Rat Adrenomedullin Research Articles

Genomic Organization and Regulation of a Human 7-Helix Transmembrane Receptor Which Is Expressed in Pulmonary Epithelial Cells and Induced in Hypoxia

The genetic, regulatory, and tissue-specific characterization of G-protein-coupled receptors is substantial since it contributes to the identification of the natural ligand which may influence basic physiological processes and cell function. Here we explored the genomic structure of a human orphan seven-transmembrane receptor which presents the human homologue of a receptor which has been controversially identified as a rat adrenomedullin receptor subtype. Based on the cDNA sequence a 3.4 kb genomic DNA fragment was isolated. Sequencing of the fragment and comparison studies revealed an intron of 544 bp in the 5′ untranslated region, followed by a second exon encoding the receptor protein of 404 amino acids. The gene is localized on chromosome 12q. The 5′ regulatory region contains several SP1, AP2, and CAAT sites as well as hypoxia responsive elements (HRE) both in the 5′ and 3′ regulatory region. RT-PCR with intron spanning primers demonstrated mRNA signals in various tissues, especially in lung. Characterizing the histological expression pattern in lung sections by nonisotopic in situ hybridization, a strong signal of receptor mRNA was identified in pulmonary epithelial cells of bronchi and alveoli. Analysis of the two human pulmonary epithelial cell lines, H23 and A549, showed significant mRNA induction of this receptor subtype in hypoxia.

Role of increased circulating and renal adrenomedullin in rats with malignant hypertension.

Although it has been reported that the circulating adrenomedullin (AM) level is elevated in hypertension and renal failure, the pathophysiological significance of circulating and intrarenal AM in malignant hypertension remains unknown. We investigated the circulating and intrarenal AM system in rats with malignant hypertension by measuring the plasma level, renal tissue level, and mRNA abundance of AM and the mRNA abundance of AM receptor. We also investigated the effects of intravenously infused calcitonin gene-related peptide (CGRP)-(8-37), an antagonist of AM, on the hemodynamics and renal tubular function. We studied the following four groups: control Wistar-Kyoto rats (WKY), control spontaneously hypertensive rats (C-SHR), salt-loaded SHR (S-SHR), and DOCA-salt SHR (D-SHR). After 3 wk of DOCA treatment, D-SHR developed malignant hypertension. D-SHR were characterized by higher blood pressure, kidney weight, urinary protein excretion and blood urea nitrogen, and lower creatinine clearance compared with the other three groups. The plasma AM level and urinary excretion of AM were markedly higher in D-SHR than in the other three groups. In the kidney, the tissue AM level and the expression of AM mRNA in the renal medulla were significantly increased in D-SHR compared with the other three groups, whereas there were no significant differences in these levels in the renal cortex among the four groups. In the renal AM receptor system, the expression of the gene for receptor activity modifying protein 3 was significantly increased in the renal medulla in D-SHR compared with the other three groups. An immunohistochemical study revealed that AM immunostaining in renal collecting duct cells and distal tubules was more intense in D-SHR than in the other three groups. After CGRP-(8-37) infusion, blood pressure increased significantly and urinary sodium excretion and urine flow decreased significantly only in D-SHR. These results suggest that the increased circulating AM and renal AM and the increased expression of the mRNA for AM and its receptor may at least partly compensate for the malignant hypertensive state in certain forms of malignant hypertension via the hypotensive, natriuretic, and diuretic actions of AM.

Expression of Adrenomedullin by Human Placental Cytotrophoblasts and Choriocarcinoma JAr Cells

Adrenomedullin is a multifunctional peptide expressed in a variety of tissues. This study was conducted to investigate the expression of adrenomedullin and its mRNA by human trophoblasts and the possible existence of adrenomedullin receptor in those cells. Human placentas in all three trimesters were obtained from patients undergoing therapeutic abortions and deliveries. Total RNA was extracted from placental trophoblastic tissues and JAr choriocarcinoma cells, and the expression of adrenomedullin mRNA was determined by RT-PCR. Immunohistochemical analysis was performed by the avidin/biotin immunoperoxidase method using a specific antibody to adrenomedullin. The secretion of adrenomedullin by JAr cells cultured in medium containing [35S]cysteine-[35S]methionine was determined by immunoprecipitation followed by PAGE. The presence of adrenomedullin receptor in JAr cells was examined using a binding assay with [125I]rat adrenomedullin. Adrenomedullin mRNA was expressed by human placental trophoblastic tissues in all three trimesters and by JAr cells. Immunohistochemical analysis revealed that adrenomedullin is expressed by cytotrophoblasts in placentas in all three trimesters, but not by syncytiotrophoblasts. The expression of adrenomedullin in the cytotrophoblast was most abundant in first trimester placenta and became less abundant during the course of pregnancy. JAr cells synthesized and secreted immunoreactive adrenomedullin. Binding assay with [125I]rat adrenomedullin demonstrated specific binding of adrenomedullin to JAr cells, indicating the existence of a specific receptor for adrenomedullin in trophoblastic cells. Adrenomedullin is transcribed and secreted by cytotrophoblastic cells that possess adrenomedullin receptor. Adrenomedullin may play a potential role as an autocrine/paracrine factor in the growth of cytotrophoblasts, especially in early gestation.

The small intestine is an important source of adrenomedullin release during polymicrobial sepsis.

Adrenomedullin (AM), a potent vasodilatory peptide, has recently been reported to be involved in the altered cardiovascular responses under various pathophysiological conditions. Although the increase in plasma AM levels is associated with upregulation of AM gene expression in various tissues, it remains unknown whether the gut is an important source of AM release under such conditions. To determine this, adult male rats were subjected to sepsis by cecal ligation and puncture (CLP) followed by fluid resuscitation. Systemic and portal blood samples were collected simultaneously at 10 and 20 h after CLP or sham operation. A portion of the jejunum was also harvested. Plasma and tissue levels of AM were then determined by RIA. The localization of AM in the intestinal tissue was examined using immunohistochemistry. In an additional group of normal rats, synthetic rat AM (8.5 microg/kg body wt) was infused for 15 min at a constant rate via the portal vein (which produces a similar level of AM as observed during sepsis). Cardiac output, stroke volume, total peripheral resistance, and microvascular blood flow in various organs were determined before and 30 min after AM administration. The results indicate that AM levels in portal blood were significantly higher than in systemic blood at 10 and 20 h after CLP. Intestinal AM was also markedly elevated. Immunohistochemical visualization shows that AM immunostainings were localized in the mucosa, submucosa, and intestinal nerve fibers, and they were increased at 10-20 h post-CLP. Because AM-immunopositive nerve fibers increase in the gut during sepsis, a nerve pathway may be involved in the regulation of vascular reactivity by this peptide. Moreover, intraportal administration of AM increased cardiac output, stroke volume, and microvascular blood flow in the liver, kidney, small intestine, and spleen. In contrast, total peripheral resistance was significantly reduced. Thus the gut plays an important role in increasing the levels of circulating AM during the progression of sepsis. Gut-derived AM appears to be a major factor in initiating the hyperdynamic response after the onset of sepsis.

Effect of adrenomedullin receptor and calcitonin gene-related peptide receptor antagonists on centrally mediated adrenomedullin renal action

To determine pharmacological specificity of intracerebroventricular (IVT) administration of adrenomedullin (AM) on water and sodium excretion, studies were performed in rats pretreated with AM (22-52), a putative AM-receptor antagonist or CGRP(8-37), a ligand that preferentially antagonizes the CGRP 1-receptor subtype. In addition, the effects of IVT injection of calcitonin-gene-related peptide (CGRP) on urinary water and electrolyte excretion was assessed. Intracerebroventricular administration of rat adrenomedullin to conscious hydrated rats resulted in a significant increase in urinary volume and sodium excretion during the 6-h period of urine collection and was most effective at 3 and 6 h. Although less effective than AM, central administration of CGRP induced diuresis and natriuresis. Pretreatment with AM (22-52) or CGRP(8-37) significantly suppressed the diuretic and natriuretic effect of IVT-AM. These data suggest that both CGRP 1 and AM receptors are involved in the centrally mediated diuretic and natriuretic action of the AM. Our results provide evidence supporting the hypothesis that endogenous AM plays a role in the central nervous control of fluid and electrolyte homeostasis.

Calcitonin gene-related peptide (CGRP) inhibits contractions of the prostatic stroma of the rat but not the guinea-pig

This study investigated the presence and effects of calcitonin gene-related peptide (CGRP) within the rat and guinea-pig prostate glands. Immunohistochemical studies demonstrated that CGRP immunoreactive nerve fibres are sparsely distributed throughout the prostatic fibromuscular stroma in both species. These CGRP immunopositive nerve fibres shared a similar distribution profile but were not colocalized with tyrosine hydroxylase immunopositive nerve fibres which also innervate the prostatic stroma of these species. Nerve terminals within rat and guinea-pig prostatic tissues were electrically field stimulated (60 V, 0.5 ms, 10 Hz, 20 pulses every 60 s). In guinea-pig preparations, application of human α-CGRP, rat adrenomedullin or rat amylin (0.1 nM–1 μM) had no effect on responses to field stimulation. In contrast, both rat and human α-CGRP (10 pM–300 nM), rat adrenomedullin (0.3 nM–1 μM) and rat amylin (3 nM–1 μM) concentration-dependently inhibited electrically evoked contractile responses in the rat prostate. The relative order of potency was rat α-CGRP=human α-CGRP>rat adrenomedullin>rat amylin. The inhibition by rat α-CGRP of field stimulation-induced contractions in the rat prostate was competitively antagonized by human CGRP (8–37) (1, 3 and 10 μM) with a pA 2 of 6.20±0.13. Rat α-CGRP (10 nM) attenuated contractile responses of the rat prostate to exogenously added noradrenaline (1–100 μM). Inhibitory concentration–response curves to rat α-CGRP in rat prostates were unaffected by preincubation in either glibenclamide (10–100 μM), N-nitro- l-arginine methyl ester ( l-NAME) (10 μM), bestatin (10 μM), captopril (10 μM) or phosphoramidon (3 μM). Our results indicate that CGRP-induced inhibition of electrically evoked contractions in the rat prostate occurs through activation of postjunctional CGRP 2 receptors which act independently of a K ATP channel or nitrergic mechanisms. Degradation of rat α-CGRP via peptidases does not appear to occur in the rat prostate.

Mechanism of the beneficial effects of pentoxifylline during sepsis: maintenance of adrenomedullin responsiveness and downregulation of proinflammatory cytokines.

Although it is known that pentoxifylline (PTX) produces various beneficial effects during sepsis, it remains unknown whether this agent has any salutary effects on the depressed vascular responsiveness to adrenomedullin (ADM), a novel potent vasodilatory peptide, under such conditions. Adult male Sprague-Dawley rats were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP). One hour after CLP, PTX (50 mg/kg body wt) or vehicle (normal saline) was infused intravenously over 90 min. Twenty hours after CLP (i.e., the late, hypodynamic stage of sepsis), the thoracic aorta and small intestine were isolated and preconstricted by norepinephrine. Rat ADM (10(-7) M) was applied, and the percentage of ADM-induced relaxation in the aortic rings and resistance vessels in the small intestine was determined. In addition, plasma ADM was determined by radioimmunoassay and tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6 levels were measured by enzyme-linked immunosorbent assay. The percentage of ADM-induced vascular relaxation in the aortic rings and resistance vessels of the isolated gut was significantly reduced 20 h after CLP. Administration of PTX early after the onset of sepsis, however, prevented the decrease in vascular ADM responsiveness at the macro- and microcirculatory levels. Plasma ADM levels increased after CLP, irrespective of PTX infusion, indicating that the effect of PTX was not mediated by altering ADM release. The upregulated TNF-alpha, IL-1beta, and IL-6 during late sepsis were, however, attenuated by PTX administration, suggesting that maintenance of ADM responsiveness by this agent appears to be due to downregulation of these cytokines. Since early administration of PTX maintains vascular ADM responsiveness even during the late stage of sepsis, this agent appears to be a useful adjunct in preventing the deterioration in hemodynamics and cardiovascular function during the progression of polymicrobial sepsis.

Diuretic and natriuretic action of adrenomedullin administered intracerebroventricularly in conscious rats

Intracerebroventricular administration of rat adrenomedullin (AM) to conscious hydrated or salt-loaded rats, resulted in a significant increase in urinary volume. The diuretic effect of adrenomedullin occurred during the 6-h period of urine collection and was most effective during the 3 and 6 h. Most remarkably, AM given IVT induced a dose-related increase in urinary sodium excretion at all periods of urine collection. In addition, AM induced significant kaliuresis. Our results strongly suggest that AM may play a significant role in the central regulation of fluid and electrolyte homeostasis, and that its diuretic and natriuretic effect may be, at least in part, centrally mediated.

A physiological role for adrenomedullin in rats; a potent hypotensive peptide in the hypothalamo-neurohypophysial system.

Adrenomedullin, a potent hypotensive peptide, was originally isolated from human phaeochromocytoma. Adrenomedullin immunoreactivity and gene expression are found not only in peripheral organs but also in the central nervous system. Adrenomedullin labelled cells were localised in the hypothalamus, including in the paraventricular and supraoptic nuclei, in rats. Abundant adrenomedullin-immunoreactive fibres and varicosities were found in the hypothalamo-neurohypophysial tract and the internal zone of the median eminence in colchicine-treated and hypophysectomized rats, whereas in control rats few adrenomedullin-labelled fibres were observed. We examined the effects of intracerebroventricular administration of adrenomedullin on neurosecretory cells in the paraventricular and supraoptic nuclei of rats, using immunohistochemistry for Fos protein and in situ hybridisation histochemistry for c-fos mRNA. Intracerebroventricular administration of adrenomedullin caused a marked induction of Fos-like immunoreactivity in the paraventricular nucleus and the dorsal part of the supraoptic nucleus. In the paraventricular and supraoptic nuclei, nuclear Fos-like immunoreactivity was predominantly in oxytocin-immunoreactive cells rather than vasopressin-immunoreactive cells. The induction of c-fos mRNA in the paraventricular and supraoptic nuclei was increased in a dose-related manner 30 min after intracerebroventricular administration of adrenomedullin. This induction was reduced by pre-treatment with the adrenomedullin receptor antagonist, human adrenomedullin-(22-52)-NH2. Intracerebroventricular administration of adrenomedullin also caused a marked increase in the plasma concentration of oxytocin. Extracellular recordings from magnocellular neurosecretory cells in the paraventricular nucleus revealed that putative oxytocin-secreting cells were activated by intracerebroventricular administration of adrenomedullin. These results suggest that central adrenomedullin preferentially stimulates the secretion of oxytocin by activating hypothalamic oxytocin-secreting cells and may have an important role in salt appetite and body fluid homeostasis in rats.

Pharmacological characterization of the CGRP receptor in the lateral line organ of Xenopus laevis.

Calcitonin gene-related peptide (CGRP) is a neurotransmitter candidate colocalized with acetylcholine in efferent fibers innervating hair cell organs. We have used the Xenopus laevis lateral line organ to investigate the responses of a hair cell organ to the CGRP family of peptides. Two isoforms of CGRP, r alpha-CGRP and r beta-CGRP, and a human analog of alpha-CGRP, h(Tyro) alpha-CGRP, produced dose-dependent increases in afferent nerve fiber discharge rate with EC50 values of approximately 1 microM. Rate increases were 31.2, 18.9, and 10.3%, respectively. The peptide fragment rCGRP8-37 a selective CGRP1 receptor antagonist, competitively inhibited the response to r alpha-CGRP. Diacetoamidomethyl cysteine CGRP (r[Cys(ACM)2,7]alpha-CGRP), a CGRP2 agonist, did not change discharge rate. Rat amylin did not increase rate until very high concentrations, and then the change was less than 7%. Rat adrenomedullin produced no increase in rate. Responses to r alpha-CGRP developed after metamorphosis. No change in spontaneous discharge rate was observed until postmetamorphic day 6, and then it was only a fraction of the maximal response. This response progressively increased until postmetamorphic day 28, when it reached its maximal value. The most straightforward interpretation of our results is that the effect of CGRP is mediated by the CGRP1 receptor and that CGRP, of the peptides presently known to exist in the CGRP family, is the most likely endogenous peptide mediating these effects.

Peripheral distribution and gene expression of adrenomedullin in the rat: possible source of blood adrenomedullin

Adrenomedullin (AM) was detected in all tissues examined with the highest concentrations in adrenal gland, lung and cardiac atrium. High concentrations of pre-proadrenomedullin mRNA were also detected in the lung, cardiac atrium, adrenal gland, thoracic aorta and mesenteric artery for the first time by solution-hybridization-RNase protection assay. The molecular forms of rat AM in various tissues and plasma were also characterized by Biogel P30gel filtration chromatography. We found no significant difference in immunoreactive AM levels between the veins draining the kidney, the lung and the adrenal and the systemic arterial blood. The very low peptide/mRNA ratio and the AM/precursor ratio in the mesenteric artery and thoracic aorta suggest that blood vessels may be the main source of plasma AM.

Potentiation of the hypotensive effect of adrenomedullin in pregnant rats

The hypotensive effect of adrenomedullin, a potent vasodilator peptide, was examined in conscious pregnant (6, 13 and 20 days of pregnancy) and non-pregnant rats. The intravenous administration of adrenomedullin (0.01–3.0 nmol/kg) produced a dose-dependent depressor response in pregnant and non-pregnant rats. At low doses (0.01–0.1 nmol/kg), the maximum decrease in blood pressure was significantly higher in pregnant rats (20 days pregnant) than in non-pregnant rats. At high doses, no significant difference was observed between the two groups. Furthermore, the administration of adrenomedullin did not significantly affect the basal mean blood pressure (MBP) at any dose when compared to the non-pregnant group at 6 and 13 days of pregnancy. In the ovariectomized rats, the depressor responses in 17β-estradiol-treated, progesterone-treated and 17β-estradiol+progesterone-treated rats were not significantly different from that in the control rats, suggesting that the augmented effect on the depressor response to adrenomedullin in pregnant rats may not be due to hormonal changes during pregnancy. The adrenomedullin receptor mRNA level of the descending thoracic aorta was significantly higher in the late-pregnancy rats (20 days of pregnancy). However, the levels did not show any difference between the early-pregnant rats (6 and 13 days of pregnancy) and the non-pregnant rats. These findings suggested that the changes in the depressor response to adrenomedullin which occur at term in pregnant rats may be mediated by changes of adrenomedullin receptor gene expression rather than by sex hormones.

Regulation of rat mesangial cell migration by platelet-derived growth factor, angiotensin II, and adrenomedullin.

This study sought to determine whether platelet-derived growth factor (PDGF) and angiotensin II (AngII) stimulate migration of cultured rat glomerular mesangial cells. After finding that this was so, the effects of adrenomedullin (ADM) and cAMP-elevating agents on basal and stimulated mesangial cell migration were examined. Two isoforms of PDGF, AB and BB, stimulated migration in a concentration-dependent manner between 1 and 50 ng/ml, while the AA isoform lacked significant effect. AngII modestly but significantly stimulated migration in a concentration-dependent manner between 10(-7) and 10(-6) mol/L. Rat ADM significantly inhibited the PDGF BB- and AngII-stimulated migration in a concentration-dependent manner between 10(-8) and 10(-7) mol/L. Inhibition by rat ADM was accompanied by an increase in cellular cAMP. cAMP agonists or inducers such as 8-bromo cAMP, forskolin, and prostaglandin I2 also significantly reduced the stimulated migration. H 89, a protein kinase A (PKA) inhibitor, attenuated the inhibitory effect of ADM, and a calcitonin gene-related peptide (CGRP) receptor antagonist, human CGRP (8-37), abolished the inhibitory effects of rat ADM. These results suggest that PDGF AB and BB as well as AngII stimulate rat mesangial cell migration and that ADM can inhibit PDGF BB- and AngII-stimulated migration, at least in part through cAMP-dependent mechanisms likely to involve specific ADM receptors with which CGRP interacts. The adenylate cyclase/cAMP/PKA system may be involved in the migration-inhibitory effect of ADM in these cells.

Acute hemodynamic and renal effects of adrenomedullin in rats with aortocaval shunt

Heart failure is characterized by increased vascular resistance and water retention. Adrenomedullin is a peptide hormone with vasodilating and diuretic properties whose efficacy in heart failure has not been well established. We used an aortocaval shunt model of moderate heart failure in rats and infused increasing doses of adrenomedullin, both as bolus injections and 20-min infusions. In controls, a clear dose-dependent 4.8±1.0 to 13.6±2.3 mm Hg decrease in arterial blood pressure was observed after injection of 1 μg to 30 μg of adrenomedullin. In rats with aortocaval shunt, the hypotensive responses were significantly diminished. The urine flow rate, which was diminished at baseline in rats with aortocaval shunt, was increased and normalized by adrenomedullin administration. The glomerular filtration rate increased after infusion of adrenomedullin (0.5 μg/kg min −1) from 2.37±0.25 to 3.47±0.43 ml/min ( P<0.01) in controls and from 1.79±0.33 to 2.58±0.49 ( P<0.05) in rats with aortocaval shunt. Similarly, renal blood flow was significantly increased by adrenomedullin in both groups. Our results indicate a beneficial effect of adrenomedullin on renal function in rats with aortocaval shunt. These data suggest that adrenomedullin might be of potential therapeutic value in heart failure, without inordinately decreasing blood pressure.

Rat-2 fibroblasts express specific adrenomedullin receptors, but not calcitonin-gene-related-peptide receptors, which mediate increased intracellular cAMP and inhibit mitogen-activated protein kinase activity

Rat-2 fibroblasts demonstrate specific binding of 125I-labelled rat adrenomedullin (KD = 0.43nM; Bmax = 50fmol/mg of protein) in the absence of 125I-labelled calcitonin-gene-related peptide (CGRP) binding. Therefore Rat-2 cells were used to examine the pharmacology and signal transduction pathways of adrenomedullin receptors. We examined the effects of adrenomedullin, the CGRP receptor antagonist CGRP-(8–37) and the amylin antagonists AC187 and AC253 on receptor binding and cAMP production. AC253, AC187 and CGRP-(8–37) inhibited 125I-adrenomedullin binding, with respective IC50 values of 25±8, 129±39 and 214±56nM. Adrenomedullin dose-dependently increased intracellular cAMP (approximate EC50 = 1.0nM). CGRP-(8–37), AC253 and AC187 antagonized adrenomedullin-stimulated cAMP production at micromolar concentrations. Using kinase-substrate assays, Mono Q FPLC and ‘phospho-specific ’ Western blotting, we found that adrenomedullin alone abolished basal mitogen-activated protein kinase (MAPK) activity and dose-dependently inhibited platelet-derived-growth-factor-stimulated MAPK activity. Radioimmunoassay for adrenomedullin of media from Rat-2 cells showed a linear release of adrenomedullin-like immunoreactivity of 3.1fmol/h per 2×106 cells. Gel-filtration chromatography showed that this adrenomedullin-like immunoreactivity co-eluted with synthetic rat adrenomedullin. Northern blotting with a rat adrenomedullin cDNA probe was used to confirm the presence of adrenomedullin mRNA. However, neither Northern blotting nor reverse transcriptase–PCR showed the presence of the cloned adrenomedullin receptor (L1). We conclude that the Rat-2 cell line expresses a specific adrenomedullin receptor (coupled to cAMP production and regulation of MAPK) and secretes adrenomedullin, which may participate in a regulatory control loop.

Rat-2 fibroblasts express specific adrenomedullin receptors, but not calcitonin-gene-related-peptide receptors, which mediate increased intracellular cAMP and inhibit mitogen-activated protein kinase activity

Rat-2 fibroblasts demonstrate specific binding of 125I-labelled rat adrenomedullin (KD=0.43 nM; Bmax=50 fmol/mg of protein) in the absence of 125I-labelled calcitonin-gene-related peptide (CGRP) binding. Therefore Rat-2 cells were used to examine the pharmacology and signal transduction pathways of adrenomedullin receptors. We examined the effects of adrenomedullin, the CGRP receptor antagonist CGRP-(8-37) and the amylin antagonists AC187 and AC253 on receptor binding and cAMP production. AC253, AC187 and CGRP-(8-37) inhibited 125I-adrenomedullin binding, with respective IC50 values of 25+/-8, 129+/-39 and 214+/-56 nM. Adrenomedullin dose-dependently increased intracellular cAMP (approximate EC50=1.0 nM). CGRP-(8-37), AC253 and AC187 antagonized adrenomedullin-stimulated cAMP production at micromolar concentrations. Using kinase-substrate assays, Mono Q FPLC and 'phospho-specific' Western blotting, we found that adrenomedullin alone abolished basal mitogen-activated protein kinase (MAPK) activity and dose-dependently inhibited platelet-derived-growth-factor-stimulated MAPK activity. Radioimmunoassay for adrenomedullin of media from Rat-2 cells showed a linear release of adrenomedullin-like immunoreactivity of 3.1 fmol/h per 2x10(6) cells. Gel-filtration chromatography showed that this adrenomedullin-like immunoreactivity co-eluted with synthetic rat adrenomedullin. Northern blotting with a rat adrenomedullin cDNA probe was used to confirm the presence of adrenomedullin mRNA. However, neither Northern blotting nor reverse transcriptase-PCR showed the presence of the cloned adrenomedullin receptor (L1). We conclude that the Rat-2 cell line expresses a specific adrenomedullin receptor (coupled to cAMP production and regulation of MAPK) and secretes adrenomedullin, which may participate in a regulatory control loop.

Cardiovascular and renal effects of adrenomedullin in rats with heart failure.

Plasma adrenomedullin (AM), a novel hypotensive peptide, has been shown to increase in heart failure (HF). This study sought to examine the cardiovascular and renal effects of intravenous infusion of AM in HF rats and sham-operated rats (control) using two doses of AM that would not induce hypotension. Rat AM-(1-50) was intravenously administered at rates of 0.01 (low) and 0.05 (high) microg. kg body wt-1. min-1. Low-dose AM increased urine flow (+21% in HF, +29% in control) and urinary sodium excretion (+109% in HF, +123% in control) without changes in any hemodynamic variables. In contrast, high-dose AM slightly decreased mean arterial pressure (-3% in HF, -5% in control) and significantly increased cardiac output (+20% in HF, +12% in control). Infusion of high-dose AM resulted in significant decreases in right ventricular systolic pressure (-11%) and right atrial pressure (-28%) only in HF rats. High-dose AM significantly increased glomerular filtration rate (+10% in HF, +16% in control) and effective renal plasma flow (+25% in HF, +46% in control) as well as urine flow and urinary sodium excretion. In summary, intravenous infusion of AM exerted diuresis and natriuresis without inducing hypotension and, in the higher dose, produced beneficial hemodynamic and renal vasodilator effects in rats with compensated HF.

Comparative Affinities of Adrenomedullin (AM) and Calcitonin Gene-Related Peptide (CGRP) for [125I] AM and [125I] CGRP Specific Binding Sites in Porcine Tissues

We have investigated the binding characteristics of rat [125I] adrenomedullin (AM) and human [125I] calcitonin gene-related peptide (CGRP) to membranes prepared from a number of porcine tissues including atrium, ventricle, lung, spleen, liver, renal cortex and medulla. These membranes displayed specific, high affinity binding for [125I] rat AM and [125I] human CGRP. Porcine lung displayed the highest density of binding sites for radiolabeled AM and CGRP followed by porcine renal cortex. Competition experiments performed with [125I] rat AM indicated that the rank order of potencies of various peptides for inhibiting [125I] rat AM binding to various tissues were rat AM ≥ human AM ≥ human AM(22–52) > hαCGRP ≥ hαCGRP(8–37) <<<< sCT except spleen, atrium, renal cortex and renal medulla where rAM and hAM were 20–300 fold more potent than hAM(22–52). When the same experiments were performed using [125I] hαCGRP as the radioligand, the rank order potencies for various peptides were rAM = hAM > hαCGRP > hαCGRP(8–37) in most of the tissues except in spleen and liver. where hαCGRP was the most potent ligand. In lung, hαCGRP was almost as potent as rAM and hAM in displacing [125I] hαCGRP binding. These data suggest the existence of distinct CGRP and AM specific binding sites in contrast to previous reports that showed that both peptides interact differently in rat tissues.

Transcriptional regulation of adrenomedullin in rat vascular smooth muscle cells

Adrenomedullin (AM), a potent vasorelaxant peptide recently identified in extracts of pheochromocytoma, is actively secreted from vascular smooth muscle cells (VSMC). AM production in rat VSMC is potently stimulated by proinflammatory cytokines including TNF α, and glucocorticoids and many vasoactive substances have also been shown to alter AM synthesis. To study AM promoter function, we cloned the 5′-flanking region of the rat AM gene, and AM promoter function was assessed in rat VSMC stably transfected with a construct containing the cloned fragment. This 1.4 kb AM promoter region includes a number of putative transcriptional control elements. The promoter activity in VSMC was stimulated by TNF α and dexamethasone, and was suppressed by 8-bromo-cAMP and forskolin. These data indicate that transcriptional regulation is an important mechanism for AM production in VSMC.

Adrenomedullin promotes epithelial restitution of rat and human gastric mucosa in vitro

We have investigated the effect of adrenomedullin (AM) on restitution of mucosal integrity following damage in rat and human gastric mucosa, measuring the potential difference (PD) on a mucosal strip mounted on an Ussing chamber. Mucosal damage was induced by 0.5, 1.0, and 2.0 M NaCl solution, and it caused an immediate and significant decrease in PD. In the rat AM group, PD recovered significantly more than in control group at 120 min after exposure to 0.5 M (p < 0.01) and 1.0 M (p < 0.05) NaCl solution. In the human AM group, PD completely recovered at 120 min after exposure to 0.5 M (p < 0.05) NaCl solution. In rat mucosa damaged by 0.5 M NaCl solution, the effect was inhibited by human (h)-CGRP(8–37) and there was no significant difference between the h-CGRP(8–37) group and control group. On immunohistochemical examination of rat gastric mucosa, AM was detected within the chief cell. AM probably promotes epithelial restitution primarily through the CGRP receptor, but it does not ameliorate more severe damage of gastric mucosa in vitro.