Sayaka Nagata

Differential regulation of angiotensin-(1-12) in plasma and cardiac tissue in response to bilateral nephrectomy.

We examined the effects of 48 h bilateral nephrectomy on plasma and cardiac tissue expression of angiotensin-(1-12) [ANG-(1-12)], ANG I, and ANG II in adult Wistar-Kyoto rats to evaluate functional changes induced by removing renal renin. The goal was to expand the evidence of ANG-(1-12) being an alternate renin-independent, angiotensin-forming substrate. Nephrectomy yielded divergent effects on circulating and cardiac angiotensins. Significant decreases in plasma ANG-(1-12), ANG I, and ANG II levels postnephrectomy accompanied increases in cardiac ANG-(1-12), ANG I, and ANG II concentrations compared with controls. Plasma ANG-(1-12) decreased 34% following nephrectomy, which accompanied 78 and 66% decreases in plasma ANG I and ANG II, respectively (P < 0.05 vs. controls). Contrastingly, cardiac ANG-(1-12) in anephric rats averaged 276 +/- 24 fmol/mg compared with 144 +/- 20 fmol/mg in controls (P < 0.005). Cardiac ANG I and ANG II values were 300 +/- 15 and 62 +/- 7 fmol/mg, respectively, in anephric rats compared with 172 +/- 8 fmol/mg for ANG I and 42 +/- 4 fmol/mg for ANG II in controls (P < 0.001). Quantitative immunofluorescence revealed significant increases in average grayscale density for cardiac tissue angiotensinogen, ANG I, ANG II, and AT(1) receptors of WKY rats postnephrectomy. Faint staining of cardiac renin, unchanged by nephrectomy, was associated with an 80% decrease in cardiac renin mRNA. These changes were accompanied by significant increases in p47(phox), Rac1, and Nox4 isoform expression. In conclusion, ANG-(1-12) may be a functional precursor for angiotensin peptide formation in the absence of circulating renin.

Localization of the novel angiotensin peptide, angiotensin-(1-12), in heart and kidney of hypertensive and normotensive rats

A low expression of angiotensinogen in the heart has been construed as indicating a circulating uptake mechanism to explain the local effects of angiotensin II on tissues. The recent identification of angiotensin-(1-12) in an array of rat organs suggests this propeptide may be an alternate substrate for local angiotensin production. To test this hypothesis, tissues from 11-wk-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats (n = 14) were stained with purified antibodies directed to the COOH terminus of angiotensin-(1-12). Robust angiotensin-(1-12) staining was predominantly found in ventricular myocytes with less staining found in the medial layer of intracoronary arteries and vascular endothelium. In addition, angiotensin-(1-12) immunoreactivity was present in the proximal, distal, and collecting renal tubules within the deep cortical and outer medullary zones in both strains. Preadsorption of the antibody with angiotensin-(1-12) abolished staining in both tissues. Corresponding tissue measurements by radioimmunoassay showed 47% higher levels of angiotensin-(1-12) in the heart of SHR compared with WKY rats (P < 0.05). In contrast, renal angiotensin-(1-12) levels were 16.5% lower in SHR compared with the WKY rats (P < 0.05). This study shows for first time the localization of angiotensin-(1-12) in both cardiac myocytes and renal tubular components of WKY and SHR. In addition, we show that increased cardiac angiotensin-(1-12) concentrations in SHR is associated with a small, but statistically significant, reduction in renal angiotensin-(1-12) levels.

An evolving story of angiotensin-II-forming pathways in rodents and humans.

Lessons learned from the characterization of the biological roles of Ang-(1-7) [angiotensin-(1-7)] in opposing the vasoconstrictor, proliferative and prothrombotic actions of AngII (angiotensin II) created an underpinning for a more comprehensive exploration of the multiple pathways by which the RAS (renin-angiotensin system) of blood and tissues regulates homoeostasis and its altered state in disease processes. The present review summarizes the progress that has been made in the novel exploration of intermediate shorter forms of angiotensinogen through the characterization of the expression and functions of the dodecapeptide Ang-(1-12) [angiotensin-(1-12)] in the cardiac production of AngII. The studies reveal significant differences in humans compared with rodents regarding the enzymatic pathway by which Ang-(1-12) undergoes metabolism. Highlights of the research include the demonstration of chymase-directed formation of AngII from Ang-(1-12) in human left atrial myocytes and left ventricular tissue, the presence of robust expression of Ang-(1-12) and chymase in the atrial appendage of subjects with resistant atrial fibrillation, and the preliminary observation of significantly higher Ang-(1-12) expression in human left atrial appendages.

Shared and separate functions of the RAMP-based adrenomedullin receptors

Adrenomedullin (AM) is a novel hypotensive peptide that exerts a variety of strongly protective effects against multiorgan damage. AM-specific receptors were first identified as heterodimers composed of calcitonin-receptor-like receptor (CLR), a G protein coupled receptor, and one of two receptor activity-modifying proteins (RAMP2 or RAMP3), which are accessory proteins containing a single transmembrane domain. RAMPs are required for the surface delivery of CLR and the determination of its phenotype. CLR/RAMP2 (AM₁ receptor) is more highly AM-specific than CLR/RAMP3 (AM₂ receptor). Although there have been no reports showing differences in intracellular signaling via the two AM receptors, in vitro studies have shed light on their distinct trafficking and functionality. In addition, the tissue distributions of RAMP2 and RAMP3 differ, and their gene expression is differentially altered under pathophysiological conditions, which is suggestive of the separate roles played by AM₁ and AM₂ receptors in vivo. Both AM and the AM₁ receptor, but not the AM₂ receptor, are crucial for the development of the fetal cardiovascular system and are able to effectively protect against various vascular diseases. However, AM₂ receptors reportedly play an important role in maintaining a normal body weight in old age and may be involved in immune function. In this review article, we focus on the shared and separate functions of the AM receptor subtypes and also discuss the potential for related drug discovery. In addition, we mention their possible function as receptors for AM2 (or intermedin), an AM-related peptide whose biological functions are similar to those of AM.

ACE2: Angiotensin II/Angiotensin-(1–7) Balance in Cardiac and Renal Injury

Our current recognition of the renin-angiotensin system is more convoluted than originally thought due to the discovery of multiple novel enzymes, peptides, and receptors inherent in this interactive biochemical cascade. Over the last decade, angiotensin-converting enzyme 2 (ACE2) has emerged as a key player in the pathophysiology of hypertension and cardiovascular and renal disease due to its pivotal role in metabolizing vasoconstrictive/hypertrophic/proliferative angiotensin II into favorable angiotensin-(1–7). This review addresses the considerable advancement in research on the role of tissue ACE2 in the development and progression of hypertension and cardiac and renal injury. We summarize the results from recent clinical and experimental studies suggesting that serum or urine soluble ACE2 may serve as a novel biomarker or independent risk factor relevant for diagnosis and prognosis of cardiorenal disease. We also review recent proceedings on novel therapeutic approaches to enhance ACE2/angiotensin-(1–7) axis.

Hemodynamic and Hormonal Changes to Dual Renin–Angiotensin System Inhibition in Experimental Hypertension

We examined the antihypertensive effects of valsartan, aliskiren, or both drugs combined on circulating, cardiac, and renal components of the renin–angiotensin system in congenic mRen2.Lewis hypertensive rats assigned to: vehicle (n=9), valsartan (via drinking water, 30 mg/kg per day; n=10), aliskiren (SC by osmotic mini-pumps, 50 mg/kg per day; n=10), or valsartan (30 mg/kg per day) combined with aliskiren (50 mg/kg per day; n=10). Arterial pressure and heart rate were measured by telemetry before and during 2 weeks of treatment; trunk blood, heart, urine, and kidneys were collected for measures of renin–angiotensin system components. Arterial pressure and left-ventricular weight/tibia length ratio were reduced by monotherapy of valsartan, aliskiren, and further reduced by the combination therapy. Urinary protein excretion was reduced by valsartan and further reduced by the combination. The increases in plasma angiotensin (Ang) II induced by valsartan were reversed by the treatment of aliskiren and partially suppressed by the combination. The decreases in plasma Ang-(1–7) induced by aliskiren recovered in the combination group. Kidney Ang-(1–12) was increased by the combination therapy whereas the increases in urinary creatinine mediated by valsartan were reversed by addition of aliskiren. The antihypertensive and antiproteinuric actions of the combined therapy were associated with marked worsening of renal parenchymal disease and increased peritubular fibrosis. The data show that despite improvements in the surrogate end points of blood pressure, ventricular mass, and proteinuria, dual blockade of Ang II receptors and renin activity is accompanied by worsening of renal parenchymal disease reflecting a renal homeostatic stress response attributable to loss of tubuloglomerular feedback by Ang II.

Plasma and tissue levels of proangiotensin-12 and components of the renin-angiotensin system (RAS) following low- or high-salt feeding in rats.

The renin-angiotensin system (RAS) is an essential regulator of the blood pressure and body fluid balance, but the processing cascade or role of the tissue RAS remains obscure. Proangiotensin-12 (proang-12), a novel angiotensin peptide recently discovered in rat tissues, is assumed to function as a factor of the tissue RAS. To investigate the tissue production of proang-12, we measured the circulating and tissue components of the RAS including proang-12 following low-, normal-, or high-salt feeding in rats. Twelve-week-old male Wistar rats were fed a low-salt 0.3% NaCl or high-salt 8% NaCl diet for 7 days and compared with those fed a normal-salt diet of 0.7% NaCl. Low-salt feeding elevated the plasma renin activity and aldosterone concentration, resulting in significant increases in Ang I and Ang II levels in the plasma or kidney tissue, as compared with the normal- or high-salt group. Despite the increases in plasma renin activity, Ang I, and Ang II, the proang-12 levels in plasma and various tissues including the kidneys, small intestine, cardiac ventricles, and brain remained unchanged following low-salt feeding. These results suggest that peptide levels of proang-12 in rat plasma and tissues are regulated in a manner independent of the circulating RAS.

Structure–function analysis of helix 8 of human calcitonin receptor-like receptor within the adrenomedullin 1 receptor

Adrenomedullin 1 (AM(1)) receptor is a heterodimer composed of calcitonin receptor-like receptor (CLR) - a family B G protein-coupled receptor (GPCR) - and receptor activity-modifying protein 2 (RAMP2). Both family A and family B GPCRs possess an eighth helix (helix 8) in the proximal portion of their C-terminal tails; however, little is known about the function of helix 8 in family B GPCRs. We therefore investigated the structure-function relationship of human (h)CLR helix 8, which extends from Glu430 to Trp439, by separately transfecting nine point mutants into HEK-293 cells stably expressing hRAMP2. Glu430, Val431, Arg437 and Trp439 are all conserved among family B GPCRs. Flow cytometric analysis revealed that Arg437Ala or Trp438Ala mutation significantly reduced cell surface expression of the receptor complex, leading to a ∼20% reduction in specific (125)I-AM binding but little change in their IC(50) values. Both mutants showed 6-8-fold higher EC(50) values for AM-induced cAMP production and ∼50% reductions in their maximum responses. Glu430Ala mutation also reduced AM signaling by ∼45%, but surface expression and (125)I-AM binding were nearly the same as with wild-type CLR. Surprisingly, Glu430Ala and Val431Ala mutations significantly enhanced AM-induced internalization of the mutant receptor complexes. Taken together, these findings suggest that within hCLR helix 8, Glu430 is crucial for Gs coupling, and Arg437 and Trp439 are involved in both cell surface expression of the hAM(1) receptor and Gs coupling. Moreover, the Glu430-Val431 sequence may participate in the negative regulation of hAM(1) receptor internalization, which is not dependent on Gs coupling.

Function of the cytoplasmic tail of human calcitonin receptor-like receptor in complex with receptor activity-modifying protein 2

Receptor activity-modifying protein 2 (RAMP2) enables calcitonin receptor-like receptor (CRLR) to form an adrenomedullin (AM)-specific receptor. Here we investigated the function of the cytoplasmic C-terminal tail (C-tail) of human (h)CRLR by co-transfecting its C-terminal mutants into HEK-293 cells stably expressing hRAMP2. Deleting the C-tail from CRLR disrupted AM-evoked cAMP production or receptor internalization, but did not affect [(125)I]AM binding. We found that CRLR residues 428-439 are required for AM-evoked cAMP production, though deleting this region had little effect on receptor internalization. Moreover, pretreatment with pertussis toxin (100ng/mL) led to significant increases in AM-induced cAMP production via wild-type CRLR/RAMP2 complexes. This effect was canceled by deleting CRLR residues 454-457, suggesting Gi couples to this region. Flow cytometric analysis revealed that CRLR truncation mutants lacking residues in the Ser/Thr-rich region extending from Ser(449) to Ser(467) were unable to undergo AM-induced receptor internalization and, in contrast to the effect on wild-type CRLR, overexpression of GPCR kinases-2, -3 and -4 failed to promote internalization of CRLR mutants lacking residues 449-467. Thus, the hCRLR C-tail is crucial for AM-evoked cAMP production and internalization of the CRLR/RAMP2, while the receptor internalization is dependent on the aforementioned GPCR kinases, but not Gs coupling.

Angiotensin-(1-12): A Chymase-Mediated Cellular Angiotensin II Substrate

The classical view of biochemical pathways for the formation of biologically active angiotensins continues to undergo significant revision as new data uncovers the existence of important species differences between humans and rodents. The discovery of two novel substrates that, cleaved from angiotensinogen, can lead to direct tissue angiotensin II formation has the potential of radically altering our understanding of how tissues source angiotensin II production and explain the relative lack of efficacy that characterizes the use of angiotensin converting enzyme inhibitors in cardiovascular disease. This review addresses the discovery of angiotensin-(1-12) as an endogenous substrate for the production of biologically active angiotensin peptides by a non-renin dependent mechanism and the revealing role of cardiac chymase as the angiotensin II convertase in the human heart. This new information provides a renewed argument for exploring the role of chymase inhibitors in the correction of cardiac arrhythmias and left ventricular systolic and diastolic dysfunction.