help button home button Endocrine Society Molecular Endocrinology ENDO 08 Sessions Library
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2006-0156
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gibbons, C.
Right arrow Articles by Caron, K. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gibbons, C.
Right arrow Articles by Caron, K. M.
Molecular Endocrinology 21 (4): 783-796
Copyright © 2007 by The Endocrine Society

Minireview

Receptor Activity-Modifying Proteins: RAMPing up Adrenomedullin Signaling

Carrie Gibbons, Ryan Dackor, William Dunworth, Kimberly Fritz-Six and Kathleen M. Caron

Department of Cell and Molecular Physiology (C.G., R.D., W.D., K.F.-S., K.M.C.), Genetics Department (R.D., W.D., K.M.C.), The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

Address all correspondence and requests for reprints to: Kathleen M. Caron, Department of Cell and Molecular Physiology, CB # 7545, 6340B MBRB, 103 Mason Farm Road, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599. E-mail: Kathleen_caron{at}med.unc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 A MULTIFUNCTIONAL PEPTIDE
 AM IN DISEASE
 AM SIGNAL TRANSDUCTION
 RAMP GENE EXPRESSION IN...
 GENETIC MOUSE MODELS
 REFERENCES
 
Adrenomedullin (AM) is a 52-amino-acid multifunctional peptide that circulates in the plasma in the low picomolar range and can exert a multitude of biological effects through an autocrine/paracrine mode of action. The mechanism by which AM transduces its signal represents a novel and pharmacologically tractable paradigm in G protein-coupled receptor signaling. Since its discovery in 1993, the study of AM has emerged into a new field of research with nearly 1800 publications that rivals the renown of other common factors like angiopoetin (1015 publications) and ghrelin (1550 publications). Despite the tremendous strides made in recent years toward unveiling the biochemical and cellular functions of AM, we are still lagging in our understanding of the essential roles of AM in normal and disease physiology. As discussed in this current review, a concerted effort to combine information from clinical, genomic, biochemical, and genetic mouse model sources can provide a focused view to help define the physiological functions of AM. Specifically, we find that certain conditions, such as pregnancy, cardiovascular disease, and sepsis, are associated with robust and dynamic changes in the expression of AM and AM receptor proteins, which together represent an elegant mechanism for altering the physiological responsiveness or function of AM. Thus, the modulation of AM signaling may be further exploited for therapeutic strategies in the management and treatment of human disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
A MULTIFUNCTIONAL PEPTIDE
 AM IN DISEASE
 AM SIGNAL TRANSDUCTION
 RAMP GENE EXPRESSION IN...
 GENETIC MOUSE MODELS
 REFERENCES
 
THE ADRENOMEDULLIN (AM) GENE (1) belongs to the calcitonin superfamily of peptides, which also includes calcitonin, calcitonin gene-related peptide (CGRP), amylin, and intermedin (2, 3). These peptides are grouped by virtue of their strict conservation of an amidated C terminus and an intramolecular disulfide bond that forms an amino acid ring structure. Both features are required for biological activity of the peptides. The mammalian AM gene remains highly conserved throughout evolution (4), implying an essential function for the peptide. Moreover, three species of teleost fish appear to have an expanded complement of AM-like peptides, and this level of gene diversity in fish supports the notion that AM plays numerous roles in the complex physiology of higher model organisms (4).

AM is most closely related to intermedin (also referred to as AM2) and CGRP. In fact, the three peptides bind and activate the same G protein-coupled receptor to elicit similar signal transduction pathways and physiological responses in a variety of cell types. Thus, it remains unclear what distinctive roles, if any, exist for these peptides in mammals. The phenotype of AM null mice (5) is categorically different than that of {alpha}CGRP null mice (6, 7), suggesting that these peptides play distinct roles in the whole animal. However, due to an evolutionary gene duplication on chromosome 7, there exist two CGRP genes in mice, so that the generation of double-knockout mice for both {alpha}CGRP and ßCGRP is needed before making this definitive conclusion. Targeted deletion of the intermedin gene has yet to be described. Thus, future gene knockout studies in mice and the study of the expanded complement of AM-like peptides in fish (8) should help to elucidate the combined physiological effects of this peptide family.

AM gene expression is broadly distributed throughout most organs during both embryonic development and adulthood and is most highly expressed in endothelial cells and vascular smooth muscle cells (9, 10). Consequently, highly vascularized tissues, such as the placenta, lung, heart, and kidney, tend to produce elevated amounts of AM peptide that can be further stimulated by inflammatory cytokines. In the blood, AM peptide circulates bound to AM-binding protein-1 (also called complement factor H) as a possible way to stabilize and direct the peptide to sites of receptor expression (11, 12). Little is known about the metabolism and clearance of AM, but current studies suggest that AM may be metabolized by neutral endopeptidase (13) and cleared in the lung (14).

The cellular signaling mechanisms through which AM mediates its functions vary among species, vascular beds, and arterial vs. venous vessels, but generally involve cAMP, NO, and Ca-dependant mechanisms (15). As depicted in the top panel of Fig. 1Go, AM binding can activate Gs to increase cellular cAMP levels and activate cAMP-dependant protein kinase in smooth muscle cells and endothelial cells. The antiapoptotic effects of AM peptide in myocardial cells have been linked to the phosphatidylinositol 3-kinase/Akt pathway. Finally, in endothelial cells, the MAPK cascade can also be activated by AM. The putative roles these signaling pathways play in cardiovascular disease have been recently reviewed (16). It is probable that combinations of second messenger pathways are responsible for mediating the complex biological effects of AM in different cell types.


Figure 1
View larger version (54K):
[in this window]
[in a new window]

  		Fig. 1. Multiple Second Messenger Responses to AM Mediate a Multitude of Biological Functions

The cellular signaling mechanisms of AM function have mostly been studied in cells of the cardiovascular system, including vascular smooth muscle cells, endothelial cells, and cardiac myocytes. These combined cellular effects lead to numerous physiological outcomes that are interrelated within the whole animal. AC, Adenylate cyclase; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A.

 

   A MULTIFUNCTIONAL PEPTIDE
TOP
 ABSTRACT
 INTRODUCTION
 A MULTIFUNCTIONAL PEPTIDE
AM IN DISEASE
 AM SIGNAL TRANSDUCTION
 RAMP GENE EXPRESSION IN...
 GENETIC MOUSE MODELS
 REFERENCES
 
The best-characterized function of AM is as a potent vasodilator (17). Infusion of AM in humans (18, 19, 20, 21, 22, 23) and in several species of experimental animals (1, 24, 25, 26, 27, 28, 29) causes a prolonged, dose-dependant vasorelaxation and hypotension. However, the precise amount of circulating AM peptide needed to elicit a systemic effect on blood pressure remains unclear. An early study in which healthy human volunteers were iv infused with AM for brief 10-min increments showed that a 40-fold increase in plasma peptide levels was required to induce a change in heart rate and blood pressure (23). Another study showed that infusing physiological levels of AM (2–10 pM) could reduce arterial blood pressure if administered over a longer period of time (21). No changes in heart rate, plasma hormone levels, urinary indices or cAMP levels were detected after this low dose, demonstrating that AM has potent and direct effects on vascular vasodilation. A subsequent study by the same group showed that slightly higher doses of AM could elevate renin and norepinephrine levels as well as increase heart rate and cardiac output and decrease arterial blood pressure (22). Taken together, these results show that modest increases in plasma AM peptide levels can have significant systemic effects, yet the dose, route, and duration of exogenous administration can significantly impact the results. Moreover, because AM naturally exists as a serum-bound paracrine hormone, it is likely that bolus infusion of native peptide may not effectively recapitulate the endogenous functions of the peptide. Consequently, it remains an important issue to determine whether basal levels of endogenous AM contribute to the establishment and maintenance of basal vascular tone and blood pressure.

The multiple physiological and cellular effects of AM peptide are summarized in the bottom panel of Fig. 1Go and have been comprehensively reviewed by others (10, 17, 30, 31). Numerous studies point to either positive or negative inotropic effects of AM on myocytes (32, 33, 34) in addition to inhibition of myocyte protein synthesis and cardiac fibroblast proliferation (35, 36), which together support a role for AM in heart contractility and development (37, 38). AM is also an angiogenic factor (39) that is induced by hypoxia (40) and can alter the permeability of vascular endothelial cells (41). The coupling of these functions with the potent effects of AM on cell migration, growth, and apoptosis have led to the hypothesis that AM may be a key player in tumor growth and metastasis (42, 43). AM may also contribute to blood volume regulation through its natriuretic and diuretic functions in the kidney and its effects on central nervous system control of thirst and salt appetite (43, 44, 45). Finally, AM is highly expressed in the skin and oral mucosa, and this expression pattern has been linked to its potent effect as an antimicrobial peptide (46). It is therefore clear that AM is a multifunctional peptide that can exert many important and interrelated biological functions under both normal and disease conditions.


   AM IN DISEASE
TOP
 ABSTRACT
 INTRODUCTION
 A MULTIFUNCTIONAL PEPTIDE
 AM IN DISEASE
AM SIGNAL TRANSDUCTION
 RAMP GENE EXPRESSION IN...
 GENETIC MOUSE MODELS
 REFERENCES
 
Soon after the discovery of AM (1), clinician scientists began measuring the plasma levels of the peptide in a multitude of different human diseases and conditions. It was rapidly recognized that elevated plasma AM was a predictive indicator of many disease states and that increases in these levels generally correlated with the severity of the disease. However, with more than 60 such clinical papers describing elevated plasma AM levels in such a wide variety of conditions, it seems unlikely that AM is playing a causative role in all of these disease processes. Therefore, we undertook a summary analysis of current clinical papers and grouped the observations into the following broad categories: cancer, cardiovascular, diabetes/obesity, endocrine, hepatic and renal, pulmonary, normal pregnancy, pregnancy complications, and sepsis. The references are provided as categorical lists published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org. The results of our analysis, shown in Fig. 2Go, allow us to draw several conclusions, which cannot be gleaned from any single clinical paper, and shed new insights into the potentially more significant functions of AM in normal and disease physiology.


Figure 2
View larger version (31K):
[in this window]
[in a new window]

  		Fig. 2. Fold Change in Plasma AM Levels in a Variety of Human Conditions

Bars indicate average fold change in circulating AM levels in various disease categories or conditions based on published human clinical data. References used for this analysis are provided in categorical lists published as supplemental data on The Endocrine Society’s Journals Online web site. The horizontal line at 2.3 represents the average fold increase in plasma AM levels across all conditions (excluding sepsis). **, P < 0.007 between pregnancy and all other disease conditions (excluding sepsis). ##, P < 0.001 between normal pregnancy and pregnancy complications. *, P < 0.05 between sepsis and all other conditions. Number on each bar indicates the number of published observations assessing plasma AM levels in each category.

 
First, the categories of cardiovascular disease and hepatic/renal disease share the greatest number of published observations that demonstrate a positive correlation between increased AM plasma levels and disease severity. Simply put, this wealth of supporting evidence provides a compelling argument that, although modest, increases in plasma AM are likely an accurate reflection of homeostatic responses elicited by AM during cardiovascular and renal stresses.

Second, we find that the mean fold change in plasma AM levels between healthy patients and those with any variety of conditions (excluding sepsis) is 2.3. Although it is unclear whether this modest increase in plasma levels is robust enough to elicit a systemic response, it is probable that it is a reflection of localized homeostatic responses to altered cardiovascular status or infection.

Third, the highest increases in plasma AM levels occur during sepsis (20- to 40-fold normal values) and during the course of a normal pregnancy (3- to 5-fold prepregnancy values). These plasma levels are well within the range in which AM elicits a systemic response and may reflect an endocrine or hormonal function of AM signaling during these conditions.

Finally, in contrast to the average 2-fold increase observed in most disease states, complications of pregnancy (including preeclampsia, fetal growth restriction, and gestational diabetes) are generally characterized by a failure of induced AM plasma levels, which normally occurs during the course of a pregnancy. Thus, unlike the homeostatic compensations described for most disease conditions, a failure of elevated AM plasma levels during pregnancy may actually be causative of disease and result in poor reproductive outcomes.


   AM SIGNAL TRANSDUCTION
TOP
 ABSTRACT
 INTRODUCTION
 A MULTIFUNCTIONAL PEPTIDE
 AM IN DISEASE
 AM SIGNAL TRANSDUCTION
RAMP GENE EXPRESSION IN...
 GENETIC MOUSE MODELS
 REFERENCES
 
The mechanism through which AM peptide transduces its cellular effects represents a novel paradigm in G protein-coupled receptor (GPCR) signaling. For many years, identifying a bona fide AM receptor was complicated because at least three different orphan receptors could bind to AM with relatively similar affinities and elicit a dose-dependant cAMP response (47, 48, 49, 50, 51, 52). However, the results for these putative receptors varied widely among different cell types and in their specificity for AM activity vs. CGRP activity. These complications were finally resolved with the identification and cloning of a novel class of GPCR activity-modifying proteins (RAMPs), which associated with the most likely candidate receptor, calcitonin receptor-like receptor (CLR, formerly called CRLR) to dictate its ligand binding specificity. As depicted in Fig. 3Go, McLatchie et al. (53) demonstrated that association of RAMP1 with CLR results in a receptor that binds preferentially to CGRP whereas association of CLR with RAMP2 or RAMP3 confers preferential AM binding. Thus, the spatial and temporal expression of RAMP proteins determines how a cell or tissue will sense and respond to either extracellular AM or CGRP. Given this new paradigm in GPCR signaling, it is not surprising that many pharmaceutical companies and receptor signaling laboratories are studying the pharmacological and biochemical properties of the RAMP-receptor interaction in the hopes of exploiting the RAMPs as potential drug targets for the treatment of human diseases related to either AM or CGRP physiology (54). Particularly exciting is BIBN4096BS, a nonpeptide CGRP antagonist that acts at the extracellular interface of the RAMP1-CLR protein interaction (55) and is currently in clinical trials for the treatment of migraine (56, 57).


Figure 3
View larger version (39K):
[in this window]
[in a new window]

  		Fig. 3. The RAMP/Receptor Paradigm for AM and CGRP Signaling

RAMPs convey receptor specificity by heterodimer formation with CLR in the endoplasmic reticulum followed by localization to the plasma membrane. The association of CLR with a RAMP determines the specificity of ligand binding. This, a CLR/RAMP1 heterodimer (green) binds preferentially to CGRP, whereas association of CLR with either RAMP2 (dark red) or RAMP3 (light yellow) results in preferential binding to AM.

 
The biochemistry and pharmacology of the RAMP-receptor interaction have recently been extensively reviewed by Hay et al. (58). Although the three mammalian RAMPs vary greatly in sequence homology (<30%) and tissue distribution (53, 59), they share a similar molecular mass (160 amino acids) and structure, which consists of a long extracellular amino terminus, a single transmembrane domain, and a short cytoplasmic carboxy terminus. The RAMPs have a broader tissue distribution than AM and CLR, which likely reflects the fact that RAMPs can interact with other class II GPCRs such as calcitonin (60), vasoactive intestinal peptide/pituitary adenylate cyclase activating peptide 1, glucagon, PTH 1 and 2 receptors (61), and a class III GPCR called calcium-sensing receptor (62). Large scale, tissue-specific analysis of the human and mouse transcriptomes reveals that RAMP2 is among the top 10 genes the expression pattern of which correlates with that of CLR, suggesting that most CLR is complexed with RAMP2 to form a functional AM receptor (4, 63).

Even though temporal and spatial gene expression profiles of the RAMPs can be a useful tool for predicting potential receptor interactions and physiological functions, pharmacological data are additionally needed to confirm the identity and function of a proposed receptor and/or peptide. To date, most pharmacological studies of RAMPs have been performed through heterologous cell culture-based systems using CLR as the GPCR of interest. The transmembrane domain of the RAMPs, the most highly conserved region across species, is required for the formation of a stable heterodimer with CLR within the endoplasmic reticulum and Golgi (64). The complex remains stable through localization at the plasma membrane, ligand binding, receptor activation, internalization, and receptor degradation or recycling (58). The use of chimeric RAMP1/2 proteins has shown that their long, extracellular N'-terminal tail confers a specific ligand-binding domain with the unusually extended N'- terminus of CLR (a feature that is common among class II GPCRs, illustrated in Fig. 2Go) (65, 66, 67). Given the relatively small size of the RAMPs, however, it is possible that these different domains do not act autonomously but rather interact together to mediate both receptor interaction and ligand binding affinities (58).

The short, intracellular C-terminal domain of the RAMPs represents a potential interface for interaction with other intracellular molecules, which may govern either receptor trafficking, degradation, or recycling processes that are distinct among the different RAMPs. McLatchie et al. (53) first proposed that the C terminus of RAMP1 and RAMP3 may contain putative endoplasmic retrieval signals (ERS), and this was functionally confirmed by Steiner et al. (64) for CLR/RAMP1. Kuwasako et al. (68) also showed that truncation of RAMP2 in the equivalent ERS domain resulted in retention of CLR/RAMP2 in the endoplasmic reticulum, whereas truncation of the RAMP1 ERS domain altered CGRP binding without affecting cell surface expression. Thus, there appear to be functional differences among the conserved domains of the RAMP C-terminal tails.

The C-terminal domain of human (h) RAMP3, unlike RAMP1 and RAMP2, also contains a type 1 PDZ recognition motif at residues 145–148, which suggests a potential role in regulating receptor trafficking after agonist stimulation (10). An elegant study was carried out by Bomberger et al. (69) in both human embryonic kidney (HEK)293 cells, transfected with bovine CLR and hRAMP3 (hRAMP3), and in human proximal tubule cells that endogenously express human CLR and hRAMP3. The hRAMP3 PDZ domain interacted in both cases with Na+/H+ exchange regulatory factor (NHERF), resulting in inhibition of receptor internalization. As expected, deletion of this domain resulted in decreased cell surface expression while not affecting agonist binding (68). This same group also found that the hRAMP3 PDZ motif could also interact with N-ethylmaleimide-sensitive factor (NSF) to promote slow recycling of bovine CLR/hRAMP3, in transfected HEK293 and in endogenously expressing rat mesangial cells as measured by radioligand binding assays and adenylate cyclase activity (70). In contrast, Kuwasako et al. (68) recently showed that overexpression of NSF did not alter cell surface expression of AM-mediated trafficking of human CLR/hRAMP3 in stably transfected HEK293 cells. Discrepancies between these two studies could be due to differences between assay techniques, stable vs. transient transfections, or the use of different species forms of CLR (bovine vs. human). Indeed, as reviewed by Kuwasako et al. (72) and demonstrated by Hay et al. (71), there is marked variation in the pharmacological properties of CLR/RAMP complexes from different species. In a broader sense, it is also worth considering that although hRAMP3 contains a conserved PDZ recognition motif, rodent RAMP3 does not, and so the effects of RAMPs on receptor trafficking may vary between species. Thus, more studies need to be performed to address the role of the C-terminal domain of RAMPs in receptor complex formation and plasma membrane targeting. In addition, potential differences in downstream signal transduction events that may be mediated by the various RAMPs have not yet been proven.


   RAMP GENE EXPRESSION IN HEALTH AND DISEASE
TOP
 ABSTRACT
 INTRODUCTION
 A MULTIFUNCTIONAL PEPTIDE
 AM IN DISEASE
 AM SIGNAL TRANSDUCTION
 RAMP GENE EXPRESSION IN...
GENETIC MOUSE MODELS
 REFERENCES
 
As recently summarized in table format by Hay et al. (58), very few studies have associated the expression of RAMP-receptor components in specific cells or tissues with peptide pharmacology or physiology. Because simple gene expression analysis is not always predictive of protein function, this type of analysis is crucial to better our understanding of RAMP-receptor function. This is particularly challenging given that multiple RAMPs may interact with multiple GPCRs within the same cell, possibly at different subcellular locations. In a similar vein, correlations between the biochemical observations previously described and the dynamic changes in the expression of RAMP gene expression that take place during disease will be equally challenging. But, with the added benefit of making these types of comparisons between normal and disease states and in the same cells and tissues in which AM peptide is elevated, some enlightening predictions may be made about the function of and responsiveness to AM signaling during normal and disease physiology.

Microarray-based, large-scale analysis of the human and mouse transcriptomes for 46 human and 45 mouse tissues [http://symatlas.gnf.org/SymAtlas/, (63)] reveals that RAMP2 is relatively equally distributed among most tissues, with the notable exception of very high expression levels in the lungs, female reproductive tissues (placenta, uterus, umbilical cord, and ovary), and adipocytes. Moreover, this pattern of tissue-specific expression is conserved between human and mouse and, as previously mentioned, correlates highly with the expression pattern of human and mouse CLR. RAMP3 gene expression in humans is also distributed evenly across most healthy tissues, but is most robust in the lung, followed by the thyroid and heart. In contrast, high levels of RAMP3 expression in mice are restricted to female reproductive tissues including the uterus, ovary, and placenta. This species-specific difference in basal RAMP3 levels may be a consequence of the endocrine status of the tissues collected for human vs. mouse samples (especially for the estrogen-sensitive, cycling uterus) or, alternatively, may reflect different physiological functions of RAMP3 between humans and mice.

Modest changes in RAMP gene expression have been demonstrated in many cell types and tissues for a variety of different cardiovascular conditions including, most notably, heart failure, myocardial infarction, hypertension, renal failure, and hypoxia. These data have been summarized in table format in two recent review articles (72, 73). For the majority of these studies the authors focused on the expression of CLR and RAMP2, because these proteins represent the most likely signaling complex for transducing AM biological activity. Although there are some variations among studies, most in vivo models of induced cardiovascular disease show a modest increase in RAMP2 gene expression levels in target tissues, and occasional changes in RAMP1 and RAMP3 expression. Hopefully, as the biochemistry of the CLR/RAMP3 complex becomes more clear, future investigations will include a comparative assessment of all known AM signaling components. Nevertheless, from the currently available data, it is tempting to speculate that modest, tissue-specific alterations in RAMP gene expression are associated with altered tissue responsiveness to locally increased AM peptide as a potential mechanism for protecting tissues against disease damage. It remains to be determined whether AM peptide itself contributes to the dynamic modulation of RAMP and CLR receptor expression.

It is worth noting that the most robust changes in RAMP and CLR expression levels coincide precisely with those situations in which plasma AM is most elevated, i.e. pregnancy and sepsis (see Fig. 2Go). For example, robust changes in RAMP gene expression occur in female reproductive tissues during the estrous cycle and pregnancy. Thota and Yallampalli (74) showed that the expression of all three RAMPs is elevated in pregnant vs. nonpregnant rat uterus. During normal pregnancy in rats, the uterine arteries and placenta were found to have high levels of CLR and RAMP1 expression, although RAMP2 and RAMP3 were not evaluated (75, 76). In contrast, in women with preeclampsia, the umbilical arteries and uterine muscle were found to have decreased levels of RAMP1 (77) and CLR and RAMP2 (78, 79) when compared with normal pregnant control samples. Using comparative microarray-based technology in wild-type and estrogen receptor knockout mice, Hewitt et al. (80) recently found that RAMP3, with a 43-fold increase, was one of the most potently induced genes in the uterus of wild-type mice treated with estrogen. Other studies have shown that regulatory regions of both the AM and RAMP3 genes contain estrogen response elements that bind estrogen receptor in chromatin immunoprecipitation assays and, furthermore, that the expression of these genes could not be stimulated by estrogen in estrogen receptor-{alpha} null mice (81). Taken together, results from these expression studies provide a molecular signaling mechanism, in part regulated by sex steroids (81, 82), that is capable of directing the complex modulation of AM responsiveness in reproductive tissues during estrous and pregnancy and, if altered, may lead to complications of pregnancy such as preeclampsia.

Recent studies by several groups have also confirmed a dynamic and robust change in the expression of RAMPs and CLR during sepsis, the pathological condition in which plasma AM is most elevated. Ono et al. (83) found that RAMP3 message levels were increased approximately 40-fold at the same time that CLR and RAMP2 expression levels were reduced by 95% in the lungs of mice treated with bacterial lipopolysaccharide (LPS) for 12 h. Using a polymicrobial model of sepsis, Ornan et al. (84) also showed that RAMP3 expression is markedly elevated in lungs during the early hyperdynamic stage of sepsis, but not in the later hypodynamic phase. This group also found that pulmonary clearance of AM is reduced in the hypodynamic phase (85). Although plasma AM levels are elevated continuously throughout sepsis, AM may be primarily acting in the early hyperdynamic response because transition to the later hypodynamic phase is associated with marked reduction in CLR and RAMP2 gene expression (86) and reduction of AM binding protein-1 (87), which ultimately result in reduced vascular responsiveness to systemic AM. Finally, Nagoshi et al. (88) found that TNF-{alpha} significantly reduced the expression of CLR, RAMP1, and RAMP2 in cultured human coronary artery smooth muscle cells in a time- and dose-dependent manner, with no effect on RAMP3 levels. Taken together, these studies indicate that during septic shock there are dynamic changes in the expression of genes involved in AM signaling, particularly involving a switch from RAMP2 to RAMP3 expression, which may underlie the marked increases in plasma AM and/or represent an altered responsiveness to AM peptide.

Thus, as illustrated in Fig. 4Go, combined clinical, genomic, biochemical, and pharmacological data can provide an elegant model for altered AM tissue responsiveness. In most human and rodent tissues that are responsive to AM peptide, RAMP2 is predominantly expressed in the basal state whereas expression levels of RAMP3 remain relatively low (89, 90) (top panel of Fig. 4Go). However, under certain conditions or disease states such as pregnancy and sepsis (when AM gene expression is markedly up-regulated), the expression of RAMP3 is dramatically increased while, at the same time, the gene expression of RAMP2, and often CLR, is markedly down-regulated in target tissues (bottom panel of Fig. 4Go). Based on biochemical evidence, possible outcomes of increased RAMP3 levels are to switch the cell from a highly AM-responsive state (RAMP2 expression) to one that has blunted AM responsiveness (RAMP3 expression) by virtue of the fact that NHERF-bound CLR/RAMP3 will remain desensitized at the plasma membrane. Perhaps in conjunction with this mechanism, NSF binding to the CLR/RAMP3 complex may be important for maintaining a low level of AM responsiveness, or alternatively, for acting as a rapid clearance mechanism for excess extracellular peptide levels through receptor recycling. It is also possible that the CLR/RAMP3 complex may elicit a different intracellular second messenger response than the CLR/RAMP2 complex, although data to confirm this hypothesis do not yet exist. Finally, it must be considered that robust up-regulation of RAMP3 gene expression may be occurring for reasons that are completely independent of AM and CLR signaling, such as modulating the function or trafficking of other GPCRs. A few recent studies have shown that the gene regulation of AM, CLR, and RAMPs can have shared stimuli such as oxygen tension, endocrine hormones, and inflammatory cytokines (40, 80, 81, 82, 91, 92). Thus, future studies in cell-based systems and animal models should continue to address the mechanisms that coordinately regulate AM, CLR, and RAMP gene expression.


Figure 4
View larger version (49K):
[in this window]
[in a new window]

  		Fig. 4. Model for Altered AM Signaling during Pregnancy and Sepsis

During basal conditions, RAMP2 (dark red) is more abundantly expressed than RAMP3 (light yellow) in most tissues. Upon AM binding, the CLR/RAMP complexes are internalized and degraded. Under certain physiological or pathological conditions, such as pregnancy and sepsis, when circulating AM levels are very high, there is a robust increase in RAMP3 expression, and often a concomitant decrease in CLR and RAMP2 expression. This may result in reduced AM responsiveness as well as changes in receptor internalization and recycling. NHERF (light purple boxes) binding to CLR/RAMP3 results in desensitized receptors that remain at the plasma membrane. In contrast, NSF (green starbursts) binding to CLR/RAMP3 has been proposed to result in receptor recycling and possibly increased clearance of AM.

 

   GENETIC MOUSE MODELS
TOP
 ABSTRACT
 INTRODUCTION
 A MULTIFUNCTIONAL PEPTIDE
 AM IN DISEASE
 AM SIGNAL TRANSDUCTION
 RAMP GENE EXPRESSION IN...
 GENETIC MOUSE MODELS
REFERENCES
 
The recent use of genetic engineering approaches in whole animal systems has validated the most predictable biological functions of AM in developmental, cardiovascular, and reproductive physiology. As described in detail below, it is perhaps no coincidence that the most readily observed phenotypes precisely parallel the predicted functions of AM that have been gleaned from clinical, genomic, and biochemical data.

AM in Embryonic Development
As predicted by the fact that the AM gene is highly conserved from starfish to humans (93), classical gene knockout approaches have proven that the AM gene is essential for survival. We found that AM–/– mice failed to survive beyond embryonic d 14.5 (E14.5). Their lethality was associated with extreme hydrops fetalis as well as developmental defects of the heart and arterial vasculature (5). The requirement of AM peptide for embryonic development was similarly supported by AM gene-targeted null mice from two other independent laboratories (94, 95).

To provide in vivo genetic evidence for the molecular identity of a bona fide AM receptor, we recently generated mice with a targeted deletion of the gene that encodes the CLR receptor, Calcrl (96). Like AM, CLR is also essential for survival because Calcrl–/– embryos die between E13.5 and E14.5. Significantly, the Calcrl–/– embryos precisely phenocopy the previously described AM–/– mice, including the presence of hydrops fetalis, thin vascular walls, and smaller hearts. Additional studies on the growth and proliferation of cardiovascular tissues were also in keeping with in vitro studies that suggested a role for AM signaling in cardiac myocyte and vascular smooth muscle cell growth and proliferation (35, 36). Thus, the Calcrl gene-targeted model provides the first in vivo and genetic evidence to support the notion that CLR functions as an AM receptor during development.

Czyzyk et al. (97) recently published another interesting report that corroborates the essential role of AM signaling during embryonic development. In their study, the authors used gene targeting to generate a mouse line lacking the gene that encodes for peptidyglycine {alpha}-amidating monooxygenase (PAM), the sole enzyme involved in peptide amidation in the mouse. Mice lacking PAM suffer from embryonic lethality, edema, and cardiovascular defects that are almost identical phenocopies of the AM and Calcrl knockout models. The authors conclude that loss of AM peptide amidation, which is normally required for full biological activity of the peptide, results in a loss of AM function, probably by reducing its ability to bind to its receptor(s). It is likely that inactivation of AM is the primary cause of the observed phenotype in PAM null mice because other knockout models for amidated peptides exist in which similar phenotypes are not observed. In conclusion, a strikingly similar phenotype is recapitulated in three separate gene knockout models that cause disruption of AM signaling, demonstrating the critical importance of AM signaling in embryonic and cardiovascular development.

AM in Pregnancy and Fertility
Many recent studies in animal models and humans point to a potential role for AM in pregnancy. These data have been extensively reviewed in two recent articles (98, 99). Pregnancy is associated with increased maternal blood volume and cardiac output and reduced vascular resistance and systemic blood pressure, which are required to maintain the low-resistance placental blood flow for appropriate maternal-fetal exchange of nutrients and wastes. The placenta itself is a highly vascularized organ, which, if defective, underlies many pathological complications of pregnancy such as preeclampsia, fetal growth restriction, placenta previa, and placenta accreta. AM is highly expressed by both the receptive maternal uterine wall and the fetal trophoectoderm at the time of implantation (100), suggesting that signals from both mother and fetus may be important for implantation and subsequent placentation. Our recent reproductive studies in female AM+/– mice have strongly supported this notion by demonstrating that a modest 50% reduction in maternal AM gene expression leads to profound defects in fertility, characterized by abnormal implantation, placentation, and fetal growth (100). The contribution of fetal AM was also substantiated because AM–/– embryos more often displayed fetal growth restriction than either AM+/– or AM+/+ embryos. Significantly, placentas from growth-restricted embryos showed defects in trophoblast cell invasion that are similar to defects that underlie human preeclampsia and placenta accreta. Finally, in our preliminary reproductive studies of Calcrl+/– females (96), we have also observed a significant decrease in litter size (Dackor, R. T., and K. M. Caron, unpublished observation), demonstrating that multiple genetic components of AM signaling contribute to normal pregnancy and fertility. The fact that these reproductive phenotypes occur on a heterozygote background raises the possibility that genetically inherited perturbations in AM signaling may affect reproductive outcomes in humans. Moreover, the pharmacological modulation of uterine AM responsiveness during implantation may prove to be an effective treatment for early pregnancy disorders.

AM in Cardiovascular Protection
Because AM–/– mice are embryonic lethal, several laboratories have focused their phenotyping efforts on the viable AM+/– mice as a model of compromised AM gene expression, as this situation may occur in the human population due to genetic polymorphisms in the AM gene (101). It is increasingly evident that an important role for endogenous AM signaling is to protect cardiovascular tissues, specifically the heart and kidneys, from organ damage after cardiovascular stresses such as hypertension and associated cardiac hypertrophy, myocardial infarction, heart failure, and atherosclerosis (72, 102). AM+/– and AM-overexpressing transgenic mice have been used to demonstrate protective effects of AM on heart and renal tissue after aortic banding (103), angiotensin II infusion (103, 104), bilateral renal artery clamping (105), and atherosclerosis (106). In general, these studies showed that AM+/– mice suffered greater degrees of cardiac hypertrophy and fibrosis and renal dysfunction with glomerular sclerosis after induced cardiovascular stress. In contrast, a transgenic mouse line that expresses rat AM from a murine preproendothelin-1 promoter results in a 2.3-fold increase in plasma AM levels that caused a decrease in blood pressure (107). In these transgenic mice, expression of rat AM in the glomeruli and arterioles of the kidney resulted in less renal damage after induced renal ischemia (105).

Another recent study characterized the effects of mRAMP2 overexpression in smooth muscle cells and found that although the transgenic mice had no differences in basal or induced blood pressure, infusion of AM peptide caused a greater degree of hypotension in the RAMP2 transgenic compared with control animals, likely due to the fact that mesenteric and cutaneous resistance vessels were sensitized to AM in the RAMP2 transgenic line (108). These studies imply that CLR/RAMP2 heterodimers may be an effective and selective target for therapeutic approaches for the treatment of hypertension and other vascular related disease.

Along those lines, Chao and colleagues (109) have shown that adenovirus-mediated gene therapy of human AM improved the cardiovascular and renal disease associated with two hypertensive rat models; the Dahl salt-sensitive hypertensive rat, and the deoxycorticosterone acetate-salt hypertensive rat (110). Taken together, these studies indicate that endogenous AM plays a protective role against organ injury after numerous types of cardiovascular stresses and provide a solid framework for the use of AM or AM-modulating compounds in the treatment of cardiovascular diseases.

AM in Sepsis
During septic shock, a systemic inflammatory condition often resulting in fatal end-organ damage, plasma AM levels are higher than in any other pathological condition (111). LPS and inflammatory cytokines induce AM gene expression in various tissues and cell types, which likely contributes to the extreme vasodilation and low blood pressure that occurs in the early stages of septic shock (112). Through its local vasodilatory effects, AM may limit hypoxic injury of tissues in addition to providing antiinflammatory (113), bactericidal (114), and positive inotropic properties (102), all of which are beneficial in the septic response. Consequently, in AM-overexpressing transgenic mice, LPS treatment induced less severe hemodynamic and inflammatory responses, less liver damage, and lower mortality rates than in control animals (107). These studies in genetically engineered animals clearly show a beneficial effect of enhanced AM production during septic shock and provide a rationale for the remarkable elevations in plasma AM levels observed in human patients with septic shock.

CONCLUDING REMARKS AND FUTURE PERSPECTIVES
Although there is a wealth of scientific exploration and literature on the topic of AM and AM signaling from clinical, genomic, biochemical, and pharmacological sources, we are only beginning to understand the ultimate function of AM in normal and disease physiology. In this article we have attempted to link clinical observations to other supportive data from biochemical signal transduction mechanisms, genomic expression analysis, and mouse models phenotypes to provide an in-depth level of analysis toward defining the more pertinent roles of AM during normal and disease physiology. Whereas it is evident that AM plays a protective role against local cardiovascular injury, it is also becoming apparent that robust induction and modulation of AM signaling may impact significantly on pregnancy and sepsis. Specifically, dynamic modulation of RAMP gene expression may provide a novel and biologically elegant mechanism for altering tissue responsiveness to AM. However, the eventual physiological significance of this modulation will be best addressed in future genetically engineered mouse models. Thus, the field of AM signaling will certainly continue to expand at an exciting pace as we learn more about the function of this multifunctional signaling system in normal and disease physiology.


   ACKNOWLEDGMENTS
 
We thank all investigators who devote their time and energy to the study of AM signaling and regret that we were unable to include all citations and opinions in this article. We also thank Dr. Ann Stuart for her careful and critical review of our manuscript.


   FOOTNOTES
 
This work was supported by The Burroughs Wellcome Fund and the National Institutes of Health (HD046970).

First Published Online October 19, 2006

Abbreviations: AM, Adrenomedullin; CGRP, calcitonin gene-related peptide; CLR, calcitonin receptor-like receptor; E14.5, embryonic d 14.5; ERS, endoplasmic retrieval signals; GPCR, G protein-coupled receptor; HEK, human embryonic kidney; LPS, lipopolysaccharide; NHERF, Na+/H+ exchange regulatory factor; NSF, N-ethylmaleimide-sensitive factor; PAM, peptidyglycine {alpha}-amidating monooxygenase; RAMP, receptor activity-modifying protein.

Received for publication April 10, 2006. Accepted for publication October 10, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 A MULTIFUNCTIONAL PEPTIDE
 AM IN DISEASE
 AM SIGNAL TRANSDUCTION
 RAMP GENE EXPRESSION IN...
 GENETIC MOUSE MODELS
 REFERENCES
 

  1. Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, Eto T 1993 Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 192:553–560[CrossRef][Medline]
  2. Muff R, Born W, Fischer JA 1995 Calcitonin, calcitonin gene-related peptide, adrenomedullin and amylin: homologous peptides, separate receptors and overlapping biological actions. Eur J Endocrinol 133:17–20[Abstract/Free Full Text]
  3. Wimalawansa SJ 1997 Amylin, calcitonin gene-related peptide, calcitonin, and adrenomedullin: a peptide superfamily. Crit Rev Neurobiol 11:167–239[Medline]
  4. Foord SM, Topp SD, Abramo M, Holbrook JD 2005 New methods for researching accessory proteins. J Mol Neurosci 26:265–276[CrossRef][Medline]
  5. Caron KM, Smithies O 2001 Extreme hydrops fetalis and cardiovascular abnormalities in mice lacking a functional adrenomedullin gene. Proc Natl Acad Sci USA 98:615–619[Abstract/Free Full Text]
  6. Oh-hashi Y, Shindo T, Kurihara Y, Imai T, Wang Y, Morita H, Imai Y, Kayaba Y, Nishimatsu H, Suematsu, Hirata Y, Yazaki Y, Nagai R, Kuwaki T, Kurihara H 2001 Elevated sympathetic nervous activity in mice deficient in {alpha}CGRP. Circ Res 89:983–990[Abstract/Free Full Text]
  7. Lu JT, Son YJ, Lee J, Jetton TL, Shiota M, Moscoso L, Niswender KD, Loewy AD, Magnuson MA, Sanes JR, Emeson RB 1999 Mice lacking {alpha}-calcitonin gene-related peptide exhibit normal cardiovascular regulation and neuromuscular development. Mol Cell Neurosci 14:99–120[CrossRef][Medline]
  8. Ogoshi M, Inoue K, Takei Y 2003 Identification of a novel adrenomedullin gene family in teleost fish. Biochem Biophys Res Commun 311:1072–1077[CrossRef][Medline]
  9. Garayoa M, Bodegas E, Cuttitta F, Montuenga LM 2002 Adrenomedullin in mammalian embryogenesis. Microsc Res Tech 57:40–54[CrossRef][Medline]
  10. Hinson JP, Kapas S, Smith DM 2000 Adrenomedullin, a multifunctional regulatory peptide. Endocr Rev 21:138–167[Abstract/Free Full Text]
  11. Elsasser TH, Kahl S, Martinez A, Montuenga LM, Pio R, Cuttitta F 1999 Adrenomedullin binding protein in the plasma of multiple species: characterization by radioligand blotting. Endocrinology 140:4908–4911[Abstract/Free Full Text]
  12. Pio R, Martinez A, Unsworth EJ, Kowalak JA, Bengoechea JA, Zipfel PF, Elsasser TH, Cuttitta F 2001 Complement factor H is a serum-binding protein for adrenomedullin, and the resulting complex modulates the bioactivities of both partners. J Biol Chem 276:12292–12300[Abstract/Free Full Text]
  13. Lisy O, Jougasaki M, Schirger JA, Chen HH, Barclay PT, Burnett Jr JC 1998 Neutral endopeptidase inhibition potentiates the natriuretic actions of adrenomedullin. Am J Physiol 275:F410–F414
  14. Dupuis J, Caron A, Ruel N 2005 Biodistribution, plasma kinetics and quantification of single-pass pulmonary clearance of adrenomedullin. Clin Sci (Lond) 109:97–102[Medline]
  15. Lopez J, Martinez A 2002 Cell and molecular biology of the multifunctional peptide, adrenomedullin. Int Rev Cytol 221:1–92[Medline]
  16. Yanagawa B, Nagaya N 2007 Adrenomedullin: molecular mechanisms and its role in cardiac disease. Amino Acids 32: 157–164
  17. Brain SD, Grant AD 2004 Vascular actions of calcitonin gene-related peptide and adrenomedullin. Physiol Rev 84:903–934[Abstract/Free Full Text]
  18. Del Bene R, Lazzeri C, Barletta G, Vecchiarino S, Guerra CT, Franchi F, La Villa G 2000 Effects of low-dose adrenomedullin on cardiac function and systemic haemodynamics in man. Clin Physiol 20:457–465[CrossRef][Medline]
  19. Nagaya N, Satoh T, Nishikimi T, Uematsu M, Furuichi S, Sakamaki F, Oya H, Kyotani S, Nakanishi N, Goto Y, Masuda Y, Miyatake K, Kangawa K 2000 Hemodynamic, renal, and hormonal effects of adrenomedullin infusion in patients with congestive heart failure. Circulation 101:498–503
  20. Oya H, Nagaya N, Furuichi S, Nishikimi T, Ueno K, Nakanishi N, Yamagishi M, Kangawa K, Miyatake K 2000 Comparison of intravenous adrenomedullin with atrial natriuretic peptide in patients with congestive heart failure. Am J Cardiol 86:94–98[CrossRef][Medline]
  21. Lainchbury JG, Cooper GJ, Coy DH, Jiang NY, Lewis LK, Yandle TG, Richards AM, Nicholls MG 1997 Adrenomedullin: a hypotensive hormone in man. Clin Sci (Lond) 92:467–472[Medline]
  22. Lainchbury JG, Troughton RW, Lewis LK, Yandle TG, Richards AM, Nicholls MG 2000 Hemodynamic, hormonal, and renal effects of short-term adrenomedullin infusion in healthy volunteers. J Clin Endocrinol Metab 85:1016–1020[Abstract/Free Full Text]
  23. Meeran K, O’Shea D, Upton PD, Small CJ, Ghatei MA, Byfield PH, Bloom SR 1997 Circulating adrenomedullin does not regulate systemic blood pressure but increases plasma prolactin after intravenous infusion in humans: a pharmacokinetic study. J Clin Endocrinol Metab 82:95–100[Abstract/Free Full Text]
  24. Khan AI, Kato J, Kitamura K, Kangawa K, Eto T 1997 Hypotensive effect of chronically infused adrenomedullin in conscious Wistar-Kyoto and spontaneously hypertensive rats. Clin Exp Pharmacol Physiol 24:139–142[Medline]
  25. Ishiyama Y, Kitamura K, Ichiki Y, Nakamura S, Kida O, Kangawa K, Eto T 1993 Hemodynamic effects of a novel hypotensive peptide, human adrenomedullin, in rats. Eur J Pharmacol 241:271–273[CrossRef][Medline]
  26. Cheng DY, DeWitt BJ, Wegmann MJ, Coy DH, Bitar K, Murphy WA, Kadowitz PJ 1994 Synthetic human adrenomedullin and ADM15–52 have potent short-lasting vasodilator activity in the pulmonary vascular bed of the cat. Life Sci 55:PL251–PL256
  27. Kato T, Bishop AT, Wood MB 1996 Effect of human adrenomedullin on a canine tibial perfusion model in the absence of vascular endothelium. J Orthop Res 14:956–961[CrossRef][Medline]
  28. Hjelmqvist H, Keil R, Mathai M, Hubschle T, Gerstberger R 1997 Vasodilation and glomerular binding of adrenomedullin in rabbit kidney are not CGRP receptor mediated. Am J Physiol 273:R716–R724
  29. Westphal M, Stubbe H, Bone HG, Daudel F, Vocke S, Van Aken H, Booke M 2002 Hemodynamic effects of exogenous adrenomedullin in healthy and endotoxemic sheep. Biochem Biophys Res Commun 296:134–138[CrossRef][Medline]
  30. Beltowski J, Jamroz A 2004 Adrenomedullin—what do we know 10 years since its discovery? Pol J Pharmacol 56:5–27[Medline]
  31. Bunton DC, Petrie MC, Hillier C, Johnston F, McMurray JJ 2004 The clinical relevance of adrenomedullin: a promising profile? Pharmacol Ther 103:179–201[CrossRef][Medline]
  32. Perret M, Broussard H, LeGros T, Burns A, Chang JK, Summer W, Hyman A, Lippton H 1993 The effect of adrenomedullin on the isolated heart. Life Sci 53:PL377–PL379
  33. Szokodi I, Kinnunen P, Ruskoaho H 1996 Inotropic effect of adrenomedullin in the isolated perfused rat heart. Acta Physiol Scand 156:151–152[CrossRef][Medline]
  34. Stangl V, Dschietzig T, Bramlage P, Boye P, Kinkel HT, Staudt A, Baumann G, Felix SB, Stangl K 2000 Adrenomedullin and myocardial contractility in the rat. Eur J Pharmacol 408:83–89[CrossRef][Medline]
  35. Tsuruda T, Kato J, Kitamura K, Kuwasako K, Imamura T, Koiwaya Y, Tsuji T, Kangawa K, Eto T 1998 Adrenomedullin: a possible autocrine or paracrine inhibitor of hypertrophy of cardiomyocytes. Hypertension 31:505–510[Abstract/Free Full Text]
  36. Horio T, Nishikimi T, Yoshihara F, Matsuo H, Takishita S, Kangawa K 1999 Effects of adrenomedullin on cultured rat cardiac myocytes and fibroblasts. Eur J Pharmacol 382:1–9[CrossRef][Medline]
  37. Kato J, Tsuruda T, Kitamura K, Eto T 2003 Adrenomedullin: a possible autocrine or paracrine hormone in the cardiac ventricles. Hypertens Res 26 (Suppl):S113–S119
  38. Nishikimi T, Matsuoka H 2005 Cardiac adrenomedullin: its role in cardiac hypertrophy and heart failure. Curr Med Chem Cardiovasc Hematol Agents 3:231–242[CrossRef][Medline]
  39. Zhao Y, Hague S, Manek S, Zhang L, Bicknell R, Rees MC 1998 PCR display identifies tamoxifen induction of the novel angiogenic factor adrenomedullin by a non estrogenic mechanism in the human endometrium. Oncogene 16:409–415[CrossRef][Medline]
  40. Nguyen SV, Claycomb WC 1999 Hypoxia regulates the expression of the adrenomedullin and HIF-1 genes in cultured HL-1 cardiomyocytes. Biochem Biophys Res Commun 265:382–386[CrossRef][Medline]
  41. Hippenstiel S, Witzenrath M, Schmeck B, Hocke A, Krisp M, Krull M, Seybold J, Seeger W, Rascher W, Schutte H, Suttorp N 2002 Adrenomedullin reduces endothelial hyperpermeability. Circ Res 91:618–625[Abstract/Free Full Text]
  42. Zudaire E, Martinez A, Cuttitta F 2003 Adrenomedullin and cancer. Regul Pept 112:175–183[CrossRef][Medline]
  43. Nikitenko LL, Fox SB, Kehoe S, Rees MC, Bicknell R 2006 Adrenomedullin and tumour angiogenesis. Br J Cancer 94:1–7[CrossRef][Medline]
  44. Serrano J, Alonso D, Fernandez AP, Encinas JM, Lopez JC, Castro-Blanco S, FernandezVizarra P, Richard A, Santacana M, Uttenthal LO, Bentura ML, Martinez-Murillo R, Martinez A, Cuttitta F, Rodrigo J 2002 Adrenomedullin in the central nervous system. Microsc Res Tech 57:76–90[CrossRef][Medline]
  45. Mukoyama M, Sugawara A, Nagae T, Mori K, Murabe H, Itoh H, Tanaka I, Nakao K 2001 Role of adrenomedullin and its receptor system in renal pathophysiology. Peptides 22:1925–1931[CrossRef][Medline]
  46. Brogden KA, Guthmiller JM, Salzet M, Zasloff M 2005 The nervous system and innate immunity: the neuropeptide connection. Nat Immunol 6:558–564[Medline]
  47. Eva C, Sprengel R 1993 A novel putative G protein-coupled receptor highly expressed in lung and testis. DNA Cell Biol 12:393–399[Medline]
  48. Aiyar N, Rand K, Elshourbagy NA, Zeng Z, Adamou JE, Bergsma DJ, Li Y 1996 A cDNA encoding the calcitonin gene-related peptide type 1 receptor. J Biol Chem 271:11325–11329[Abstract/Free Full Text]
  49. Han ZQ, Coppock HA, Smith DM, Van Noorden S, Makgoba MW, Nicholl CG, Legon S 1997 The interaction of CGRP and adrenomedullin with a receptor expressed in the rat pulmonary vascular endothelium. J Mol Endocrinol 18:267–272[Abstract/Free Full Text]
  50. Harrison JK, Barber CM, Lynch KR 1993 Molecular cloning of a novel rat G-protein-coupled receptor gene expressed prominently in lung, adrenal, and liver. FEBS Lett 318:17–22[CrossRef][Medline]
  51. Kapas S, Catt KJ, Clark AJ 1995 Cloning and expression of cDNA encoding a rat adrenomedullin receptor. J Biol Chem 270:25344–25347[Abstract/Free Full Text]
  52. Kapas S, Clark AJ 1995 Identification of an orphan receptor gene as a type 1 calcitonin gene-related peptide receptor. Biochem Biophys Res Commun 217:832–838[CrossRef][Medline]
  53. McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N, Solari R, Lee MG, Foord SM 1998 RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 393:333–339[CrossRef][Medline]
  54. Julian M, Cacho M, Garcia MA, Martin-Santamaria S, de Pascual-Teresa B, Ramos A, Martinez A, Cuttitta F 2005 Adrenomedullin: a new target for the design of small molecule modulators with promising pharmacological activities. Eur J Med Chem 40:737–750[CrossRef][Medline]
  55. Doods H, Hallermayer G, Wu D, Entzeroth M, Rudolf K, Engel W, Eberlein W 2000 Pharmacological profile of BIBN4096BS, the first selective small molecule CGRP antagonist. Br J Pharmacol 129:420–423[CrossRef][Medline]
  56. Doods H 2001 Development of CGRP antagonists for the treatment of migraine. Curr Opin Investig Drugs 2:1261–1268[Medline]
  57. Moreno MJ, Abounader R, Hebert E, Doods H, Hamel E 2002 Efficacy of the non-peptide CGRP receptor antagonist BIBN4096BS in blocking CGRP-induced dilations in human and bovine cerebral arteries: potential implications in acute migraine treatment. Neuropharmacology 42:568–576[CrossRef][Medline]
  58. Hay DL, Poyner DR, Sexton PM 2006 GPCR modulation by RAMPs. Pharmacol Ther 109:173–197[CrossRef][Medline]
  59. Sexton PM, Albiston A, Morfis M, Tilakaratne N 2001 Receptor activity modifying proteins. Cell Signal 13:73–83[CrossRef][Medline]
  60. Christopoulos G, Perry KJ, Morfis M, Tilakaratne N, Gao Y, Fraser NJ, Main MJ, Foord SM, Sexton PM 1999 Multiple amylin receptors arise from receptor activity-modifying protein interaction with the calcitonin receptor gene product. Mol Pharmacol 56:235–242[Abstract/Free Full Text]
  61. Christopoulos A, Christopoulos G, Morfis M, Udawela M, Laburthe M, Couvineau A, Kuwasako K, Tilakaratne N, Sexton PM 2003 Novel receptor partners and function of receptor activity-modifying proteins. J Biol Chem 278:3293–3297[Abstract/Free Full Text]
  62. Bouschet T, Martin S, Henley JM 2005 Receptor-activity-modifying proteins are required for forward trafficking of the calcium-sensing receptor to the plasma membrane. J Cell Sci 118:4709–4720[Abstract/Free Full Text]
  63. Su AI, Cooke MP, Ching KA, Hakak Y, Walker JR, Wiltshire T, Orth AP, Vega RG, Sapinoso RM, Mogrich A, Patapoutian A, Hampton GM, Schultz PG, Hogenesch JB 2002 Large-scale analysis of the human and mouse transcriptomes. Proc Natl Acad Sci USA 99:4465–4470[Abstract/Free Full Text]
  64. Steiner S, Muff R, Gujer R, Fischer JA, Born W 2002 The transmembrane domain of receptor-activity-modifying protein 1 is essential for the functional expression of a calcitonin gene-related peptide receptor. Biochemistry 41:11398–11404[CrossRef][Medline]
  65. Zumpe ET, Tilakaratne N, Fraser NJ, Christopoulos G, Foord SM, Sexton PM 2000 Multiple ramp domains are required for generation of amylin receptor phenotype from the calcitonin receptor gene product. Biochem Biophys Res Commun 267:368–372[CrossRef][Medline]
  66. Fraser NJ, Wise A, Brown J, McLatchie LM, Main MJ, Foord SM 1999 The amino terminus of receptor activity modifying proteins is a critical determinant of glycosylation state and ligand binding of calcitonin receptor-like receptor. Mol Pharmacol 55:1054–1059[Abstract/Free Full Text]
  67. Kuwasako K, Kitamura K, Nagoshi Y, Cao YN, Eto T 2003 Identification of the human receptor activity-modifying protein 1 domains responsible for agonist binding specificity. J Biol Chem 278:22623–22630[Abstract/Free Full Text]
  68. Kuwasako K, Cao YN, Chu CP, Iwatsubo S, Eto T, Kitamura K 2006 Functions of the cytoplasmic tails of the human receptor activity-modifying protein components of calcitonin gene-related peptide and adrenomedullin receptors. J Biol Chem 281:7205–7213[Abstract/Free Full Text]
  69. Bomberger JM, Spielman WS, Hall CS, Weinman EJ, Parameswaran N 2005 Receptor activity-modifying protein (RAMP) isoform-specific regulation of adrenomedullin receptor trafficking by NHERF-1. J Biol Chem 280:23926–23935[Abstract/Free Full Text]
  70. Bomberger JM, Parameswaran N, Hall CS, Aiyar N, Spielman WS 2005 Novel function for receptor activity-modifying proteins (RAMPs) in post-endocytic receptor trafficking. J Biol Chem 280:9297–9307[Abstract/Free Full Text]
  71. Hay DL, Howitt SG, Conner AC, Doods H, Schindler M, Poyner DR 2002 A comparison of the actions of BIBN4096BS and CGRP(8–37) on CGRP and adrenomedullin receptors expressed on SK-N-MC, L6, Col 29 and Rat 2 cells. Br J Pharmacol 137:80–86[CrossRef][Medline]
  72. Kuwasako K, Cao YN, Nagoshi Y, Kitamura K, Eto T 2004 Adrenomedullin receptors: pharmacological features and possible pathophysiological roles. Peptides 25:2003–2012[CrossRef][Medline]
  73. Udawela M, Hay DL, Sexton PM 2004 The receptor activity modifying protein family of G protein coupled receptor accessory proteins. Semin Cell Dev Biol 15:299–308[CrossRef][Medline]
  74. Thota C, Yallampalli C 2005 Progesterone upregulates calcitonin gene-related peptide and adrenomedullin receptor components and cyclic adenosine 3'5'-monophosphate generation in Eker rat uterine smooth muscle cell line. Biol Reprod 72:416–422[Abstract/Free Full Text]
  75. Gangula PR, Thota C, Wimalawansa SJ, Bukoski RD, Yallampalli C 2003 Mechanisms involved in calcitonin gene-related peptide-induced relaxation in pregnant rat uterine artery. Biol Reprod 69:1635–1641[Abstract/Free Full Text]
  76. Dong YL, Vegiraju S, Chauhan M, Yallampalli C 2003 Expression of calcitonin gene-related peptide receptor components, calcitonin receptor-like receptor and receptor activity modifying protein 1, in the rat placenta during pregnancy and their cellular localization. Mol Hum Reprod 9:481–490[Abstract/Free Full Text]
  77. Dong YL, Green KE, Vegiragu S, Hankins GD, Martin E, Chauhan M, Thota C, Yallampalli C 2005 Evidence for decreased calcitonin gene-related peptide (CGRP) receptors and compromised responsiveness to CGRP of fetoplacental vessels in preeclamptic pregnancies. J Clin Endocrinol Metab 90:2336–2343[Abstract/Free Full Text]
  78. Knerr I, Dachert C, Beinder E, Metzler M, Dotsch J, Repp R, Rascher W 2002 Adrenomedullin, calcitonin gene-related peptide and their receptors: evidence for a decreased placental mRNA content in preeclampsia and HELLP syndrome. Eur J Obstet Gynecol Reprod Biol 101:47–53[CrossRef][Medline]
  79. Makino Y, Shibata K, Makino I, Kangawa K, Kawarabayashi T 2001 Alteration of the adrenomedullin receptor components gene expression associated with the blood pressure in pregnancy-induced hypertension. J Clin Endocrinol Metab 86:5079–5082[Abstract/Free Full Text]
  80. Hewitt SC, Collins J, Grissom S, Deroo B, Korach KS 2005 Global uterine genomics in vivo: microarray evaluation of the estrogen receptor {alpha}-growth factor cross-talk mechanism. Mol Endocrinol 19:657–668[Abstract/Free Full Text]
  81. Watanabe H, Takahashi E, Kobayashi M, Goto M, Krust A, Chambon P, Iguchi T 2006 The estrogen-responsive adrenomedullin and receptor-modifying protein 3 gene identified by DNA microarray analysis are directly regulated by estrogen receptor. J Mol Endocrinol 36:81–89[Abstract/Free Full Text]
  82. Ikeda K, Arao Y, Otsuka H, Kikuchi A, Kayama F 2004 Estrogen and phytoestrogen regulate the mRNA expression of adrenomedullin and adrenomedullin receptor components in the rat uterus. Mol Cell Endocrinol 223:27–34[CrossRef][Medline]
  83. Ono Y, Okano I, Kojima M, Okada K, Kangawa K 2000 Decreased gene expression of adrenomedullin receptor in mouse lungs during sepsis. Biochem Biophys Res Commun 271:197–202[CrossRef][Medline]
  84. Ornan DA, Chaudry IH, Wang P 2002 Saturation of adrenomedullin receptors plays an important role in reducing pulmonary clearance of adrenomedullin during the late stage of sepsis. Biochim Biophys Acta 1586:299–306[Medline]
  85. Ornan DA, Chaudry IH, Wang P 1999 Pulmonary clearance of adrenomedullin is reduced during the late stage of sepsis. Biochim Biophys Acta 1427:315–321[Medline]
  86. Wang P, Yoo P, Zhou M, Cioffi WG, Ba ZF, Chaudry IH 1999 Reduction in vascular responsiveness to adrenomedullin during sepsis. J Surg Res 85:59–65[CrossRef][Medline]
  87. Cui Y, Ji Y, Wu R, Zhou M, Wang P 2006 Adrenomedullin binding protein-1 is downregulated during polymicrobial sepsis in the rat. Int J Mol Med 17:925–929[Medline]
  88. Nagoshi Y, Kuwasako K, Cao YN, Imamura T, Kitamura K, Eto T 2004 Tumor necrosis factor-{alpha} downregulates adrenomedullin receptors in human coronary artery smooth muscle cells. Peptides 25:1115–1121[CrossRef][Medline]
  89. Chakravarty P, Suthar TP, Coppock HA, Nicholl CG, Bloom SR, Legon S, Smith DM 2000 CGRP and adrenomedullin binding correlates with transcript levels for calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) in rat tissues. Br J Pharmacol 130:189–195[CrossRef][Medline]
  90. Nagae T, Mukoyama M, Sugawara A, Mori K, Yahata K, Kasahara M, Suganami T, Makino H, Fujinaga Y, Yoshioka T, Tanaka I, Nakao K 2000 Rat receptor-activity-modifying proteins (RAMPs) for adrenomedullin/CGRP receptor: cloning and upregulation in obstructive nephropathy. Biochem Biophys Res Commun 270:89–93[CrossRef][Medline]
  91. Kitamuro T, Takahashi K, Totsune K, Nakayama M, Murakami O, Hida W, Shirato K, Shibahara S 2001 Differential expression of adrenomedullin and its receptor component, receptor activity modifying protein (RAMP) 2 during hypoxia in cultured human neuroblastoma cells. Peptides 22:1795–1801[CrossRef][Medline]
  92. Nikitenko LL, Smith DM, Bicknell R, Rees MC 2003 Transcriptional regulation of the CRLR gene in human microvascular endothelial cells by hypoxia. FASEB J 17:1499–1501[Abstract/Free Full Text]
  93. Martinez A, Unsworth EJ, Cuttitta F 1996 Adrenomedullin-like immunoreactivity in the nervous system of the starfish Marthasterias glacialis. Cell Tissue Res 283:169–172[CrossRef][Medline]
  94. Shindo T, Kurihara Y, Nishimatsu H, Moriyama N, Kakoki M, Wang Y, Imai Y, Ebihara A, Kuwaki T, Ju KH, Minamino N, Kangawa K, Ishikawa T, Fukuda M, Akimoto Y, Kawakami H, Imai T, Morita H, Yazaki Y, Nagai R, Hirata Y, Kurihara H 2001 Vascular abnormalities and elevated blood pressure in mice lacking adrenomedullin gene. Circulation 104:1964–1971
  95. Shimosawa T, Shibagaki Y, Ishibashi K, Kitamura K, Kangawa K, Kato S, Ando K, Fujita T 2002 Adrenomedullin, an endogenous peptide, counteracts cardiovascular damage. Circulation 105:106–111
  96. Dackor RT, Fritz-Six K, Dunworth WP, Gibbons CL, Smithies O, Caron KM 2006 Hydrops fetalis, cardiovascular defects, and embryonic lethality in mice lacking the calcitonin receptor-like receptor gene. Mol Cell Biol 26:2511–2518[Abstract/Free Full Text]
  97. Czyzyk TA, Ning Y, Hsu MS, Peng B, Mains RE, Eipper BA, Pintar JE 2005 Deletion of peptide amidation enzymatic activity leads to edema and embryonic lethality in the mouse. Dev Biol 287:301–313[CrossRef][Medline]
  98. Di Iorio R, Marinoni E, Letizia C, Cosmi EV 2003 Adrenomedullin in perinatal medicine. Regul Pept 112:103–113[CrossRef][Medline]
  99. Wilson C, Nikitenko LL, Sargent IL, Rees MC 2004 Adrenomedullin: multiple functions in human pregnancy. Angiogenesis 7:203–212[CrossRef][Medline]
  100. Li M, Yee D, Magnuson T, Smithies O, Caron K 2006 Reduced maternal expression of adrenomedullin disrupts fertility, placentation and fetal growth in mice. J Clin Invest 116:2653–2662[CrossRef][Medline]
  101. Ishimitsu T, Tsukada K, Minami J, Ono H, Matsuoka H 2003 Variations of human adrenomedullin gene and its relation to cardiovascular diseases. Hypertens Res 26 (Suppl):S129–S134
  102. Nishikimi T, Yoshihara F, Mori Y, Kangawa K, Matsuoka H 2003 Cardioprotective effect of adrenomedullin in heart failure. Hypertens Res 26(Suppl):S121–S127
  103. Niu P, Shindo T, Iwata H, Iimuro S, Takeda N, Zhang Y, Ebihara A, Suematsu Y, Kangawa K, Hirata Y, Nagai R 2004 Protective effects of endogenous adrenomedullin on cardiac hypertrophy, fibrosis, and renal damage. Circulation 109:1789–1794
  104. Niu P, Shindo T, Iwata H, Ebihara A, Suematsu Y, Zhang Y, Takeda N, Iimuro S, Hirata Y, Nagai R 2003 Accelerated cardiac hypertrophy and renal damage induced by angiotensin II in adrenomedullin knockout mice. Hypertens Res 26:731–736[CrossRef][Medline]
  105. Nishimatsu H, Hirata Y, Shindo T, Kurihara H, Kakoki M, Nagata D, Hayakawa H, Satonaka H, Sata M, Tojo A, Suzucki E, Kangawa K, Matsuo H, Kitamura T, Nagai R 2002 Role of endogenous adrenomedullin in the regulation of vascular tone and ischemic renal injury: studies on transgenic/knockout mice of adrenomedullin gene. Circ Res 90:657–663[Abstract/Free Full Text]
  106. Imai Y, Shindo T, Maemura K, Sata M, Saito Y, Kurihara Y, Akishita M, Osuga J, Ishibashi S, Tobe K, Morita H, Oh-hashi Y, Suzuki T, Maekawa H, Kangawa K, Minamino N, Yazaki Y, Nagai R, Kurihara H 2002 Resistance to neointimal hyperplasia and fatty streak formation in mice with adrenomedullin overexpression. Arterioscler Thromb Vasc Biol 22:1310–1315[Abstract/Free Full Text]
  107. Shindo T, Kurihara H, Maemura K, Kurihara Y, Kuwaki T, Izumida T, Minamino N, Ju KH, Morita H, Oh-hashi Y, Kumada M, Kangawa K, Nagai R, Yazaki Y 2000 Hypotension and resistance to lipopolysaccharide-induced shock in transgenic mice overexpressing adrenomedullin in their vasculature. Circulation 101:2309–2316
  108. Tam CW, Husmann K, Clark NC, Clark JE, Lazar Z, Ittner LM, Gotz J, Douglas G, Grant AD, Sugden D, Poston L, Poston R, McFadzean I, Marber MS, Fischer JA, Born W, Brain SD 2006 Enhanced vascular responses to adrenomedullin in mice overexpressing receptor-activity-modifying protein 2. Circ Res 98:262–270[Abstract/Free Full Text]
  109. Zhang JJ, Yoshida H, Chao L, Chao J 2000 Human adrenomedullin gene delivery protects against cardiac hypertrophy, fibrosis, and renal damage in hypertensive dahl salt-sensitive rats. Hum Gene Ther 11:1817–1827[CrossRef][Medline]
  110. Dobrzynski E, Wang C, Chao J, Chao L 2000 Adrenomedullin gene delivery attenuates hypertension, cardiac remodeling, and renal injury in deoxycorticosterone acetate-salt hypertensive rats. Hypertension 36:995–1001[Abstract/Free Full Text]
  111. Hirata Y, Mitaka C, Sato K, Nagura T, Tsunoda Y, Amaha K, Marumo F 1996 Increased circulating adrenomedullin, a novel vasodilatory peptide, in sepsis. J Clin Endocrinol Metab 81:1449–1453[Abstract]
  112. Nishio K, Akai Y, Murao Y, Doi N, Ueda S, Tabuse H, Miyamoto S, Dohi K, Minamino N, Shoji H, Kitamura K, Kangawa K, Matsuo H 1997 Increased plasma concentrations of adrenomedullin correlate with relaxation of vascular tone in patients with septic shock. Crit Care Med 25:953–957[CrossRef][Medline]
  113. Isumi Y, Kubo A, Katafuchi T, Kangawa K, Minamino N 1999 Adrenomedullin suppresses interleukin-1ß-induced tumor necrosis factor-{alpha} production in Swiss 3T3 cells. FEBS Lett 463:110–114[CrossRef][Medline]
  114. Allaker RP, Zihni C, Kapas S 1999 An investigation into the antimicrobial effects of adrenomedullin on members of the skin, oral, respiratory tract and gut microflora. FEMS Immunol Med Microbiol 23:289–293[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J EndocrinolHome page
M. M White and W. K Samson
A possible relationship between brain-derived adrenomedullin and oxytocin in the regulation of sodium balance
J. Endocrinol., November 1, 2009; 203(2): 253 - 262.
[Abstract] [Full Text] [PDF]

Home page
 JNMHome page
J. Dupuis, F. Harel, Y. Fu, Q. T. Nguyen, M. Letourneau, A. Prefontaine, and A. Fournier
Molecular Imaging of Monocrotaline-Induced Pulmonary Vascular Disease with Radiolabeled Linear Adrenomedullin
J. Nucl. Med., July 1, 2009; 50(7): 1110 - 1115.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
M. Li, Y. Wu, and K. M. Caron
Haploinsufficiency for Adrenomedullin Reduces Pinopodes and Diminishes Uterine Receptivity in Mice
Biol Reprod, December 1, 2008; 79(6): 1169 - 1175.
[Abstract] [Full Text] [PDF]

Home page
 JNMHome page
F. Harel, Y. Fu, Q. T. Nguyen, M. Letourneau, L. P. Perrault, A. Caron, A. Fournier, and J. Dupuis
Use of Adrenomedullin Derivatives for Molecular Imaging of Pulmonary Circulation
J. Nucl. Med., November 1, 2008; 49(11): 1869 - 1874.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
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]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gibbons, C.
Right arrow Articles by Caron, K. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gibbons, C.
Right arrow Articles by Caron, K. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals