Mariko Tokashiki

Increased plasma levels of the mature and intermediate forms of adrenomedullin in obesity.

Adrenomedullin (AM) is a cardiovascular protective peptide produced in various organs and tissues including adipose tissue. In the present study, we measured the plasma AM levels of subjects with or without obesity by two assay methods to separately evaluate the biologically active AM-NH(2) and the intermediate form of AM-glycine (AM-Gly). We measured the total AM and AM-NH(2) levels of plasma in 52 obese and 172 non-obese residents of a Japanese community, who received regular health check-ups and had no overt cardiovascular disease. AM-Gly values were obtained by subtracting AM-NH(2) levels from those of total AM. Both the AM-NH(2) and AM-Gly levels of the subjects with obesity were higher than those without obesity, and significant relationships were noted between body mass index (BMI) and the plasma levels of the two molecular forms of AM in a simple regression analysis. Moreover, the significant factors identified by multivariate analyses were BMI and serum triglyceride for AM-NH(2) and diastolic blood pressure, insulin, high-density lipoprotein-cholesterol, and plasma renin activity for AM-Gly. These results suggest active roles for the two molecular forms of AM in metabolic disorders associated with obesity in subjects without overt cardiovascular disease.

Big angiotensin-25: a novel glycosylated angiotensin-related peptide isolated from human urine.

The renin-angiotensin system (RAS), including angiotensin II (Ang II), plays an important role in the regulation of blood pressure and body fluid balance. Consequently, the RAS has emerged as a key target for treatment of kidney and cardiovascular disease. In a search for bioactive peptides using an antibody against the N-terminal portion of Ang II, we identified and characterized a novel angiotensin-related peptide from human urine as a major molecular form. We named the peptide Big angiotensin-25 (Bang-25) because it consists of 25 amino acids with a glycosyl chain and added cysteine. Bang-25 is rapidly cleaved by chymase to Ang II, but is resistant to cleavage by renin. The peptide is abundant in human urine and is present in a wide range of organs and tissues. In particular, immunostaining of Bang-25 in the kidney is specifically localized to podocytes. Although the physiological function of Bang-25 remains uncertain, our findings suggest it is processed from angiotensinogen and may represent an alternative, renin-independent path for Ang II synthesis in tissue.

Biological properties of adrenomedullin conjugated with polyethylene glycol

Adrenomedullin (AM) is a vasodilator peptide with pleiotropic effects, including cardiovascular protection and anti-inflammation. Because of these beneficial effects, AM appears to be a promising therapeutic tool for human diseases, while intravenous injection of AM stimulates sympathetic nerve activity due to short-acting potent vasodilation, resulting in increased heart rate and renin secretion. To lessen these acute reactions, we conjugated the N-terminal of human AM peptide with polyethylene glycol (PEG), and examined the biological properties of PEGylated AM in the present study. PEGylated AM stimulated cAMP production, an intracellular second messenger of AM, in cultured human embryonic kidney cells expressing a specific AM receptor in a dose-dependent manner, as did native human AM. The pEC50 value of PEGylated AM was lower than human AM, but no difference was noted in maximum response (Emax) between the PEGylated and native peptides. Intravenous bolus injection of 10nmol/kg PEGylated AM lowered blood pressure in anesthetized rats, but the acute reduction became significantly smaller by PEGylation as compared with native AM. Plasma half-life of PEGylated AM was significantly longer than native AM both in the first and second phases in rats. In summary, N-terminal PEGylated AM stimulated cAMP production in vitro, showing lessened acute hypotensive action and a prolonged plasma half-life in comparison with native AM peptide in vivo.

Gender-related alterations in plasma adrenomedullin level and its correlation with body weight gain

Plasma levels of adrenomedullin (AM), a bioactive peptide produced in adipose tissue, have been shown to be higher in obese patients than in non-obese patients, but little is known about gender differences in plasma AM levels. The aims of this study were to clarify gender-related alterations in plasma AM levels and to examine the body weight (BW) gain–plasma AM relationship in the general population. We measured plasma AM levels of 346 local residents (62.0±8.9 years, mean±s.d.) in the Kiyotake area, Japan, who underwent a regular health check-up, by a specific fluorescence immunoassay. Plasma AM levels in the female residents were lower than that in the males, and multiple regression analysis revealed a possible gender difference in plasma AM. The AM levels were significantly correlated with BMI or waist circumference in women, but such a relationship was not seen in men. When the subjects were divided into two groups by results of a questionnaire about BW gain of 10 kg or more since the age of 20 years, the plasma AM level of women with BW gain ≧10 kg was significantly higher than that in those without BW gain, although no difference was noted between the men with and without BW gain. In conclusion, possible gender differences were noted in the plasma AM levels and in the BW gain–plasma AM relationship in the general population. The plasma AM levels in the female residents without BW gain seem partly attributable to the lower AM of women.

Augmented Blood Pressure Variability in Hypertension Induced by Angiotensin II in Rats.

BACKGROUND Augmented blood pressure (BP) variability is associated with cardiovascular diseases in some clinical conditions including hypertension. Drugs that effectively reduce BP variability need to be identified, while few animal models are currently available to study BP variability. Here, we report that hypertension induced by continuous infusion of angiotensin II (Ang II) was accompanied by increased BP variability in rats. METHODS Ang II was subcutaneously infused at a rate of 240 pmol/kg/min into male Wistar rats undergoing intraperitoneal implantation of a transmitter connected to an abdominal aortic catheter. BP was continuously monitored via a telemetry system before and after the Ang II infusion in a conscious, unrestrained condition. BP variability was evaluated by coefficient of variation (CV) of BP levels measured every 15 minutes. In addition, spontaneously hypertensive and Wistar-Kyoto rats (SHR and WKY) were subjected to the BP monitoring experiment at 15 weeks of age. RESULTS Both systolic and diastolic BP levels were significantly elevated following the Ang II infusion. Similarly, CVs of systolic and diastolic BP in the Ang II infusion group were significantly higher than in the vehicle group upon 1 and 2 weeks of the infusion. Meanwhile, CVs of systolic and diastolic BP of SHR were in a range similar to those of WKY despite significantly higher BP than in WKY. CONCLUSIONS Hypertension induced by the continuous infusion of Ang II was accompanied by augmented BP variability in rats, an effect assumed to be at least in part, independent of BP elevation.

Long-Term Therapy with Nifedipine-CR Improves Arterio-Sclerosis Related Markers in Patients with Untreated Essential Hypertension

Increased arteriosclerosis is associated with high risk of cardiovascular events. Several non-invasive markers for arteriosclerosis have been introduced, such as pulse wave velocity (PWV), augmentation index (AI), and carotid prop- erties assessed by echogram, to estimate the current risk and therapeutic merit of antihypertensives. In this study, 17 hy- pertensive patients were treated with nifedipine-CR alone for one year, and the non-invasive markers were simultaneously monitored every 3 months. Nifedipine-CR treatment achieved stable blood pressure control, and PWV and AI improved in parallel with the blood pressure. Interestingly, the elastic property of the carotid artery progressively decreased and there was a significant difference between the results at 3 and 12 months (85.8 ± 6.1 vs 72.4 ± 5.0 kPa, P = 0.009). In- tima-media thickness of the carotid artery also decreased. In conclusion, nifedipine-CR demonstrated a stable anti-sclerotic quality in hypertensive patients and seems to be prominent in large arteries such as the carotid.

Plasma levels of natriuretic peptides and year-by-year blood pressure variability: a population-based study

Augmented blood pressure (BP) variability over various time periods has been recognized as a risk factor for cardiovascular diseases. Both atrial and B-type natriuretic peptides (ANP and BNP) are secreted in response to volume or pressure overload to the heart, exerting natriuretic and vasodilator actions. In this study, we examined the relationships between year-by-year BP variability and plasma levels of ANP and BNP in the general population. Study subjects were local residents receiving an annual heath checkup, who had an estimated glomerular filtration rate of >30 ml min−1 per 1.73 m2 and no history of heart disease. Of those, we selected 314 subjects that received checkups at least five times over the past 6 years. BP variability year-by-year was retrospectively evaluated by s.d., coefficient of variation, average real variability and variation independent of the mean of BP values of 6 or 7 time points. The four parameters of BP variability were each found to significantly correlate with logarithmically transformed ANP and BNP levels by simple regression. When classified by quartiles of s.d. of systolic BP, the highest quartile group showed significantly higher levels of the natriuretic peptides compared with other groups. Multivariate analyses revealed that BP variability was an independent determinant for the ANP and BNP levels. In conclusion, augmented year-by-year BP variability over the past 6 years was associated with elevation of plasma levels of ANP and BNP, suggesting a possible relationship between the BP variability and cardiac load, in the general population.

PS 07-20 STRUCTURAL ANALYSIS OF GLYCOSYL CHAIN AT 14TH AMINO ACID OF ANGIOTENSINOGEN IN HUMAN PLASMA

Objective: The Renin angiotensin system (RAS) is a major regulator of body fluid balance and control of blood pressure. The protease enzyme renin secreted from kidneys cleaves specifically angiotensinogen (Aogen) circulating in the blood to produce angiotensin I (Ang I). Human Aogen is a heterogeneous glycoprotein constitutively secreted by the liver. In addition, human Aogen contains four putative asparagine-linked glycosylation sites (Asn 14, 137, 271, 295) and contains four cysteines (Cys 18, 138, 232, 308), with Cys18 and Cys138 linked by a disulfide bridge. The glycosyl chains and cysteines position are very important for binding of the renin. Big angiotensin-25 (Bang-25) is a consisting of 25 amino acids with glycosyl chain (14th amino asid) and added cysteine (18th amino acid), which we recently isolated from human urine. To compare glycosyl chain of Bang-25 and Aogen, we analyzed of structure glycosyl chain at position 14th amino acid of human Aogen in plasma. Design and Method: To determine of glycosyl chain at position 14th amino acid, we performed lysyl endopeptidase digestion and reduction on human plasma Aogen. Then, Aogen after digest was purified by reverse-phase high-performance liquid chromatograpy (RP-HPLC), and glycosyl chain structure analyzed by the two-/three-dimensional HPLC mapping method. Results: We show that plasma Aogen has three types of glycosyl chain at position 14th amino acid. One glycosyl chain structure is identical to Bang-25 in urine. N-linked glycosylation on 14th amino acid of Aogen plays an important role about renin reaction. In addition, Bang-25 is rapidly cleaved by chymase to Ang II, but is resistant to cleave by renin. Conclusions: These results suggest that the structure of the glycosyl chain at position 14th amino acid of the human Aogen may be involved in the substrate specificity for renin or chymase.

[PS 01-11] BIOCHEMICAL PROPERTIES OF THE N-TERMINALLY PALMITOYLATED ADRENOMEDULLIN

Objective: Adrenomedullin(AM) is a vasodilator peptide having pleiotropic effects including cardiovascular protection and angiogenesis. Because of these beneficial effects, AM appears to be a promising therapeutic tool for human diseases such as myocardial infarction or peripheral artery disease. However, intravenous injection of AM stimulates sympathetic nerve activity due to the short-acting potent vasodilation resulting in increased heart rate and renin secretion. To lessen those acute unfavorable actions, we conjugated the N-terminal palmitoylation human adrenomedullin, and examined biological of palmitoylation AM in the present study. Design and Method: N-termial synthesized human AM peptide was conjugated with palmitic acid, and then palmitoylation AM was obtained-by purification with HPLC. Biological effects in vitro stimulating intracellular cAMP,a major second messenger of AM,were examined using cultured human embryonic (HEK)-239 cells stably expressing a specific AM receptor. Blood pressure-lowering effects in vivo were tested by intravenous injections of palmitoylated AM or native AM peptides into anesthetized rats. Plasma disappearance curve of peptides were evaluated by the two compartment model. Results: Palmitoylated AM stimulated intracellular accumulation of cAMP in cultured HEK-293 cells, as did native human AM peptide, in a dose-dependent manner. pEC50 of palmitoylatted AM was lower than native AM (8.49 ± 0.12 vs. 9.17 ± 0.12mean ± SEM, P < 0.05),but no difference was noted in the maximum response of cAMP (579.9 ± 24.5 vs. 667.2 ± 24.5 pmol/well) . The first and second plasma half-lives of native AM were 0.0046 min and 13.36 min,while those of palmitoylated AM were 1.15 min and 599.03 min,respectively. Both half-lives of the palmitoylated peptide were significantly prolonged, as compared with the native peptide (P < 0.05). Conclusions: N-terminally palmitoylated AM stimulated cAMP production in vitro, showing smaller acute hypotensive action and a prolonged plasma half-life in comparison of native AM peptide in vivo. The present results suggest a possibility for palmitoylated AM as a therapeutic tool with lessened unfavorable effect of acute hyportensin of native AM.