Peptides (v.22, #11)

Adrenomedullin (AM), identified from pheochromocytoma and having 52 amino acids, elicits a long-lasting vasodilatation and diuresis. AM is mainly mediated by the intracellular adenylate cyclase coupled with cyclic adenosine monophosphate (cAMP) and nitric oxide (NO) -cyclic guanosine monophosphate (cGMP) pathway through its specific receptor. The calcitonin receptor-like receptor (CLCR) and receptor-activity modifying protein (RAMP) 2 or RAMP3 models have been proposed as the candidate receptor. AM is produced mainly in cardiovascular tissues in response to stimuli such as shear stress and stretch, hormonal factors and cytokines. Recently established AM knockout mice lines revealed that AM is essential for development of vitelline vessels of embryo. Plasma AM levels elevate in cardiovascular diseases such as heart failure, hypertension and septic shock, where AM may play protective roles through its characteristic biological activities. Human AM gene delivery improves hypertension, renal function, cardiac hypertrophy and nephrosclerosis in the hypertensive rats. AM decreases cardiac preload and afterload and improves cardiac contractility and diuresis in patients with heart failure and hypertension. Advances in gene engineering and receptor studies may contribute to further understandings of biological implication and therapeutic availability of AM.
Keywords: Adrenomedullin (AM); Proadrenomedullin N-terminal 20 peptide (PAMP); Vasoactive substance; Vasodilatation; Diuresis; Heart failure; Hypertension; Cardiac remodeling;

Adrenomedullin (11–26): a novel endogenous hypertensive peptide isolated from bovine adrenal medulla by Kazuo Kitamura; Eizaburo Matsui; Jhoji Kato; Fumi Katoh; Toshohiro Kita; Tetsuo Tsuji; Kenji Kangawa; Tanenao Eto (1713-1718).
Adrenomedullin (AM) is a potent hypotensive peptide originally isolated from pheochromocytoma tissue. Both the ring structure and the C-terminal amide structure of AM are essential for its hypotensive activity. We have developed an RIA which recognizes the ring structure of human AM. Using this RIA, we have characterized the molecular form of AM in bovine adrenal medulla. Gel filtration chromatography revealed that three major peaks of immunoreactive AM existed in the adrenal medulla. The peptide corresponding to Mr 1500 Da was further purified to homogeneity. The peptide was determined to be AM (11–26) which has one intramolecular disulfide bond. Amino acid sequences of bovine AM and its precursor were deduced from the analyses of cDNA encoding bovine AM precursor. The synthetic AM (11–26) produced dose-dependent strong pressor responses in unanesthetized rats in vivo. The hypertensive activity lasted about one minute, and a dose dependent increase in heart rate was also observed. The present data indicate that AM (11–26) is a major component of immunoreactive AM in bovine adrenal medulla and shows pressor activity.
Keywords: Adrenomedullin (11–26); Vasopressor peptide; Purification; Sequence determination; cDNA; Bovine adrenal medulla;

Cancer and diabetes: two pathological conditions in which adrenomedullin may be involved by Rubén Pı́o; Alfredo Martı́nez; Frank Cuttitta (1719-1729).
Adrenomedullin (AM) is a regulatory peptide involved in several physiological processes. Among them, AM has been implicated in the regulation of growth, both with mitogenic and antiproliferative activities on normal cells. AM is widely expressed during embryogenesis and may have a significant role in the proliferation and differentiation processes associated with development. AM is also expressed by cancer cell lines and tumors and has been implicated in the growth of malignant cells. Some additional activities associated with AM (antiapoptotic capabilities, angiogenic potential, and upregulation in hypoxic conditions), together with its wide distribution in cancer, suggest that AM may be an important factor in carcinogenesis. Besides its implication in growth, embryogenesis and tumor biology, AM is also involved in pancreatic regulation and diabetes. AM regulates insulin secretion and is overexpressed in the plasma of diabetic patients. Several findings indicate that AM may participate in the pathogenesis and/or clinical complications of this disease.
Keywords: Adrenomedullin; Binding protein; Cancer; Diabetes; Embryogenesis; Factor H; Growth; Insulin secretion;

Human adrenomedullin gene delivery protects against cardiovascular remodeling and renal injury by Julie Chao; Kazuo Kato; Jenny J. Zhang; Eric Dobrzynski; Cindy Wang; Jun Agata; Lee Chao (1731-1737).
We investigated the potential roles of adrenomedullin (AM) in cardiovascular and renal function by somatic gene delivery. We showed that a single intravenous injection of the human AM gene under the control of cytomegalovirus promoter/enhancer induces a prolonged delay in blood pressure rise for several weeks in spontaneously hypertensive rats, Dahl salt-sensitive, DOCA-salt, and two-kidney one-clip hypertensive rats as compared to their respective controls injected with a reporter gene. Expression of the human AM transcript was identified in the heart, kidney, lung, liver and aorta of the rat after adenovirus-mediated AM gene delivery by RT-PCR followed by Southern blot analysis. Immunoreactive human AM levels were measured in rat plasma and urine following AM gene delivery. AM gene delivery induced significant reduction of left ventricular mass in these hypertensive animal models. It also reduces urinary protein excretion and increases glomerular filtration rate, renal blood flow and urinary cAMP levels. AM gene transfer attenuated cardiomyocyte diameter and interstitial fibrosis in the heart, and reduced glomerular sclerosis, tubular disruption, protein cast accumulation and renal cell proliferation in the kidney. In the rat model with myocardial ischmia/reperfusion injury, AM gene delivery significantly reduced myocardial infarction, apoptosis, and superoxide production. Furthermore, local AM gene delivery significantly inhibited arterial thickening, promoted re-endothelialization and increased vascular cGMP levels in rat artery after balloon angioplasty. Collectively, these results indicate that human AM gene delivery attenuates hypertension, myocardial infarction, renal injury and cardiovascular remodeling in animal models via cAMP and cGMP signaling pathways. These findings provide new insights into the role of AM in cardiovascular and renal function.
Keywords: Adrenomedullin; Gene delivery; Hypertension; Renal injury; Myocardial infarction; Balloon angioplasty; Cardiac hypertrophy; Fibrosis;

Chromosomal sublocalization and microsatellite DNA polymorphism of human adrenomedullin gene by Toshihiko Ishimitsu; Kazuyoshi Hosoya; Kohju Tsukada; Junichi Minami; Hidehiko Ono; Masami Ohrui; Jun Hino; Kenji Kangawa; Hiroaki Matsuoka (1739-1744).
Adrenomedullin (AM) is a hypotensive peptide widely produced in the cardiovascular organs and tissues such as the heart, kidney and vascular cells. We have cloned and sequenced genomic DNA encoding the human AM gene. In this study, we determined that the AM gene was located in the short arm of chromosome 11 (p15.1–3). The 3′-end of the gene is flanked by a microsatellite marker of cytosine adenine (CA) repeats. Moreover, we analyzed this DNA variation in the AM gene in the general Japanese population. Genomic DNA was obtained from the peripheral leukocytes of healthy normotensive subjects, 327 men and 149 women, aged 51 ± 8 years (mean ± SD). The genomic DNA was subject to PCR using a fluorescence-labeled primer, and the number of CA repeats were determined via polyacrylamide gel electrophoresis (PAGE). Plasma AM concentration was measured by RIA and compared with respect to the number of CA repeats adjacent to the AM gene. In Japanese, four types of alleles with different CA-repeat numbers; 11, 13, 14 and 19, appear to exist. The frequencies of these alleles were as follows: 11 repeats, 28.8%; 13 repeats, 33.1%; 14 repeats, 35.0% and 19 repeats, 3.1%. This DNA variation does not seem to affect the transcription of the AM gene, because plasma concentrations of AM were not significantly different between the genotypes.
Keywords: Adrenomedullin; Chromosome 11; Gene polymorphism; Microsatellite repeats; Genetic markers;

Bioactivity of adrenomedullin and proadrenomedullin N-terminal 20 peptide in man by M.Gary Nicholls; John G Lainchbury; Lynley K Lewis; David O McGregor; A.Mark Richards; Richard W Troughton; Timothy G Yandle (1745-1752).
Although the biological effects of adrenomedullin (AM) and PAMP have been reported extensively in animal studies and from in-vitro experiments, relatively little information is available on responses to the hormone administered to man. This review summarizes data from the few studies carried out in man. In healthy volunteers, i.v. infusion of AM reduces arterial pressure, probably at a lower rate of administration than is required to elicit other responses. AM stimulates heart rate, cardiac output, plasma levels of cAMP, prolactin, norepinephrine and renin whilst inhibiting any concomitant response in plasma aldosterone. Little or no increase in urine volume or sodium excretion has been observed. Patients with essential hypertension differ only in showing a greater fall in arterial pressure and in the development of facial flushing and headache. In patients with heart failure or chronic renal failure, i.v. AM has similar effects to those seen in normal subjects but also induces a diuresis and natriuresis, depending on the dose administered. Infusion of AM into the brachial artery results in a dose-related increase in forearm and skin blood flow, more prominent and more dependent on endogenous nitric oxide in healthy volunteers than in patients with cardiac failure. When infused into a dorsal hand vein, AM partially reversed the venoconstrictor action of norepinephrine. Although much more information is required to clarify the role of AM under physiological and pathophysiological circumstances, it is clear that it has prominent hemodynamic and neurohormonal effects, though generally lesser urinary effects when administered short-term in doses sufficient to raise its levels in plasma to those seen in a number of clinical disorders. The only study of PAMP in man showed that its skeletal muscle vasodilator potency, when infused into the brachial artery of healthy volunteers, was less than one hundredth that of AM, and it was without effect on skin blood flow.

Adrenomedullin receptors: molecular identity and function by Debbie L Hay; David M Smith (1753-1763).
Since its discovery in 1993 adrenomedullin (AM) has been the subject over 600 published articles. This multifunctional peptide has powerful vasodilator actions and recent evidence from AM gene-deleted mice suggest that AM plays an essential role in vascular development. However the lack of valid AM receptor clones and non-peptide receptor ligands has considerably slowed research progress on this important peptide. In this review we have focused on the proposition that the calcitonin receptor-like receptor (CRLR) is a receptor both for AM and the related vasoactive peptide calcitonin gene-related peptide (CGRP). The receptor activity modifying proteins (RAMPs) that are essential for defining CRLR pharmacology will also be discussed. We will describe how AM receptors have been reported to signal and be regulated and to consider whether further receptors for AM beyond CRLR/RAMP combinations might exist.
Keywords: Adrenomedullin receptors; CGRP receptors; CRLR; RAMPs;

Adrenomedullin and related peptides: receptors and accessory proteins by Roman Muff; Walter Born; Jan A Fischer (1765-1772).
Adrenomedullin (AM), α- and β-calcitonin gene-related peptide (CGRP), amylin and calcitonin (CT) are structurally and functionally related peptides. The structure of a receptor for CT (CTR) was elucidated in 1991 through molecular cloning, but the structures of the receptors for the other three peptides had yet to be elucidated. The discovery of receptor-activity-modifying proteins (RAMP) 1 and -2 and their co-expression with an orphan receptor, calcitonin receptor-like receptor (CRLR) has led to the elucidation of functional CGRP and AM receptors, respectively. RAMP1 and -3 which are co-expressed with CTR revealed two amylin receptor isotypes. Molecular interactions between CRLR and RAMPs are involved in their transport to the cell surface. Heterodimeric complexes betweeen CRLR or CTR and RAMPs are required for ligand recognition.
Keywords: Adrenomedullin; Amylin; Calcitonin gene related peptide; Complex; Heterodimer; Receptor activity modifying protein;

The role of the CGRP-receptor component protein (RCP) in adrenomedullin receptor signal transduction by Marya A Prado; Bornadata Evans-Bain; Kevin R Oliver; Ian M Dickerson (1773-1781).
G protein-coupled receptors are usually thought to act as monomer receptors that bind ligand and then interact with G proteins to initiate signal transduction. In this study we report an intracellular peripheral membrane protein named the calcitonin gene-related peptide (CGRP)-receptor component protein (RCP) required for signal transduction at the G protein-coupled receptor for adrenomedullin. Cell lines were made that expressed an antisense construct of the RCP cDNA, and in these cells diminished RCP expression correlated with loss of adrenomedullin signal transduction. In contrast, loss of RCP did not diminish receptor density or affinity, therefore RCP does not appear to act as a chaperone protein. Instead, RCP represents a novel class of protein required to couple the adrenomedullin receptor to the cellular signal transduction pathway. A candidate adrenomedullin receptor named the calcitonin receptor-like receptor (CRLR) has been described, which forms high affinity adrenomedullin receptors when co-expressed with the accessory protein receptor-activity modifying protein 2 (RAMP2). RCP co-immunoprecipitated with CRLR and RAMP2, indicating that a functional adrenomedullin receptor is composed of at least three proteins: the ligand binding protein (CRLR), an accessory protein (RAMP2), and a coupling protein for signal transduction (RCP).
Keywords: Adrenomedullin; CGRP; RCP; Receptor; Signal transduction;

Regulation of adrenomedullin secretion from cultured cells by Yoshio Tomoda; Yoshitaka Isumi; Takeshi Katafuchi; Naoto Minamino (1783-1794).
Characterization of immunoreactive adrenomedullin (AM) secreted from cultured human vascular smooth muscle cells and 7 other cells indicates that AM is synthesized and secreted from all cultured cells we surveyed. The secretion rate of AM measured ranges from 0.001–6.83 fmol/105 cells/h, and endothelial cells, vascular smooth muscle cells and fibroblasts generally secrete AM at high rates. Based on the results of regulation of AM secretion from vascular wall cells, fibroblasts, macrophages and other cells measured in this and previous studies, AM secretion is found to be generally stimulated by inflammatory cytokines, lipopolysaccharide (LPS) and hormones. Especially, vascular smooth muscle cells and fibroblasts elicited uniform and strong stimulatory responses of AM secretion to tumor necrosis factor (TNF), interleukin-1 (IL-1), LPS and glucocorticoid, but endothelial cells did not elicit such prominent responses. AM secretion of monocyte-macrophage was mainly regulated by the degree of differentiation into macrophage and activation by LPS and inflammatory cytokines including interferon-γ. The other examined cells showed weaker responses to LPS and IL-1. Although cultured cells may have been transformed as compared with those in the tissue, these data indicate that AM is widely synthesized and secreted from most of the cells in the body and functions as a local factor regulating inflammation and related reactions in addition to as a potent vasodilator. The responses of AM secretion to LPS and inflammatory cytokines suggest that fibroblasts, vascular smooth muscle cells and macrophage are the major sources of AM in the septic shock.
Keywords: Adrenomedullin; Endothelial cell; Vascular smooth muscle cell; Fibroblast; Macrophage; Cytokine; Endotoxin; Inflammation; Sepsis;

Differential expression of adrenomedullin and its receptor component, receptor activity modifying protein (RAMP) 2 during hypoxia in cultured human neuroblastoma cells by Tomomi Kitamuro; Kazuhiro Takahashi; Kazuhito Totsune; Masaharu Nakayama; Osamu Murakami; Wataru Hida; Kunio Shirato; Shigeki Shibahara (1795-1801).
Adrenomedullin is a potent vasodilator peptide originally isolated from a pheochromocytoma. Recently, a novel adrenomedullin receptor has been identified as a complex consisting of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP) 2. To explore possible pathophysiological roles of adrenomedullin and its receptor component RAMP2 in hypoxic tissues, we studied effects of hypoxia on expression of adrenomedullin and RAMP2 in two human neuroblastoma cell lines, IMR-32 and NB69, by radioimmunoassay and Northern blot analysis. Expression levels of adrenomedullin were increased by hypoxia in both cell lines. Treatment with cobalt chloride or desferrioxamine mesylate also increased expression levels of adrenomedullin mRNA. On the other hand, expression levels of RAMP2 mRNA were decreased in IMR-32 cells and were not changed in NB69 cells by hypoxia. Treatment with cobalt chloride or desferrioxamine mesylate decreased expression levels of RAMP2 mRNA in both IMR-32 and NB69 cells. These findings indicate that adrenomedullin expression is induced during hypoxia in IMR-32 and NB69 neuroblastoma cells, but RAMP2 expression is rather suppressed under the same conditions. The decreased expression of RAMP2 and the ADM expression induction under hypoxia may constitute one mechanism of cellular adaptation to hypoxic stress.
Keywords: Adrenomedullin; Hypoxia; RAMP2; Brain; Neuroblastoma;

Adrenomedullin gene products have been localized to neurons in brain that innervate sites known to be important in the regulation of cardiovascular function. Those sites also have been demonstrated to possess receptors for the peptide and central administrations of adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) elevate blood pressure and heart rate in both conscious and anesthetized animals. The accumulated evidence points to a role of the sympathetic nervous system in these cardiovascular effects. These sympathostimulatory actions of AM and PAMP have been hypothesized to be cardioprotective in nature and to reflect the central nervous system (CNS) equivalent of the direct cardiostimulatory effects of the peptides in the periphery. This review summarizes the most recent data on the CNS actions of the adrenomedullin gene-derived peptides and suggests future strategies for the elucidation of the physiologic relevance of the already demonstrated, pharmacologic actions of these peptides.

Poadrenomedullin N-terminal 20 peptide (PAMP) is a hypotensive peptide derived from the precursor of adrenomedullin. We identified novel actions of proadrenomedullin N-terminal 20 peptide (PAMP) on blood glucose, food intake and gastric emptying after exogenous administration. PAMP elevated blood glucose levels after central injection in fasted mice. PAMP had affinity for bombesin (BN) receptor and the hyperglycemic effect of PAMP was blocked by a BN antagonist, indicating that the elevation of blood glucose after central administration of PAMP was mediated by BN receptor. Centrally administered PAMP inhibited food intake and gastric emptying in fasted conscious mice. However, studies using a BN antagonist and BN receptor knockout mice suggested that the inhibitory effects of PAMP on feeding and gastric emptying were mediated not via BN receptor but via another receptor specific for PAMP. In the present review, we summarize these effects of PAMP and report other novel actions of PAMP on body temperature and oxygen consumption. In addition, the mechanism underlying the cardiovascular functions of PAMP is discussed.
Keywords: Proadrenomedullin N-terminal 20 peptide (PAMP); Blood glucose; Food intake; Gastric emptying; Body temperature; Oxygen consumption; Bombesin;

Action sites of adrenomedullin in the rat brain: functional mapping by Fos expression by Yoichi Ueta; Yuko Hara; Kazuo Kitamura; Kenji Kangawa; Tanenao Eto; Yukio Hattori; Hiroshi Yamashita (1817-1824).
The effects of intracerebroventricular (icv) administration of adrenomedullin (AM) and proadrenomedullin NH2-terminal 20 peptide (PAMP) on the expression of Fos in the central nervous system (CNS) were examined in conscious rats, using immunohistochemistry. Fos-like immunoreactivity (LI) was detected in various brain areas of the rats, including the supraoptic nucleus, the paraventricular nucleus, the locus coeruleus, the area postrema and the nucleus of the tractus solitarius 90 min after icv administration of AM. Few cells with Fos-LI were found in the CNS 90 min after icv administration of saline. Fos-LI was also detected in the various hypothalamic areas after icv administration of PAMP. These results suggest that centrally administered AM and PAMP may cause physiological responses through the activation of a neural network in the hypothalamus and the brainstem.
Keywords: Adrenomedullin; Brainstem; Fos; Immunohistochemistry; Locus coeruleus; PAMP; Paraventricular nucleus; Supraoptic nucleus;

Adrenomedullin in the cerebral circulation by Béla Kis; Csongor S. Ábrahám; Mária A. Deli; Hideyuki Kobayashi; Akihiko Wada; Masami Niwa; Hiroshi Yamashita; Yoichi Ueta (1825-1834).
The central nervous system requires an effective autoregulation of cerebral circulation in order to meet the critical and unusual demands of the brain. In addition, cerebral microvessels has a unique feature, the formation of the blood-brain barrier, which contributes to the stability of the brain parenchymal microenvironment. Many factors are known to be involved in the regulation of cerebral circulation and blood-brain barrier functions. In the last few years a new potential candidate, adrenomedullin, a hypotensive peptide was added to this list. Adrenomedullin has a potent vasodilator effect on the cerebral vasculature, and it may be implicated in the pathologic mechanism of cerebrovascular diseases. In this review, we describe current knowledge about the origin and possible role of adrenomedullin in the regulation of cerebral circulation and blood-brain barrier functions.
Keywords: Adrenomedullin; Blood-brain barrier; Cerebral circulation; Cerebral endothelial cells; Production; Receptors;

The typical cardiovascular response to polymicrobial sepsis is characterized by an early, hyperdynamic phase followed by a late, hypodynamic phase. Although the factors and/or mediators responsible for producing the transition from the hyperdynamic to the hypodynamic stage are not fully understood, recent studies have suggested that adrenomedullin (AM), a potent vasodilatory peptide, appears to play an important role in initiating the hyperdynamic response following the onset of sepsis. In addition, the reduced vascular responsiveness to AM may result in the transition from the early, hyperdynamic phase to the late, hypodynamic phase of sepsis. It is possible that changes in newly reported AM receptors calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein-2 or -3 (RAMP2, RAMP3) as well as AM binding protein-1 (AMBP-1) may also play distinct roles in the biphasic cardiovascular response observed during sepsis. Although it remains unknown whether AM gene delivery or a chronic increase in vascular AM production in transgenic animals attenuates the development of hypodynamic sepsis and septic shock, it has been shown that modulation of AM vascular responsiveness with pharmacologic agents reduces sepsis-induced mortality. It has been recently demonstrated that AMBP-1 enhances AM’s physiologic effects and plasma levels of AMBP-1 decrease following infections. We therefore propose that downregulation of AMBP-1 and the reduced AM receptor responsiveness are crucial factors responsible for the transition from the hyperdynamic phase to the hypodynamic phase of sepsis.
Keywords: Hemodynamic responses; Vascular responsiveness; Proinflammatory cytokines; Adrenomedullin receptors; Hyperdynamic sepsis; Septic shock; Cecal ligation and puncture;

Regulation of cardiac adrenomedullin in heart failure by Michihisa Jougasaki; J.Aaron Grantham; Margaret M. Redfield; John C. Burnett (1841-1850).
Adrenomedullin (ADM), a potent natriuretic and vasorelaxing peptide with inotropic properties, is elevated in plasma in human and experimental congestive heart failure (CHF). Recent studies suggest that angiotensin II stimulates ADM production and secretion from cardiac myocytes and fibroblasts. In the present study, we investigated cardiac ADM in experimental CHF, and tested the hypothesis that angiotensin converting enzyme (ACE) inhibition modulates cardiac ADM in CHF. Cardiac tissue ADM immunoreactivity and gene expression were assessed by radioimmunoassay, immunohistochemistry, in situ hybridization and Northern blot analysis in normal and CHF dogs in the presence and absence of ACE inhibition. Experimental CHF was produced by progressive rapid ventricular pacing and characterized by increased ventricular ADM concentrations as well as increased ventricular ADM gene expression. ACE inhibition abolished the increases in ventricular ADM concentrations and ventricular ADM gene expression in CHF. Ventricular ADM gene expression was localized to ventricular myocytes and correlated with left ventricular mass index, suggesting that ventricular ADM is a marker for ventricular hypertrophy. In contrast, atrial ADM concentrations and gene expression did not change in CHF with or without ACE inhibition. Increased plasma ADM concentrations in CHF were also abolished with ACE inhibition. The present study demonstrates that circulating and ventricular ADM are activated in pacing-induced experimental CHF and that ACE inhibition reverses ventricular ADM activation in CHF. This study also indicates that cardiac ADM gene expression is differently regulated between atrium and ventricle in CHF.
Keywords: Gene expression; Heart failure; Peptides; Hormone; Heart hypertrophy;

Adrenomedullin (AM) is a multifunctional peptide with a range of cardiovascular functions including direct effects on cardiomyocytes. Despite the demonstration of high numbers of AM binding sites in rat heart, the receptor responsible for cardiac AM action has not been characterized. We show here that the hearts of adult and neonatal rats, and neonatal cardiomyocyte cultures coexpress mRNA transcripts for the calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs). RAMP2 mRNA predominated over RAMP1 in adult (20:1 ratio) and neonatal heart (10:1 ratio), and in cardiomyocyte cultures (1.7:1 ratio), though the relative abundance of these transcripts appears to be developmentally regulated. Transient transfection of cardiomyocytes using a cAMP-responsive element (CRE)-luciferase reporter gene as an indirect measure of cAMP activation, demonstrated that overexpression of either CRLR or RAMP2 potentiated the AM signalling response. Overexpression of CRLR and RAMP2 together led to an additive effect on AM signalling that was approximately 4-fold greater than control cardiomyocytes stimulated with AM. Furthermore, the AM-mediated induction of CRE-luciferase activity was abolished by coincubation with the receptor antagonist CGRP(8–37) or by overexpression of a CRLR antisense construct. These data demonstrate that AM action in the rat cardiomyocyte requires CRLR and RAMP2 to constitute functional AM receptors.
Keywords: Adrenomedullin; CRLR; RAMP; Cardiomyocyte;

Effects of adrenomedullin on hypertrophic responses induced by angiotensin II, endothelin-1 and phenylephrine by Marja Luodonpää; Olli Vuolteenaho; Sinikka Eskelinen; Heikki Ruskoaho (1859-1866).
We examined whether adrenomedullin (AM), a vasoactive peptide with significant expression and binding sites in the heart, modulates the hypertrophic response in cultured neonatal rat ventricular myocytes. Myocyte hypertrophy was induced by treating the cells with angiotensin II (Ang II), endothelin-1 (ET-1) or α-adrenergic agonist, L-phenylephrine (PHE). All treatments resulted in a hypertrophic response as reflected by increased protein synthesis and expression of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) genes. AM treatment resulted in a complete inhibition of the Ang II-induced increase in ANP and BNP gene expression and secretion. In contrast, no inhibitory effect was seen in either ET-1-induced natriuretic peptide gene expression or PHE-induced ANP and BNP gene expression and secretion. AM had only a modest effect on basal levels of natriuretic peptide secretion and gene expression. When AM mRNA levels in isolated neonatal rat myocytes treated for 48 h with Ang II, ET-1 or PHE were measured, only Ang II induced a consistent increase in AM gene expression. These results indicate that AM is not invariably associated with attenuation of the hypertrophic response but its effect is dependent on the stimulus activating myocyte hypertrophy. AM may form an important autocrine/paracrine growth-inhibitory loop in Ang II-induced myocyte hypertrophy.
Keywords: Adrenomedullin; Natriuretic peptides; Ventricular hypertrophy; Angiotensin II; Endothelin-1; Catecholamines;

Two molecular forms of plasma adrenomedullin during tilt test in healthy subjects by Toshio Nishikimi; Minami Junichi; Takanori Yasu; Suzuki Takeshi; Kenji Kangawa; Hiroaki Matsuoka (1867-1872).
There is accumulating evidence suggesting that adrenomedullin (AM) may participate in the regulation of circulatory homeostasis and pathophysiology of cardiovascular disease. A recent study revealed that two molecular forms of AM, an active form of mature AM (AM-m) and an intermediate inactive form of glycine-extended AM (AM-Gly), circulate in human plasma. The object of the present study was to evaluate the effect of orthostasis on a time course of two molecular forms of plasma AM and to compare them with the behavior of other vasoactive hormones. Twelve healthy male volunteers were studied. The experimental protocol consisted of 20 min of supine rest, tilting at 70° for 20 min, and then 20 min of supine rest. Blood pressure and heart rate were measured every minute. Blood samples were obtained before, at 2 and 18 min during the tilt test, and 2 and 18 min after the test for the measurements of vasoacting hormones and hematocrit. Blood pressure and heart rate were slightly increased earlier during tilting and then remained elevated until the end of the test. The increase in heart rate and blood pressure returned to normal levels early after the tilt test. Plasma epinephrine and norepinephrine significantly increased during the tilt test. These hormones returned to normal levels 18 min after the test. The plasma renin activity, antidiuretic hormone and dopamine were also increased by the end of the tilt test, whereas plasma atrial natriuretic peptide was significantly decreased after the tilt test. Hematocrit increased slightly in the early phase of the tilt test and was further increased by the end of the test. In contrast, plasma AM-Gly or AM-m did not change during the tilt test or the recovery period. Nitric oxide metabolites did not change, either. There were no significant relationships between plasma catecholamines and AM. Plasma brain natriuretic peptide did not change during the tilt test or the recovery period, either. These results suggest that the two molecular forms of AM, AM-m and AM-Gly in plasma, did not respond to the short term tilting stress. These findings may support the hypothesis that plasma AM is secreted in a constitutive manner from the vascular wall.
Keywords: Adrenomedullin; Tilt-test; Brain natriuretic peptide; Atrial natriuretic peptide;

Association of plasma adrenomedullin with carotid atherosclerosis in chronic ischemic stroke by Kaori Shinomiya; Koji Ohmori; Hideo Ohyama; Naohisa Hosomi; Tsutomu Takahashi; Kunihiko Osaka; Masakazu Kohno (1873-1880).
Adrenomedullin is a potent vasodilator peptide exerting anti-atherosclerotic actions in vitro. We investigated the impact of the severity of atherosclerosis on plasma mature-adrenomedullin (m-AM) levels in 38 patients with chronic ischemic stroke. The variables of carotid artery atherosclerosis assessed using ultrasound measurement, blood pressure, and risk factors were related to m-AM levels. Severe atherosclerosis was associated with a further elevation of the increased m-AM level in patients with high systolic blood pressure. Even in patients with fewer risk factors, the presence of severe atherosclerosis was associated with an increased m-AM level. Thus, atherosclerosis elevates m-AM independent of the blood pressure level or presence of risk factors.
Keywords: Adrenomedullin; Atherosclerosis; Ultrasound; Carotid artery; Risk factors;

Studies of the microvascular effects of adrenomedullin and related peptides by Duc Quyen Chu; David M Smith; Susan D Brain (1881-1886).
Adrenomedullin (ADM) exerts potent vasoactive effects in the microvasculature. These activities have been most extensively studied in the cutaneous microcirculation. In this review we examine the knowledge gained to date of the ability of ADM to influence microvascular effects that include increased blood flow, microvascular permeability (leading to edema formation), neutrophil accumulation and cutaneous thermal hyperalgesia. ADM is structurally related to the vasodilator neuropeptide calcitonin gene-related peptide (CGRP). The peptides are considered to act via a family of receptor activity modifying proteins (RAMPs) that interact with a G-protein linked receptor, calcitonin receptor-like receptor (CRLR). A correlation of microvascular activity with effects mediated via CRLR and RAMP is discussed.
Keywords: Adrenomedullin; ADM; CGRP; Microvasculature; Vasodilation; Increased microvascular permeability; Neutrophil accumulation; Edema formation; CRLR; RAMPs;

We have reported that the rat mesenteric resistance artery has innervation of calcitonin gene-related peptide (CGRP)-containing vasodilator nerves (CGRPergic nerves). We also demonstrated that adrenomedullin (AM) causes mesenteric vasodilation through activation of CGRP receptors. The present study was designed to examine the effect of AM on neurotransmission of CGRPergic nerves in rat mesenteric arteries. In preconstricted preparations without endothelium, periarterial nerve stimulation (PNS, 1 and 2 Hz) induced a frequency-dependent vasodilation. A bolus injection of CGRP (10 pmol) into the perfusate also caused a vasodilation. AM (0.1 to 10 nM) concentration-dependently caused 40% to 60% inhibition of the PNS-induced vasodilation, but AM did not attenuate vasodilation induced by exogenous CGRP injection. The inhibitory effect of AM (10 nM) on PNS-induced vasodilation was further potentiated by CGRP [8–37] (CGRP receptor antagonist, 50 nM), which attenuated the vasodilator response to the CGRP injection. Combined perfusion of AM [22–52] (AM receptor antagonist, 10 to 100 nM) resulted in further inhibition of PNS-induced neurogenic vasodilation without affecting the vasodilator response to the CGRP injection. CGRP [8–37] but not AM [22–52] antagonized vasodilation induced by AM perfusion. These findings suggest that AM presynaptically inhibits neurotransmission of CGRPergic nerves, probably decreasing CGRP release, via receptors different from CGRP receptors.
Keywords: Adrenomedullin; Calcitonin gene-related peptide (CGRP); Adrenomedullin receptor; CGRP receptors; Neurotransmission of CGRPergic nerves; Rat mesenteric resistance artery;

Adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP) in adrenal chromaffin cells by Hideyuki Kobayashi; Toshihiko Yanagita; Hiroki Yokoo; Akihiko Wada (1895-1901).
Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are peptides having multiple physiological functions and are most abundantly expressed in the adrenal medulla. In addition to PAMP, PAMP12, a 12 amino acid peptide with sequence identity to PAMP between amino acids 9–20, has also been shown to be expressed in the adrenal medulla. AM, PAMP and PAMP12 are released along with catecholamines by regulated exocytosis upon stimulation of adrenal chromaffin cells. PAMP and PAMP12 regulate catecholamine release and synthesis by interfering with nicotinic cholinergic receptors in these chromaffin cells. AM may also cause gradual release of catecholamine from these cells. AM, PAMP and PAMP12 are endogenous peptides that modulate chromaffin cell function via different mechanisms.
Keywords: Adrenomedullin; PAMP; Cyclic AMP; Adrenal medulla; Chromaffin cells; Catecholamine; Cholinergic receptors;

Adrenomedullin and calcitonin gene-related peptide receptors in the rat adrenal cortex by Supriya Kapas; Derek Renshaw; Mark Carroll; Joy P Hinson (1903-1907).
The actions of calcitonin gene-related peptide (CGRP) and adrenomedullin on steroid hormone secretion from the rat zona glomerulosa are controversial, with reports in the literature of both stimulatory and inhibitory effects. It appears that these results previously obtained may depend on the nature of the receptors expressed by zona glomerulosa cells. The present study was designed to characterize CGRP and adrenomedullin binding in the rat adrenal zona glomerulosa. Specific binding for both peptides was observed, with two CGRP receptor sites found, and a single population of adrenomedullin receptors, but approximately twice the number of adrenomedullin binding sites. Messenger RNA analysis of the candidate genes for CGRP and adrenomedullin receptors revealed an abundance of both CRLR and RAMP1 mRNA, suggesting that these genes encode one of the CGRP receptors in this tissue. Much less RAMP2 expression was observed, however, which suggests that another gene product may account for adrenomedullin binding. There were very low levels of RAMP3 expression, but abundant L1 mRNA present, which may suggest that this rather controversial receptor has a role in the adrenal. The finding of distinct and specific adrenomedullin and CGRP binding in this tissue may account for the different effects these peptides appear to exert on adrenal function.
Keywords: CGRP; adrenomedullin; RAMPs; adrenal; zona glomerulosa;

Proadrenomedullin N-terminal 20 peptide (PAMP) enhances proliferation of rat zona glomerulosa cells by activating MAPK cascade by Piera Rebuffat; Lucia Gottardo; Ludwik K Malendowicz; Giuliano Neri; Gastone G Nussdorfer (1909-1912).
The effect of proadrenomedullin N-terminal 20 peptide (PAMP) on the proliferative activity of rat zona glomerulosa (ZG) cells has been investigated. Dispersed rat ZG cells were cultured in vitro for 24 h and then exposed to PAMP for an additional 24 h, and the proliferation rate was assessed by the 5-bromo-2′-deoxyuridine (BrdU) incorporation technique. PAMP dose-dependently increased the percentage of BrdU-positive cells, with a maximal effective concentration observed at 10−8 M. The tyrosine kinase (TK) inhibitor, tyrphostin-23, and the p42/p44 MAPK inhibitor, PD-98059, abolished the proliferogenic effect of PAMP, while the protein kinase (PK) A inhibitor, H-89, and the PKC inhibitor, calphostin-C, were ineffective in blocking the response to PAMP. PAMP (10−8 M) enhanced TK and MAPK activity of dispersed rat ZG cells. The stimulatory action of PAMP on TK activity was annulled by tyrphostin-23, while that on MAPK activity was abolished by either tyrphostin-23 or PD-98059. Taken together, these data indicate that PAMP enhances proliferation of cultured rat ZG cells, through the TK-dependent activation of p42/p44 MAPK cascade.
Keywords: PAMP; Adrenal zona glomerulosa; Cell proliferation; MAPK cascade;

Effects of intracavernous administration of adrenomedullin on erectile function in rats by Hiroaki Nishimatsu; Yasunobu Hirata; Hiroshi Hayakawa; Daisuke Nagata; Hiroshi Satonaka; Etsu Suzuki; Shigeo Horie; Takumi Takeuchi; Nobutaka Ohta; Yukio Homma; Shigeru Minowada; Ryozo Nagai; Kazuki Kawabe; Tadaichi Kitamura (1913-1918).
We have reported that adrenomedullin (AM)-induced vasodilation is at least in part nitric oxide (NO)-cGMP-dependent in the rat. Although it is well known that NO is much involved in the erectile function, it is controversial as to whether AM influences the erectile function. Thus, we examined the effects of AM on intracavernous pressure (ICP) during penile erection. The left carotid artery of rats was cannulated to monitor of mean arterial pressure (MAP). Bipolar electrodes were positioned on the cavernous nerve. The right cavernous body was cannulated with a needle connected to a pressure transducer to monitor ICP. Electrical stimulation (ES) increased ICP in a voltage-dependent manner. Elevation of ICP continued during ES. The intracavernous injection of 0.5 nmol AM significantly potentiated ES-induced increases in both maximal developed ICP/MAP and area under the curve (ICP trace; AUC). Since AM slightly lowered MAP, ICP was normalized by MAP. i.v. administration of Nω-nitro-L-arginine, a NO synthase inhibitor, markedly decreased AM/ES-induced ICP elevation. However, in the presence of E-4021, a cGMP-specific phosphodiesterase inhibitor, AM further increased both ICP/MAP and AUC. These results suggest that a NO-cGMP pathway is involved in the regulation of AM-induced rat cavernous vasorelaxation.
Keywords: Adrenomedullin; Vascular endothelium; Nitric oxide; Erectile dysfunction;

Cellular and molecular actions of adrenomedullin in glomerular mesangial cells by Narayanan Parameswaran; Wojciech Nowak; Carolyn S Hall; Harvey V Sparks; William S Spielman (1919-1924).
Adrenomedullin (AM), a potent vasodilatory and hypotensive peptide produces several biological outcomes in glomerular mesangial cells. Mesangial cells are important in the pathogenesis of glomerulonephritis, and therefore the actions of AM on mesangial cells have important clinical and therapeutic implications. This minireview describes the various actions of AM on mesangial cell function and the signal transduction mechanisms involved. As in other systems, most actions of AM can be explained by increase in cAMP levels in the cell, although a few exceptions remain. The fact that most data obtained to date has been in culture, the physiological significance of the actions of AM in mesangial cells is discussed.

Role of adrenomedullin and its receptor system in renal pathophysiology by Masashi Mukoyama; Akira Sugawara; Tetsuya Nagae; Kiyoshi Mori; Hiroyuki Murabe; Hiroshi Itoh; Issei Tanaka; Kazuwa Nakao (1925-1931).
Adrenomedullin (AM), a potent vasorelaxing, natriuretic and cell growth-modulating peptide, is thought to act as an autocrine/paracrine regulator in renal glomeruli and tubules. AM receptors comprise the calcitonin receptor-like receptor (CRLR) and a family of receptor-activity-modifying proteins (RAMPs 1–3); however, the pathophysiological role of AM and its receptor system in the kidney remains to be clarified. We examined the regulation of their expression in a rat model of renal injury and found that RAMP1, RAMP2 and CRLR expressions were markedly upregulated upon induction of fibrosis during obstructive nephropathy. Since AM exerts potent antiproliferative effects in various cell types, upregulation of the AM receptor system may play important roles in modulating the progression of renal diseases.
Keywords: Adrenomedullin; Adrenomedullin receptor; Receptor-activity-modifying proteins (RAMPs); Calcitonin receptor-like receptor (CRLR); Mesangium; Obstructive nephropathy;

Adrenomedullin and its receptor complexes in remnant kidneys of rats with renal mass ablation: decreased expression of calcitonin receptor-like receptor and receptor-activity modifying protein-3 by Kazuhito Totsune; Kazuhiro Takahashi; Harald S Mackenzie; Zenei Arihara; Fumitoshi Satoh; Masahiko Sone; Osamu Murakami; Sadayoshi Ito; Barry M Brenner; Toraichi Mouri (1933-1937).
Adrenomedullin (AM) has vasodilator and diuretic actions, similarly to natriuretic peptides. AM receptor complexes are composed of calcitonin receptor-like receptor (CRLR) and receptor-activity modifying protein-2 (RAMP2), or CRLR and RAMP3. We aimed to know whether gene expression of AM and AM receptor complexes are regulated in kidneys under pathophysiological conditions. Expression of AM, RAMP2, RAMP3 and CRLR mRNA was studied in the remnant kidney of rats with renal mass ablation using competitive quantitative RT-PCR techniques. Partial cloning was performed to determine the rat RAMP3 nucleotide sequence. In normal rat kidneys, expression levels of RAMP2, RAMP3, CRLR and AM mRNAs were 26.5 ± 1.9 mmol/mole of GAPDH, 7.7 ± 0.9 mmol/mole of GAPDH, 3.6 ± 0.2 mmol/mole of GAPDH and 0.57 ± 0.03 mmol/mole of GAPDH (mean ± SE, n = 6), respectively. RAMP3 mRNA levels decreased significantly to about 50% and about 70% of control (sham-operated rats) 4 days and 14 days after 5/6 nephrectomy, respectively. CRLR mRNA levels also decreased significantly to about 30% and about 43% of control. Sodium intake restriction had no significant effects on the RAMP3 and CRLR gene expression. On the other hand, RAMP2 mRNA expression in the kidney was suppressed by sodium intake restriction regardless of nephrectomy, while RAMP2 levels in the remnant kidney were not significantly changed by 5/6 nephrectomy. Neither 5/6 nephrectomy or sodium intake restriction had any significant effects on the AM gene expression in the kidney. The present study showed that expression of mRNAs encoding AM, RAMP2, RAMP3 and CRLR were differentially regulated in remnant kidneys of rats with renal mass ablation.
Keywords: Adrenomedullin; Calcitonin receptor-like receptor; Receptor-activity modifying protein; Renal failure; Kidney; RT-PCR;

Corticotrophin-releasing hormone (CRH) is a 41 amino acid neuropeptide that is expressed in the hypothalamus and the human placenta. Placental CRH production has been linked to the determination of gestational length in the human. Although encoded by a single copy gene, CRH expression in the placenta is regulated differently to the hypothalamus. Glucocorticoids stimulate CRH promoter activity in the placenta but inhibit it’s activity in the hypothalamus, via mechanisms involving different regions of the CRH promoter. We discuss how various stimuli alter CRH promoter activity and why these responses are unique to the placenta.
Keywords: cAMP; Glucocorticoids; Steroid hormones; AP-1; Ecdysone;