(Hypertension. 1998;31:505.)
© 1998 American Heart Association, Inc.
Scientific Contributions |
Adrenomedullin: A Possible Autocrine or Paracrine Inhibitor of Hypertrophy of Cardiomyocytes
From the First Department of Internal Medicine, Miyazaki Medical College, Kihara, Kiyotake, Miyazaki 889-16, Japan; the *Diagnostic Science Department, Shionogi & Co., Ltd., Mishima, Settsu, Osaka 566, Japan (T.T.); and the Department of Biochemistry, National Cardiovascular Center Research Institute, Fujishinodai, Suita, Osaka 565, Japan (K.K.).
Correspondence to Tanenao Eto, MD, First Department of Internal Medicine, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-16, Japan
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Abstract |
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Adrenomedullin (AM), a potent vasodilator peptide, exists in the cardiac ventricle; however, the role of AM in the ventricular tissue remains unknown. In the present study, we investigated the production and secretion of AM in cultured neonatal rat cardiomyocytes, and we examined the effect of AM on de novo protein synthesis in these cells by measuring [14C]phenylalanine incorporation. The cardiomyocytes cultured with serum-free media secreted AM into the media in a time-dependent manner at the rate of 12.2±0.5 fmol/105 cells/48 hours (mean±SEM). Angiotensin II (1µmol/L) or 10% fetal bovine serum significantly (P<.01) increased the AM secretion by 115% and 305%, respectively. In addition, Northern blot analysis of total RNA extracted from the myocytes disclosed the expression of prepro-AM mRNA of 1.6 kb. Synthetic AM at 1 µmol/L significantly reduced the 10-6 mol/L angiotensin II-and 10% fetal bovine serum-stimulated [14C]phenylalanine incorporation into the cells, by 16% (P<.05) and 20% (P<.01), respectively. The inhibitory effect of AM on the angiotensin II-stimulated [14C]phenylalanine incorporation was abolished dose-dependently by a calcitonin gene-related peptide receptor antagonist, CGRP(8–37). Furthermore, blockade of the action of endogenous AM by either 10-6 mol/L CGRP(8–37) or anti-AM monoclonal antibody significantly enhanced the basal and 10-6 mol/L angiotensin II-stimulated [14C]phenylalanine incorporation. In summary, cultured neonatal rat cardiomyocytes produce and secrete AM, and the secreted AM inhibits the protein synthesis of these cells. Thus, AM may act on cardiomyocytes as an autocrine or a paracrine factor modulating the cardiac growth.
Key Words: adrenomedullin • cardiomyocytes • autocrine • paracrine • cardiac hypertrophy • monoclonal antibody
Abbreviations: AM = adrenomedullin • Ang II = angiotensin II • CGRP = calcitonin gene-related peptide • DMEM = Dulbecco’s modified Eagle’s medium • FBS = fetal bovine serum • HPLC = high-performance liquid chromatography • ir = immunoreactive • RIA = radioimmunoassay
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Introduction |
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Many efforts have been made to clarify the mechanisms of growth regulation of the cardiac myocardium because of complications of the heart caused by cardiac hypertrophy. At the cellular level, cardiac myocytes are known to compensate for increased workload by an increase in their size but not in their number because these cells are unable to divide later in life.1 Multiple factors such as hemodynamic overload and humoral factors were shown to be involved in the process of the cardiac hypertrophy,2–4 but the detailed mechanism remains to be elucidated.
AM, a potent vasodilator peptide first detected in human pheochromocytoma, has slight homology with CGRP.5 In addition to the direct vasodilator activity. AM has been shown to possess a broad spectrum of biological actions such as diuresis, inhibition of aldosterone secretion, and inhibition of proliferation of vascular smooth muscle cells.6–8 A specific RIA revealed that AM circulates in the blood and is present in the adrenal medulla, kidney, lung and cardiac ventricle of humans and rats.9,10 The plasma AM concentration in patients with essential hypertension or primary aldosteronism was reported to be higher than that in normotensive control subjects, suggesting a possible role of AM in acting against further elevation of blood pressure.11,12 In the cardiac ventricle, AM mRNA is expressed at a level comparable to that of the adrenal medulla,13,14 and both the AM content and mRNA expression are increased in Dahl salt-sensitive and renovascular hypertensive rats compared to respective controls.15,16 However, at present, it remains unknown whether the cardiac myocytes secrete AM, and what the role of AM is in the cardiac tissue.
In the first part of this study, we examined the production and secretion of AM from cultured neonatal cardiac myocytes. In the second, we investigated the effect of AM on the de novo protein synthesis in these cells by measuring [14C]phenylalanine incorporation, and we evaluated the action of endogenous AM by using a peptide analogue of CGRP and anti-AM monoclonal antibody.
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Methods |
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Chemicals
Ang II, rat AM, human AM(22–52), and human CGRP(8–37) were purchased from Peptide Institute, Inc and L-[14C]phenylalanine from DuPont-New England Nuclear. Holo-transferrin (human), collagenase type IV, trypsin, and insulin (bovine pancreas) were purchased from Sigma Chemical Co.
Cell Culture
Primary cultures of cardiac myocytes were prepared from cardiac ventricles of 1- to 3-day-old Wistar rats according to the method described by Simpson and Savion17 with some modifications. After digestion of minced ventricles with 0.12% trypsin and 0.03% collagenase, cells were collected and preincubated for 30 minutes at 37°C in culture dish to obtain the medium enriched with cardiomyocytes. Cells not attached to the bottom of the culture dish were plated onto collagen-coated 24-well plates (Sumitomo Bakelite Co) at 1.0x105 cells/cm2 and cultured for 48 hours with DMEM containing 15 mmol/L HEPES (pH 7.4), 10%FBS, 10 µg/mL insulin, 10 µg/mL holo-transferrin, 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B in 5% CO2/95% humidified air at 37°C. Bromodeoxyuridine was added to the medium at 0.1 mmol/L during this 48-hour incubation period to prevent proliferation of the remaining nonmyocytes. At 48 hours after seeding, the culture medium was changed to serum-free medium, a DMEM containing the above-listed chemicals without bromodeoxyuridine, and the cells were cultured for 48 hours further. This purification procedure of cardiomyocytes has well been established,17,18 and in fact more than 90% of the cells we obtained by this method were beating when assessed carefully by a microscopic examination. In addition, the number of myocytes per culture remained unchanged during the experiments.
Measurement of AM in Conditioned Medium
One milliliter of the conditioned medium of the cardiac myocytes was collected and immediately acidified with acetic acid to a final concentration of 1.0 N. The media was heated at 100°C for 10 minutes to inactivate proteases and applied to a Sep-Pak C18 cartridge (Millipore-Waters). After the cartridge was washed with 10% CH3CN in 0.1% trifluoroacetic acid, the absorbed materials were eluted with 50% CH3CN in 0.1% trifluoroacetic acid and lyophilized for storage at -30°C. Recovery of AM for this extraction procedure was 82%, a rate apparently constant. The AM in the media extract was measured by a specific RIA for AM as described previously.9 The antibody used in this RIA recognizes the C-terminal portion of AM with the amide structure and has no cross-reactivity with CGRP or amylin.9
Characterization of Secreted AM
For examination of the molecular forms of ir-AM, the extracts of the conditioned media were analyzed by reverse-phase HPLC with a TSK ODS 120A column (Tosoh). A linear gradient of 10% to 60% acetonitrile was made in 0.1% trifluoroacetic acid, and the ir-AM in each fraction was measured with the RIA. The recovery of ir-AM in this HPLC was greater than 64%.
Northern Blot Analysis
Thirty micrograms of the total RNA extracted from the cultured myocytes by the acid guanidinium thiocyanate-phenol-chloroform method was denatured by glyoxal and dimethyl sulfoxide. The denatured RNA was electrophoresed on 1.0% agarose gel and transferred to a nylon membrane (
probe, Bio-Rad). The membrane was hybridized with32P-labeled rat prepro-AM cDNA at 42°C in 50% formamide, 6 x SSPE, 0.5x Denhardt’s solution, 1.0% sodium dodecyl sulfate, and 0.5 mg/ml salmon sperm DNA. Then the hybridized membrane was washed with 0.1x SSC at 55°C and scanned with a Fuji BAS 2000 Bio-imaging analyzer (Fuji Photo Film Co).
Measurement of de Novo Protein Synthesis
The rate of de novo protein synthesis was assessed by measuring the radioactivity incorporated into the cultured cells after exposure to 0.1 µCi/ml L[14C]phenylalanine for 24 hours. In brief, after the incubation with serum-free medium for 48 hours as described above, the cardiomyocytes on a 24-well plate were treated with the agents described in the absence or presence of AM for 24 hours. The treated cells were washed with cold phosphate-buffered saline three times and were incubated with 10% trichloroacetic acid at 4°C for 60 minutes to precipitate protein. After the cell residues were rinsed with 95% ethanol, the dried materials were solubilized in 0.5 N NaOH overnight. The radioactivity in the solubilized samples was determined by a liquid scintillation counter (LSC-5100, Aloka). The cardiomyocytes incorporated [14C]phenylalanine time-dependently at a constant rate up to 25 hours by this procedure. The results are expressed as percentages normalized by the mean counts per minute of control cells for each experiment.
Preparation of Anti-AM Monoclonal Antibody
Synthetic human AM(46–52), a C-terminal fragment, was conjugated to bovine thyroglobulin (Sigma) by the carbodiimide coupling procedure.9 Five-week-old female BALB/c mice were immunized by subcutaneous injections with the conjugate containing 4.7 µg of the peptide emulsified in Freund’s complete adjuvant eight times at intervals of 3 weeks. Fusion of spleen cells of the immunized mice with a mouse myeloma cell line, X63-Ag8.653, was performed at the ratio of 5:1 with 50% polyethylene glycol 4000 (Merck) according to the method described by Galfrè et al.19 Culture media of the hybridoma were periodically screened for capacity to bind 125I-AM. Cells from the well giving the highest titer were cloned by limiting dilution and injected intraperitoneally into BALB/c mice. The monoclonal antibody in ascites was available for an RIA for AM at a final dilution of 1:2,720,000 with a Ka value of 0.74x1011 mmol/L-1. The monoclonal antibody obtained belonged to the immunoglobulin G1 subclass, when determined by the Ouchterlony technique, and equally cross-reacts with rat AM(1–50), but has no cross-reactivity with CGRP or amylin.
Statistical Analysis
Student’s t test was used for comparison between two variables. Multiple comparison was made with one-way ANOVA followed by Scheffé’s test. All data are expressed as mean±SEM, and P values of <.05 was considered significant.
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Secretion and Production of AM in Cardiomyocytes
To examine whether the cultured neonatal rat cardiac myocytes secrete AM, we measured the AM concentrations in the conditioned media by using the RIA. The cardiomyocytes cultured with serum-free media secreted AM into the media in a time-dependent manner at a rate of 12.2±0.5 fmol/1x105 cells for 48 hours (Fig 1). As shown in the Table, the addition of 10-6 mol/L Ang II or 10% FBS significantly (both, P<.01) increased the AM secretion, by 115% and 305%, respectively. Molecular forms of ir-AM secreted into the medium were characterized by reverse-phase HPLC (Fig 2). The ir-AM was composed of one major and some minor peaks, and the major peak appeared at an elution position identical to that of synthetic rat AM(1–50)-NH2, a whole active molecule of the rat AM peptide. For examination of the expression of mRNA for the AM precursor by the cells, 30 µg of total RNA extracted from the myocytes was analyzed by the Northern blot method with a prepro-rat AM cDNA probe. As shown in Fig 3, a single band was observed at a molecular weight of 1.6 kb, a size identical to prepro-AM mRNA of the other rat tissues.13
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AM Effect on Protein Synthesis in Cardiomyocytes
For examination of the effect of AM on de novo protein synthesis of the cardiac myocytes, [14C]phenylalanine incorporation was measured in the cells cultured with serum-free media with or without Ang II or FBS in the absence or presence of AM. Synthetic rat AM at 1 µmol/L significantly attenuated 10-6 mol/L Ang II- and 10% FBS-stimulated [14C]phenylalanine incorporation into the cells, by 16% (P<.05) and 20% (P<.01), respectively (Fig 4A). As shown in Fig 4B, AM inhibited the Ang II-stimulated [14C]phenylalanine incorporation in a concentration-dependent manner. Then, we tested the effects of two peptide analogues on the AM action: one was CGRP(8–37), which is a CGRP type I receptor antagonist,20 and the other was human AM(22–52), an N-terminal-deleted form of AM. When incubated in serum-free media with these analogues, CGRP(8–37) attenuated the inhibitory AM effect on the protein synthesis in a dose-dependent manner (Fig 5A), whereas AM(22–52) had no effect on the AM action (Fig 5B).
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Action of Endogenous AM
In the examination of the effect of endogenous AM secreted from the cells, the cardiac myocytes were incubated with CGRP(8–37), which inhibited the action of synthetic AM, or with anti-AM monoclonal antibody, which specifically binds to the C-terminal structure of AM, an important portion for the biological activity.21 As shown in Fig 6A, 10-6 mol/L CGRP(8–37) significantly increased the basal and 10-6 mol/L Ang II-stimulated [14C]phenylalanine incorporation, by 17% (P<.05) and 11% (P<.05), respectively. Similarly, the addition of 10 µL/well of the anti-AM monoclonal antibody significantly enhanced the basal and 10-6 mol/L Ang II-stimulated [14C]phenylalanine incorporation, by 49% (P<.01) and 73% (P<.05), respectively (Fig 6B).
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Discussion |
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AM is a potent vasodilating peptide originally detected in human pheochromocytoma.5 Development of an RIA revealed that AM circulates in the blood at considerable concentration and is present in the cardiac ventricular tissue of humans and rats,9,10 but no direct evidence of the secretion of AM from cardiac myocytes has been presented. The present study clearly demonstrated that cultured neonatal rat cardiomyocytes produce and secrete AM into the media and that the secretion is markedly increased in the presence of either Ang II or FBS. In addition, reverse-phase HPLC analysis disclosed that the elution position of the secreted AM was identical to rat AM(1–50), a whole peptide molecule of the active AM.13 In contrast to the amount of secreted AM, the intracellular AM concentration in the cultured myocytes was too low to be detected in the present study (data not shown), despite the substantial expression of AM mRNA. This finding appears to be consistent with the much lower tissue concentration of AM in the cardiac ventricle than that in the adrenal medulla in spite of a comparable AM mRNA expression of these two tissues.9,10,13,14 We reported that both cultured vascular endothelial and smooth muscle cells actively secrete AM with much lower intracellular concentration than that secreted.22,23 Thus, most AM produced in the cells seems to be secreted with a very small proportion in intracellular storage.
Many mechanisms, including hemodynamic overload and humoral factors, have been shown to be involved in the process of cardiac ventricular hypertrophy. Among the hormonal mechanisms, Ang II is an important growth factor-inducing cardiac hypertrophy,4 as also shown clinically by the regression of the ventricular hypertrophy observed after treatment with angiotensin-converting enzyme inhibitors in hypertensive patients.24 Consistent with this, in the present study, Ang II as well as FBS, which contains a variety of growth factors, stimulated the de novo protein synthesis evaluated by [14C]phenylalanine incorporation into the cardiac myocytes. The present study revealed that AM had a significant inhibitory effect on the de novo protein synthesis stimulated by Ang II or FBS. Moreover, the effect occurred dose-dependently and was significantly abolished by the presence of CGRP(8–37), an N-terminal-deleted CGRP known as a CGRP type I receptor antagonist.20 The concentration of synthetic AM used in this study, which significantly inhibited the protein synthesis, was much higher than that in the plasma or in the ventricular tissue. However, when the action of endogenous AM secreted from the cultured myocytes was blocked by the addition of either CGRP(8–37), or anti-AM monoclonal antibody, not only the basal but also the Ang II-stimulated [14C]phenylalanine incorporation was significantly enhanced. These findings suggest an inhibitory role of endogenous AM on the de novo protein synthesis. Recently, Sato et al25 reported that AM inhibits gene expression of atrial natriuretic peptide, a well-established marker for hypertrophic response, in cultured cardiac myocytes. Thus, AM may have an important role as an autocrine or a paracrine factor in inhibiting hypertrophy of cardiac myocytes.
AM was discovered by monitoring an activity increasing the cAMP concentration in rat platelets.5 In accord with this, AM has been shown to dilate blood vessels via elevation of cAMP in vascular smooth muscle cells.21 In the meantime, Ikenouchi et al26 reported that AM has a negative inotropic action on isolated rabbit cardiomyocytes via NO-mediated increase of intracellular cGMP. Some studies suggested the AM effects are through CGRP type 1 receptors,27,28 but others showed that the action of AM is not mediated by this receptor subtype.29,30 In the present study, the inhibitory action on the protein synthesis of the myocytes was attenuated by CGRP(8–37), but not by AM(22–52). Similarly, Champion et al31 reported that AM(22–52) failed to antagonize the vasodilator action of AM in the hindlimb vascular bed of cat. On the other hand, this AM analogue has been shown to displace 125I-AM binding and inhibit AM-stimulated cAMP formation in cultured vascular smooth muscle cells of rat.21 There have been a number of reports on the cloning of AM and CGRP receptors,32–35 but none of them seem to clearly explain these differences or discrepancies. Further experiments seem to be needed to understand the receptor subtypes and receptor-mediated intra-cellular action of AM.
We reported that the plasma concentration of AM in patients with essential hypertension or primary aldosteronism is elevated progressively in relation to the severity of the disease,11,12 suggesting a role of plasma AM in acting to prevent further elevation of blood pressure through its potent vasodilator and natriuretic action. Both the tissue AM concentration and AM mRNA expression have been shown to be increased in the hypertrophied left ventricle of Dahl salt-sensitive and renovascular hypertensive rats compared to those in the respective normotensive control.15,16 Taken together with the present results, AM may act not only systematically to reduce blood pressure but also within ventricular tissue to modulate the growth of cardiomyocytes.
In summary, cultured neonatal cardiac myocytes of rats synthesize and secrete AM, and Ang II stimulates the secretion of AM. AM secreted from the myocytes inhibits the de novo protein synthesis of these cells. These findings suggest a role of this novel vasodilator peptide in regulating the cardiac growth as an autocrine or a paracrine factor.
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Acknowledgments |
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This study was supported in by Grants-in-Aid for Scientific Research from the Ministry of Education in Japan. We thank Dr. Hideyuki Kobayashi, Department of Pharmacology, Miyazaki Medical College, for his technical advice on culturing cells.
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H. Okumura, N. Nagaya, T. Itoh, I. Okano, J. Hino, K. Mori, Y. Tsukamoto, H. Ishibashi-Ueda, S. Miwa, K. Tambara, et al. Adrenomedullin Infusion Attenuates Myocardial Ischemia/Reperfusion Injury Through the Phosphatidylinositol 3-Kinase/Akt-Dependent Pathway Circulation, January 20, 2004; 109(2): 242 - 248. [Abstract] [Full Text] [PDF]
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 H. Yin, L. Chao, and J. Chao Adrenomedullin Protects Against Myocardial Apoptosis After Ischemia/Reperfusion Through Activation of Akt-GSK Signaling Hypertension, January 1, 2004; 43(1): 109 - 116. [Abstract] [Full Text] [PDF]
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T. Nishikimi, F. Yoshihara, S. Horinaka, N. Kobayashi, Y. Mori, K. Tadokoro, K. Akimoto, N. Minamino, K. Kangawa, and H. Matsuoka Chronic Administration of Adrenomedullin Attenuates Transition From Left Ventricular Hypertrophy to Heart Failure in Rats Hypertension, November 1, 2003; 42(5): 1034 - 1041. [Abstract] [Full Text] [PDF]
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K. Kato, H. Yin, J. Agata, H. Yoshida, L. Chao, and J. Chao Adrenomedullin gene delivery attenuates myocardial infarction and apoptosis after ischemia and reperfusion Am J Physiol Heart Circ Physiol, October 1, 2003; 285(4): H1506 - H1514. [Abstract] [Full Text] [PDF]
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T. Nishikimi, K. Tadokoro, Y. Mori, X. Wang, K. Akimoto, F. Yoshihara, N. Minamino, K. Kangawa, and H. Matsuoka Ventricular Adrenomedullin System in the Transition From LVH to Heart Failure in Rats Hypertension, March 1, 2003; 41(3): 512 - 518. [Abstract] [Full Text] [PDF]
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 N. Fukai, M. Shichiri, N. Ozawa, M. Matsushita, and Y. Hirata Coexpression of Calcitonin Receptor-Like Receptor and Receptor Activity-Modifying Protein 2 or 3 Mediates the Antimigratory Effect of Adrenomedullin Endocrinology, February 1, 2003; 144(2): 447 - 453. [Abstract] [Full Text] [PDF]
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 E. Dobrzynski, D. Montanari, J. Agata, J. Zhu, J. Chao, and L. Chao Adrenomedullin improves cardiac function and prevents renal damage in streptozotocin-induced diabetic rats Am J Physiol Endocrinol Metab, December 1, 2002; 283(6): E1291 - E1298. [Abstract] [Full Text] [PDF]
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R. Nakamura, J. Kato, K. Kitamura, H. Onitsuka, T. Imamura, K. Marutsuka, Y. Asada, K. Kangawa, and T. Eto Beneficial effects of adrenomedullin on left ventricular remodeling after myocardial infarction in rats Cardiovasc Res, December 1, 2002; 56(3): 373 - 380. [Abstract] [Full Text] [PDF]
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 T. Tsuruda and J. C. Burnett Jr Adrenomedullin: An Autocrine/Paracrine Factor for Cardiorenal Protection Circ. Res., April 5, 2002; 90(6): 625 - 627. [Full Text] [PDF]
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 K Tambara, M Fujita, N Nagaya, S Miyamoto, A Iwakura, K Doi, G Sakaguchi, K Nishimura, K Kangawa, and M Komeda Increased pericardial fluid concentrations of the mature form of adrenomedullin in patients with cardiac remodelling Heart, March 1, 2002; 87(3): 242 - 246. [Abstract] [Full Text] [PDF]
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M. Iemitsu, T. Miyauchi, S. Maeda, S. Sakai, T. Kobayashi, N. Fujii, H. Miyazaki, M. Matsuda, and I. Yamaguchi Physiological and pathological cardiac hypertrophy induce different molecular phenotypes in the rat Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2001; 281(6): R2029 - R2036. [Abstract] [Full Text] [PDF]
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G. Foldes, M. Suo, I. Szokodi, Z. Lako-Futo, R. deChatel, O. Vuolteenaho, P. Huttunen, H. Ruskoaho, and M. Toth Factors Derived from Adrenals Are Required for Activation of Cardiac Gene Expression in Angiotensin II-Induced Hypertension Endocrinology, October 1, 2001; 142(10): 4256 - 4263. [Abstract] [Full Text] [PDF]
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D. J Autelitano, R. Ridings, and F. Tang Adrenomedullin is a regulated modulator of neonatal cardiomyocyte hypertrophy in vitro Cardiovasc Res, August 1, 2001; 51(2): 255 - 264. [Abstract] [Full Text] [PDF]
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 C. Wang, E. Dobrzynski, J. Chao, and L. Chao Adrenomedullin gene delivery attenuates renal damage and cardiac hypertrophy in Goldblatt hypertensive rats Am J Physiol Renal Physiol, June 1, 2001; 280(6): F964 - F971. [Abstract] [Full Text] [PDF]
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Y. Tomoda, K. Kikumoto, Y. Isumi, T. Katafuchi, A. Tanaka, K. Kangawa, K. Dohi, and N. Minamino Cardiac fibroblasts are major production and target cells of adrenomedullin in the heart in vitro Cardiovasc Res, March 1, 2001; 49(4): 721 - 730. [Abstract] [Full Text] [PDF]
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H. Romppanen, J. Puhakka, G. Foldes, I. Szokodi, O. Vuolteenaho, H. Tokola, M. Toth, and H. Ruskoaho Endothelin-1-Independent and Angiotensin II-Independent Induction of Adrenomedullin Gene Expression Hypertension, January 1, 2001; 37(1): 84 - 90. [Abstract] [Full Text] [PDF]
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H. Yamakawa, T. Imamura, T. Matsuo, H. Onitsuka, Y. Tsumori, J. Kato, K. Kitamura, Y. Koiwaya, and T. Eto Diastolic wall stress and ANG II in cardiac hypertrophy and gene expression induced by volume overload Am J Physiol Heart Circ Physiol, December 1, 2000; 279(6): H2939 - H2946. [Abstract] [Full Text] [PDF]
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E. Dobrzynski, C. Wang, J. Chao, and L. Chao Adrenomedullin Gene Delivery Attenuates Hypertension, Cardiac Remodeling, and Renal Injury in Deoxycorticosterone Acetate-Salt Hypertensive Rats Hypertension, December 1, 2000; 36(6): 995 - 1001. [Abstract] [Full Text] [PDF]
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R. W. Troughton, L. K. Lewis, T. G. Yandle, A. M. Richards, and M. G. Nicholls Hemodynamic, Hormone, and Urinary Effects of Adrenomedullin Infusion in Essential Hypertension Hypertension, October 1, 2000; 36(4): 588 - 593. [Abstract] [Full Text] [PDF]
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 F. Piquard, A. Charloux, B. Mettauer, E. Epailly, E. Lonsdorfer, S. Popescu, J. Lonsdorfer, and B. Geny Exercise-Induced Increase in Circulating Adrenomedullin Is Related to Mean Blood Pressure in Heart Transplant Recipients J. Clin. Endocrinol. Metab., August 1, 2000; 85(8): 2828 - 2831. [Abstract] [Full Text]
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P. Kinnunen, I. Szokodi, M. G. Nicholls, and H. Ruskoaho Impact of NO on ET-1- and AM-induced inotropic responses: potentiation by combined administration Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2000; 279(2): R569 - R575. [Abstract] [Full Text] [PDF]
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T. Tsuruda, J. Kato, K. Kitamura, T. Imamura, Y. Koiwaya, K. Kangawa, I. Komuro, Y. Yazaki, and T. Eto Enhanced Adrenomedullin Production by Mechanical Stretching in Cultured Rat Cardiomyocytes Hypertension, June 1, 2000; 35(6): 1210 - 1214. [Abstract] [Full Text] [PDF]
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 J. P. Hinson, S. Kapas, and D. M. Smith Adrenomedullin, a Multifunctional Regulatory Peptide Endocr. Rev., April 1, 2000; 21(2): 138 - 167. [Abstract] [Full Text]
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N. Nagaya, T. Nishikimi, F. Yoshihara, T. Horio, A. Morimoto, and K. Kangawa Cardiac adrenomedullin gene expression and peptide accumulation after acute myocardial infarction in rats Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2000; 278(4): R1019 - R1026. [Abstract] [Full Text] [PDF]
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F. Yoshihara, T. Nishikimi, T. Horio, C. Yutani, N. Nagaya, H. Matsuo, T. Ohe, and K. Kangawa Ventricular adrenomedullin concentration is a sensitive biochemical marker for volume and pressure overload in rats Am J Physiol Heart Circ Physiol, February 1, 2000; 278(2): H633 - H642. [Abstract] [Full Text] [PDF]
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 W. K. Samson, Z. T. Resch, T. C. Murphy, T. T. Vargas, and D. A. Schell Adrenomedullin: Is There Physiological Relevance in the Pathology and Pharmacology? Physiology, December 1, 1999; 14(6): 255 - 259. [Abstract] [Full Text] [PDF]
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T. Tsuruda, J. Kato, K. Kitamura, M. Kawamoto, K. Kuwasako, T. Imamura, Y. Koiwaya, T. Tsuji, K. Kangawa, and T. Eto An autocrine or a paracrine role of adrenomedullin in modulating cardiac fibroblast growth Cardiovasc Res, September 1, 1999; 43(4): 958 - 967. [Abstract] [Full Text] [PDF]
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T. Horio, T. Nishikimi, F. Yoshihara, N. Nagaya, H. Matsuo, S. Takishita, and K. Kangawa Production and Secretion of Adrenomedullin in Cultured Rat Cardiac Myocytes and Nonmyocytes: Stimulation by Interleukin-1{beta} and Tumor Necrosis Factor-{alpha} Endocrinology, November 1, 1998; 139(11): 4576 - 4580. [Abstract] [Full Text] [PDF]
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 K. M. Caron and O. Smithies Extreme hydrops fetalis and cardiovascular abnormalities in mice lacking a functional Adrenomedullin gene PNAS, January 16, 2001; 98(2): 615 - 619. [Abstract] [Full Text] [PDF]
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