failure and/or cerebral vasospasms and
aneurysms. Again, knowledge of the production sites
predicts biologic activities. The major site of ET-1 production is the endothelium, where on release it causes
vasoconstriction. Additional production sites include the
brain, uterus, kidney mesangial cells, Sertoli cells, and
breast epithelial cells. ET-3 production occurs mainly
within the CNS, where a role for the peptide in neuronal
and astroglial development and proliferation has been
suggested. What little ET-2 is produced in the body is
found in kidney, intestine (hence, the alternative name
vasoactive intestinal constrictor), myocardium, and
uterus. The ETs have a relatively short half-life in
plasma, about 4–7 min, and are released primarily in
response to hypoxia, ischemia, and shear stress.
3.3. Site and Mechanisms of Action
Two mammalian ET receptor subtypes have been
cloned, and they are members of the G protein–linked,
seven-transmembrane-spanning domain superfamily
of biologic receptors (Fig. 3). The ET-A receptor displays a rank order of binding affinity with ET-1 being
the preferred ligand (ET-1 ⱖ ET-2 >> ET-3). This
receptor predominates in VSMCs and cardiac
myocytes. Activation of the receptor results, depending on tissue site, in the activation of a variety of signaling cascades, including in the lung the production
of prostanoids via stimulation of PLD and PLA2 activities resulting in bronchoconstriction and in the scenario
of hypersecretion, pulmonary hypertension (Fig. 4). In
VSMCs, ET stimulates contraction and mitogenesis
via multiple signaling pathways, including activation
of PLC with the resultant formation of diacylglycerol
(DAG) and inositol triphosphate (IP 3). The DAG
formed activates the kinase cascade via protein kinase
C (PKC), and IP3 mobilizes intracellular calcium, initiating the contractile event. ET-A receptor activation
also in these cells has been reported to open potassium
channels and to activate adenylyl cyclase. In the myocardium, the ET-A receptor is thought to be activated
by endogenous ET released in response to ischemia
following myocardial infarction. The resultant opening of potassium channels causes a decrease in the electrical activity of the myocyte and closes the chloride
channel, resulting in a suppression of catecholamine
activation of contractile function.
The ET-B receptor predominates in the endothelium itself and in the CNS. This receptor binds all three
isoforms equally and is responsible for the activity of
circulating ET to stimulate a transient vasodilatory
response in the periphery, via acute release of vasodilators such as NO and CNP. The signaling cascade that
follows activation of the ET-B receptor is multifaceted.
G protein–coupled activation of PLC results in PKC
activation and mobilization of intracellular calcium. NO
synthase activity is stimulated with the resultant production of the potent vasodilator NO, which can act
within the endothelial cell to activate GC or diffuse
across to the smooth muscle cells to perform the same
function. Additionally, ET-B activation results in opening of the sodium-hydrogen antiporter and inhibition of
adenylyl cyclase. The mitogenic effects of ET are
thought to be transduced via PKC activation and tyrosine phosporylation–intiated activation of the mitogenactivated protein kinase (MAPK) system. In mesangial
cells, the mitogenic effect of ET is mediated via transcriptional activation of immediate early genes. Activation of Ras proteins and downstream induction of the
kinase activity of Raf-1 result in transcriptional induction of the c-fos serum response element, perhaps providing a mechanism for the mitogenic effect of ET. One
hallmark characteristic of the biologic effects of the ETs
is their profound tachyphylaxis. Although some data
indicate this to be the result of chronic membrane effects
or overloading of the cytosolic calcium pool, evidence
also exists for rapid internalization of the ligand-receptor complex and continued signaling from the internalized aggregate.
3.4. Biologic Actions
Although multiple pharmacologic effects of the ETs
have been reported, there is a need to establish which of
Chapter 21 / Cardiovascular Hormones
329
Fig. 4. Summary of biologic actions of ETs.
those have biologic significance and physiologic relevance. In this case, multiple pharmacologic tools are
available, such that selective antagonism of the ET-A
receptor is possible and isoform-specific activation of
the ET-B receptor can now be accomplished. Also available are antagonists that affect both the ET-A and ETB receptor, and a new generation of relatively specific
ET-B antagonists. Much interest continues regarding
the possible existence of a unique, ET-3-selective ET-C
receptor in mammals similar to that found in frog melanophores, and it is hoped that eventual cloning of that
protein will permit generation of similarly selective
antagonists.
Surprising results from molecular approaches have
provided new insight into the biology of the ETs. As
discussed below, it was anticipated that these potent
vasoconstrictive peptides would be found to play an
important role in the development of hypertension;
however, gene knockout homozygotes have, if anything, slightly elevated blood pressure, not the expected
hypotension. Unexpected results accrued from these
null mutation strategies. Mice lacking expression of the
normal ET-1 gene are born with severe craniofacial
malformations, suggesting a developmental role of the
peptide in pharyngeal arch structures. Additionally,
these animals succumb to respiratory failure, suggesting an important embryonic role of the peptide in the
preparation of respiratory structures for postnatal life.
Knockouts of the genes encoding the ET-B receptor or
ET-3 itself result in a postnatal phenotype similar to
that observed in Hirschsprung disease (congenital
megacolon), suggesting the importance of ET in the
development of the intrinsic nervous system of the gut
and the regulation of gastrointestinal smooth muscle
function. Multiple CNS actions of the ETs have been
reported, ranging from mitogenic effects on astrocytes
mediated via the ET-B receptor to effects on descending sympathetic activity. The presence of the ET peptide and ET receptors during fetal development
indicates a potential embryonic role in CNS structures,
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Part IV / Hypothalamic–Pituitary
which may mirror the situation uncovered by the ET-3
and ET-B receptor knockouts in the intrinsic nervous
system of the gut. Loss of the neurotropic effects of the
ETs may be responsible in part for the respiratory failure seen in the immediate postpartum interval. Regarding neuromodulatory effects of the ETs, antagonist
studies have revealed the physiologic relevance of the
antidipsogenic effects of ET; however, the day-to-day
significance of the neuroendocrine actions of the peptide (including activation of the hypothalamic mechanisms controlling anterior pituitary function and
vasopressin secretion) and the effects of the peptides on
sympathetic function have yet to be established.
A final CNS consequence of the administration of ET
is potentially disastrous. In some species, ET infusion
results in rupture of the basal artery on the ventral surface of the Pons, and a role for endogenous ET in vasospasm subsequent to subarachinoid hemorrhage has been
established by the observation that pretreatment with an
ET antagonist can prevent this event. This has led to the
hypothesis that ET antagonists may prove therapeutically advantageous to prevent or lessen ischemic damage downstream from damaged, hypoxic endothelium
of the cerebral vessels after thrombosis or infarct.
3.5. Pathophysiology of the ETs
Because of the multiple pharmacologic activities of
the ETs, their involvement in numerous pathologic
states has been hypothesized. These hypotheses were
based largely on elevated circulating ET levels or
responses seen in these conditions and, under many
circumstances, it remains unclear whether the associations are causal or coincidental. The most predicted
role for the ETs in pathophysiology was that in hypertensive states. Indeed, plasma levels are not consistently
found to correlate with blood pressure, and although
they may be elevated in some animal models of hypertension, null mutant mice actually have elevated blood
pressures. Similarly controversial is the potential role
for ET in reperfusion injury; one group using an isolated perfused rat heart model argued against a causative role and another utilizing isolated ventricular
myocytes argued in favor. In vivo evidence favoring a
role for endogenous ET in reperfusion injury comes
from studies in pig in which both myocardial ischemia
and infarction resulted in significantly elevated ET
production and release. In rats receiving infusion of an
antiserum directed against ET-1 prior to coronary artery
ligation, damage distal to the ligation was markedly
reduced. Similar results were obtained with an ET-A
receptor antagonist in a canine myocardial infarction
model. These data provide the best evidence for a role
for ET in reperfusion injury and ischemia.
ET antagonists improve renal function in a genetic
model of hypertension, the spontaneously hypertensive
rat, and ET has been invoked in the pathogenesis of
acute renal failure (postischemia renal failure). Cyclosporine A–induced nephrotoxicity has been identified
to be owing at least in part to ET-induced vasoconstriction of the afferent arteriole, since the renal toxicity of
immunosuppressant therapy was blocked by ET antagonist pretreatment. The mitogenic actions of ET are
thought to underlie the development of graft arteriosclerosis in cardiac allografts, the establishment of
atherosclerotic plaques, and the development of diabetes-related vascular lesions. Its role in vasospasm
secondary to subarachnoid hemorrhage has been established in animal models. Within the lung, roles for ET
in asthma and pulmonary hypertension have been proposed. The current literature favors the therapeutic use
of ET antagonists in a variety of pathologic situations.
However, clinical trials have not always been successful in establishing their clinical value.
Several patient trials were conducted to determine
the efficacy of ET antagonists in the setting of pulmonary arterial hypertension (PAH). The rational for these
trials was that lung levels of ET mRNA are increased in
PAH, and a positive correlation between serum ET levels and measured pulmonary vascular resistance had
been described. Although the nonselective ET antagonist, bosentan, was effective in reducing pulmonary
artery pressure and pulmonary vascular resistance in
one study when administered with another agent (a prostaglandin included to increase pulmonary blood flow),
deaths occurred in the treatment group. In another trial,
bosentan did improve exercise tolerance in patients with
severe PAH. This led to the approval by the Food and
Drug Administration of the use of low-dose bosentan in
PAH; however, liver function tests were abnormal in a
significant portion of the treatment group, and, thus, the
possible negative side effect of hepatotoxicity must be
weighed against the benefit obtained. Hepatotoxicity
was also a problem in trials employing the selective ETA antagonist sitaxsentan.
Again in human trials, this time in the setting of CHF,
bosentan treatment resulted in a significant incidence of
liver abnormalities. On the other hand, tezosentan, a
nonselective ET antagonist, showed some benefit in
acute heart failure patients in one trial but failed to provide protection in others. The selective ET-A antagonist
darusentan improved cardiac index in patients with New
York Heart Association stage III heart failure, but there
were more adverse events in the treatment group than in
placebo control subjects. Thus, in heart failure trials, ET
antagonists have not been proven to be safe alternatives
to conventional therapies. The same can be said of trials
Chapter 21 / Cardiovascular Hormones
331
Fig. 5. Posttranslational processing of the preproadrenomedullin protein results in the formation of two biologically active peptides,
AM and PAMP.
examining the efficacy of the ET antagonist in essential
hypertension. Only recent, preliminary trials in patients
with coronary artery disease are promising. Because ET
is a vasoconstrictor and stimulates smooth muscle proliferation, neutrophil adhesion, and platelet aggregation,
it was hypothesized that ET antagonists would be effective in the treatment of coronary artery disease. Indeed,
ET antagonists increased coronary artery diameter and
prevented vasoconstriction after percutaneous coronary
angiography. However, these trials need to be repeated
and extended with a careful examination of liver function in patients before any conclusions can be made.
4. ADRENOMEDULLIN GENE PRODUCTS
4.1. Gene Structure and Regulation
Utilizing a bioassay system that monitored accumulation of cAMP in platelets, Japanese investigators identified in 1993 a novel vasoactive hormone in extracts of
a human pheochromocytoma. The peptide (Fig. 5) is
produced in normal chromaffin cells of the adrenal
gland, as well as a variety of other tissues, including
brain, kidney, endothelial cells, and VSMCs. Posttranslational processing of the 185-amino-acid prohormone results in the production and secretion of
the mature 52-amino-acid form, designated AM, and a
20-amino-acid fragment from the N-terminus designated proadrenomedullin N-terminal 20 peptide
(PAMP), a peptide that shares some, but not all, of the
biologic activities of AM. Although activation of
adenylyl cyclase was used as a screening bioassay in
the initial phases of discovery, the hallmark bioassay
for AM is the potent hypotensive action when infused
intravenously (Fig. 6).
AM production in VSMCs and endothelial cells is
regulated at the transcriptional level by a variety of cytokines, including IL-1α, IL-1β, TNF-α, and TNF-β. Production of AM in these cells is also stimulated by
thrombin, aldosterone, cortisol, retinoic acid, and thyroid hormones. To a lesser degree, stimulation of production in VSMCs was reported also in response to Ang
II, epinephrine, platelet-derived growth factor, EGF, and
FGF. TGF-β and cAMP inhibit production.
332
Part IV / Hypothalamic–Pituitary
Fig. 6. Summary of biologic actions of AM. CRH = corticotropin-releasing hormone; ACTH = adrenocorticotropin; AVP = vasopressin; OT = oxytocin; CO = cardiac output; HR = heart rate; ANP = atrial natriuretic peptide; UV = urine volume; UNaV = urinary
sodium excretion; ET = endothelin.
4.2. Hormone Secretion
Many of the factors that stimulate hormone production also stimulate secretion in isolated cell systems,
leading to the hypothesis that AM may be responsible
for the hypotension of inflammation, septic shock, and
atherosclerosis. Circulating levels in humans are similar to those of other vasoactive hormones (about 3 fmol/
mL of plasma), suggesting that like other vasoactive
substances, the actions of AM may be predominantly
autocrine or paracrine in nature. One group failed to
observe elevations in plasma AM concentrations during
hypertensive attacks in patients with pheochromocytomas; however, cosecretion of AM and catecholamines
has been observed from cultured, bovine adrenal medullary cells. In fact, the cosecretion of PAMP and catecholamines from these cells is calcium dependent and
induced by carbachol activation of nicotinic receptors.
Those studies also pointed to an autocrine or paracrine
action of the peptide, since PAMP acted as an anticholinergic inhibiting sodium influx and reducing the
magnitude of catecholamine response to carbachol.
4.3. Sites of Action
Some of the pharmacologic actions of AM can be
blocked by the calcitonin gene–related peptide (CGRP)
receptor blocker CGRP8–37, which is not surprising
because AM and CGRP share considerable structural
homology. Both activate adenylyl cyclase in a variety of
tissues and can displace each other in binding assays.
Mesenteric vasodilatory responses to AM are blocked
by CGRP8–37 in vitro, but the in vivo vasodilatory
responses are not. Additionally, the increase in cAMP
levels observed in response to AM in endothelial cells in
culture is not blocked byCGRP8–37. Both AM and CGRP
bind the calcitonin receptor–like receptor (CRLR),
which is a unique G protein–coupled receptor. When
Chapter 21 / Cardiovascular Hormones
CRLR associates with an accessory protein (receptor
activity modifying protein-1 [RAMP-1]), it functions as
a selective CGRP receptor, one that can be blocked by
CGRP8–37. On the other hand, when CRLR associates
with the homologous RAMP-2 and RAMP-3, the complex is more selective for AM binding. This may explain
the apparent similarity in action of CGRP and AM in
some systems. Not all of the biologic activities of AM
can be explained by the presence of the CRLR/RAMP2 or RAMP-3 receptor complexes; thus, additional yetto-be described receptors may exist. In bovine aortic
endothelial cells, AM binding leads to activation of
adenylyl cyclase via a cholera toxin–sensitive G protein mechanism, with concomitant stimulation of PLC
activity. As a result, cyctosolic calcium levels increase
and NO is generated. In mesangial cells, AM inhibits
MAPK, an effect that may underlie the peptide’s
antimitogenic actions. In VSMCs, AM activates a proline-rich tyrosine kinase. Very little is understood about
the identity of the PAMP receptor. PAMP inhibits catecholamine release induced by periarterial nerve stimulation, possibly by a direct membrane action mediated
via a pertussis toxin–sensitive G protein. Pertussis toxin
also blocks the ability of PAMP to inhibit opening of
voltage-gated (N-type) calcium channels in pheochromocytoma cells (PC-12 cells), a cell line in which PAMP
also opens inwardly rectifying potassium channels. A
potassium channel also appears to mediate the ability of
PAMP to inhibit corticotropin-releasing hormone–
stimulated adrenocorticotropin release from cultured
anterior pituitary cells in vitro.
4.4. Biologic Actions
AM and PAMP exert profound effects on cardiovascular function and fluid and electrolyte homeostasis by actions in a variety of tissues (Fig. 6). The
hallmark action of AM is vasodilation by an action on
endothelial cells to generate NO and by direct activation of adenylyl cyclase in VSMCs. PAMP, on the other
hand, exerts its hypotensive action not by an action on
the endothelial cells or VSMCs, but, instead, by presynaptic inhibition of electrical activity in the sympathetic fibers that innervate the blood vessels. These
vascular effects appear to be physiologically relevant
because heterozygote AM gene knockout mice (missing one copy of the AM gene) display only 50% of the
normal circulating levels of AM and are hypertensive
compared with wild-type (normal two-gene copy)
mice. The vasodilatory effect of AM is even more pronounced in circumstances of high levels of Ang II,
such as in preconstricted vessels in vitro or human
hypertensives in vivo. This has led to the hypothesis
that the physiologic action of AM is to act as a coun-
333
terbalance to the renin-angiotensin-aldosterone system. Indeed, this may be the case because both AM and
PAMP inhibit Ang II–stimulated aldosterone secretion in vivo and block activation of the hypothalamicpituitary-adrenal axis at the level of the anterior
pituitary gland. AM is produced in cardiomyocytes and
acts locally to inhibit fibrosis stimulated by increased
Ang II and aldosterone levels. It acts to increase coronary blood flow, a function that is thought to have clinical relevance in recovery of the ischemic myocardium
following infarction. This is one area where a therapeutic action of AM may hold promise. Additionally,
AM exerts positive inotropic and chronotropic actions
in the heart, effects that may be beneficial in patients
in CHF. Finally, AM upregulates expression of the
vasodilatory peptide ANP in the heart, while downregulating ET expression in the vasculature.
The AM gene is expressed abundantly in kidney,
where the peptide exerts numerous effects. It exerts
direct natriuretic and diuretic actions (prostacyclin
mediated) in renal tubule and is an important determinant of renal perfusion pressure. Even when administered intravenously, AM maintains renal blood flow in
the face of profound decreases in mean arterial pressure. AM accomplishes this paradox by exerting a
vasodilatory effect (NO dependent) on the afferent
arteriole, thus maintaining glomerular filtration and
urine flow. Although in pharmacologic studies the
threshold dose in humans for the renal effects exceeded
that required to observe the cardiovascular actions, it is
unlikely that circulating AM explains the observed in
vitro or in vivo actions of AM. Locally produced peptide instead appears to act in an autocrine/paracrine
fashion to control sodium and water handling by the
tubule and perhaps even glomerular filtration. The
same pharmacologic administration of AM in patients
displaying already high levels of plasma AM (renal
failure) did improve renal function in one study, and
it appears that administration of exogenous AM can
improve survival in septic crisis (a hypotensive state in
which endogenous AM levels are already elevated).
The AM gene is highly expressed in the CNS, and
significant effects of the peptide in brain have been
demonstrated, many related to the regulation of fluid
and electrolyte homeostasis. The natriuretic and diuretic
actions of AM in kidney appear to be mirrored by CNS
effects to inhibit thirst and sodium appetite. Both of
these CNS actions have been demonstrated to be physiologically relevant. AM also acts in brain to stimulate
sympathetic tone and to stimulate the release of vasopressin and oxytocin. Although the actions to elevate
peripheral blood pressure and circulating levels of AVP
may seem counter to the peptide’s renal and vascular
334
Part IV / Hypothalamic–Pituitary
effects (natriurestis, diuresis, and vasodilation), they
may reflect brain actions that are cardioprotective in
nature, just as those exerted by AM in the heart may
have evolved to protect against cardiovascular collapse.
Other actions of AM include a stimulatory effect on
progesterone secretion and a quiescent effect in uterus.
The fact that the knockout of the AM gene resulted in
embryonic death at approx d 14 of mouse gestation
points to significant effects of the peptide during
embryogeneis as well. Indeed, the cause of embryo
demise appeared to be a constriction of the umbilical
arteries and veins. The knockout also provided insight
into the physiologic relevance of another pharmacologic
effect of AM. Heterozygote (one gene copy) animals
develop a late-onset diabetic phenotype, suggesting that
the ability of AM to inhibit insulin secretion has physiologic relevance.
4.5. Pathophysiology of AM
The multiple pharmacologic effects of AM have predicted that the peptide or its analogs would be beneficial in a variety of disease states. The ability of AM to
inhibit Ang II–mediated cardiomyocyte hypertrophy
and fibroblast proliferation predicted a role for the peptide in protection against hypertension-induced ventricular hypertrophy and interstitial fibrosis of the heart.
In addition, AM exerts antimigratory and antiproliferative effects in VSMCs, promising a possible role in
the prevention of atherosclerosis and angiogenesis.
This is supported by observations in heterozygote
knockout animals in which perivascular fibrosis and
intimal hyperplasia were exaggerated compared with
wild-type controls following salt loading or chronic
administration of Ang II. The protective effect of AM
is also supported by results from transgenic mice in
which overexpression of AM has been engineered.
Much less interstitial fibrosis and a milder form of
hypertrophy were observed in these animals compared
with controls in several models of hypertension. In
experimental animals, overexpression of AM reduces
the magnitude of arterial thickening and promotes
reendothelialization following balloon angioplasty. In
one study, administration of AM immediately following myocardial infarction enhanced ejection fraction
and improved coronary sinus blood flow. Thus, administration of AM may provide both acute and chronic
benefit in this patient population.
Plasma AM levels are elevated in CHF. Because of
the peptide’s vasodilatory and renotropic effects, it was
thought that exogenous administration might provide
some benefit in these patients. Initial results were disappointing. Increases in forearm blood flow to iv administration of AM were attenuated in CHF patients
compared with control subjects, as were the blood pressure–lowering effects. Later it was determined that the
failure of AM in these models was owing to an impairment in NO generation in these patients. However, more
promising were the observations that administration of
AM decreased pulmonary capillary wedge pressure as
well as pulmonary arterial pressure in patients with CHF.
Furthermore, AM infusion increased heart rate, stroke
volume, ejection fraction, and cardiac index in patients
with CHF and in another study increased urine volume
and sodium excretion. These were short-term infusion
protocols; longer-term clinical studies are needed. However, chronic infusion studies in experimental animals
have demonstrated the ability of AM to reduce renin
levels in models of renovascular hypertension and to
decrease renal injury in hypertensive animals. Similarly,
animal models have demonstrated that administration of AM improves blood pressure in spontaneously
hypertensive (SHR) rats, exerting a more profound
effect in those animals than in their normotensive
(Wistar-Kyoto) control counterparts. This should not
be surprising because the vasodilatory effect of AM is
more pronounced in preconstricted vessels. In one clinical trial in patients with essential hypertension, administration of AM reduced both systolic and diastolic
blood pressure and also reduced total peripheral resistance.
Therapeutically, AM is already being employed for
the treatment of septic crisis. It had been observed that
plasma AM levels correlate positively with the severity of sepsis, which initially suggested that AM was the
causative agent for the observed hypotension, since
proinflammatory cytokines stimulate AM production
and release. However, it was subsequently reported that
there was also a direct, positive correlation between
absolute levels of plasma AM and survival, in that
patients with highest AM levels in circulation had a
greater chance of surviving the septic event. This was
observed to be owing to a preservation of renal function in the high AM group, mirroring the ability of
exogenous AM to maintain renal perfusion even in the
face of profound hypotension.
5. CARDIOVASCULAR HORMONES AS
DIAGNOSTIC AND THERAPEUTIC TOOLS
The three families of cardiovascular hormones discussed in this chapter have proven to be potent regulators of cardiovascular and renal function. Acting as
either true endocrine or autocrine/paracrine hormones,
they exert a wide variety of physiologically and pathologically relevant actions. Although the effects of the
ETs are predominantly pathologic in nature, this still
provides promise for the use of antagonists to block
Chapter 21 / Cardiovascular Hormones
those deleterious actions in a variety of disease states
characterized by overproduction or secretion of the peptides. Results from the initial trials with these antagonists are discussed above. More promising are the
beneficial effects of long-term administration of the
natriuretic peptides or AM because their actions in general appear organ specific and protective. In addition,
pathologic secretion of these two classes of peptides
may reflect the recruitment of compensatory mechanisms within the body that can hallmark the onset of
disease and therefore provide diagnostic advantage. Just
as important are the basic biomedical lessons learned
from the discovery and characterization of the actions of
these peptide hormones. Emerging now is an integrated
view of how these hormones can coordinate endocrine,
cardiovascular, and renal mechanisms that protect
against postischemia proliferative disease and tissue
damage caused by volume over- or underload. The roles
played by these peptides in inflammatory disease are
now being recognized and their importance in normal
glucose metabolism and bone health is better understood.
In summary, the roles played by these potent cardiovascular hormones in the maintenance of cardiovascular function and fluid and electrolyte homeostasis
have taught investigators a great deal about integra-
335
tive, systems biology. These peptides promise to open
new avenues into cellular and molecular control
mechanisms underlying other normal and pathologic
systems as well.
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