Inhibited cardiac hypertrophy in proadrenomedullin NH2-terminal 20 peptide-transgenic rat with unilateral nephrectomy and high salt loading

AJH–May 2005–VOL. 18, NO. 5, PART 2

P-656 INHIBITED CARDIAC HYPERTROPHY IN PROADRENOMEDULLIN NH2-TERMINAL 20 PEPTIDE-TRANSGENIC RAT WITH UNILATERAL NEPHRECTOMY AND HIGH SALT LOADING Yuanning Cao, Kazuo Kitamura, Kenji Kuwasako, Johji Kato, Kensaku Nishihira, Yujiro Asada, Tanenao Eto. First Department of Internal Medicine, Faculty of Medicine, University of Miyazaki, Kiyotake, Miyazaki, Japan; First Department of Pathology, Faculty of Medicine, University of Miyazaki, Kiyotake, Miyazaki, Japan. Proadrenomedullin NH2-Terminal 20 peptide (PAMP) is synthesized from the same preproadrenomedullin gene of adrenomedullin (AM). Unlike AM, little is known about PAMP. We developed transgenic rats by constructing a PAMP transgene incorporating the potent chicken ␤-actin promoter and then microinjected into fertilized eggs from superovulated Wistar rats. Potential transgenic founders were screened by PCR of genomic DNA from rat tail tips. Significant increase of human PAMP levels was confirmed in both plasma and tissues by ELISA method. PAMP-transgenic (Tg) or wild (W) rats were fed a high salt diet (8% NaCl) but with free access to drinking water for 5 weeks after unilateral nephrectomy. Rats were sacrificed and plasma and tissue were sampled at the end of 5 week. Rat heart tissue was either evaluated by immunohistochemistry or by real-time quantitative PCR methods for gene quantification. Heart rate did not significantly differ in two groups. However, systolic blood pressure, weekly measured by tail-cuff method, was attenuated in Tg rats compared with wild ones (p⬍0.05). Meanwhile, the cardiac hypertrophy in wild rat was significantly ameliorated in the Tg rat with heart weight (mg) to body weight (g) ratio of 3.5⫾0.1 to 2.99⫾0.11 (p⬍0.05). The cardiac fibrosis observed in wild rat was significantly attenuated by 50% in Tg rat (p⬍0.05). Further evaluation of the local renin-angiotensin system (RAS) related genes in the left ventricle by real-time PCR method showed that ACE gene transcription in Tg rat was downregulated to 60% of wild rat (p⬍0.05). Renin gene expression, however, was about 2.5-fold upregulated in Tg rat (p⬍0.05), reflecting the subsequently reduced angiotensin II production in the heart. This is also consistent with previous report that renin gene transcription is negatively regulated by angiotensin II. In contrast to local renin changes in heart, plasma renin activity was not significantly different in the two groups, though both were inhibited by salt loading. In conclussion, PAMP possesses hypotensive and anti-hypertrophy effect in this model implicating its clinical application of hypertension and organ damage. Key Words: Adrenomedullin, Hypertension, Peptide

P-657 ACUTE ORAL PEGYLATED HUMAN BNP ACTIVATES CGMP AND DECREASES MEAN ARTERIAL PRESSURE IN NORMAL DOGS Alessandro Cataliotti, John A Schirger, Fernando L Martin, Horng H Chen, Kenneth D James, Mark A Miller, Navdeep B Malkar, Nnoch N Ekwuribe, John C Burnett. Internal Medicine, Mayo Clinic, Rochester, MN; Nobex Corporation, Durham, NC. The objective of this study was to address the feasibility and the biological activity of orally administered human BNP (hBNP). Proprietary technology (Nobex, Durham NC) has developed amphiphilic oligomers covalently attached to peptides, which protect peptides from proteolysis. Pegylated peptides have an improved pharmacokinetic profile and may enable oral administration. We hypothesized that novel pegylated oral hBNP (PEG-hBNP) increases plasma hBNP, activates cGMP, and reduces mean arterial pressure (MAP). This randomized crossover designed study tested the biological activity of oral PEG-hBNP compared to oral native hBNP in normal dogs. Measurements of MAP, plasma hBNP, and cGMP were made at Baseline © 2005 by the American Journal of Hypertension, Ltd. Published by Elsevier Inc.

POSTERS: Vasoactive Hormones/Autacoids

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(BL) and repeated at 10, 30, 60, 120, 180, and 240 min after oral administration. Plasma hBNP was not detectable in dogs at BL. Plasma hBNP was detected after native hBNP and PEG-HBNP administration. However, plasma hBNP concentration was significantly higher after PEG-hBNP than after native hBNP administration (p⫽0.0374 between groups). Plasma cGMP increased after PEG-hBNP for 60 min (from 10.8⫾3 to 36.8⫾26 pmol/ml, p⬍0.05), while it did not change after native hBNP (p⫽0.001 between groups). MAP decreased at 10 minutes and remained decreased for 60 minutes after PEG-hBNP (from 113⫾8 to 101⫾12 after 10 min to 97.5⫾10 after 30 min to 99⫾13 mmHg after 60 min), while remained unchanged after native hBNP (p⫽0.0387 between groups). This study reports for the first time that novel pegylated oral BNP activates cGMP and significantly reduces MAP, thus implying an efficacious coupling of PEG-hBNP to the natriuretic receptor-A. This data may advance a new concept of orally administered chronic BNP therapy for cardiovascular diseases. Key Words: Blood Pressure, cGMP, Oral Pegylated BNP

P-658 A COMPARISON STUDY OF ADRENOMEDULLIN (AM) AND ADRENOMEDULLIN 2 (AM2) IN DISTRIBUTION OF RAT TISSUE Kazuo Kitamura, Mariko Tokashiki, Cao Yuan-Ning, Kenji Kuwasako, Toshihiro Kita, Johji Kato, Tanenao Eto. First Department of Internal Medicine, Miyazaki Medical College, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki, Japan. Adrenomedullin (AM) is a potent vasodilator peptide that was originally isolated from human pheochromocytoma AM is widely distributed in various tissues, including the cardiovascular system. Very recently, adrenomedullin 2 (AM2) is discovered independently by two groups. It consists of 47 amino acids and shows homology to AM. Intravenous infusion of AM2 induced potent cardiovascular and renal effects in mice and rat. Both AM2 and AM are thought to belong to the same superfamily. In the present study, the radioimmunoassay (RIA) for AM2 has been established. And we compared the tissue distribution of AM and AM2 in rat to elucidate the biological roles of AM and AM2. The peptide, AM2(35-47)CONH2 was conjugated to thyroglobulin and used as the antigen for immunization. The antiserum to AM2 recognized the peptide with high affinity at a final dilution of 1:50,000. The half maximal inhibition of radioiodinated ligand binding was observed at 32 fmol/tube. The antiserum had 100% crossreactivity with human AM2 but less than 0.1% crossreactivity with AM and PAMP, respectively. High concentration of immunoreractive (ir-) AM was detected in the adrenal gland (5.4⫹/-3.6 fmol/mg wet weight, mean ⫹/- SD), lung (9.8⫹/-3.0) and atrium (2.1⫹/-0.47) and ubiquitously found in all tissue examined. However the ir-AM2 was very low in the adrenal gland (⬍0.2fmol/mg) and the atrium(⬍0.2fmol/mg). The ir-AM2 in the lung (1.85⫹/-0.19) is significantly lower than that of AM. The concentration of AM and AM-2 were comparable in the ventricle (AM;0.63⫹/-0.09, AM2; 0.72⫹/-0.15) and the kidney (AM;0.83⫹/-0.09, AM2; 0.78⫹/0.29). Interestingly, ir-AM2 in brain (0.60⫹/-0.21) and submaxillary gland (0.27⫹/-0.08) were significantly higher than those of ir-AM. Ir-AM was also detectable in testis (0.71⫹/-0.02) and fat tissue (0.33⫹/0.08), but AM2 was not detectable (⬍0.2fmol/mg) in these tissues. In addition, the plasma level of AM2 seems to be very low (⬍0.5 fmol/ml), in comparison, the plasma level of AM is measurable (3.2⫹/-0.16 fmol/ ml). In conclusion, AM2 is ubiquitously distributed in rat tissues, though it is different from that of AM. Similar distribution of AM2 and AM in 0895-7061/05/$30.00