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Department of Molecular and Cellular Biology (J.P.H.) and Clinical Sciences Research Centre (S.K.), St. Bartholomew’s and the Royal London School of Medicine and Dentistry, Queen Mary and Westfield College, University of London, London E1 4NS; and Department of Metabolic Medicine (D.M.S.), Division of Investigative Sciences, Imperial College School of Medicine, Hammersmith Hospital, London W12 0NN United Kingdom
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Abstract |
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 Top Abstract I. IntroductionII. Synthesis and Secretion...III. Receptors and Signal...IV. Biological Actions of...V. Unresolved Issues and...References |
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I. Introduction |
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TopAbstractI. IntroductionII. Synthesis and Secretion...III. Receptors and Signal...IV. Biological Actions of...V. Unresolved Issues and...References |
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The growth of interest in adrenomedullin has been exponential, with more than 600 papers published in this field to date including a number of reviews (5 6 7 8 9 10 ). This review aims to summarize the present state of our knowledge of adrenomedullin biology, and to focus on issues that are currently unresolved, with an indication of likely areas for future research.
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II. Synthesis and Secretion of Adrenomedullin |
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TopAbstractI. IntroductionII. Synthesis and Secretion...III. Receptors and Signal...IV. Biological Actions of...V. Unresolved Issues and...References |
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The gene encoding preproadrenomedullin is termed the adrenomedullin
gene and has been mapped and localized to a single locus of chromosome
11 (16 ). The human adrenomedullin gene comprises 4 exons and 3 introns,
with TATA, CAAT and GC boxes in the 5'-flanking region (16 ) (Fig. 2). There are several binding sites for
activator protein-2 (AP-2) and a cAMP-regulated enhancer element (16 ).
It has also been found that there are nuclear factor-
B (NF-B)
sites on the promoter of the adrenomedullin gene (16 ). The organization
and chromosomal localization of the murine adrenomedullin gene have
also been elucidated (17 ).
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). In addition, many tumor cell
lines express the adrenomedullin gene or have been shown to synthesize
the immunoreactive peptide (58 ). Although adrenomedullin is often
described as a ubiquitous regulatory peptide, it is worth noting that
certain cell types and cell lines do not appear to express the
adrenomedullin gene (Table 2). It is
widely recognized that adrenomedullin is a product of vascular
endothelial cells, but it appears that not all vascular endothelial
cells synthesize the peptide. In some tissues, although adrenomedullin
immunostaining has been demonstrated in certain cells, the vascular
endothelial cells and smooth muscle cells remain unstained. This is
particularly noticeable in the rat adrenal gland (25 29 ).
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B. Circulating adrenomedullin: adrenomedullin assays
After the initial report of picomolar levels of adrenomedullin
circulating in plasma (60 ), several research groups have developed
in-house assays to measure plasma adrenomedullin levels. In general
these assays appear to have been carefully validated, with evidence
presented from HPLC analysis to show that immunoreactive adrenomedullin
from human plasma coelutes with authentic human
adrenomedullin1–52 (60 61 62 ). There is a remarkable
consistency between these different methods in terms of the absolute
concentrations of adrenomedullin reported in the circulation of healthy
controls (see Table 3). We can therefore
conclude with some certainty that the normal plasma concentration of
adrenomedullin is in the range of 1 to 10 pM, with most
values between 2 and 3.5 pM. There do not appear to be
significant differences between males and females or between different
age groups, although to date these questions have not been directly
addressed. It has been suggested that the Peninsula assay
(Peninsula Laboratories, Inc., Belmont, CA) may
overestimate the levels of adrenomedullin (63 ), and although a
comparison of the values obtained with the Peninsula assay in different
laboratories shows that these tend to be higher than values obtained
with other assays (see Table 3), these values are still within the
range of 1 to 10 pM. The sole exception is the value
reported by Hata and co-workers (77 ), which is clearly outside the
normal range, although the reason for this is unclear.
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Adrenomedullin has also been measured in rat plasma, using an antiserum that recognizes both rat and human forms of adrenomedullin. Adrenomedullin levels in rat plasma were comparable with those measured in man, at 3.6 ± 0.34 pM (81 ).
C. Circulating adrenomedullin in disease
Adrenomedullin has been measured in a wide range of disease states
(see Table 4). In many cardiovascular
disorders, plasma adrenomedullin is reported to be elevated, possibly
suggesting that increased adrenomedullin is part of the homeostasis of
blood pressure, released to compensate for elevated blood pressure. The
finding that adrenomedullin is lower in preeclampsia compared with
uncomplicated pregnancies may suggest that adrenomedullin is involved
in the pathogenesis of this disorder (77 ). These findings have been
questioned, however, on the basis of the exceptionally high
adrenomedullin levels measured in the healthy controls in this
study (77 ) (see Table 3
). A more recent study has reported no
difference in plasma adrenomedullin between preeclamptic and
normotensive pregnant women, although adrenomedullin concentrations in
amniotic fluid were found to be higher in preeclampsia (89 ).
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Of all the conditions investigated, the greatest increment in plasma adrenomedullin has been observed in septic shock (75 106 ). It appears that adrenomedullin has a key role in the pathophysiology of septic shock. This is the only pathological condition in which plasma levels of adrenomedullin approach the levels required for receptor activation (see Section III below). The plasma levels of adrenomedullin observed in patients with sepsis are likely to be directly responsible for the hypotension characteristic of septic shock, as a correlation has been demonstrated between plasma adrenomedullin concentrations and relaxation of vascular tone in this condition (108 ). One corollary of this is that the actions of adrenomedullin under normal conditions must therefore be autocrine or paracrine in nature.
D. Origins of circulating adrenomedullin
Adrenomedullin is synthesized in most tissues of the body (see
Table 1). Although the gene encoding adrenomedullin is very highly
expressed in the adrenal gland, in both zona glomerulosa and the
adrenal medulla (25 109 ), there is considerable evidence against the
adrenal as the major source of circulating peptide. Adrenal venous
sampling reveals levels of adrenomedullin that are not significantly
different from arterial plasma, in contrast to epinephrine and
norepinephrine, which show marked trans-adrenal gradients (110 ).
Insulin-induced hypoglycemia has been used in an attempt to provoke the
release of medullary adrenomedullin, and while plasma epinephrine
levels were increased 20-fold, no significant change in circulating
adrenomedullin was observed (75 ). Furthermore, in a patient with a
pheochromocytoma, no change in plasma adrenomedullin concentration was
seen during a hypotensive attack, although both epinephrine and
norepinephrine concentrations increased significantly (111 ). As it has
been shown that adrenomedullin is cosecreted with catecholamines, at
least by bovine chromaffin cells in culture (112 ), these data suggest
that the adrenal medulla is unlikely to be a significant source of
circulating adrenomedullin.
Selective arterial and venous sampling across various vascular beds (including heart, lungs, kidney, and adrenal) in patients with a variety of cardiovascular pathologies, failed to identify a site of significant adrenomedullin production (111 ). In congestive heart failure, however, significant cardiac secretion of adrenomedullin has been reported (88 ). It has been suggested that the increased plasma adrenomedullin seen in pregnancy (see below) may derive from the placenta, but in the study that measured both venous and arterial umbilical cord plasma, no difference in adrenomedullin concentrations was found, suggesting that no net production or clearance of adrenomedullin occurs in the placenta (113 ). In certain disease states, notably cerebrovascular disease, the reported increase in plasma adrenomedullin concentration is thought to reflect the degree of endothelial cell damage (86 ).
In measuring plasma adrenomedullin concentrations in disease states, it is unclear whether the generally elevated levels reflect increased production or decreased clearance. This question has not been directly addressed. However, in septic shock there is evidence for increased adrenomedullin production by several different cell types (19 34 35 36 57 59 ). In congestive heart failure there is also evidence for increased cardiac production of adrenomedullin (67 ).
E. Metabolic clearance of adrenomedullin
The plasma half-life of adrenomedullin has been reported to be
22.0 ± 1.6 min with a MCR of 27.4 ± 3.6 ml/kg·min and
with an apparent volume of distribution of 880 ± 150 ml/kg (74 ).
The effects of plasma membrane enzymes on adrenomedullin have been
investigated. It appears likely that adrenomedullin is degraded
initially by metalloproteases to yield adrenomedullins 8–52, 26–52,
and 33–52, followed by an aminopeptidase action to yield
adrenomedullins 2–52, 27–52, and 28–52 (114 ). It has been suggested
that the lung may be a major site of adrenomedullin clearance in man
(111 ).
F. Adrenomedullin in other biological fluids
In addition to peripheral plasma, significant levels of
adrenomedullin have been measured in urine (66 115 ), milk (48 ),
cerebrospinal fluid (CSF) (116 117 ) saliva (S. Kapas, unpublished
data), amniotic fluid (89 ), sweat (50 ), and in umbilical vein blood
(89 ). Using healthy subjects, urinary adrenomedullin concentrations
have been reported to be approximately 6-fold higher than plasma levels
(66 ). The authors suggest that the lack of correlation between urinary
and plasma levels argues against the kidney as a major site of
adrenomedullin excretion (66 ). However, urinary adrenomedullin is
reported to be decreased in various renal disorders, such as IgA
nephropathy (99 ), with an increase in plasma peptide levels, possibly
suggesting impaired excretion. In healthy subjects, however, the high
urinary adrenomedullin concentrations relative to plasma may suggest
that the kidney itself is the major source of urinary adrenomedullin
(66 ). The concentration of adrenomedullin in CSF is lower than that in
plasma, and while plasma adrenomedullin increases in pregnancy, no
change in CSF concentration is seen, suggesting independent regulation
of adrenomedullin in the two compartments (116 ).
Although the presence of adrenomedullin in murine milk has been
demonstrated (48 ), no data are presently available on the
concentration, so it is unclear whether it is actively secreted into
milk. In urine (66 ) and sweat (50 ) the concentration of adrenomedullin
appears to be significantly higher than in plasma, and there is
evidence for production of adrenomedullin in both skin and kidney (see
Table 1).
G. Regulation of adrenomedullin gene expression and peptide
synthesis in vivo
The effects of various physiological manipulations on plasma
adrenomedullin concentrations have been investigated in both man and
other species. Exercise has been reported by some studies (83 ), but not
others (118 119 ), to increase plasma adrenomedullin in man, with a
correlation between plasma adrenomedullin and blood pressure (83 ).
Moving from low to high altitude was also associated with an increase
in plasma adrenomedullin probably related to the degree of hypoxia
experienced by the subjects (69 ). In dogs, as in man, hemorrhagic shock
causes an increase in plasma adrenomedullin (120 ), and endotoxic shock
increases adrenomedullin gene expression in blood vessels (12 ). In rat
and mouse a consistent finding is that experimentally induced sepsis
increases adrenomedullin gene expression and plasma concentration
(121 122 123 124 125 ). In rats, a period of fasting causes an increase in
adrenomedullin concentration in the gastrointestinal tract (126 ).
Pregnancy is associated with increased circulating adrenomedullin
concentrations in both rats (127 ) and women (77 89 116 ). The plasma
concentration of adrenomedullin has been reported to increase
progressively from the first to third trimester, with a further
increase postpartum, although these data have been questioned due to
the excessively high concentrations of adrenomedullin found in this
study (see Table 3) (63 128 ). A more recent study reported that, while
plasma adrenomedullin was increased on average 5-fold in pregnant women
compared with nonpregnant women, there was no correlation with
gestational age, and within 48 h post partum plasma adrenomedullin
concentrations had significantly decreased (89 ). An interesting
observation is that babies delivered by the vaginal route had
significantly higher umbilical cord adrenomedullin concentrations than
babies delivered by elective cesarean section (129 ). In rats it is
possible to mimic the effects of pregnancy on plasma adrenomedullin
concentrations by the administration of a progesterone derivative
(127 ), suggesting that the increased adrenomedullin has a role in the
cardiovascular changes of pregnancy. It has been shown that
adrenomedullin mRNA in the rat uterus is significantly increased in
pregnancy (130 ), suggesting that the uterus itself may be the source of
plasma adrenomedullin.
The effects of various endocrine manipulations have also been investigated. Hyperthyroid rats were found to have increased plasma adrenomedullin concentrations and also an increased adrenomedullin mRNA level in the lung (131 ). Glucocorticoids are also implicated in the regulation of adrenomedullin: patients with Addison’s disease (primary adrenal insufficiency) had their plasma adrenomedullin levels reduced by glucocorticoid replacement (68 ). However, insulin-induced hypoglycemia, a potent stimulus to glucocorticoid secretion, had no effect on plasma adrenomedullin concentrations (75 ). In rats with septic shock, dexamethasone did not alter plasma adrenomedullin levels, but in control adrenalectomized animals dexamethasone significantly increased both lung mRNA levels and plasma adrenomedullin (122 ). In the rat ventral prostate adrenomedullin expression is highly androgen dependent, with a 25-fold reduction in mRNA after castration, which is fully reversible by androgen administration (132 ).
Adrenomedullin is implicated in the regulation of fluid and electrolyte status (5 ), and it has been shown that adrenomedullin concentrations are reduced by hemodialysis in patients with renal disease (71 ). Altering the renin-angiotensin system by the use of captopril or furosemide was found to have no effect on plasma adrenomedullin in normal subjects (133 ), and an infusion of ACTH was also without effect (133 ). Similarly it has been found that feeding rats a diet either high (4%) or low (0.02%) in salt has no effect on renal adrenomedullin gene expression (40 ). In the Dahl salt-sensitive rat strain, however, those on a high-salt diet had increased plasma and ventricular adrenomedullin by comparison with those on a control diet (134 ). In human subjects, changes in salt intake, either acute or chronic, had no effect on plasma adrenomedullin in either normotensive or hypertensive subjects (135 ).
It has been demonstrated that an infusion of atrial natriuretic peptide increases plasma adrenomedullin levels in healthy control subjects (73 ). In this study blood was taken for adrenomedullin measurement at 30-min intervals for 5 h. During this time there was apparently no change in plasma peptide levels in the control subjects, while the test subjects showed an elevated plasma adrenomedullin concentration for only the 60-min duration of the infusion. A steady-state 4-fold increase was achieved within 20 min of the onset of the infusion, and levels returned to basal within 30 min of the cessation of the infusion (73 ). Two models of pressure overload have also been used to investigate the regulation of adrenomedullin in the rat: hormonally induced overload, using either arginine vasopressin (AVP) or angiotensin II, resulted in an increase in cardiac adrenomedullin mRNA and peptide (136 ). No effect on adrenomedullin expression was seen in the surgical model, however, despite a marked increase in atrial natriuretic peptide (137 ).
From the data outlined above it is difficult to describe the exact mechanisms that regulate adrenomedullin synthesis and secretion in vivo. The question is clouded by the fact that these data were obtained from several different species and using different techniques. However, the major consistent findings are of increased adrenomedullin in two conditions: sepsis and pregnancy. It also appears likely that adrenomedullin is not, in general, subject to regulation by electrolyte balance, although in some conditions of altered blood pressure, adrenomedullin levels appear to change in a manner consistent with the possible role of this peptide in a compensatory mechanism. The data concerning hormonal regulation of adrenomedullin in vivo are, at present, conflicting. It also appears likely that specific regulatory mechanisms may exist in different tissues for the local control of adrenomedullin production.
H. Experimental regulation of adrenomedullin gene expression and
peptide synthesis in vitro
The in vitro regulation of adrenomedullin gene
transcription and peptide synthesis has been studied in a number of
comprehensive papers by Kangawa and co-workers (19 20 22 ),
using either rat vascular smooth muscle cells (VSMCs), or rat
endothelial cells. Adrenomedullin production by vascular smooth muscle
cells is increased by a range of cytokines, growth factors, and
hormones, including tumor necrosis factor 
and ß, interleukin-1
and ß, (19 20 ), dexamethasone, cortisol, aldosterone, retinoic acid,
and thyroid hormone (138 139 ). Other hormones and growth factors were
found to have little effect, including fibroblast growth factor,
epidermal growth factor, platelet-derived growth factor, progesterone,
estradiol, and testosterone (19 138 139 ). Other studies on VSMCs have
shown that oxidative stress, induced by diethyldithiocarbamate, also
increases adrenomedullin production (140 ). The regulation of fibroblast
adrenomedullin gene expression is essentially the same as that
of VSMCs (57 ), but there are some differences between vascular
endothelial cells and VSMCs, notably in their response to thrombin and
-interferon (59 ). -Interferon also increases adrenomedullin
expression by rat astrocytes (32 ), while interleukin-1ß, tumor
necrosis factor-, and dexamethasone stimulate cardiac myocytes to
produce adrenomedullin (24 141 ). An interesting observation on human
aortic endothelial cells is the finding that shear stress
down-regulates adrenomedullin gene expression (142 ).
In mouse and human macrophages, lipopolysaccharide, -interferon,
tumor necrosis factor , retinoic acid, and the phorbol ester phorbol
12-myristate 13-acetate increase adrenomedullin gene
transcription and secretion (34 35 36 ). Synergistic effects were found
when retinoic acid was added in combination with other effectors (36 ).
It has also been shown that the antiestrogen, tamoxifen, induces
adrenomedullin synthesis in endometrial macrophages (47 ).
Lipopolysaccharide is a potent stimulus to adrenomedullin secretion by macrophages (34 35 36 ), VSMCs (19 ), fibroblasts (57 ), and endothelial cells (59 ). It is clear that the induction of adrenomedullin transcription and synthesis by lipopolysaccharide and cytokines gives this peptide a significant role in sepsis and inflammatory states. However, more tissue-specific regulatory mechanisms also exist. In rat granulosa cells, for example, adrenomedullin gene expression is decreased by FSH treatment (44 ), and there is evidence that adrenomedullin is differentially regulated in renal mesangial and glomerular epithelial cells (37 ). In general, it appears that cAMP-mediated effects decrease adrenomedullin, while activation of the phospholipase C-protein kinase C pathway stimulates adrenomedullin (37 143 ). Studies on transcriptional regulation of both human and rat adrenomedullin gene suggest that the effects of the cytokines are mediated by the NF-IL-6 regulatory element in the promoter region of the adrenomedullin gene (143 144 ).
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III. Receptors and Signal Transduction |
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TopAbstractI. IntroductionII. Synthesis and Secretion...III. Receptors and Signal...IV. Biological Actions of...V. Unresolved Issues and...References |
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A. Do CGRP receptors mediate the effects of adrenomedullin?
Adrenomedullin receptors have always been closely associated with
receptors for the related peptide, CGRP (145 ). CGRP receptors have been
classified into two subtypes on the basis of the potency
(pA2) of the CGRP receptor antagonist fragment,
CGRP8–37 (146 147 ). Some
CGRP1 receptors, at least in the rat, are
antagonized by the fragment with a pA2 of about
8.0 while CGRP2 receptors require higher
concentrations (pA2 
6.0) (148 149 150 ).
CGRP2 receptors have also been characterized by
the ability of the CysACM analog (-[acetimidomethyl-Cys2,
7]hCGRP) to act as an agonist at these receptors but
not at CGRP1 receptors (151 ), but this has
recently been disputed (152 ).
Initial pharmacology seemed to indicate that the vascular effects of adrenomedullin were directly mediated by a well characterized CGRP1 receptor mechanism (153 154 ). Nuki et al. (155 ) showed that the vasodilator effects of adrenomedullin and CGRP on the rat mesenteric vascular bed (a prototypic CGRP1 preparation) could be blocked by CGRP8–37. Similar effects of CGRP8–37 on CGRP- or adrenomedullin-induced vasodilation were shown in the isolated rat heart preparation (156 ) and in the rat and hamster microvasculature (157 ). Since publication of these studies, a large body of evidence suggests that some adrenomedullin effects can be blocked by CGRP8–37 (130 157 158 159 160 161 162 163 164 ), but here we will consider only the receptors. Adrenomedullin certainly can bind with high affinity to and activate CGRP receptors in SK-N-MC neuroblastoma cells, commonly used as a model of CGRP1 receptors (156 162 ). In the study of Zimmermann et al. (162 ) adrenomedullin was only 7 times weaker in affinity than CGRP (IC50= 0.3 vs. 2 nM). Adrenomedullin has also been shown to compete with 125I-CGRP binding in rat lung and heart membranes (165 ), rat brain (166 ), SK-N-MC (156 162 167 ), L6 myoblasts (163 ), rat spinal cord (168 ), rat aorta (169 ), rat uterus (130 ), guinea-pig vas deferens (170 ), and rat hypothalamus (158 ). These data in general support the idea of high-affinity (low nanomolar) binding of adrenomedullin to all CGRP receptors with an affinity of about one-tenth to one-hundredth that of CGRP itself.
Interestingly, adrenomedullin has a low affinity (IC50=129 nM) for 125I-CGRP binding sites in guinea-pig vas deferens (170 ), a model of CGRP2 receptors, indicating perhaps that adrenomedullin has a lower affinity at these receptors than at CGRP1 receptors. Some caution should be used when interpreting the inhibitory effect of CGRP8–37 on adrenomedullin actions as evidence of CGRP receptor involvement. In some studies very high concentrations of the antagonist are used, which may bind to specific adrenomedullin receptors (e.g., Ref. 56 ; IC50 for CGRP8–37 binding was 214 nM at adrenomedullin receptors in Rat-2 cells, where 10 µM CGRP inhibited cAMP elevation via specific adrenomedullin receptors) and cloud the interpretation. Binding studies showing an affinity of CGRP8–37 for 125I-CGRP sites similar to the concentrations used to inhibit adrenomedullin effects (e.g., Ref. 163 ) are very useful although not always possible in animal experiments. In vivo experiments that show a lack of effect of CGRP8–37 on adrenomedullin effects but antagonism of CGRP effects at the same concentration are convincing evidence for specific adrenomedullin effects (169 171 172 173 174 175 ).
B. Are there specific adrenomedullin receptors?
Thus, CGRP receptors mediate at least some of the effects of
adrenomedullin. However, later experiments using
125I-adrenomedullin showed that specific
adrenomedullin receptors existed. Eguchi et al. (176 )
demonstrated binding of 125I-rat adrenomedullin
to rat VSMCs and that this binding could be competed by rat
adrenomedullin [dissociation constant (KD)
= 13 nM] and CGRP [
inhibition constant (Ki ) = 300
nM (176 )]. This 23 times
greater affinity for adrenomedullin over CGRP would not be expected for
a CGRP receptor. Surprisingly, the rat adrenomedullin-mediated
stimulation of cAMP levels seen in these VSMCs was inhibited by
CGRP8–37, albeit at high concentrations
[IC50 = 300 nM (176 )]. In
another study on rat VSMCs, human adrenomedullin increased
intracellular cAMP with an EC50 of 20
nM compared with 8.5 nM for
CGRP with CGRP8–37 blocking the action of
adrenomedullin (IC50=93 nM)
(177 ). However, 125I-human adrenomedullin binding
in these cells (IC50=73 nM)
was not inhibited by either CGRP or CGRP8–37 at
concentrations up to 10 µM (177 ).
C. The pharmacology of specific adrenomedullin receptors
The data of Ishizaka et al. (177 ) and Eguchi et
al. (176 ) indicate the presence of receptors with a higher
affinity for adrenomedullin than CGRP, distinguishing these from any
known CGRP receptor (148 150 ). Examination of specific
125I-adrenomedullin binding sites in rat tissues
(165 ) showed high levels of specific binding in heart, lung, spleen,
liver, skeletal muscle (soleus, diaphragm, and gastrocnemius), and
spinal cord. CGRP receptors are also abundant in highly vascular
tissues such as lung, heart, and spleen (145 150 ). Apart from spinal
cord, binding in the central nervous system (CNS) was low in contrast
to CGRP binding, which is widespread and abundant in the brain (7 145 ). Binding in adrenal and kidney membranes was low but as binding
was measured in membrane preparations from whole tissues/glands, this
in no way negates the large bodies of evidence for important roles for
adrenomedullin in these tissues acting via highly localized receptors
(5 25 178 ).
Binding sites in heart and lung were further characterized. These sites
showed saturation dissociation and competition as would be expected of
receptor binding sites. Rat and human adrenomedullin competed at both
sites with rat adrenomedullin showing the greater affinity (Table 5). Competition by CGRP, amylin, and
calcitonin was approximately 3 orders of magnitude less than rat
adrenomedullin, indicating a high level of specificity. Other reports
of 125I-adrenomedullin binding also show a
low affinity of CGRP at this site [VSMCs (176 177 ), NG108–15
neuroblastoma-glioma cells (179 ), Swiss 3T3 mouse fibroblasts (57 180 ), rat hypothalamus (158 ), rat spinal cord (168 ), rat blood vessels
(169 ), L6 myoblasts (163 ), rat uterus (130 ), human brain (181 ), bovine
endothelial cells (182 ), mouse astrocytes (183 ), human oral
keratinocytes (184 ), rat-2 fibroblasts (56 ), rabbit kidney glomeruli
(174 ), guinea-pig vas deferens (170 ), rat adrenal zona glomerulosa
cells (25 ), and human skin cells (50 )]. These results are summarized
in Table 5. It is clear from the table that high-affinity (mean
affinity = 6 nM)
125I-adrenomedullin binding sites can be detected
in tissues and cells from a number of species with differing
methodologies. These sites all show low affinity for CGRP and, where
measured, amylin and calcitonin, and therefore appear highly specific.
The human adrenomedullin fragment,
adrenomedullin22–52, has been used as a specific
adrenomedullin receptor antagonist (164 185 188 189 190 191 192 193 ) in a similar
way that CGRP8–37 is used for
CGRP1 receptors. In rabbit aortic endothelial
cells [Ki for adrenomedullin-stimulated cAMP was
2.6 nM with no effect on CGRP-stimulated cAMP (188 )] and
rat cerebral blood vessels [5 µg/kg/min infusion inhibited
adrenomedullin-mediated vasodilation (193 )]
adrenomedullin22–52 was an effective antagonist.
Some specificity was demonstrated by its lack of effect in T47 D cells
[calcitonin receptor, 1000 nM (190 )] or L6 myocytes
[CGRP receptor, 1000 nM (192 )]. However, in rat mesangial
cells (IC50 for inhibition of
adrenomedullin-stimulated cAMP: 70 nM compared with 50
nM for CGRP8–37) and human
neuroblastoma TGW cells [DNA synthesis stimulated by adrenomedullin
(190 )], there was no difference in potency between the effects of
adrenomedullin22–52 and
CGRP8–37. In rat VSMCs [half-maximal antagonism
of adrenomedullin-stimulated cAMP was 4000 nM and
Ki for binding 1600 nM (185 )],
adrenomedullin22–52 was a very weak antagonist.
Worse still, in rat cardiac cells [adrenomedullin-stimulated cAMP
(189 )] and the hindlimb vascular bed of the cat [vasodilation —
effect of 30 nmol adrenomedullin22–52 (194 )],
adrenomedullin22–52 was inactive against
adrenomedullin but inhibited CGRP effects. Thus, better antagonists
need to be developed. It has been suggested that human
adrenomedullin26–52 is a more specific
antagonist (195 ). The use of either rat or human adrenomedullin as
radioligand appears not to affect the results and the two labels
cross-react across species (170 174 177 179 182 185 186 ). We have
found that human adrenomedullin is associated with a much higher
nonspecific binding than rat adrenomedullin and therefore prefer this
radioligand (nonspecific binding was 8% for rat adrenomedullin and
23% for human adrenomedullin in rat lung (A. A. Owji and D.
M. Smith, unpublished observation). One obvious conclusion from this
binding data is that circulating levels of adrenomedullin,
approximately 3–6 pM in man (60 62 ) and 3 pM
in rat (81 ), cannot mediate the physiological effects of adrenomedullin
by these specific receptors or by CGRP receptors, placing it firmly as
a paracrine/autocrine factor.
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One aspect of this binding data that remains puzzling is the lack of competition of 125I-adrenomedullin binding by CGRP in tissues and cells that express both adrenomedullin and CGRP binding, since adrenomedullin will bind effectively to 125I-CGRP sites. One explanation is that if the affinity of adrenomedullin for CGRP receptors is at least 10-fold less than for adrenomedullin receptors, then binding of the low concentration of 125I-adrenomedullin radioligand to CGRP receptors may not be apparent in competition studies. Another possible explanation is that the 125I-adrenomedullin is specific for adrenomedullin sites, whereas adrenomedullin binds to CGRP sites as well. This was supported by the lack of competition of nonradioactive iodoadrenomedullin with 125I-CGRP (165 ). This requires further investigation, perhaps involving the development of new adrenomedullin probes such as fluorosceinated/biotinylated adrenomedullin. 3H-adrenomedullin would be effective in tissues where receptor numbers are high, such as lung, but would be limited by its low specific activity. One possible improvement on this would be the use of higher specific activity metabolically labeled 14C-adrenomedullin.
D. Receptor biochemistry: chemical cross-linking of adrenomedullin
and CGRP receptors
Chemical cross-linking experiments using
125I-rat adrenomedullin showed relative molecular
weights (Mr) for adrenomedullin binding
site-ligand complexes (in this section minor bands are shown in
italics separated from the major band by a slash
mark) in rat VSMCs of 120,000 and 70,000 (176 ) and in rat tissues
of 83,000/105,000 and 94,000 (165 ). In neither case were the
labeled bands competed by CGRP. In further experiments specific
adrenomedullin binding site-ligand complexes were demonstrated with
Mrs, of 83,000 in rat 2 fibroblasts (56 ), of
84,000/122,000 in rat spinal cord (199 ) of 76,000 in L6
myoblasts (163 ), and of 89,000/105,000 in rat aorta (169 ).
These complexes can be compared with cross-linked
125I-CGRP sites that vary in
Mr: 70,000/110,000 in rat skeletal
muscle, 55,000/44,000 in rat liver (200 ), 70,000 and 120,000
in porcine ventricle (201 ), 75,000–90,000 in rat spleen (202 ), and
60,000–70,000 in rat VSMCs (203 ). Thus, on average, CGRP binding sites
have an Mr of about 70,000 with adrenomedullin
binding site about 85,000. This assertion should be treated with some
caution as cross-linked bands are often broad, making comparison across
studies difficult [e.g., in rat liver Stangl et
al. (202 ) found Mr = 74,000 and 68,000
compared with Mr = 55,000 by Chantry et
al. (200 )]. Also the same receptor protein may be subject to
large variations in size due to differential glycosylation in different
tissues and species.
In rat spinal cord and L6 myoblasts, a direct comparison of the cross-linked adrenomedullin and CGRP bands was made (163 199 ). In spinal cord 125I-CGRP complexes showed an Mr= 74,000 and 61,000 compared with Mr = 84,000/122,000 for 125I-adrenomedullin complexes. Deglycosylation of 125I-adrenomedullin complexes in spinal cord, heart, and lung resulted in a number of complexes with the lowest Mr being 52,000, 47,000, and 43,000, respectively (199 ). In L6 myoblasts 125I-CGRP complexes showed an Mr = 82,000 compared with Mr = 76,000 for 125I-adrenomedullin complexes. Thus, on the whole, although there is some evidence for CGRP receptors showing a lower Mr on SDS-PAGE than adrenomedullin receptors, this remains an outstanding question. Also a number of cross-linking studies of both adrenomedullin and CGRP binding sites (165 169 176 199 200 ) show second bands of higher mol wt than the major band, indicating the possibility of a further complex in addition to that of the ligand and binding site. On the whole, these second bands are not large enough to be receptor dimers (204 ).
E. Receptor biochemistry: molecular characterization of
adrenomedullin and CGRP receptors
Examination of the pattern of binding of
125I-adrenomedullin in rat tissues led us to
reconsider the role of the L1 orphan receptor (205 ) [also known as
G10d from rat liver (206 ), a 395-amino acid seven-transmembrane
receptor, GenBank accession number L04672], which is expressed in
lung, adrenal, heart, and spleen (4 ). When transfected into COS-7
cells, this receptor bound 125I-adrenomedullin
(KD = 8.2 nM) and gave
adrenomedullin-mediated increases in intracellular cAMP
(ED50 = 7 nM) that were only
inhibited by high concentrations of CGRP8–37
(Ki = 1 µM) (4 ). The human homolog
of this receptor was then cloned but not expressed and found to show
73% similarity by amino acid sequence, which is not high for a species
homolog (404 amino acids, EMBL accession number Y13583) (207 ). The
identification of both rat and human sequences as adrenomedullin
receptors has recently been questioned (208 ). No binding of rat or
human 125I-adrenomedullin followed transfection
of either sequence into COS-7 cells, despite the presence of mRNA and
expression of the protein at the cell surface (208 ). The most closely
related receptor to L1 is a dog 7-transmembrane receptor called RDC-1
(49 ) (GenBank accession number X14048). Expression of this receptor in
COS-7 cells gave a pharmacology typical of a
CGRP1 receptor with CGRP-stimulated cAMP
generation (EC50 = 3 nM) potently
inhibited by CGRP8–37 (209 ). Adrenomedullin also
stimulated cAMP levels with an EC50 of 100
nM, as would be expected of a CGRP1
receptor. Binding studies showed a similar affinity for CGRP and
CGRP8–37 (9 and 13 nM respectively) (209 ). RDC-1 mRNA expression is high in vascular tissues
such as lung and liver, which express high levels of
125I-CGRP binding (49 ).
An interesting study from Luebke et al. (210 ) showed the expression cloning of a hydrophilic 146-amino acid protein from guinea pig organ of Corti, which conferred CGRP receptor activity on Xenopus oocytes (210 ) (GenBank accession number U50188). This protein, called receptor component protein (RCP), is expressed in human and mouse mainly in testis, with smaller amounts in human in prostate, ovary, small intestine, and spleen (211 ). RCP conferred CGRP (10 nM) effects on oocytes but not calcitonin or amylin (100 nM) effects. Using in situ hybridization in the guinea pig CNS, RCP was shown to be abundant in the cerebellum and hippocampus (212 ). Adrenomedullin effects via RCP have not been tested, but the limited distribution of RCP means it can only account for a small subset of adrenomedullin receptors at best unless other RCPs are yet to be cloned.
The other side of the CGRP/adrenomedullin receptor story relates to another orphan receptor called calcitonin receptor-like receptor (CRLR). This was originally cloned by two groups, Legon and co-workers (213 ) (GenBank accession number X70658) using a PCR strategy amplifying rat hypothalamic mRNA with primers based on the porcine calcitonin and opossum PTH receptors and Chang et al. (214 ), who also described the identification of a CRF receptor (GenBank accession number L27487). The full sequence of the human homolog of CRLR from cerebellum was reported by Fluhmann et al. (215 ) (GenBank accession number U17473). Human CRLR, expressed mainly in lung, heart, and kidney, is a 461-amino acid seven-transmembrane protein with 91% homology to its rat homolog and 51% similarity to the human calcitonin receptor. This receptor expressed in COS-7 cells did not bind any member of the calcitonin family of peptides and was considered an orphan receptor. However, in 1996 Aiyar and co-workers (216 ) showed that hCRLR stably transfected into human embryonic kidney (HEK) 293 cells exhibited the pharmacology of a CGRP1 receptor (CGRP Kd = 19 pM, CGRP8–37 pA2 = 7.57, CysACM-CGRP ineffective up to 1 µM) (216 ). Adrenomedullin showed binding and stimulation of cAMP, albeit weak, in these cells. These results were confirmed using the rCRLR stably transfected into HEK 293 cells (217 ) and later the porcine CRLR as well (218 ). CRLR mRNA is extremely abundant in the rat lung [as is specific 125I-adrenomedullin binding (165 )] and was shown by in situ hybridization studies to be associated with blood vessels (213 ). CRLR protein was also shown by immunocytochemistry to be associated with vascular endothelial cells (217 ). This fits well with a role for adrenomedullin as a pulmonary vasodilator (see Section IV.A) and the presence of adrenomedullin binding on endothelial cells (197 ) but disagrees with the previous localization by in situ PCR to alveolar cells (216 ).
The question now became what was the factor in HEK 293 cells that was not present in COS-7 cells that allowed CGRP receptor expression? The surprising answer was provided by Foord’s group using a Xenopus oocyte/cystic fibrosis transmembrane regulator system, similar to that used to clone RCP, where increases in intracellular cAMP can be detected as chloride currents. They cloned a receptor-activity modifying protein (RAMP-1) of 148 amino acids with a single transmembrane domain that conferred CGRP1 receptor activity (no response to adrenomedullin, amylin, or calcitonin but inhibited by CGRP8–37) to the oocytes (186 ). When transiently cotransfected with hCRLR into HEK 293 cells, RAMP-1 conferred 125I-CGRP binding properties to these cells, but no binding was seen with either RAMP-1 or CRLR alone. The pharmacology of this combination was very similar to that of CGRP receptors in SK-N-MC cells (186 ). The failure of expression of CGRP binding by CRLR in COS-7 cells is therefore probably due to the lack of endogenous RAMP-1. The action of RAMP-1 was shown to involve transport of CRLR to the cell surface. 125I-CGRP could be cross-linked to proteins of Mr = 66,000 (CRLR) and 17,000 (RAMP-1). This Mr of 66,000 compares well with that reported for CGRP receptors in tissues (see previous section). Cross-linking of CGRP to RAMP-1 allows for the intriguing possibility that RAMP-1 forms part of the binding site for CGRP. CRLR is present in HEK 293 cells as a Mr = 58,000 glycosylated receptor, which in the presence of RAMP-1 is further glycosylated to Mr = 66,000, consistent with RAMPs acting to transport CRLR.
Two further members of the RAMP family that did not confer CGRP receptor activity were also identified, but overall the three RAMPs showed 31% identity in amino acids (186 ). The three RAMP mRNAs have widespread and different distributions in human tissues. RAMP-2 and RAMP-3 also facilitated expression of CRLR on the cell surface but as a Mr = 58,000 glycoprotein. Coexpression of CRLR and RAMP-2 in oocytes or HEK 293 cells resulted in a typical specific adrenomedullin receptor pharmacology with no effects of CGRP (186 ). Thus, the RAMP hypothesis offers an extremely interesting explanation of CGRP/adrenomedullin receptor pharmacology, i.e., CRLR/RAMP-1 = CGRP1 and CRLR/RAMP-2 = adrenomedullin.
Since the discovery of RAMPs, some aspects of the hypothesis have been confirmed. Kamitani et al. (219 ) showed that RAMP-2 is expressed in human VSMCs and endothelial cells, and a combination of RAMP-2/CRLR transfected into HeLa EBNA or 293 EBNA cells led to adrenomedullin-stimulated cAMP with no effect of CGRP. Transfection of RAMP-1/CRLR conferred CGRP and adrenomedullin-stimulated increases in cAMP, which differs from the oocyte studies by McLatchie et al. (186 ) but agrees with the binding data for CGRP1 receptors (see above). Muff et al. (188 ) showed that rabbit endothelial cells express RAMP-2 and CRLR and that adrenomedullin stimulated cAMP (EC50=0.18 nM). When these cells are transfected with RAMP-1, they then express CGRP-stimulated cAMP (EC50 = 0.41 nM) which is inhibited by CGRP8–37 (100 nM), indicating that CRLR can be converted from an adrenomedullin-specific receptor to a CGRP receptor by a dominant effect of RAMP-1. This effect was further investigated by the same group using rCRLR and RAMPs expressed in UMR-106 rat osteoblast-like cells and COS-7 cells (187 ). UMR-106 cells transiently transfected with CRLR express 125I-adrenomedullin binding, which was enhanced by RAMP-2 cotransfection. Here 125I-CGRP binding required transfection of RAMP-1 but was unaffected by RAMP-2. Similar results were shown in COS-7 but, as expected, since COS-7 lack RAMPs, adrenomedullin binding required RAMP-2 cotransfection. The amino terminus of the RAMPs has been shown to be the major factor controlling glycosylation and ligand binding using chimeric RAMP-1/2 proteins (220 ). RAMPs 2 and 3 appear indistinguishable in terms of CRLR glycosylation and adrenomedullin binding in this study. Thus, the RAMP hypothesis appears correct but has not yet been totally proven, and some aspects of it need to be investigated. What is the purpose of RAMP-3/CRLR if it yields an identical pharmacology with RAMP-2/CRLR? In rat tissues, adrenomedullin receptors appear by cross-linking to be equal to or larger than CGRP receptors (see previous section), whereas the RAMP hypothesis predicts that CGRP receptors should be larger than adrenomedullin receptors. Do partners other than CRLR exist for RAMP, e.g., the calcitonin receptor (221 )? Do CRLR and RAMP-1/2 account for all adrenomedullin and CGRP binding?
There has been little study of whether the presence of RDC-1, L1, and CRLR correlate with CGRP/adrenomedullin binding. In the rat brain, the distributions of the three putative receptors were compared by in situ hybridization (222 ). RDC-1 mRNA was mainly associated with the dentate gyrus, hippocampal CA3, choroid plexus, and blood vessels. L1 was very weakly expressed except in cells of the pia mater. CRLR was expressed in the caudate putamen and the central and basolateral amygdaloid nuclei. These data match well in some areas but not at all in others with CGRP or adrenomedullin binding in the rat brain (145 181 223 ). None of the three mRNAs was present in spinal cord (222 ) despite high levels of CGRP and adrenomedullin binding (168 ). All three mRNAs were expressed in adult rat heart and neonatal cardiac myocytes, with RDC-1 being most abundant followed by CRLR with L1 being of low abundance (224 ). In rat aortic VSMCs, RDC-1, but not L1 or CRLR, mRNA was detected using specific RNase protection assays (225 ). Binding experiments using 125I-adrenomedullin and fragments of adrenomedullin and CGRP showed two subtypes of adrenomedullin receptor in astrocytes and NG108–15 cells (179 ). Chemical cross-linking of 125I-adrenomedullin binding sites in rat tissues also shows heterogeneity of mol wt (165 168 ). Thus, there are indications that CRLR/RAMP and/or L1/RDC-1 do not account for all CGRP/adrenomedullin binding sites.
F. Signal transduction pathways activated by adrenomedullin
It is now clear from a vast number of studies that the major
effect on adrenomedullin-stimulated cells is an elevation of cAMP (25 33 38 39 44 56 58 59 156 162 163 164 174 176 177 179 180 182 183 184 185 189 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 ). This is typical of the calcitonin family of
peptides, all of which have been shown to elevate cAMP levels in
various tissues and cells (8 150 ). It should not be forgotten that
this was the property that was used to discover adrenomedullin (1 ).
Also, all of the cloned receptors, regardless of whether they are
actually adrenomedullin/CGRP receptors in vivo, are
associated with increased cAMP when transfected into cells (4 186 188 209 216 217 ). Thus, the initial mechanism of action of
adrenomedullin (and CGRP) is in most cases via G-protein linked
receptor activation of Gs, adenylyl cyclase, and protein kinase A (PKA)
(163 ). In this section we will examine other possible mechanisms and
some consequences of elevated cAMP. Most of the studies on
adrenomedullin signaling have been performed using primary cells
(especially VSMCs) or cell lines. Since adrenomedullin will bind to
both specific adrenomedullin and CGRP receptors and these are often
expressed together in cells, the failure to define which receptor is
actually mediating the effect is a problem. This can be addressed by
use of inhibitors such as CGRP8–37 or
adrenomedullin22–52, but these are not
especially potent or specific, and better antagonists would greatly
advance this work. At present, the only conclusive results are obtained
by careful use of antagonists, use of cells expressing only one type of
binding, or use of transfected cells. Of course the transfection
approach has problems associated with heterologous expression and also
the doubt surrounding which receptor should actually be transfected.
The effects of adrenomedullin on calcium signaling mechanisms have been investigated since endothelial NO has been implicated in adrenomedullin-mediated vasodilation (see Section IV.A), and logically this should be the result of an increase in intracellular calcium ([Ca2+]i) activating endothelial cell nitric oxide synthase (NOS). In the bovine aortic endothelial cell-specific 125I-adrenomedullin binding (IC50 = 10 nM) and adrenomedullin-mediated cholera toxin-sensitive increases in cAMP were observed (EC50=0.17 nM) (182 ). Here adrenomedullin also directly increased [Ca2+]i (EC50 = 3 nM) with an initial peak followed by a prolonged increase. The initial effect was blocked by thapsigargin, and the prolonged effect by EGTA and nifedipine. Pretreatment of cells with U-73122, the phospholipase C (PLC) inhibitor, but not its inactive analog U-73343, blocked all calcium responses to adrenomedullin. Similarly, cholera toxin, but not H89 (PKA inhibitor) or pertussis toxin, blocked all effects. As expected for a PLC-mediated effect, adrenomedullin (100 nM) increased the intracellular levels of ITP. Adrenomedullin also increased intracellular cGMP. This very detailed study offers an interesting account of how adrenomedullin might increase NO production and thereby vasodilation. One omission in this study was the effects of CGRP and the action of CGRP8–37 on the adrenomedullin effects. Another problem is that Barker et al. (247 ) were able to show increases in cAMP but unable to show any [Ca2+]i effects in bovine endothelial cells . Adrenomedullin did not affect [Ca2+]i but did increase cAMP in Swiss 3T3 cells that express only specific adrenomedullin receptors and not CGRP receptors (180 ). Similar results were obtained using cultured rat astrocytes (179 ). Adrenomedullin was shown to decrease [Ca2+]i and calcium sensitivity in porcine coronary artery strips, possibly by a direct cAMP-mediated mechanism (248 ). In favor of adrenomedullin increasing [Ca2+]i are results using KG-1C human oligodendroglial cells where adrenomedullin and CGRP both increased cAMP and [Ca2+]i (228 ). Unfortunately the effects of CGRP8–37 or 125I-ligand binding were not investigated. Also, in the perfused rat heart, adrenomedullin enhanced cardiac contractility by a mechanism that was independent of cAMP but involved changes in [Ca2+]i (249 ). In the L6 skeletal muscle cell line, both adrenomedullin and CGRP receptors were present, but increases in intracellular cAMP were mediated only via CGRP receptor binding (163 ).
Reports of effects of adrenomedullin on growth and mitogenesis (see Section IV.B) have led to investigation of the regulation of mitogen-activated protein kinase (MAPK) by adrenomedullin. In rat glomerular mesangial cells, adrenomedullin increased cAMP and PKA but inhibited proliferation (both of quiescent and platelet-derived growth factor (PDGF)-stimulated cells) and MAPK activity (241 ). Also in mesangial cells, adrenomedullin (and other agents that increased cAMP) inhibited endothelin-1 (ET-1)-stimulated MAPK (but not, in this case, basal levels) and MAPK kinase (250 ) and stimulated expression of a MAPK phosphatase (251 ). Chini et al. (230 ) also showed that adrenomedullin reduced PDGF-stimulated MAPK activity and mitogenesis in rat VSMCs, effects that were blocked by the PKA inhibitor, H89. However, in quiescent rat VSMCs, adrenomedullin increased DNA synthesis, cell proliferation, tyrosine phosphorylation, MAPK activity, and expression of the immediate-early gene, c-fos. These effects could be blocked by CGRP8–37 and the tyrosine kinase inhibitor, genistein, but not by cAMP or PKA antagonists, indicating a cAMP-independent effect (252 ). Interestingly, in these VSMCs, adrenomedullin had no effect on [Ca2+]i or ITP. In the Rat-2 fibroblast cell line, which expresses specific adrenomedullin but not CGRP receptors, adrenomedullin stimulated cAMP and inhibited basal and PDGF-stimulated MAPK (56 ).
Adrenomedullin has also been shown to activate other signal transduction mechanisms including K+-ATP channels (253 ) and c-fos expression (229 254 ). Adrenomedullin augmented interleukin-1ß-stimulated NO synthesis in rat VSMCs by a cAMP-dependent mechanism (239 ). Desensitization of adrenomedullin receptors has not been widely investigated, but Iwasaki et al. (227 ) showed that adrenomedullin pretreatment caused a loss of adrenomedullin-stimulated adenylyl cyclase activity in rat aortic VSMCs. Drake et al. (226 ) showed that in SK-N-MC cells, preexposure to CGRP or adrenomedullin desensitized the cells to a subsequent CGRP stimulus, but preexposure to CGRP or adrenomedullin did not affect a subsequent exposure to adrenomedullin. This is interesting since if CRLR is responsible for cAMP stimulation with both CGRP and adrenomedullin in SK-N-MC cells, then they would be expected to give similar desensitization patterns. It seems then that we still need to learn a lot more about adrenomedullin signaling before its mechanisms of action in each of its different roles can be deduced.
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IV. Biological Actions of Adrenomedullin |
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TopAbstractI. IntroductionII. Synthesis and Secretion...III. Receptors and Signal...IV. Biological Actions of...V. Unresolved Issues and...References |
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A. Vascular actions
In rat, cat, sheep, and man, intravenous infusion of
adrenomedullin results in a potent and sustained hypotension (101 174 255 256 257 258 259 260 261 262 ), mainly via NO generation in the vasculature (261 263 264 )
and is comparable to that of CGRP (1 265 ). Initial studies of the
hemodynamic effects of human adrenomedullin used anesthetized rats
(266 267 ). Acute or chronic administration of adrenomedullin resulted
in a significant decrease in total peripheral resistance accompanied by
a fall in blood pressure. This is concomitant with a rise in heart
rate, cardiac output, and stroke volume (266 267 268 269 ). Similar effects are
seen in both conscious (270 ) and hypertensive rats (266 268 ). The
hypotensive effect of adrenomedullin on mean arterial pressure in the
anesthetized rat is not inhibited by CGRP8–37,
suggesting this effect is not mediated via CGRP receptors (169 ). The
vascular beds in which adrenomedullin is effective are listed in Table 6.
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Adrenomedullin is vasodilatory in the systemic vascular system of the cat (171 255 287 290 ), an effect that is antagonized by CGRP8–37. However, in the hindlimb vascular bed of the cat, adrenomedullin induces a vasodilatory effect not altered by the CGRP1 receptor antagonist (171 194 275 ). The mechanism by which adrenomedullin reduces vascular resistance in the cat hindlimb circulation is not clear, but it is possible that adrenomedullin may relax vascular smooth muscle by inducing an increase in cAMP levels (176 177 185 291 ). It is also possible that vasodilation induced by adrenomedullin may be mediated via NO release or arachidonic acid metabolism from the endothelium. It has been previously shown that NO mediates responses to adrenomedullin in the renal vascular bed of the dog and the rat pulmonary and hindquarter vascular beds (261 272 278 ). However, in the cat, NOS inhibitors were without effect and, in fact, duration of the vasodilator response to adrenomedullin was significantly increased after administration of rolipram, a type IV phosphodiesterase inhibitor, suggesting a cAMP-mediated mechanism of action (287 290 ).
In pig renal artery smooth muscle strips stimulated by phenylephrine, adrenomedullin caused a fall in tension that was concomitant with a decrease in [Ca2+]i (273 ). Using the intact canine kidney it has been demonstrated that adrenomedullin caused an increase in renal blood flow (RBF) that was attenuated by NOS inhibitors (263 ). Similar effects were seen in isolated perfused rat kidneys (264 274 ) and in rabbits (174 ). In all cases CGRP8–37 was without effect.
The feline model has been used in investigating the physiology and pharmacology of penile erection (292 293 ). Champion and co-workers (287 288 289 ) have used this model to study the effects of adrenomedullin on the erectile response. Adrenomedullin caused significant dose-dependent increases in intracavernous pressure and penile length when injected directly into the corpus cavernosum. Responses to adrenomedullin were comparable to those induced by intracavernous injection of a standard triple-drug combination composed of papaverine, phentolamine, and PGE1. The mechanism of the erectile response to adrenomedullin is unclear but is unlikely to be NO dependent since administration of NG-nitro-L-arginine methyl ester (L-NAME) was without effect.
In the intact cat pulmonary vascular bed, adrenomedullin has no effect on resting arterial pressure; however, in the presence of a thromboxane A2 agonist, adrenomedullin caused a decrease in pulmonary arterial pressure (276 277 ). Using the isolated perfused rat lung preparation, it has been shown that human adrenomedullin causes decreases in preconstricted vascular tone with no effect on resting tone (173 ). Neither CGRP8–37 nor NOS inhibitors had any effect, suggesting that in the lung at least, vasodilation occurs via an NO-independent mechanism. This finding, however, contradicts a study using pulmonary arterial rings whereby NO produced by the vascular endothelium was required for relaxation in response to adrenomedullin (294 ). In conditions of hypoxia, the vasodilatory effect appears to be mediated via PG synthesis rather than NO production (294 ).
PGs have been implicated in another regional vascular system. Adrenomedullin is 16 times more potent than PGI2 as a vasodilator in the uterine circulation. Using nonpregnant, oophorectomized ewes, Friedman et al. (279 ) infused either adrenomedullin (0.01 to 3 µg/min) or PGI2 (0.03 to 10 µg/min) for a period of 5 min. At the doses administered there were no changes in heart rate, cardiac output, or blood pressure; however, there was a significant and dose-dependent increase in uterine blood flow. Clearly, local levels of adrenomedullin, released from endothelial cells in the uterine vasculature, may result in significant vasodilation, suggesting that adrenomedullin may play a role in pregnancy-associated vasodilation (295 ).
Adrenomedullin appears to have direct effects on the heart and coronary circulation. As mentioned above, canine heart and cultured rat cardiac myocytes express adrenomedullin immunoreactivity and mRNA. The vasodilatory effect of adrenomedullin on porcine coronary arteries is mediated via CGRP receptors and is abolished upon removal of the vascular endothelium (156 248 280 ). Szokodi et al. (249 ) reported that adrenomedullin exhibits inotropic effects by increasing heart contractility via a specific adrenomedullin, Ca2+-dependent mechanism in the rat heart. The same group, however, had previously demonstrated that the inotropic effect of adrenomedullin was inhibited by CGRP8–37 (296 ). In rabbit cardiac myocytes, adrenomedullin has a negative inotropic effect which appears to be mediated via NO (297 ).
Kato and co-workers (281 ) designed studies to investigate the effect of adrenomedullin on the pressor response to exogenous norepinephrine using the isolated canine tibia as a model for bone circulation. Bolus administration of adrenomedullin (1 nmol) reduced the pressor response to norepinephrine significantly, and this effect was long lasting. The effect was not altered by indomethacin, which blocks PG synthesis. However, infusion of L-NMMA blocked the effect of adrenomedullin on norepinephrine-induced pressor responses, suggesting a role for NO in this action (281 298 ).
Until recently, the effects and mechanisms of action of adrenomedullin in cerebral vessels have been poorly defined. Baskaya et al. demonstrated that the vasodilator effects of adrenomedullin on conducting arteries of the cerebral circulation were inhibited by CGRP8–37 and that prior administration of CGRP prevented subsequent adrenomedullin-induced relaxation (282 ). This effect was not mediated via NO or vasodilator prostanoids, but because intracisternal administration of adrenomedullin caused an increase in cAMP levels in CSF, these authors speculated that vasodilation in response to adrenomedullin involved an adenylyl cyclase-dependent mechanism. A study of the effect of adrenomedullin on cat cerebral parenchymal microvessels reported that adrenomedullin caused a significant increase in cerebral blood volume, but had no effect on maintaining the resting tone of intracerebral parenchymal vessels (284 ). These effects were not significantly inhibited by CGRP8–37 but were attenuated by adrenomedullin22–52. A recent study by Lang et al. (285 ) demonstrates that adrenomedullin-induced dilation of pial arteries is not subject to tachyphylaxis, but involves the opening of both ATP-sensitive and Ca2+-dependent K+ channels. The precise physiological role of adrenomedullin in the cerebral circulation remains unclear. However, its clinical significance may be related to a role in the mechanism of injury from focal cerebral ischemia (283 286 299 ).
B. Growth and development
Adrenomedullin was originally purified from a human adrenal tumor
(1 ). Cuttitta’s group extended their initial observation of
adrenomedullin and L1 receptors in pulmonary tumors (43 300 ) to study
the expression of adrenomedullin in human tumor cell lines in general
(58 ). This opened up the possibility of adrenomedullin being an
autocrine/paracrine growth factor in tumors and possibly normal cells.
A neutralizing antiadrenomedullin monoclonal antibody was growth
inhibitory to these cells, which also showed both
125I-adrenomedullin binding (L1 receptor mRNA was
also present) and adrenomedullin-stimulated cAMP. In Swiss 3T3 cells
adrenomedullin increased DNA synthesis in a dose-dependent manner by a
mechanism involving specific adrenomedullin receptors and increased
cAMP/PKA (180 ). These findings have been confirmed, and Swiss 3T3 cells
were shown to produce correctly processed adrenomedullin, which is
regulated by cytokines and growth factors (57 ). In normal and malignant
skin, adrenomedullin and the L1 receptor (263 ) were detected and
adrenomedullin increased 3H-thymidine uptake
(50 ). Adrenomedullin also stimulated DNA and cAMP synthesis in human
oral keratinocytes (184 ). The effect on DNA synthesis was inhibited by
an adenylyl cyclase inhibitor (SQ22, 36) and mimicked by
(Bu)2cAMP (184 ). In quiescent rat VSMCs,
adrenomedullin and CGRP stimulated DNA synthesis and cell proliferation
(252 ). These results provide strong evidence for a growth-promoting
effect of adrenomedullin, possibly mediated via cAMP.
However, agents that increase cAMP are often associated with inhibition of cell proliferation. In human normal glial cells and glial cell tumors, adrenomedullin suppressed cell growth and increased intracellular cAMP (183 301 ). Growth of human and rat astrocytomas and human glioblastomas, as well as cultured glioblastoma-derived cell lines, was inhibited by adrenomedullin (33 183 301 ). However, this contrasts with studies using C6 gliomal cell cultures. Moody et al. (229 ) reported that adrenomedullin exerted mitogenic effects on these cells that correlated with increases in cAMP and c-fos expression. In rat mesangial cells, adrenomedullin suppressed mitogenesis by a cAMP-dependent mechanism (241 251 302 ). A similar result was obtained in TGW human neuroblastoma cells where the inhibition was blocked by both CGRP8–37 and adrenomedullin22–52 (191 ). In bovine aortic endothelial cells, addition of a monoclonal antibody to adrenomedullin increased DNA synthesis and cAMP, but no effect was seen in rat mesangial or VSMCs, even though all three cell types released adrenomedullin (38 ). Using rat VSMCs, adrenomedullin was shown to inhibit serum-stimulated 3H-thymidine uptake, which could be blocked by CGRP8–37 (303 ). These effects indicate an inhibition of growth by adrenomedullin as might be expected with increased intracellular cAMP, but some of these results are contradictory to those above (9 252 ). This needs to be investigated further in vascular cells to establish whether adrenomedullin is a vasodilator and inhibitor of proliferation, which might counteract the effects of vasoconstrictor/proliferators such as endothelin and angiotensin II.
In addition to the possible antiproliferative effects of adrenomedullin, it may also inhibit coronary artery smooth muscle cell migration (232 304 ), perhaps with the two effects combining to inhibit vascular remodeling. Adrenomedullin has also been shown to inhibit hypertrophy in cultured neonatal cardiac myocytes [inhibition of angiotensin II stimulated 14C-phenylalanine incorporation (23 )] and in the right ventricle of pulmonary hypertensive rats [right ventricle weight of monocrotaline-treated rats (305 )]. Adrenomedullin has also been shown to be angiogenic in the chick chorio-allantoic membrane assay and to increase human umbilical vein endothelial cell number (47 ). This finding, combined with the tamoxifen induction of the adrenomedullin gene in endometrial stromal cells, may indicate a role for adrenomedullin in uterine growth and vascularization (47 ). Adrenomedullin has also been proposed as an important factor in embryogenesis and differentiation (36 53 306 307 308 ) and as an apoptosis survival factor for rat endothelial cells (233 ). Taken together, although there is some debate on the exact effects of adrenomedullin, there is little doubt that these findings indicate a role for adrenomedullin in cell and tumor growth, and this might be expected to be a productive area of research in the near future.
C. Endocrine effects
1. The pituitary. In 1995, two groups described the effects of
adrenomedullin on the pituitary. In the first study primary cultures of
rat anterior pituitary cells were exposed to adrenomedullin and
accumulation of ACTH in the medium measured (309 ). Adrenomedullin
inhibited ACTH release from these cells in a dose-dependent manner and
also attenuated CRH-stimulated ACTH production. It appeared that
adrenomedullin did not affect basal or CRH-stimulated cAMP responses in
these cells, which suggested that adrenomedullin was exerting its
effect through an adenylyl cyclase-independent mechanism. Samson
et al. (309 ) also demonstrated the ability of angiotensin II
to antagonize the actions of adrenomedullin. In this report, the
workers did not observe any changes in levels of LH or GH from isolated
pituitary cells in response to adrenomedullin. The second study by
Parkes and May (260 ) describes the effects of intravenous infusion of
adrenomedullin into conscious sheep. These workers observed a
significant reduction in plasma ACTH levels from 50 pg/ml to 21 pg/ml,
which continued to fall to a level of 14 pg/ml 1 h after cessation
of the infusion. Taken together, these studies suggest that
adrenomedullin has a role in inhibiting ACTH release.
2. The adrenal gland. Like other regulatory peptides present in the adrenal gland (310 311 ), adrenomedullin affects the secretory activity of the adrenal cortex in both rat and human. Yamaguchi et al. (312 ) studied the effect of adrenomedullin on aldosterone production in the rat in vivo. Secretion of aldosterone was stimulated by placing rats either on a sodium-deficient diet or by performing a bilateral nephrectomy. In both cases adrenomedullin was administered by injection, and adrenal renin activity and aldosterone concentration were measured. These workers demonstrated that adrenomedullin significantly inhibited aldosterone production in response to either of these manipulations, but had no effect on adrenal or PRA, plasma corticosterone, or K+ levels. The results of such experiments are difficult to interpret since many different factors interact to maintain aldosterone levels. For example, pituitary peptides have a significant influence in the response to sodium depletion, and these were not measured in the study by Yamaguchi et al. (312 ). As adrenomedullin inhibits ACTH secretion in the sheep (260 ), it would be expected that this may have been a factor in the aldosterone studies.
Previously this group has reported that adrenomedullin significantly inhibited aldosterone secretion in response to angiotensin II, K+, and the calcium ionophore, A23187, from dispersed rat adrenal zona glomerulosa cells, but that aldosterone stimulated by either ACTH or (Bu)2cAMP was not affected (313 ). These data are entirely consistent with an agonist working through cAMP generation as we have previously shown that agonists activating cAMP-dependent and inositol trisphosphate-dependent pathways are mutually antagonistic in the rat adrenal gland (314 ).
Several different adrenal tissue preparations have been used to investigate the effects of adrenomedullin on steroid secretion. These studies have proven to be rather contradictory. The first studies, using collagenase-dispersed zona glomerulosa cells, demonstrated the inhibitory effect of adrenomedullin on angiotensin II-stimulated aldosterone secretion in rat and man (315 316 ). This was blocked by CGRP8–37, suggesting that adrenomedullin exerts this effect via a CGRP1 receptor in this tissue, but the high concentration of CGRP8–37 used (1 µM) may favor nonselective effects. Other studies, using intact capsular tissue, to which the zona glomerulosa cells adhere, or slices of human adrenal tissue have shown a stimulatory effect of adrenomedullin on aldosterone secretion (236 298 316 ). Most recently it has been demonstrated that adrenomedullin, acting through specific adrenomedullin receptors, stimulates zona glomerulosa cells to produce aldosterone (25 317 ). In addition, it has been reported that CGRP and adrenomedullin exert opposite effects on aldosterone secretion (317 ). Thus it is likely that the differences between responses of different rat zona glomerulosa cells are dependent on whether adrenomedullin is acting through CGRP or adrenomedullin receptors in this tissue.
Using the isolated perfused in situ rat adrenal preparation developed in our laboratory, it has been shown that adrenomedullin causes an increase in perfusion medium flow rate (160 298 318 ). In intact rats adrenomedullin also causes an increase in adrenal blood flow (266 ). The observation that adrenomedullin acts as a vasodilator in the adrenal vascular bed, as in other tissues, strongly suggests that adrenomedullin can stimulate corticosterone secretion by the rat adrenal. Indeed, studies on the intact perfused rat adrenal preparation suggest this is the case (298 315 316 ). Studies of the direct action of adrenomedullin on collagenase-dispersed rat zonae-fasciculata/reticularis cells in vitro suggest that adrenomedullin does not stimulate corticosterone secretion (298 ). This suggests that the observations in the perfused adrenal preparation are likely to be secondary to vascular events. However, infusion of adrenomedullin (100 µg/h) into sheep results in a 55% decrease in cortisol production with a significant increase in PRA (260 ). In this study there was also a 58% decrease in ACTH levels, and it is likely that the decrease in cortisol levels was a consequence of the drop in ACTH, rather than a direct effect on the adrenal gland (260 ).
We, and others, have demonstrated that adrenomedullin is abundant in adrenal medullary cells (25 178 ), and it appears to be cosecreted with catecholamines in response to nicotinic receptor stimulation (112 ). In cultured bovine adrenomedullary cells, adrenomedullin does not affect basal catecholamine release, but increases Ca2+ efflux, presumably by stimulating Na+/Ca2+ exchange (319 ). Masada et al. (320 ) have recently reported the results of their study of adrenomedullin on adrenal catecholamine release in dogs. In this study, catecholamine release was evoked by splanchnic nerve stimulation and by injecting cholinergic agonists into the adrenal gland of conscious dogs via the phrenicoabdominal artery. Adrenomedullin (1–10 ng/kg/min) had no effect on basal catecholamine output; neither did it alter catecholamine levels in response to splanchnic nerve stimulation or acetylcholine administration. Even at high doses (100 ng/kg/min), adrenomedullin did not affect catecholamine levels in response to nerve stimulation even though blood pressure was significantly lowered and there was an increase in adrenal plasma flow. However, the possibility exists that endogenous adrenomedullin has a maximal effect on adrenal catecholamine release physiologically and, therefore, masks the effects of exogenous adrenomedullin, since adrenomedullin levels in the adrenal medulla have been demonstrated to be more than 30-fold higher than other tissues (6 ).
3. Reproductive effects. Reproduction is a complex and finely programmed process involving extensive tissue remodelling and changes in blood flow (321 322 ). The mechanisms underlying normal functioning of reproductive tissue are being unraveled; however, it is clear there is cross-talk involving endocrine, paracrine, and autocrine factors. In light of the known roles of adrenomedullin, it has been suggested that it may have important regulatory roles in reproductive tissues. Adrenomedullin has been shown to be present throughout the female reproductive tract/system (46 323 ). Studies from our laboratory tested the effect of adrenomedullin on galanin-stimulated contractile responses of rat uterine muscles (130 ). Adrenomedullin significantly attenuated contractility in response to galanin, suggesting a role for adrenomedullin in uterine function. This may have some relevance in the events that occur in late pregnancy for example (63 77 128 324 ).
As described above, plasma levels of adrenomedullin are elevated in normal pregnancy. The physiological significance of this secretion remains to be established, but a number of studies provide some indications. Marinoni et al. (45 ) suggested that adrenomedullin may be involved in the process of adaptation of the vascular system to pregnancy. The presence of adrenomedullin in placenta and fetoplacental tissues (45 306 307 325 ) supports a role for adrenomedullin in control of vascular tone at the local level to regulate uteroplacental-fetal circulation. Furthermore, adrenomedullin has been demonstrated to inhibit PDGF- and thrombin-induced ET-1 production (245 ). In the placenta ET-1 is localized to trophoblasts and endothelial vessels of villi (326 ). Taken together, these findings suggest adrenomedullin may modulate vascular tone by inhibiting/stimulating vasoactive agents during normal and pathological states of pregnancy, such as preeclampsia and intrauterine growth restriction. In this light, a very recent study by Makino et al. (327 ) investigated the effect of adrenomedullin during preeclampsia. In this study an animal model of preeclampsia was used. Other workers have previously demonstrated that L-NAME-treated pregnant rats show preeclampsia-like symptoms consisting of hypertension, intrauterine growth restriction, proteinuria, and renal glomeruli injury (328 329 ). There is also increased fetal mortality (327 330 ). Makino and co-workers (327 ) demonstrated that infusion of adrenomedullin (3–10 pmol/h) reversed hypertension and decreased pup mortality induced by L-NAME when given to rats during late gestation (day 14 of pregnancy), but not in animals in early gestation or in nonpregnant rats. Furthermore, adrenomedullin appeared not to affect basal blood pressure or pup mortality in normal pregnant animals nor did it have any effect on L-NAME-induced hypertension after delivery. These findings suggest that adrenomedullin may have an important regulatory role in the utero-placental cardiovascular system.
Expression of adrenomedullin and its receptor have been found in mouse mammary glands (48 ). Apart from effects on growth, further studies are required to determine the role of adrenomedullin in ductal homeostasis, immune/innate defense, and neonatal nutritional supplement.
4. The pancreas. Mulder et al. (28 ) first reported the stimulatory effects of adrenomedullin (1–100 nM) on insulin secretion from isolated rat islets (in the presence of either 3.3 or 8.3 M glucose). In direct contrast to this, Martínez et al.(54 ) clearly demonstrated the inhibitory role of adrenomedullin on insulin secretion in vitro. These results were further strengthened by the observations that a blocking adrenomedullin antibody neutralized both the endogenous and exogenous effects of adrenomedullin on insulin secretion. These workers then went on to study the in vivo effects of adrenomedullin: adrenomedullin attenuated and delayed the insulin response to oral glucose challenge, resulting in initial elevated glucose levels. The vasodilatory effect of adrenomedullin may also have some influence on insulin secretion by elevating pancreatic perfusion rate, but this remains to be proven. However, the existence of a constitutively inhibitory tone in pancreatic islets may play a role in the homeostasis of this organ.
D. Renal effects
Circulating adrenomedullin can affect renal function, and evidence
exists for a role for locally produced adrenomedullin in tubular
function. The first reported studies on renal function involved
intrarenal arterial perfusion in anesthetized dogs (274 ).
Adrenomedullin administration had no effect on heart rate or mean
arterial blood pressure, but increased RBF, urine output, and urinary
Na+ excretion in a dose-dependent manner,
indicative of direct preglomerular and postglomerular arteriolar
effects (274 ). Subsequent studies found this effect to be mediated via
an endothelial, NO-dependent mechanism (263 264 ). In the anesthetized
rat, intrarenal adrenomedullin infusion leads to increases in RBF,
arterial conductance, glomerular filtration rate (GFR),
Na+ excretion, and urine flow (264 291 331 ).
These effects are not inhibited by CGRP8–37.
Bolus administration of adrenomedullin peripherally significantly
lowers mean arterial pressure and raises RBF, GFR, and urine flow; the
latter three responses were significantly attenuated in the presence of
L-NAME (264 332 ).
Studies have been carried out recently in a rat model of heart failure. In this series of experiments, Nagaya and co-workers (333 ) demonstrated that intravenous infusion of a low dose of adrenomedullin to normal rats or those with heart failure led to significantly increased urine volume and Na+ excretion without changing GFR, RBF or any other hemodynamic parameter (333 ). High-dose adrenomedullin infusion decreased mean arterial pressure and increased cardiac output in both rat groups. They also showed that adrenomedullin significantly reduced right ventricular systolic pressure in heart failure rats with pulmonary hypertension. In addition, adrenomedullin did not increase urinary cGMP levels, suggesting that the renal actions of adrenomedullin may not be mediated totally by the NO pathway in these rats (67 238 263 ).
Contrary to this, Rademaker et al. showed that intravenous administration of adrenomedullin increased Na+ excretion without an increase in urine flow or creatine clearance in an ovine model of heart failure (334 ). The discrepancy between these studies may be explained by the differences in renal perfusion pressure; however, it is unlikely that circulating levels of adrenomedullin regulate renal function physiologically. This is due to the fact that the threshold levels for cardiovascular actions are much lower than those required for renal effects (21 258 ). Recently, however, it has been suggested that neutral endopeptidase (NEP) can potentiate the renal natriuretic and diuretic actions of intrarenal adrenomedullin infusion (335 ). NEP is a membrane-bound metalloproteinase that cleaves endogenous peptides at the amino side of the hydrophobic residues. This ectoenzyme is localized in a number of tissues but is found predominantly in the kidney (336 337 ). Substrates for NEP include bradykinin, AVP, and substance P, and the study described by Lisy et al. (335 ) concludes that adrenomedullin is also a substrate for this ectoenzyme. Inhibiting systemic NEP raises plasma adrenomedullin levels significantly, supporting the conclusion that adrenomedullin is a substrate for NEP. NEP inhibition also potentiates an increase in Na+ excretion in the absence of an increase in GFR or further increases in RBF in response to exogenous adrenomedullin. This indicates that a decrease in tubular Na+ resorption is the mechanism for natriuresis. The identification of adrenomedullin in the inner medullary ducts correlates with the ability of this tissue to increase its permeability to water in response to adrenomedullin (39 ).
There is also evidence for a role for adrenomedullin in mesangial cell
biology. In addition to its capability to modulate mesangial cell
contraction, adrenomedullin also inhibits ET-1 production in response
to PDGF in these cells (241 ). This mirrors the inhibitory effect
adrenomedullin has on PDGF-induced MAPK and cell proliferation (241 251 302 ). Parameswaran et al. (338 ) have shown that
adrenomedullin stimulates hyaluronic acid (an extracellular matrix
component) release from cultured rat mesangial cells via p38 kinase and
phosphatidylinositol-3-kinase pathways. These data imply there may be a
role for adrenomedullin in the pathophysiology of mesangial cell
proliferation and matrix biology. Adrenomedullin may also have a role
in protecting the kidney glomeruli from inflammatory reactions or
immune injuries. It has been demonstrated that the proinflammatory
cytokines, tumor necrosis factor- and interleukin-1ß, stimulate
adrenomedullin production from mesangial cells and because it is known
that adrenomedullin attenuates the generation of free radicals in these
cells and in macrophages, parallels have been drawn (230 ).
Adrenomedullin could also play a regulatory role in the endocrine function of the kidney. Whole animal studies, described by Jensen et al. (234 ) suggest that adrenomedullin elevated plasma renin levels in rats, a response thought to be secondary to the hypotensive action of adrenomedullin. Subsequent studies, using the isolated perfused kidney preparation, suggest that, in the absence of changes in perfusion pressure or renal nerve activity, adrenomedullin stimulates intrarenal renin release. Renin release was shown to be from juxtaglomerular granulosa cells (234 ).
E. Other peripheral effects
1. Gastric function. The central actions of adrenomedullin on
gastric function are described below. Adrenomedullin has been shown to
have profound effects on gastrointestinal motor and secretory functions
in several species and experimental models. In conscious rats,
intravenous injection of adrenomedullin (150–600 pmol) decreased
gastric emptying of a noncalorific meal in a dose-dependent manner.
These actions were reversed by administration of
CGRP8–37 (339 ). Attenuation of gastric emptying
appears to be unrelated to cardiovascular changes since the doses of
adrenomedullin required to inhibit gastric emptying are lower than
those necessary to evoke changes in cardiovascular parameters (266 339 340 ). Motility effects of adrenomedullin have not been studied,
but phasic and tonic intraluminal pressure in the gastric corpus may be
affected (341 ).
Peripheral infusion of adrenomedullin into conscious rats with gastric cannulae inhibits both basal and pentagastrin- and 2-deoxy-D-glucose-stimulated gastric acid secretion (342 ). In contrast, in conscious rats with pylorus ligation, a bolus intravenous injection of adrenomedullin stimulated gastric acid output due to increases in the volume of secretion and elevated pepsin levels (339 340 ). This discrepancy may be due to the different experimental models used.
Very recently, a study by Fukuda et al. (343 ) investigated the effects of adrenomedullin on gastric mucosa integrity. Gastric mucosal restitution involves the rapid reestablishment of epithelial integrity as part of the important mechanism of host defense/protection. In this study rat and human gastric mucosa were damaged by applying various concentrations of NaCl (with low concentrations of NaCl causing less mucosal damage and high NaCl causing extensive damage to the submucosa). Restitution was assessed by measuring the transmucosal potential difference and it was found that 1 µM adrenomedullin had a significant restitutional effect at low concentrations (0.5–1.0 M) of NaCl. These workers also studied the effect of adrenomedullin on Na+ absorption and Cl- secretion in rat colonic mucosa, but found adrenomedullin had little effect on ion transport. Adrenomedullin did, however, cause relaxation of colonic smooth muscle contraction in response to KCl. This remained persistent particularly when using high (100 nM) concentrations of adrenomedullin.
2. Bone. Studies by Montuenga and co-workers (306 307 ) have supported a role for adrenomedullin beyond that of the cardiovascular system and fluid homeostasis. Expression of adrenomedullin, and its receptor, was seen in osteoblasts during the later stages of rodent embryogenesis and in maturing chrondocytes of fetal mice. Further evidence for a physiological role of adrenomedullin in bone metabolism comes from Cornish et al. (344 ). In a series of elegant experiments, these workers have demonstrated that adrenomedullin acts on fetal and adult rodent osteoblasts to increase cell growth comparable to those of known osteoblast growth factors such as transforming growth factor-ß. Also, adrenomedullin increased protein synthesis in vitro and the area of mineralized and unmineralized bone in vivo. Taken together, these data suggest that adrenomedullin might play a paracrine regulatory role in skeletal growth throughout life. This has important implications clinically; for example, one of the major challenges in osteoporosis research is to develop a therapy that increases bone mass via osteoblastic stimulation.
3. Lung. In addition to causing pulmonary vasodilation, adrenomedullin inhibits bronchoconstriction induced by histamine or acetylcholine (294 ). This suggests there may be a relevant role for the increase in circulating levels of adrenomedullin seen during acute attacks of asthma (93 345 ). The adrenomedullin-relaxant response of the vasculature may also provide a protective role, for example, in the pulmonary circulation of patients with pulmonary hypertension (119 346 ). Additionally, adrenomedullin may have an antiinflammatory role in the lung. Macrophages secrete neutrophil chemoattractants in response to chemotaxis as part of the inflammatory process, particularly in the lung (347 ). Adrenomedullin significantly inhibits alveolar macrophage release of neutrophil chemoattractants in response to lipopolysaccharide, in a dose-dependent manner (244 ).
4. Innate immunity/mucosal defense. Studies from our laboratory, and others, have shown that adrenomedullin is expressed in key mucosal surfaces, such as the skin, lung, gut, and oral cavity (348 349 ). The epithelium provides a first line of defense against potentially pathogenic microorganisms. Many antimicrobial peptides with a broad spectrum of activity have been identified in the mucosal epithelia of mammals (350 351 ). Data obtained from our laboratory (349 ) and that of Walsh et al. (348 ) provide evidence that adrenomedullin also has antimicrobial properties against both Gram-positive and -negative bacteria isolated from skin, oral cavity, respiratory tract, and the gut. The concentration of adrenomedullin required to kill/inhibit bacterial growth is higher than those levels found in the circulation; however, in certain circumstances, such as sepsis, elevated plasma adrenomedullin levels obtained during this condition may contribute to the response to bacterial challenge.
F. CNS effects
Adrenomedullin and its receptor exist in the CNS and its cellular
components (see above). Focal brain ischemia is the most common event
leading to stroke in humans, and a role for adrenomedullin in this
condition has been suggested. Using the rat focal stroke model of
middle cerebral artery occlusion (MCAO), Wang et al. (286 )
demonstrated increases in adrenomedullin mRNA expression in the
ischemic cortex. This occurred 3 h after MCAO and remained
elevated for up to 15 days. Using immunocytochemistry, adrenomedullin
was localized to the ischemic neuronal processes. These workers also
demonstrated that administration of adrenomedullin (8
nM) before and after MCAO led to an increase in
the degree of focal ischemia injury (286 299 ). This study contrasts
with one reported by Dogan et al. (193 ). Using spontaneously
hypertensive rats pre- and postinfusion of adrenomedullin (1
µg/kg/min) attenuated the reduction in regional blood flow after MCAO
and decreased the degree of ischemic brain injury. These workers
concluded that adrenomedullin may have a role in preventing ischemic
brain injury by increasing collateral circulation. Furthermore,
adrenomedullin administration into the CNS has a number of effects. The
first such study was published in 1995 by Parkes and May (260 ).
Adrenomedullin (100 µg/h) was administered to sheep for 60 min by
intracerebroventricular (icv) infusion. No significant changes were
observed in any cardiovascular parameter measured, although there was a
trend for heart rate and cardiac output to decrease. There was no
effect on plasma ACTH, cortisol, basal AVP, or renin (260 352 ).
However, an inhibitory effect on stimulated AVP release was observed
(260 ). In contrast, Charles et al. recently described the
infusion of adrenomedullin (3.3 pmol/kg/min for 90 min) into the
lateral cerebral ventricle of healthy sheep (353 ). These workers found
that plasma AVP levels rose by 50%, while plasma levels of ACTH and
cortisol increased 3- to 4-fold in response to adrenomedullin. No
changes in arterial pressure, heart rate, or cardiac output were
observed, in accordance with the findings of Parkes and May (260 352 353 354 ).
Yokoi et al. (355 ) showed that icv administration of adrenomedullin into conscious rats attenuated AVP increases induced by giving large volumes of saline (hyperosmolarity) or by reducing plasma volume by giving polyethylene glycol (hypovolemia). Since icv injection of adrenomedullin cannot reach the posterior pituitary (356 ), the exact site(s) of adrenomedullin action is not clear. However, the paraventricular nuclei and supraoptic nuclei are the most likely sites of action; previous studies have shown adrenomedullin immunoreactivity and binding sites exist here (109 357 ). Under conditions of volume excess it makes sense physiologically for adrenomedullin to have an inhibitory effect on AVP production consistent with its renal actions.
For many vasoactive peptides, CNS actions tend to go hand-in-hand with their peripheral effects (358 ). For example, the calcitonin family of peptides and atrial natriuretic factor are known to exert natriuretic and diuretic effects and also act within the CNS to reduce food and water intake (158 350 359 360 361 362 363 ). The hypothalamus has been identified as the main target for inducing anorexia (364 ), and it is known that adrenomedullin, and its receptor, are found here. Based on these observations it was predicted that adrenomedullin actions in the CNS would match the peripheral effects of inhibiting water drinking (365 ) and salt appetite (366 ). In the former study, Murphy and Samson reported that adrenomedullin had significant inhibitory effects on pharmacologically induced (icv administration of angiotensin II or hyperosmotic challenge) or physiologically induced (overnight dehydration) water drinking in the rat (365 ). These actions were exerted with no significant effect on mean arterial blood pressure lending further support to a CNS site of action. Water uptake by rats is not affected by icv administration of adrenomedullin; however, adrenomedullin significantly attenuated saline drinking in response to isotonic hypovolemia in a dose-dependent manner (366 ). The CNS actions of adrenomedullin on water drinking and salt appetite are the first biological effects shown to have physiological relevance. Use of antiadrenomedullin antibodies to passively neutralize endogenous adrenomedullin results in an exaggerated intake of water and salt (366 ). Very recently, an attempt has been made to determine whether endogenous, brain-derived adrenomedullin plays a role in salt appetite in rats. Using antisense oligonucleotide technology, Samson and co-workers (367 ) tried to compromise adrenomedullin production in the brain. This antisense approach significantly reduced production of adrenomedullin; however, it did not result in a decrease of anterior pituitary levels of adrenomedullin, probably due to the diffusion of substances from the site of injection (lateral ventricle). With limited data available it appears antisense injection had an effect on the early phase of saline drinking, i.e., exaggerated drinking was observed.
Intracerebroventricular injection of adrenomedullin also causes a dose-dependent reduction in feeding in rats (158 ). This effect was attenuated by administration of CGRP8–37 indicating that the adrenomedullin action is mediated, in part, by CGRP receptors. Low doses of adrenomedullin administration, which reduced food intake, had no effect on blood pressure when given icv, similar to other studies in the rat described by Samson and Murphy (366 ) and in sheep (260 ). In fact, larger doses of icv adrenomedullin administration are required to induce hypertension than to attenuate food or water intake in rats, suggesting that the receptors mediating hypertension are further from the ventricular system (158 365 366 ).
Intracerebroventricular administration of adrenomedullin has also been shown to prevent reserpine-induced gastric ulcers in rats in a dose-related manner (368 ). It appears, however, that this action is mediated via CGRP receptors since administration of CGRP8–37 abolishes this effect. This is in agreement with studies by Martínez et al. (339 ) who reported that adrenomedullin and CGRP inhibit gastric emptying in rats by acting on the autonomic nervous system via a common mechanism. When given intravenously, adrenomedullin does not modify reserpine-induced gastric damage, lending further support for a central mode of action for adrenomedullin.
The hypotensive effect of adrenomedullin in the periphery is not paralleled in the brain. In fact icv administration of high doses of adrenomedullin provokes hypertension and increased heart rate in conscious, unrestrained rats (369 370 ). These CNS hypertensive effects have also been reported from studies using rats anesthetized with urethane (364 ) but not inactin-anesthetized animals (365 ). The central hypertensive actions of adrenomedullin in conscious rats are dose dependent and not antagonized by CGRP8–37 (370 ). Direct effects of adrenomedullin on sympathetic nerve activity in conscious rats were observed (370 ), and these studies are consistent with the observations of Takahashi et al. (364 ) who showed, in anesthetized rats, that icv adrenomedullin induced an increase in preganglionic sympathetic discharge. Electrical activity of neurons within the area postrema of the medulla are directly affected by adrenomedullin in brain slice preparations (371 372 ).
The temporal aspects of CNS-induced hypertension by adrenomedullin parallel those seen after administration of angiotensin II (369 373 ). This suggests that there is a common mechanism underlying the hypertensive action of both these peptides in the brain. For example, phentolamine blocks the actions of both adrenomedullin and angiotensin II in the CNS, lending further support for adrenomedullin and angiotensin II to act within the brain to stimulate sympathetic nervous system function. Additionally, this type of mechanism can explain the CNS effect of adrenomedullin-induced inhibition of gastric emptying (339 ).
The central hypertensive actions of adrenomedullin may be cardioprotective in that it acts to protect against major cardiovascular collapse such as events encountered during sepsis. To understand completely the mechanism by which CNS administration of adrenomedullin controls cardiovascular parameters, further studies must be carried out to determine whether there is a physiological role of adrenomedullin in this process and what factors regulate adrenomedullin transcription in the brain.
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V. Unresolved Issues and Future Perspectives |
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TopAbstractI. IntroductionII. Synthesis and Secretion...III. Receptors and Signal...IV. Biological Actions of...V. Unresolved Issues and...References |
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Adrenomedullin binding has been demonstrated in most cell types and tissues of the body. The effects of adrenomedullin may be mediated by both specific adrenomedullin binding sites and by CGRP binding sites. The actions of adrenomedullin have been inhibited using CGRP8–37 and adrenomedullin22–52, a CGRP1 receptor antagonist and an adrenomedullin receptor antagonist, respectively. However, neither of these peptides is a very potent inhibitor, and some doubts have been expressed as to their specificity. What is clearly required to further investigate the physiology of adrenomedullin receptors are potent and specific antagonists, preferably small molecule, nonpeptide antagonists.
Two receptor clones have been proposed to have specific adrenomedullin binding properties, L1 and the CRLR-RAMP2 combination. There are, however, situations in which neither of these candidates appears to account for specific adrenomedullin binding. Clearly there are receptors for adrenomedullin that remain to be cloned.
Adrenomedullin has a range of biological actions including vasodilatation, cell growth, regulation of hormone secretion, natriuresis, and antimicrobial effects. Its mechanism of action, however, remains unclear. cAMP is the second messenger in the majority of adrenomedullin actions, but other systems must be involved. The role of NO remains to be elucidated, as does the mechanism of the growth-stimulatory effect of cAMP.
Clearly there are many questions remaining in this field. The answers are likely to contribute, not only to our understanding of adrenomedullin biology, but also to our fundamental understanding both of receptor biology and the regulation of cellular growth.
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Footnotes |
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1 Work in the authors laboratories was supported by the Medical
Research Council, The Wellcome Trust, The British Heart Foundation, and
The Royal Society. 
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TopAbstractI. IntroductionII. Synthesis and Secretion...III. Receptors and Signal...IV. Biological Actions of...V. Unresolved Issues and...References |
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 D. Stolz, M. Christ-Crain, N. G. Morgenthaler, D. Miedinger, J. Leuppi, C. Muller, R. Bingisser, J. Struck, B. Muller, and M. Tamm Plasma Pro-Adrenomedullin But Not Plasma Pro-Endothelin Predicts Survival in Exacerbations of COPD Chest, August 1, 2008; 134(2): 263 - 272. [Abstract] [Full Text] [PDF]
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R. Morimoto, F. Satoh, O. Murakami, T. Hirose, K. Totsune, Y. Imai, Y. Arai, T. Suzuki, H. Sasano, S. Ito, et al. Expression of adrenomedullin 2/intermedin in human adrenal tumors and attached non-neoplastic adrenal tissues J. Endocrinol., July 1, 2008; 198(1): 175 - 183. [Abstract] [Full Text] [PDF]
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A. Geambasu and T. L. Krukoff Adrenomedullin acts in the lateral parabrachial nucleus to increase arterial blood pressure through mechanisms mediated by glutamate and nitric oxide Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2008; 295(1): R38 - R44. [Abstract] [Full Text] [PDF]
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S. Nobata, M. Ogoshi, and Y. Takei Potent cardiovascular actions of homologous adrenomedullins in eels Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2008; 294(5): R1544 - R1553. [Abstract] [Full Text] [PDF]
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 H. Nishida, T. Sato, M. Miyazaki, and H. Nakaya Infarct size limitation by adrenomedullin: protein kinase A but not PI3-kinase is linked to mitochondrial KCa channels Cardiovasc Res, January 15, 2008; 77(2): 398 - 405. [Abstract] [Full Text] [PDF]
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 H.W.F. van Eijndhoven, R. Aardenburg, M.E.A. Spaanderman, J.G.R. De Mey, and L.L.H. Peeters Pregnancy Enhances the Prejunctional Vasodilator Response to Adrenomedullin in Selective Regions of the Arterial Bed of Wistar Rats Reproductive Sciences, December 1, 2007; 14(8): 771 - 779. [Abstract] [PDF]
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 C. Ertmer, A. Morelli, S. Rehberg, M. Lange, C. Hucklenbruch, H. Van Aken, M. Booke, and M. Westphal Exogenous adrenomedullin prevents and reverses hypodynamic circulation and pulmonary hypertension in ovine endotoxaemia Br. J. Anaesth., December 1, 2007; 99(6): 830 - 836. [Abstract] [Full Text] [PDF]
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 X.-H. Zhang, G.-R. Li, and J.-P. Bourreau The effect of adrenomedullin on the L-type calcium current in myocytes from septic shock rats: signaling pathway Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H2888 - H2893. [Abstract] [Full Text] [PDF]
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 K. Tsujikawa, K. Yayama, T. Hayashi, H. Matsushita, T. Yamaguchi, T. Shigeno, Y. Ogitani, M. Hirayama, T. Kato, S.-i. Fukada, et al. Hypertension and dysregulated proinflammatory cytokine production in receptor activity-modifying protein 1-deficient mice PNAS, October 16, 2007; 104(42): 16702 - 16707. [Abstract] [Full Text] [PDF]
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 M. Christ-Crain and B. Muller Biomarkers in respiratory tract infections: diagnostic guides to antibiotic prescription, prognostic markers and mediators Eur. Respir. J., September 1, 2007; 30(3): 556 - 573. [Abstract] [Full Text] [PDF]
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 D. Ribatti, M. T. Conconi, and G. G. Nussdorfer Nonclassic Endogenous Novel Regulators of Angiogenesis Pharmacol. Rev., June 1, 2007; 59(2): 185 - 205. [Abstract] [Full Text] [PDF]
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 S. Q. Khan, R. J. O'Brien, J. Struck, P. Quinn, N. Morgenthaler, I. Squire, J. Davies, A. Bergmann, and L. L. Ng Prognostic Value of Midregional Pro-Adrenomedullin in Patients With Acute Myocardial Infarction: The LAMP (Leicester Acute Myocardial Infarction Peptide) Study J. Am. Coll. Cardiol., April 10, 2007; 49(14): 1525 - 1532. [Abstract] [Full Text] [PDF]
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C. Gibbons, R. Dackor, W. Dunworth, K. Fritz-Six, and K. M. Caron Receptor Activity-Modifying Proteins: RAMPing up Adrenomedullin Signaling Mol. Endocrinol., April 1, 2007; 21(4): 783 - 796. [Abstract] [Full Text] [PDF]
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 G. Fejes-Toth and A. Naray-Fejes-Toth Early Aldosterone-Regulated Genes in Cardiomyocytes: Clues to Cardiac Remodeling? Endocrinology, April 1, 2007; 148(4): 1502 - 1510. [Abstract] [Full Text] [PDF]
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V. Ramachandran, T. Arumugam, R. F. Hwang, J. K. Greenson, D. M. Simeone, and C. D. Logsdon Adrenomedullin Is Expressed in Pancreatic Cancer and Stimulates Cell Proliferation and Invasion in an Autocrine Manner via the Adrenomedullin Receptor, ADMR Cancer Res., March 15, 2007; 67(6): 2666 - 2675. [Abstract] [Full Text] [PDF]
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K. Caron, J. Hagaman, T. Nishikimi, H.-S. Kim, and O. Smithies Adrenomedullin gene expression differences in mice do not affect blood pressure but modulate hypertension-induced pathology in males PNAS, February 27, 2007; 104(9): 3420 - 3425. [Abstract] [Full Text] [PDF]
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 E. Gonzalez-Rey, A. Chorny, F. O'Valle, and M. Delgado Adrenomedullin Protects from Experimental Arthritis by Down-Regulating Inflammation and Th1 Response and Inducing Regulatory T Cells Am. J. Pathol., January 1, 2007; 170(1): 263 - 271. [Abstract] [Full Text] [PDF]
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L. J Pearson, C. Rait, M G. Nicholls, T. G Yandle, and J. J Evans Regulation of adrenomedullin release from human endothelial cells by sex steroids and angiotensin-II. J. Endocrinol., October 1, 2006; 191(1): 171 - 177. [Abstract] [Full Text] [PDF]
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 L. L. Nikitenko, T. Cross, L. Campo, H. Turley, R. Leek, S. Manek, R. Bicknell, and M. C.P. Rees Expression of Terminally Glycosylated Calcitonin Receptor-Like Receptor in Uterine Leiomyoma: Endothelial Phenotype and Association with Microvascular Density. Clin. Cancer Res., October 1, 2006; 12(19): 5648 - 5658. [Abstract] [Full Text] [PDF]
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E. Marinoni, C. Zacharopoulou, A. Di Rocco, C. Letizia, M. Moscarini, and R. Di Iorio Effect of Betamethasone In Vivo on Placental Adrenomedullin in Human Pregnancy Reproductive Sciences, September 1, 2006; 13(6): 418 - 424. [Abstract] [PDF]
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N. Schwarz, D. Renshaw, S. Kapas, and J. P Hinson Adrenomedullin increases the expression of calcitonin-like receptor and receptor activity modifying protein 2 mRNA in human microvascular endothelial cells. J. Endocrinol., August 1, 2006; 190(2): 505 - 514. [Abstract] [Full Text] [PDF]
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 E Gonzalez-Rey, A Fernandez-Martin, A Chorny, and M Delgado Therapeutic effect of urocortin and adrenomedullin in a murine model of Crohn's disease Gut, June 1, 2006; 55(6): 824 - 832. [Abstract] [Full Text] [PDF]
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E. Gonzalez-Rey, A. Chorny, N. Varela, G. Robledo, and M. Delgado Urocortin and Adrenomedullin Prevent Lethal Endotoxemia by Down-Regulating the Inflammatory Response Am. J. Pathol., June 1, 2006; 168(6): 1921 - 1930. [Abstract] [Full Text] [PDF]
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S. Mittra and J.-P. Bourreau Gs and Gi coupling of adrenomedullin in adult rat ventricular myocytes Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1842 - H1847. [Abstract] [Full Text] [PDF]
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 L. L. Nikitenko, N. Blucher, S. B. Fox, R. Bicknell, D. M. Smith, and M. C. P. Rees Adrenomedullin and CGRP interact with endogenous calcitonin-receptor-like receptor in endothelial cells and induce its desensitisation by different mechanisms. J. Cell Sci., March 1, 2006; 119(Pt 5): 910 - 922. [Abstract] [Full Text] [PDF]
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 A. Al-Ghafra, N.M. Gude, S.P. Brennecke, and R.G. King Increased adrenomedullin protein content and mRNA expression in human fetal membranes but not placental tissue in pre-eclampsia Mol. Hum. Reprod., March 1, 2006; 12(3): 181 - 186. [Abstract] [Full Text] [PDF]
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N. G. Morgenthaler, J. Struck, C. Alonso, and A. Bergmann Measurement of Midregional Proadrenomedullin in Plasma with an Immunoluminometric Assay Clin. Chem., October 1, 2005; 51(10): 1823 - 1829. [Abstract] [Full Text] [PDF]
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 J. M. Bomberger, W. S. Spielman, C. S. Hall, E. J. Weinman, and N. Parameswaran Receptor Activity-modifying Protein (RAMP) Isoform-specific Regulation of Adrenomedullin Receptor Trafficking by NHERF-1 J. Biol. Chem., June 24, 2005; 280(25): 23926 - 23935. [Abstract] [Full Text] [PDF]
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 N. Cuesta, A. Martinez, F. Cuttitta, and E. Zudaire Identification of Adrenomedullin in Avian Type II Pneumocytes: Increased Expression after Exposure to Air Pollutants J. Histochem. Cytochem., June 1, 2005; 53(6): 773 - 780. [Abstract] [Full Text] [PDF]
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 D. L. Hay, G. Christopoulos, A. Christopoulos, D. R. Poyner, and P. M. Sexton Pharmacological Discrimination of Calcitonin Receptor: Receptor Activity-Modifying Protein Complexes Mol. Pharmacol., May 1, 2005; 67(5): 1655 - 1665. [Abstract] [Full Text] [PDF]
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 K. Boussery, C. Delaey, and J. Van de Voorde The Vasorelaxing Effect of CGRP and Natriuretic Peptides in Isolated Bovine Retinal Arteries Invest. Ophthalmol. Vis. Sci., April 1, 2005; 46(4): 1420 - 1427. [Abstract] [Full Text] [PDF]
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J. M. Bomberger, N. Parameswaran, C. S. Hall, N. Aiyar, and W. S. Spielman Novel Function for Receptor Activity-modifying Proteins (RAMPs) in Post-endocytic Receptor Trafficking J. Biol. Chem., March 11, 2005; 280(10): 9297 - 9307. [Abstract] [Full Text] [PDF]
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M. Westphal, M. Booke, and A.T. Dinh-Xuan Adrenomedullin: a smart road from pheochromocytoma to treatment of pulmonary hypertension Eur. Respir. J., October 1, 2004; 24(4): 518 - 520. [Full Text] [PDF]
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N. Ozawa, M. Shichiri, N. Fukai, T. Yoshimoto, and Y. Hirata Regulation of Adrenomedullin Gene Transcription and Degradation by the c-myc Gene Endocrinology, September 1, 2004; 145(9): 4244 - 4250. [Abstract] [Full Text] [PDF]
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 S. D. Brain and A. D. Grant Vascular Actions of Calcitonin Gene-Related Peptide and Adrenomedullin Physiol Rev, July 1, 2004; 84(3): 903 - 934. [Abstract] [Full Text] [PDF]
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 I. Haulica, W. Bild, C. Mihaila, D. N Serban, L. Serban, D. Boisteanu, T. Ionita, and O. Radasanu Comparative study of the inhibitory effects of adrenomedullin on angiotensin II contraction in rat conductance and resistance arteries Journal of Renin-Angiotensin-Aldosterone System, June 1, 2004; 5(2): 79 - 83. [Abstract] [PDF]
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M. Luodonpaa, H. Leskinen, M. Ilves, O. Vuolteenaho, and H. Ruskoaho Adrenomedullin modulates hemodynamic and cardiac effects of angiotensin II in conscious rats Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2004; 286(6): R1085 - R1092. [Abstract] [Full Text] [PDF]
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 S. W. Olson, L. E. Deal, and M. Piesman Epinephrine-Secreting Pheochromocytoma Presenting with Cardiogenic Shock and Profound Hypocalcemia Ann Intern Med, May 18, 2004; 140(10): 849 - 851. [Full Text] [PDF]
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W. Huang, L. Wang, M. Yuan, J. Ma, and Y. Hui Adrenomedullin Affects Two Signal Transduction Pathways and the Migration in Retinal Pigment Epithelial Cells Invest. Ophthalmol. Vis. Sci., May 1, 2004; 45(5): 1507 - 1513. [Abstract] [Full Text] [PDF]
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 P. Niu, T. Shindo, H. Iwata, S. Iimuro, N. Takeda, Y. Zhang, A. Ebihara, Y. Suematsu, K. Kangawa, Y. Hirata, et al. Protective Effects of Endogenous Adrenomedullin on Cardiac Hypertrophy, Fibrosis, and Renal Damage Circulation, April 13, 2004; 109(14): 1789 - 1794. [Abstract] [Full Text] [PDF]
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 S. Kapas, K. Pahal, A.T. Cruchley, E. Hagi-Pavli, and J.P. Hinson Expression of Adrenomedullin and its Receptors in Human Salivary Tissue Journal of Dental Research, April 1, 2004; 83(4): 333 - 337. [Abstract] [Full Text] [PDF]
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 J. Balasch, M. Guimera, O. Martinez-Pasarell, J. Ros, J. A. Vanrell, and W. Jimenez Adrenomedullin and vascular endothelial growth factor production by follicular fluid macrophages and granulosa cells Hum. Reprod., April 1, 2004; 19(4): 808 - 814. [Abstract] [Full Text] [PDF]
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K. M. I. Caron, L. R. James, H.-S. Kim, J. Knowles, R. Uhlir, L. Mao, J. R. Hagaman, W. Cascio, H. Rockman, and O. Smithies Cardiac hypertrophy and sudden death in mice with a genetically clamped renin transgene PNAS, March 2, 2004; 101(9): 3106 - 3111. [Abstract] [Full Text] [PDF]
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K. Boussery, C. Delaey, and J. Van de Voorde Influence of Adrenomedullin on Tone of Isolated Bovine Retinal Arteries Invest. Ophthalmol. Vis. Sci., February 1, 2004; 45(2): 552 - 559. [Abstract] [Full Text] [PDF]
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 E. Hagi-Pavli, P. M. Farthing, and S. Kapas Stimulation of adhesion molecule expression in human endothelial cells (HUVEC) by adrenomedullin and corticotrophin Am J Physiol Cell Physiol, February 1, 2004; 286(2): C239 - C246. [Abstract] [Full Text]
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 G. M. Kravtsov, I. S. S. Hwang, and F. Tang The Inhibitory Effect of Adrenomedullin in the Rat Ileum: Cross-Talk with {beta}3-Adrenoceptor in the Serotonin-Induced Muscle Contraction J. Pharmacol. Exp. Ther., January 1, 2004; 308(1): 241 - 248. [Abstract] [Full Text] [PDF]
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 H. Li, J. Dakour, S. Kaufman, L. J. Guilbert, B. Winkler-Lowen, and D. W. Morrish Adrenomedullin Is Decreased in Preeclampsia Because of Failed Response to Epidermal Growth Factor and Impaired Syncytialization Hypertension, November 1, 2003; 42(5): 895 - 900. [Abstract] [Full Text] [PDF]
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E. S. Dettmann, I. Vysniauskiene, R. Wu, J. Flammer, and I. O. Haefliger Adrenomedullin-Induced Endothelium-Dependent Relaxation in Porcine Ciliary Arteries Invest. Ophthalmol. Vis. Sci., September 1, 2003; 44(9): 3961 - 3966. [Abstract] [Full Text] [PDF]
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 D. Yoshikawa, F. Kawahara, N. Okano, H. Hiraoka, Y. Kadoi, N. Fujita, T. Morita, and F. Goto Increased Plasma Concentrations of the Mature Form of Adrenomedullin During Cardiac Surgery and Hepatosplanchnic Hypoperfusion Anesth. Analg., September 1, 2003; 97(3): 663 - 670. [Abstract] [Full Text] [PDF]
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 R. P. Allaker and S. Kapas Adrenomedullin Expression by Gastric Epithelial Cells in Response to Infection Clin. Vaccine Immunol., July 1, 2003; 10(4): 546 - 551. [Abstract] [Full Text] [PDF]
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N. Fukai, M. Shichiri, N. Ozawa, M. Matsushita, and Y. Hirata Coexpression of Calcitonin Receptor-Like Receptor and Receptor Activity-Modifying Protein 2 or 3 Mediates the Antimigratory Effect of Adrenomedullin Endocrinology, February 1, 2003; 144(2): 447 - 453. [Abstract] [Full Text] [PDF]
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P. Hasbak, O. S. Opgaard, K. Eskesen, S. Schifter, H. Arendrup, J. Longmore, and L. Edvinsson Investigation of CGRP Receptors and Peptide Pharmacology in Human Coronary Arteries. Characterization with a Nonpeptide Antagonist J. Pharmacol. Exp. Ther., January 1, 2003; 304(1): 326 - 333. [Abstract] [Full Text] [PDF]
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K. Marutsuka, K. Hatakeyama, A. Yamashita, Y. Sato, A. Sumiyoshi, and Y. Asada Adrenomedullin augments the release and production of tissue factor pathway inhibitor in human aortic endothelial cells Cardiovasc Res, January 1, 2003; 57(1): 232 - 237. [Abstract] [Full Text] [PDF]
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 S. Hippenstiel, M. Witzenrath, B. Schmeck, A. Hocke, M. Krisp, M. Krull, J. Seybold, W. Seeger, W. Rascher, H. Schutte, et al. Adrenomedullin Reduces Endothelial Hyperpermeability Circ. Res., October 4, 2002; 91(7): 618 - 625. [Abstract] [Full Text] [PDF]
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G. Neri, S. Bova, L. K. Malendowicz, G. Mazzocchi, and G. G. Nussdorfer Simulated microgravity impairs aldosterone secretion in rats: possible involvement of adrenomedullin Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2002; 283(4): R832 - R836. [Abstract] [Full Text] [PDF]
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T. Tokudome, T. Horio, F. Yoshihara, S.-i. Suga, Y. Kawano, M. Kohno, and K. Kangawa Adrenomedullin Inhibits Doxorubicin-Induced Cultured Rat Cardiac Myocyte Apoptosis via a cAMP-Dependent Mechanism Endocrinology, September 1, 2002; 143(9): 3515 - 3521. [Abstract] [Full Text] [PDF]
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M. Zhou, Z. F. Ba, I. H. Chaudry, and P. Wang Adrenomedullin binding protein-1 modulates vascular responsiveness to adrenomedullin in late sepsis Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2002; 283(3): R553 - R560. [Abstract] [Full Text] [PDF]
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D. R. Poyner, P. M. Sexton, I. Marshall, D. M. Smith, R. Quirion, W. Born, R. Muff, J. A. Fischer, and S. M. Foord International Union of Pharmacology. XXXII. The Mammalian Calcitonin Gene-Related Peptides, Adrenomedullin, Amylin, and Calcitonin Receptors Pharmacol. Rev., June 1, 2002; 54(2): 233 - 246. [Abstract] [Full Text] [PDF]
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 H. Ruan, N. Hacohen, T. R. Golub, L. Van Parijs, and H. F. Lodish Tumor Necrosis Factor-{alpha} Suppresses Adipocyte-Specific Genes and Activates Expression of Preadipocyte Genes in 3T3-L1 Adipocytes: Nuclear Factor-{kappa}B Activation by TNF-{alpha} Is Obligatory Diabetes, May 1, 2002; 51(5): 1319 - 1336. [Abstract] [Full Text] [PDF]
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L'H. Ouafik, S. Sauze, F. Boudouresque, O. Chinot, C. Delfino, F. Fina, V. Vuaroqueaux, C. Dussert, J. Palmari, H. Dufour, et al. Neutralization of Adrenomedullin Inhibits the Growth of Human Glioblastoma Cell Lines in Vitro and Suppresses Tumor Xenograft Growth in Vivo Am. J. Pathol., April 1, 2002; 160(4): 1279 - 1292. [Abstract] [Full Text] [PDF]
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A. T. Baumer, C. Schumann, B. Cremers, G. Itter, W. Linz, F. Jockenhovel, and M. Bohm Gene expression of adrenomedullin in failing myocardium: comparison to atrial natriuretic peptide J Appl Physiol, March 1, 2002; 92(3): 1058 - 1063. [Abstract] [Full Text] [PDF]
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J. J. Miret, L. Rakhilina, L. Silverman, and B. Oehlen Functional Expression of Heteromeric Calcitonin Gene-related Peptide and Adrenomedullin Receptors in Yeast J. Biol. Chem., February 22, 2002; 277(9): 6881 - 6887. [Abstract] [Full Text] [PDF]
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 V. A. Cameron, D. J. Autelitano, J. J. Evans, L. J. Ellmers, E. A. Espiner, M. G. Nicholls, and A. M. Richards Adrenomedullin expression in rat uterus is correlated with plasma estradiol Am J Physiol Endocrinol Metab, January 1, 2002; 282(1): E139 - E146. [Abstract] [Full Text] [PDF]
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 G. S. Filippatos, N. Gangopadhyay, O. Lalude, N. Parameswaran, S. I. Said, W. Spielman, and B. D. Uhal Regulation of apoptosis by vasoactive peptides Am J Physiol Lung Cell Mol Physiol, October 1, 2001; 281(4): L749 - L761. [Abstract] [Full Text] [PDF]
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D. J Autelitano, R. Ridings, and F. Tang Adrenomedullin is a regulated modulator of neonatal cardiomyocyte hypertrophy in vitro Cardiovasc Res, August 1, 2001; 51(2): 255 - 264. [Abstract] [Full Text] [PDF]
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L.L. Nikitenko, N.S. Brown, D.M. Smith, I.Z. MacKenzie, R. Bicknell, and M.C.P. Rees Differential and cell-specific expression of calcitonin receptor-like receptor and receptor activity modifying proteins in the human uterus Mol. Hum. Reprod., July 1, 2001; 7(7): 655 - 664. [Abstract] [Full Text] [PDF]
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 C. Wang, E. Dobrzynski, J. Chao, and L. Chao Adrenomedullin gene delivery attenuates renal damage and cardiac hypertrophy in Goldblatt hypertensive rats Am J Physiol Renal Physiol, June 1, 2001; 280(6): F964 - F971. [Abstract] [Full Text] [PDF]
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D. Naot, K. E. Callon, A. Grey, G. J. S. Cooper, I. R. Reid, and J. Cornish A Potential Role for Adrenomedullin as a Local Regulator of Bone Growth Endocrinology, May 1, 2001; 142(5): 1849 - 1857. [Abstract] [Full Text]
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T. Udono, K. Takahashi, M. Nakayama, A. Yoshinoya, K. Totsune, O. Murakami, Y. K. Durlu, M. Tamai, and S. Shibahara Induction of Adrenomedullin by Hypoxia in Cultured Retinal Pigment Epithelial Cells Invest. Ophthalmol. Vis. Sci., April 1, 2001; 42(5): 1080 - 1086. [Abstract] [Full Text]
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P. Rocchi, F. Boudouresque, A. J. Zamora, X. Muracciole, E. Lechevallier, P.-M. Martin, and L'H. Ouafik Expression of Adrenomedullin and Peptide Amidation Activity in Human Prostate Cancer and in Human Prostate Cancer Cell Lines Cancer Res., February 1, 2001; 61(3): 1196 - 1206. [Abstract] [Full Text]
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 M. Fujioka, K. Nishio, T. Sakaki, N. Minamino, and K. Kitamura Adrenomedullin in Patients With Cerebral Vasospasm After Aneurysmal Subarachnoid Hemorrhage Stroke, December 1, 2000; 31 (12): 3079 - 3083. [Full Text]
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A. Ladoux and C. Frelin Coordinated Up-regulation by Hypoxia of Adrenomedullin and One of Its Putative Receptors (RDC-1) in Cells of the Rat Blood-Brain Barrier J. Biol. Chem., December 15, 2000; 275(51): 39914 - 39919. [Abstract] [Full Text] [PDF]
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R. Pio, A. Martinez, E. J. Unsworth, J. A. Kowalak, J. A. Bengoechea, P. F. Zipfel, T. H. Elsasser, and F. Cuttitta Complement Factor H Is a Serum-binding Protein for Adrenomedullin, and the Resulting Complex Modulates the Bioactivities of Both Partners J. Biol. Chem., April 6, 2001; 276(15): 12292 - 12300. [Abstract] [Full Text] [PDF]
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K. M. Caron and O. Smithies Extreme hydrops fetalis and cardiovascular abnormalities in mice lacking a functional Adrenomedullin gene PNAS, January 16, 2001; 98(2): 615 - 619. [Abstract] [Full Text] [PDF]
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R. D. Reidelberger, L. Kelsey, and D. Heimann Effects of amylin-related peptides on food intake, meal patterns, and gastric emptying in rats Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2002; 282(5): R1395 - R1404. [Abstract] [Full Text] [PDF]
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