Adrenomedullin Gene Expression Research Articles

Differential regulation of cardiac adrenomedullin and natriuretic peptide gene expression by AT1 receptor antagonism and ACE inhibition in normotensive and hypertensive rats.

To study the effects of long-term treatment with the type 1 angiotensin (AT1) receptor antagonist losartan and the angiotensin-converting enzyme (ACE) inhibitor enalapril, on cardiac adrenomedullin (ADM), atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) gene expression. Spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were given losartan (15 mg/kg per day) or enalapril (4 mg/kg per day) orally for 10 weeks. The effects of drugs on systolic blood pressure, cardiac hypertrophy, ANP, BNP and ADM mRNA and immunoreactive-ANP (IR)-ANP, IR-BNP and IR-ADM levels in the left ventricle and atria were compared. Losartan and enalapril treatments completely inhibited the increase of systolic blood pressure occurring with ageing in SHR. The ratio of heart to body weight was reduced in both losartan- and enalapril-treated SHR and WKY rats. Treatment with losartan or enalapril reduced left ventricular ANP mRNA and IR-ANP in both strains, and ventricular BNP mRNA levels in SHR rats. Inhibition of ACE, AT1 receptor antagonism, changes in blood pressure or cardiac mass had no effect on left ventricular ADM gene expression in SHR and WKY rats. In addition, atrial IR-ANP and IR-ADM levels increased in SHR whereas IR-BNP levels decreased in WKY and SHR rats in response to drug treatments. Our results show that ventricular ADM synthesis is an insensitive marker of changes in haemodynamic load or cardiac hypertrophy. Furthermore, the expression of ADM, ANP and BNP genes is differently regulated both in the left ventricle and atria in response to AT1 receptor antagonism and ACE inhibition.

The distribution and gene expression of adrenomedullin in the rat brain: peptide/mRNA and precursor/active peptide relationships

The highest immunoreactive adrenomedullin (AM) concentrations were found in the pituitary gland, hypothalamus and brainstem. Rat preproadrenomedullin mRNA levels were highest in the pituitary, followed by the hypothalamus. The peptide to mRNA ratios are higher in the neurointermediate lobe, brainstem and hypothalamus. Immunocytochemical study showed discrete localization of AM immunostaining in various brain regions including the cerebral cortex, hypothalamus and brainstem and in the pituitary gland. By gel filtration chromatography, the precursor peaks were low in the pituitary, hypothalamus and brainstem but high in other brain regions.

Production and secretion of adrenomedullin in cultured rat cardiac myocytes and nonmyocytes: stimulation by interleukin-1beta and tumor necrosis factor-alpha.

The present study investigated the secretion level and gene expression of adrenomedullin (AM), a novel vasorelaxant peptide, in cultured neonatal rat cardiac myocytes and nonmyocytes, and the effects of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF alpha) on its production and secretion in these cells. Under serum-free conditions, both myocytes and nonmyocytes secreted immunoreactive (ir-) AM into the culture medium in a time-dependent manner. The secretion rates of ir-AM from myocytes and nonmyocytes per 10(5) cells were almost equivalent. The expression of AM messenger RNA was also observed in cultured myocytes and nonmyocytes. The peptide secretion and messenger RNA level of AM in cardiac myocytes were increased after stimulation with IL-1beta. In nonmyocytes, IL-1beta and TNF alpha remarkably augmented both the release of ir-AM into the medium and AM gene expression after 24 and 48 h of incubation. These observations indicate that cardiac ventricular cells (i.e. myocytes and nonmyocytes) actively produce AM and also suggest that cytokines such as IL-1beta and TNF alpha regulate the gene expression and secretion of this peptide in the ventricles. On the basis of these results and the findings that IL-1beta and TNF alpha are involved in heart failure and cardiac hypertrophy, AM may play a role as an autocrine/paracrine modulator in some cardiac disorders.

Production and Secretion of Adrenomedullin in Cultured Rat Cardiac Myocytes and Nonmyocytes: Stimulation by Interleukin-1 and Tumor Necrosis Factor-

The present study investigated the secretion level and gene expression of adrenomedullin (AM), a novel vasorelaxant peptide, in cultured neonatal rat cardiac myocytes and nonmyocytes, and the effects of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) on its production and secretion in these cells. Under serum-free conditions, both myocytes and nonmyocytes secreted immunoreactive (ir-) AM into the culture medium in a time-dependent manner. The secretion rates of ir-AM from myocytes and nonmyocytes per 105 cells were almost equivalent. The expression of AM messenger RNA was also observed in cultured myocytes and nonmyocytes. The peptide secretion and messenger RNA level of AM in cardiac myocytes were increased after stimulation with IL-1β. In nonmyocytes, IL-1β and TNFα remarkably augmented both the release of ir-AM into the medium and AM gene expression after 24 and 48 h of incubation. These observations indicate that cardiac ventricular cells (i.e. myocytes and nonmyocytes) actively produce AM and also suggest that cytokines such as IL-1β and TNFα regulate the gene expression and secretion of this peptide in the ventricles. On the basis of these results and the findings that IL-1β and TNFα are involved in heart failure and cardiac hypertrophy, AM may play a role as an autocrine/paracrine modulator in some cardiac disorders.

Shear stress down-regulates gene transcription and production of adrenomedullin in human aortic endothelial cells

Vascular endothelial cells are potent modulators of vascular tone in response to shear stress. Levels of vasoactive peptides such as adrenomedullin (AM), endothelin-1 (ET-1), C-type natriuretic peptide (CNP), and nitric oxide (NO) are affected by fluid shear stress. AM, a potent vasodilator and suppressor of smooth muscle cell proliferation, contains the shear stress responsive element (SSRE) "GAGACC" in its promoter region. To examine the role of AM in the shear stress response, cultured human aortic endothelial cells (HAoECs) were exposed to fluid shear stresses of 12 and 24 dynes/cm2 in a cone-plate shear stress loading apparatus for various time periods, and the levels of AM gene expression and peptide secretion from HAoECs were measured by Northern blotting analysis and radioimmunoassay (RIA), respectively. Both AM gene transcription and AM peptide levels were down-regulated by fluid shear stress in a time- and magnitude-dependent manner. Our results demonstrate that the normal level of arterial shear stress down-regulates AM expression in HAoECs, suggesting that AM participates in the modulation of vascular tone by fluid shear stress.

Distribution and expression of adrenomedullin in human gastrointestinal tissue.

Adrenomedullin (AM) is a biologically active peptide recently isolated from phaeochromocytoma. We report here the distribution and characterization of immunoreactive AM and gene expression of AM in human gastrointestinal tissue. Using a sensitive radioimmunoassay system for the peptide, immunoreactive human AM was detected in the stomach, duodenum, jejunum, ileum and colon. The AM concentration of these tissues was about 0.4-0.8 pmol/g wet tissue. Reverse phase and gel filtration high-performance liquid chromatographies showed that most of the immunoreactive AM in stomach and jejunum was identical to authentic human AM. By northern blot analysis, human AM mRNA was found to be expressed ubiquitously in the human gastrointestinal tissues. Furthermore, an immunohistochemical study revealed that immunoreactive AM cells were present in the gastrointestinal glands. These results suggest that AM may play some role as a gastrointestinal hormone.

Ventricular gene expression of adrenomedullin is increased in experimental heart failure

Ventricular gene expression of adrenomedullin is increased in experimental heart failure

Up-regulation of a novel potent vasodilatory peptide adrenomedullin during polymicrobial sepsis.

A large number of studies have been and are being carried out to examine the role of nitric oxide in the hyperdynamic and hypodynamic stages of sepsis. It remains unknown, however, whether adrenomedullin (ADM), a novel potent vasodilatory peptide, is up-regulated during hyperdynamic sepsis and, if so, whether its production is sustained during hypodynamic sepsis. To determine this, rats were subjected to sepsis by cecal ligation and puncture (CLP), followed by administration of 3 mL/100 g body weight normal saline to these and sham-operated animals. Blood samples were taken at 1, 1.5, 2, 5, and 10 h (2-10 h post-CLP represents the hyperdynamic stage of sepsis) or at 20 and 30 h after CLP (i.e., the hypodynamic stage). Plasma levels of ADM were measured by radioimmunoassay. Adrenomedullin gene expression in various tissues was examined at 2, 10, or 20 h after CLP by reverse transcription-polymerase chain reaction (RT-PCR). The results indicated that plasma levels of ADM did not increase at 1 and 1.5 h after CLP but increased significantly at 2 h after the onset of sepsis. Moreover, circulating ADM increased progressively at 5-20 h and remained elevated at 30 h after CLP. The increased levels of plasma ADM during sepsis were correlated with up-regulation of ADM mRNA in the small intestine, left ventricle, and thoracic aorta. In contrast, ADM gene expression in renal and hepatic tissues was not significantly altered following the onset of sepsis. The association between the up-regulated ADM and the occurrence of hyperdynamic circulation during the early stage of sepsis (both occur at 2 h after CLP) may indicate a possible cause and effect relationship between the two events. Since we have previously shown that ADM-induced vascular relaxation decreased at 20 h after CLP, it appears that the down-regulation of ADM receptors may be responsible for the transition from the hyperdynamic stage to the hypodynamic stage of sepsis.

Coronary pressure as a determinant of B-type natriuretic peptide gene expression in isolated perfused adult rat heart

The role of coronary flow in the regulation of ventricular B-type natriuretic peptide (BNP) gene expression was studied in isolated perfused rat heart preparation. The increase of coronary flow from 5 ml/min to 20 ml/min for 2 h resulted in a 132 ± 6 mm Hg increase in aortic perfusion pressure. The changes in BNP mRNA and immunoreactive BNP (IR-BNP) levels in response to hemodynamic stress were compared to those of c-fos and adrenomedullin (ADM) gene expression. The increase of coronary flow resulted in 1.5-fold increases in the left ventricular BNP mRNA ( P < 0.001) and IR-BNP ( P < 0.05) levels in 2-month old rats. There was also a 1.5-fold ( P < 0.05) increase in ventricular c-fos mRNA levels, whereas ADM mRNA levels decreased by 74% ( P < 0.001) in the left ventricle. In 18-month old rats, the increase in coronary flow decreased left and right ventricular BNP mRNA levels by 18% ( P < 0.05) and 39 % ( P < 0.001), respectively. There were no changes in IR-BNP peptide and c-fos mRNA levels, whereas ADM mRNA levels decreased by 46% ( P < 0.001) in the left ventricles. The results show that increased aortic perfusion pressure results in differential expression of cardiac genes including up-regulation of ventricular BNP and c-fos gene expression and down-regulation of ADM gene expression. Furthermore, aging seems to elevate the threshold at which hemodynamic stress of the heart results in a response at BNP gene level.

Adrenomedullin Gene Expression Is Developmentally Regulated and Induced by Hypoxia in Rat Ventricular Cardiac Myocytes

Adrenomedullin is a recently discovered hypotensive peptide that is expressed in a variety of cell and tissue types. Using the technique of differential display, the adrenomedullin gene was observed to be differentially expressed in developing rat heart. Reverse transcription-polymerase chain reaction analysis revealed that the level of adrenomedullin mRNA was significantly higher in adult ventricular cardiac muscle as compared with embryonic day 17 ventricular cardiac muscle. Adrenomedullin receptor mRNA was constitutively expressed throughout development of the ventricular heart. Two potential hypoxia-inducible factor-1 (HIF-1) consensus binding sites were identified in the mouse adrenomedullin promoter at -1095 and -770 nucleotides from the transcription start site. Exposure of cultured adult rat ventricular cardiac myocytes to hypoxia (1% O2) resulted in a significant, time-dependent increase in adrenomedullin mRNA levels. Transfection studies revealed that the 5'-flanking sequence of adrenomedullin was capable of mediating a hypoxia-inducible increase in transcription. Mutation of the putative HIF-1 consensus binding sites revealed that the major regulatory sequence that mediates the hypoxia-inducible transcriptional response is located at -1095. These data demonstrate that the adrenomedullin gene is developmentally regulated in ventricular cardiomyocytes, that adrenomedullin transcription can be induced by hypoxia, and that this response is primarily mediated by HIF-1 consensus sites in the adrenomedullin promoter.

Novel Sites of Adrenomedullin Gene Expression in Mouse and Rat Tissues

Novel Sites of Adrenomedullin Gene Expression in Mouse and Rat Tissues

Novel sites of adrenomedullin gene expression in mouse and rat tissues.

Adrenomedullin (AM) was originally identified in pheochromocytoma tissue and was characterized as a hypotensive peptide. The tissue distribution and cellular localization of AM messenger RNA (mRNA) were determined in mouse and rat tissues by in situ hybridization. Three probes were used: two nonoverlapping probes to the pro-AM N-terminal 20 peptide (PAMP) and AM peptide regions of mouse pro-AM, and a larger complementary DNA (cDNA) probe spanning both the PAMP- and AM peptide-coding regions. The most intense expression of AM mRNA was in endometrium and epithelial cells lining the uterus and mouse adrenal medulla. Moderate levels of expression were detected in kidney glomerulus and cortical distal tubules, ovarian corpus luteum and follicles, epithelial cells lining the bronchioles, cardiac atrium and ventricle, posterior pituitary (particularly in female rats), stomach, small intestine (microvilli, mucosa and submucosa), spleen, and pancreas. Lower levels were observed in pulmonary alveoli, anterior pituitary, and submandibular gland. No expression was detected in the testis, thymus, skeletal muscle, or liver. The localization of AM mRNA in epithelial cells lining the uterus, bronchioles, and gastrointestinal tract indicates novel roles for AM, possibly as an antimicrobial agent. The strong expression of AM in uterus, ovary, and posterior pituitary suggests that AM plays a role in female reproduction.

Differential Regulation of Ventricular Adrenomedullin and Atrial Natriuretic Peptide Gene Expression in Pressure and Volume Overload in the Rat

1. Adrenomedullin is a recently discovered vasodilating and natriuretic peptide whose physiological and pathophysiological roles remain to be established. Like atrial natiuretic peptide adrenomedullin is expressed in the left ventricle. Ventricular expression of atrial natriuretic peptide is known to be markedly increased by volume or pressure overload. In this study we investigated whether ventricular expression of adrenomedullin is similarly stimulated under such conditions. 2. Ventricular adrenomedullin and atrial natriuretic peptide mRNA levels as well as those of a loading control mRNA (glyceraldehyde-3-phosphate dehydrogenase) were quantified by Northern blot analysis in (a) rats with severe post-infarction heart failure induced by left coronary ligation at 30 days post-surgery and (b) in rats with pressure-related cardiac hypertrophy induced by aortic banding at several time points (0.5, 1 and 4 h, and 1, 4, 7 and 28 days) after surgery. Levels were compared with those in matched sham-operated controls. 3. The mRNA level of atrial natriuretic peptide was markedly increased (8-10-fold) in the left ventricle of animals with post-infarction heart failure. In contrast, there was only a modest (40%) increase in the level of adrenomedullin mRNA. In rats with pressure-induced cardiac hypertrophy the ventricular level of atrial natriuretic peptide mRNA was again markedly increased (maximum 10-fold). The increase was first noticeable at 24 h post-banding and persisted until 28 days. In contrast, there was no change in adrenomedullin mRNA level compared with sham-operated rats at any time point. 4. Despite having similar systemic effects, the expression of adrenomedullin and atrial natriuretic peptide in the left ventricle is differently regulated. The findings imply distinct roles for the two peptides. The results do not support an important role for ventricular adrenomedullin expression in the remodelling process that occurs during the development of cardiac hypertrophy but suggest that ventricular adrenomedullin participates in the local and/or systemic response to heart failure.

Gene Transcription and Synthesis of Adrenomedullin by Cultured Human Renal Cells

Adrenomedullin (ADM) is a novel 52 amino acid peptide with a potent vasodilator effect. Gene expression of ADM is found in human kidney but the exact cell source in the kidney is uncertain. Its plasma level is raised in association with changes in sympathetic nervous activity and body fluid volume in hypertension and chronic renal failure. Herein, we examined the presence of mRNA encoding for ADM in cultured human glomerular cells. Adrenomedullin in cell culture supernatant was measured by a radio-immunoassay (with a detection level of 3.2 pmol/l). Adrenomedullin mRNA was found in cultured mesangial and glomerular epithelial cells as well as in vascular endothelial cells. Supernatant levels of ADM for cultured mesangial and glomerular epithelial cells were 21.2 and < 3.2 pmol/l respectively. Contrary to vascular smooth muscle cells, the gene expression for ADM in mesangial cells was up-regulated when incubated with increasing concentration of TNF-α or fetal bovine serum (FBS) but this effect was not observed with very high concentration. Parallel results were observed in adrenomedullin levels in supernatant from mesangial cell cultures. Forskolin, captopril, or TGF-β had no effect on the transcription or synthesis of ADM in mesangial cells. The gene expression for ADM in glomerular epithelial cells was down-regulated when incubated with increasing concentration of TNF-α, forskolin or FBS. The ADM levels in all supernatant from resting glomerular epithelial cell cultures were < 3.2 pmol/l. Recent murine data show that ADM stimulates the release of cAMP but suppresses mitogenesis in cultured mesangial cells. Our results suggest ADM is synthesized by mesangial cells in an autocrine fashion and the peptide may potentially be involved in intra-renal blood pressure control.

Transcriptional Regulation of Human Adrenomedullin Gene in Vascular Endothelial Cells

Adrenomedullin (AM) is a recently identified vasodilator peptide produced in cardiovascular organs such as the heart, vascular wall, kidney and lung. Because plasma levels of AM are not different between various portions of the cardiovascular system, vascular cells are supposed to be the main source of circulating AM. To elucidate the regulatory mechanism of human AM gene expression, functional elements of 5′-flanking region of AM gene were studied in human aortic endothelial cells (HAEC). Northern blot analysis revealed considerable AM mRNA expression in cultured HAEC, and 2 transcription start sites were recognized at 21 and 25 bp downstream from the TATA box. The 1534 bp 5′-flanking region DNA inserted in the luciferase vector showed significant promoter activity when transfected into HAEC using liposome. Serial deletion of the inserted 5′-flanking region DNA length resulted in reduction in promoter activity by 41% between 110 and 90 bp and further reduction by 42% between 66 and 29 bp. The 19 bp DNA fragment without TATA box had almost no promoter activity. Gel shift assay revealed presence of HAEC nuclear proteins specifically bound to nuclear factor for interleukin-6 expression (NF-IL6) consensus sequence existing from −85 to −93 bp of the AM gene 5′-flanking region and activator protein 2 (AP-2) consensus sequence clustered from −33 to −68 bp. Furthermore, mutation of NF-IL6 consensus sequence in the 5′-flanking region resulted in 42% reduction in the expression of luciferase activity. These findings suggest that NF-IL6 and AP-2 sites in the promoter region are the functional elements in the transcriptional regulation of human AM gene in vascular endothelial cells.

Upregulation of endothelin-1 and adrenomedullin gene expression in the mouse endotoxin shock model.

Septic shock is a life-threatening disorder caused by lipopolysaccharide (LPS) and other bacterial products. Accumulating evidence indicates a role for vasoactive substances and cytokines in this disease process. In this study we examined the effect of LPS on the gene expression of endothelin-1 (ET-1) and adrenomedullin (AM), two major vasoactive peptides predominantly produced by vascular endothelial cells, to investigate their role in the pathophysiology of septic shock. LPS induced ET-1 and AM gene expression in the heart, lung, kidney, liver, and aorta within 6 h. In the liver, whereas basal ET-1 and AM mRNA were hardly detectable, ET-1 and AM gene expression and peptide production were markedly increased by LPS. This LPS-induced upregulation of ET-1 and AM expression is greatly potentiated by D-galactosamine (D-GalN), although D-GalN alone could not induce ET-1 and AM gene expression. These results, together with the previous findings that liver injury induced by LPS and D-GalN is mainly mediated by tumor necrosis factor-alpha (TNF-alpha), suggest that the LPS-cytokine pathway may cause upregulation of ET-1 and AM production, leading to dysregulation of systemic and regional vascular tone.

Dexamethasone-induced Up-regulation of Adrenomedullin and Atrial Natriuretic Peptide Genes in Cultured Rat Ventricular Myocytes

To determine whether adrenomedullin (AM), a novel 51-residue vasodilator peptide originally isolated from human pheochromocytoma, is expressed by the heart, and whether the expression of cardiac AM gene is regulated by glucocorticoids, the effect of dexamethasone (DEX) on the expression of protein and mRNA of AM and atrial natriuretic peptide (ANP) was tested in cultured ventricular myocytes prepared from the neonatal rats. Northern blot analysis of rat ventricular myocytes with cDNAs for rat AM and ANP as probes revealed distinct bands corresponding to the sizes of rat AM mRNA (1.6 kb) and rat ANP mRNA (1 kb), respectively. Dexamethasone increased steady-state levels of both AM and ANP mRNA as well as secretion of both AM and ANP immunoreactivity in a time- and dose-dependent manner. The stimulatory effects of dexamethasone were completely abolished by a glucocorticoid antagonist (RU38486) and a RNA synthesis inhibitor (actinomycin D). The approximate half-lives of basal and dexamethasone-induced AM mRNA were about 4 h, suggesting that dexamethasone-induced up-regulation of AM mRNA was unlikely due to its decreased degradation. These data suggest that glucocorticoids directly stimulate gene expression of AM as well as ANP in rat ventricular myocytes.

Adrenomedullin gene expression in the rat heart is stimulated by acute pressure overload: blunted effect in experimental hypertension.

The levels of adrenomedullin (ADM), a newly discovered vasodilating and natriuretic peptide, are elevated in plasma and ventricular myocardium in human congestive heart failure suggesting that cardiac synthesis may contribute to the plasma concentrations of ADM. To examine the time course of induction and mechanisms regulating cardiac ADM gene expression, we determined the effect of acute and short-term cardiac overload on ventricular ADM mRNA and immunoreactive ADM (ir-ADM) levels in conscious rats. Acute pressure overload was produced by infusion of arginine8-vasopressin (AVP, 0.05 microg/kg/min, i.v.) for 2 h into 12-week-old hypertensive TGR(mREN-2)27 rats and normotensive Sprague-Dawley (SD) rats. Hypertension and marked left ventricular hypertrophy were associated with 2.2-times higher ir-ADM levels in the left ventricular epicardial layer (178 +/- 36 vs. 81 +/- 23 fmol/g, P<0.05) and 2.6-times higher ir-ADM levels in the left ventricular endocardial layer (213 +/- 23 vs. 83 +/- 22 fmol/g, P<0.01). The infusion of AVP for 2 h in normotensive rats produced rapid increases in the levels of left ventricular ADM mRNA (epicardial layer: 1.6-fold, P<0.05) and ir-ADM (endocardial layer: from 83 +/- 22 to 140 +/- 12 fmol/g, P<0.05), whereas ventricular ADM mRNA and ir-ADM levels did not change significantly in hypertensive rats. Short-term cardiac overload, induced by administration of angiotensin II (33.3 microg/kg/h, s.c., osmotic minipumps) for two weeks in normotensive SD rats resulted in left ventricular hypertrophy (3.05 +/- 0.17 vs. 2.75 +/- 0.3 mg/g, P<0.05) and a 1.5-fold increase (P<0.05) in ventricular ADM mRNA levels. In conclusion, the present results show that pressure overload acutely stimulated ventricular ADM gene expression in conscious normotensive rats suggesting a potential beneficial role for endogenous ADM production in the heart against cardiac overload. Since pressure overload-induced increase in ADM synthesis was attenuated in hypertensive rats, alterations in the ADM system may contribute to the pathogenesis of hypertension in the TGR(mREN-2)27 rat.

Adrenomedullin Gene Expression in the Rat Heart is Stimulated by Acute Pressure Overload: Blunted Effect in Experimental Hypertension

Adrenomedullin Gene Expression in the Rat Heart is Stimulated by Acute Pressure Overload: Blunted Effect in Experimental Hypertension

Use of differential display reverse transcription – polymerase chain reaction for discovery of induced adrenomedullin gene expression in focal stroke

Differential display and other techniques such as subtractive library screening and representational difference analysis have been developed and widely used for novel gene discovery in pathological and specific physiological conditions. In the present work, we illustrate the utilization of the differential display method for the investigation of the differentially expressed genes in a rat model of focal brain ischemia. Adrenomedullin, a recently discovered vasodilator peptide, was identified to be upregulated after focal brain ischemia and further demonstrated to play a pathophysiological role in exacerbating ischemic damage. The data suggest a high potential for the application of mRNA differential display, as well as other techniques, for the discovery of novel therapeutic targets.