High-glucose-induced regulation of intracellular ANG II synthesis and nuclear redistribution in cardiac myocytes

The prevailing paradigm is that cardiac ANG II is synthesized in the extracellular space from components of the circulating and/or local renin-angiotensin system. The recent discovery of intracrine effects of ANG II led us to determine whether ANG II is synthesized intracellularly in neonatal rat ventricular myocytes (NRVM). NRVM, incubated in serum-free medium, were exposed to isoproterenol or high glucose in the absence or presence of candesartan, which was used to prevent angiotensin type 1 (AT(1)) receptor-mediated internalization of ANG II. ANG II was measured in cell lysates and the culture medium, which represented intra- and extracellularly synthesized ANG II, respectively. Isoproterenol increased ANG II concentration in cell lysates and medium of NRVM in the absence or presence of candesartan. High glucose markedly increased ANG II synthesis only in cell lysates in the absence and presence of candesartan. Western analysis showed increased intracellular levels of angiotensinogen, renin, and chymase in high-glucose-exposed cells. Confocal immunofluorocytometry confirmed the presence of ANG II in the cytoplasm and nucleus of high-glucose-exposed NRVM and along the actin filaments in isoproterenol-exposed cells. ANG II synthesis was dependent on renin and chymase in high-glucose-exposed cells and on renin and angiotensin-converting enzyme in isoproterenol-exposed cells. In summary, the site of ANG II synthesis, intracellular localization, and the synthetic pathway in NRVM are stimulus dependent. Significantly, NRVM synthesized and retained ANG II intracellularly, which redistributed to the nucleus under high-glucose conditions, suggesting a role for an intracrine mechanism in diabetic conditions.     

Local renin-angiotensin systems

The existence of a local cardiovascular renin-angiotensin system (RAS) is often invoked to explain the long-term beneficial effects of RAS inhibitors in heart failure and hypertension. The implicit assumption is that all components of the RAS are synthesized in situ, so that local angiotensin II formation may occur independently of the circulating RAS. Evidence for this assumption however is lacking. The angiotensin release from isolated perfused rat hearts or hindlimbs depends on the presence of renal renin. When calculating the in vivo angiotensin production at tissue sites in humans and pigs, taking into account the extensive regional angiotensin clearance by infusing radiolabeled angiotensin I or II, it was found that angiotensin production correlated closely with plasma renin activity. Moreover, in pigs the cardiac tissue levels of renin and angiotensin were directly correlated with their respective plasma levels, and both in tissue and plasma the levels were undetectably low after nephrectomy. Similarly, rat vascular renin and angiotensin decrease to low or undetectable levels within 48 h after nephrectomy. Aortic renin has a longer half life than plasma renin, suggesting that renin may be bound by the vessel wall. In support of this assumption, both renin receptors and renin-binding proteins have been described. Like ACE, renin was enriched in a purified membrane fraction prepared from cardiac tissue. Binding of renin to cardiac or vascular membranes may therefore be part of a mechanism by which renin is taken up from plasma. It appears that the concept of a local RAS needs to be reassessed. Local angiotensin formation in heart and vessel wall does occur, but depends, at least under normal circumstances, on the uptake of renal renin from the circulation. Tissues may regulate their local angiotensin concentrations by varying the number of renin receptors and/or renin-binding proteins, the ACE level, the amount of metabolizing enzymes and the angiotensin receptor density.Abbreviations RAS renin-angiotensin system - ANG angiotensin - ACE angiotensin-converting enzyme - PRA plasma renin activity     

High glucose increases extracellular matrix production in pancreatic stellate cells by activating the renin-angiotensin system

Pancreatic stellate cells (PSCs) are involved in pancreatic inflammation and fibrosis. Recent studies have shown that blocking the renin-angiotensin system (RAS) attenuates pancreatic inflammation and fibrosis. However, there are few data about the direct effects of high glucose on extracellular matrix (ECM) protein synthesis and angiotensin II (Ang II) induction in PSCs. PSCs were isolated from male Sprague-Dawley rats and cultured in medium containing 5.5 mM (LG group) or 27 mM D-glucose (HG group). Levels of Ang II and transforming growth factor-beta (TGF-beta) in culture media were measured and Ang II-positive cells were counted. We used real-time polymerase chain reaction (PCR) to detect Ang II receptor expression and Western blot analysis for the expression of ECM proteins such as connective-tissue growth factor (CTGF) and collagen type IV. Cells were also treated with an Ang II-receptor antagonist (candesartan, 10 microM) or angiotensin-converting enzyme (ACE) inhibitor (ramiprilat, 100 nM). Thymidine uptake by PSCs increased fourfold with high glucose treatment. Ang II levels and the proportion of Ang II-positive PSCs were significantly increased after 6 h under high-glucose conditions. TGF-beta concentrations also increased significantly with high glucose. After 72 h, the expression of CTGF and collagen type IV proteins in high-glucose cultures increased significantly and this increase was effectively attenuated by the candesartan or the ramiprilat. All together, high glucose induced PSCs proliferation and ECM protein synthesis, and these effects were attenuated by an Ang II-receptor antagonist. The data suggest that pancreatic inflammation and fibrosis aggravated by hyperglycemia, and Ang II play an important role in this pathogenesis.     

Renin released from mast cells activated by circulating MCP-1 initiates the microvascular phase of the systemic inflammation of alveolar hypoxia

Reduced alveolar Po(2) in rats produces a rapid systemic inflammation characterized by reactive O(2) species generation, mast cell (MC) degranulation, leukocyte-endothelial interactions, and increased vascular permeability. The inflammation is not initiated by the low systemic Po(2) but rather by the release of monocyte chemoattractant protein-1 (MCP-1) from alveolar macrophages (AMO) activated by alveolar hypoxia. Circulating AMO-borne MCP-1 induces MC degranulation, which activates the local renin-angiotensin system (RAS) and mediates the microvascular inflammation. This study was directed to determine the mechanism of RAS activation by MCP-1-induced MC degranulation. Experiments in isolated rat peritoneal MCs showed the following: 1) Western blots and immunocytochemistry demonstrated the presence of renin and angiotensin-converting enzyme (ACE) in MCs and their release upon degranulation; 2) MCP-1-induced degranulation of MCs incubated in plasma produced an increase in angiotensin II (ANG II) concentration; and 3) this increase was inhibited completely by the following agents: the MCP-1 receptor antagonist RS-102895, the specific rat renin inhibitor WFML, or the ACE inhibitor captopril administered separately. Captopril also inhibited ANG II generation by MCs incubated in culture medium plus ANG I. The results show that peritoneal MCs contain active renin, which activates the RAS upon degranulation, and that peritoneal MCs are a source of ACE and suggest that conversion of ANG I to ANG II is mediated predominantly by ACE. This study provides novel evidence of the presence of active renin in rat peritoneal MCs and helps explain the mechanism of activation of the RAS during alveolar hypoxia.     

Intracardiac intracellular angiotensin system in diabetes

The renin-angiotensin system (RAS) has mainly been categorized as a circulating and a local tissue RAS. A new component of the local system, known as the intracellular RAS, has recently been described. The intracellular RAS is defined as synthesis and action of ANG II intracellularly. This RAS appears to differ from the circulating and the local RAS, in terms of components and the mechanism of action. These differences may alter treatment strategies that target the RAS in several pathological conditions. Recent work from our laboratory has demonstrated significant upregulation of the cardiac, intracellular RAS in diabetes, which is associated with cardiac dysfunction. Here, we have reviewed evidence supporting an intracellular RAS in different cell types, ANG II's actions in cardiac cells, and its mechanism of action, focusing on the intracellular cardiac RAS in diabetes. We have discussed the significance of an intracellular RAS in cardiac pathophysiology and implications for potential therapies.     

High Na intake increases renal angiotensin II levels and reduces expression of the ACE2-AT(2)R-MasR axis in obese Zucker rats

High sodium intake is known to regulate the renal renin-angiotensin system (RAS) and is a risk factor for the pathogenesis of obesity-related hypertension. The complex nature of the RAS reveals that its various components may have opposing effects on natriuresis and blood pressure regulation. We hypothesized that high sodium intake differentially regulates and shifts a balance between opposing components of the renal RAS, namely, angiotensin-converting enzyme (ACE)-ANG II-type 1 ANG II receptor (AT(1)R) vs. AT(2)-ACE2-angiotensinogen (Ang) (1-7)-Mas receptor (MasR), in obesity. In the present study, we evaluated protein and/or mRNA expression of angiotensinogen, renin, AT(1A/B)R, ACE, AT(2)R, ACE2, and MasR in the kidney cortex following 2 wk of a 8% high-sodium (HS) diet in lean and obese Zucker rats. The expression data showed that the relative expression pattern of ACE and AT(1B)R increased, renin decreased, and ACE2, AT(2)R, and MasR remained unaltered in HS-fed lean rats. On the other hand, HS intake in obese rats caused an increase in the cortical expression of ACE, a decrease in ACE2, AT(2)R, and MasR, and no changes in renin and AT(1)R. The cortical levels of ANG II increased by threefold in obese rats on HS compared with obese rats on normal salt (NS), which was not different than in lean rats. The HS intake elevated mean arterial pressure in obese rats (27 mmHg) more than in lean rats (16 mmHg). This study suggests that HS intake causes a pronounced increase in ANG II levels and a reduction in the expression of the ACE2-AT(2)R-MasR axis in the kidney cortex of obese rats. We conclude that such changes may lead to the potentially unopposed function of AT(1)R, with its various cellular and physiological roles, including the contribution to the pathogenesis of obesity-related hypertension.     

Modulation of renin angiotensin system components by high glucose levels in the culture of collecting duct cells

Diabetes mellitus and its complications have become a major health concern in Western countries. Increased activity of the intrarenal renin–angiotensin system (RAS) contributes to diabetic nephropathy (DN). We previously reported that in mesangial cells, the high glucose concentration (HG) leads to upregulation of angiotensin-converting enzyme (ACE) messenger RNA, suggesting that ACE was modulated by angiotensin II (Ang II) release. However, this relation in the collecting duct has not yet been studied. We, therefore, aimed to evaluate RAS modulation in inner medullary collecting duct cells (IMCD) exposed to HG. The IMCD were divided into normal glucose (5 mM D -glucose, NG), high glucose (30 mM, HG), and mannitol (30 mM, M) groups. The cells were cultured 48 hr in their respective media. The intracellular and extracellular ACE activity was measured using hippuryl-His-Leu as substrate via a fluorimetric assay and expression was analyzed using western blot analysis. ACE activity, intracellular (27%) and extracellular (22%), was significantly lower in the HG group than in NG and M. ACE2 activity and Ang 1–7 levels were higher in the intracellular compartment. Our data suggest that the HG cannot modify ACE synthesis in IMCD cells but can modulate its activity. The decrease in ACE activity may result in decreased levels of Ang II to protect the IMCD against proliferative and inflammatory deleterious effects of this peptide. Conversely, the increase of ACE2 generating high levels of Ang 1–7, a vasodilator peptide, suggesting that this peptide can induce glucose uptake and protect cells against oxidative stress, which can elicit insulin resistance.     

Autocrine regulation of internal anal sphincter tone by renin-angiotensin system: comparison with phasic smooth muscle

The myogenic control mechanisms that govern the basal tone in the internal anal sphincter (IAS) are not known. The present studies determined the autocrine regulation of ANG II in the IAS. The studies were performed in the freshly isolated smooth muscle cells (SMC) of the IAS. We determined the presence of ANG II precursor angiotensinogen (Angen), and the enzymes that convert it into ANG II, using functional, molecular biology, and immunocytochemical studies in rats. ANG II levels in the SMC were determined using ELISA. The IAS SMC generate ANG II at a rate severalfold higher than those from the adjoining smooth muscle of rectum (RSM). RT-PCR data show that IAS exclusively expresses significant higher levels of renin, Angen, and angiotensin-converting enzyme (ACE). These data were confirmed using Western blot analyses and immunocytochemistry. In the IAS SMC, H-77 (10 microM; renin inhibitor) and captopril (1 microM; ACE inhibitor) decreased the basal as well as Angen-increased levels of ANG II. The following functional data corroborate the role of renin-angiotensin system (RAS) in the IAS tone. Angen produced concentration-dependent shortening of the IAS SMC that was inhibited by H-77 and captopril. In addition, H-77 or captopril caused a concentration-dependent fall in the IAS tone vs. nontonic tissues. Basal tone in IAS is partially under the autocrine control of cellular RAS evident by the expression of mRNA coding Angen, renin, and ACE and translation to the respective proteins in the SMC.     

Reciprocal changes in renal ACE/ANG II and ACE2/ANG 1-7 are associated with enhanced collecting duct renin in Goldblatt hypertensive rats

Alterations in the balance between ANG II/ACE and ANG 1-7/ACE2 in ANG II-dependent hypertension could reduce the generation of ANG 1-7 and contribute further to increased intrarenal ANG II. Upregulation of collecting duct (CD) renin may lead to increased ANG II formation during ANG II-dependent hypertension, thus contributing to this imbalance. We measured ANG I, ANG II, and ANG 1-7 contents, angiotensin-converting enzyme (ACE) and ACE2 gene expression, and renin activity in the renal cortex and medulla in the clipped kidneys (CK) and nonclipped kidneys (NCK) of 2K1C rats. After 3 wk of unilateral renal clipping, systolic blood pressure and plasma renin activity increased in 2K1C rats (n = 11) compared with sham rats (n = 9). Renal medullary angiotensin peptide levels were increased in 2K1C rats [ANG I: (CK = 171 ± 4; NCK = 251 ± 8 vs. sham = 55 ± 3 pg/g protein; P < 0.05); ANG II: (CK = 558 ± 79; NCK = 328 ± 18 vs. sham = 94 ± 7 pg/g protein; P < 0.001)]; and ANG 1-7 levels decreased (CK = 18 ± 2; NCK = 19 ± 2 pg/g vs. sham = 63 ± 10 pg/g; P < 0.001). In renal medullas of both kidneys of 2K1C rats, ACE mRNA levels and activity increased but ACE2 decreased. In further studies, we compared renal ACE and ACE2 mRNA levels and their activities from chronic ANG II-infused (n = 6) and sham-operated rats (n = 5). Although the ACE mRNA levels did not differ between ANG II rats and sham rats, the ANG II rats exhibited greater ACE activity and reduced ACE2 mRNA levels and activity. Renal medullary renin activity was similar in the CK and NCK of 2K1C rats but higher compared with sham. Thus, the differential regulation of ACE and ACE2 along with the upregulation of CD renin in both the CK and NCK in 2K1C hypertensive rats indicates that they are independent of perfusion pressure and contribute to the altered content of intrarenal ANG II and ANG 1-7.     

Inhibition of intracellular Angiotensin II formation blocks high glucose effect on mesangial matrix

High glucose causes increased matrix synthesis by glomerular mesangial cells and angiotensin II (Ang II) has been shown to mediate this effect of glucose. These studies investigate whether inhibition of Ang II formation can block high glucose-induced increase in mesangial matrix. Human mesangial cells were incubated with 25 mM glucose (HG) along with captopril, an ACE inhibitor, to block Ang II formation. In other experiments, cells were nucleofected with siRNA to knockdown angiotensinogen (Agt), the precursor of Ang II, and then exposed to high glucose. Captopril blocked high glucose-induced increase in Ang II levels in the cell media (extracellular) but failed to inhibit it in the cell lysate (intracellular). Moreover, captopril treatment did not block the stimulatory effect of high glucose on TGF-β1 and fibronectin. In contrast, knockdown of the Agt gene prevented high glucose-induced increase in both extracellular and intracellular Ang II levels, and was accompanied by normalization of TGF-β1 and fibronectin. These data suggest that intracellular Ang II may play an important role in the mediation of the high glucose effect on matrix and that ACE inhibitors may not be effective in blocking intracellular Ang II formation in mesangial cells.     

Angiotensin II enhances adenylyl cyclase signaling via Ca2+/calmodulin. Gq-Gs cross-talk regulates collagen production in cardiac fibroblasts

Cardiac fibroblasts regulate formation of extracellular matrix in the heart, playing key roles in cardiac remodeling and hypertrophy. In this study, we sought to characterize cross-talk between Gq and Gs signaling pathways and its impact on modulating collagen synthesis by cardiac fibroblasts. Angiotensin II (ANG II) activates cell proliferation and collagen synthesis but also potentiates cyclic AMP (cAMP) production stimulated by beta-adrenergic receptors (beta-AR). The potentiation of beta-AR-stimulated cAMP production by ANG II is reduced by phospholipase C inhibition and enhanced by overexpression of Gq. Ionomycin and thapsigargin increased intracellular Ca2+ levels and potentiated isoproterenol- and forskolin-stimulated cAMP production, whereas chelation of Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid/AM inhibited such potentiation. Inhibitors of tyrosine kinases, protein kinase C, or Gbetagamma did not alter this cross-talk. Immunoblot analyses showed prominent expression of adenylyl cyclase 3 (AC3), a Ca2+-activated isoform, along with AC2, AC4, AC5, AC6, and AC7. Of those isoforms, only AC3 and AC5/6 proteins were detected in caveolin-rich fractions. Overexpression of AC6 increased betaAR-stimulated cAMP accumulation but did not alter the size of the ANG II potentiation, suggesting that the cross-talk is AC isoform-specific. Isoproterenol-mediated inhibition of serum-stimulated collagen synthesis increased from 31 to 48% in the presence of ANG II, indicating that betaAR-regulated collagen synthesis increased in the presence of ANG II. These data indicate that ANG II potentiates cAMP formation via Ca2+-dependent activation of AC activity, which in turn attenuates collagen synthesis and demonstrates one functional consequence of cross-talk between Gq and Gs signaling pathways in cardiac fibroblasts.     

Profibrotic influence of high glucose concentration on cardiac fibroblast functions: effects of losartan and vitamin E

Long-standing diabetes can result in the development of cardiomyopathy, which can be accompanied by myocardial fibrosis. Although exposure of cultured kidney and skin fibroblasts to high glucose (HG) concentration is known to increase collagen synthesis, little is known about cardiac fibroblasts (CFs). Therefore, we determined the influence of HG conditions on CF functions and the effects of losartan and vitamin E in these responses. We cultured rat CFs in either normal glucose (NG; 5.5 mM) or HG (25 mM) media and assessed changes in protein and collagen synthesis, matrix metalloproteinase (MMP) activity, and levels of mRNA for ANG II type 1 (AT(1)) receptors. Results indicate that HG-level CFs synthesized more protein and collagen, and these effects were not due to changes in osmotic pressure. The addition of ANG II stimulated protein and collagen synthesis in NG-concentration but not HG-concentration CFs. Interestingly, losartan pretreatment blocked the HG- or ANG II-induced increases in both protein and collagen synthesis. HG or ANG II decreased total MMP activity. Decreases in MMP activity were blocked by losartan. AT(1) mRNA levels were upregulated with HG concentration. Vitamin E pretreatment blocked the effects of HG on total protein synthesis and stimulated MMP activity. Results suggest that HG levels may promote fibrosis by increasing CF protein and collagen synthesis and decreasing MMP activity. HG levels may cause these effects via the upregulation of AT(1) receptors, which can be blocked by losartan. However, vitamin E can alter HG concentration-induced changes in CF functions independently of AT(1) mRNA levels.     

Regulation of ACE2 in cardiac myocytes and fibroblasts

Angiotensin-converting enzyme 2 (ACE2) preferentially forms angiotensin-(1-7) [ANG-(1-7)] from ANG II. We showed that cardiac ACE2 is elevated following treatment of coronary artery-ligated rats with AT1 receptor blockers (ARBs). Cardiac myocytes and fibroblasts were isolated from neonatal rats to determine the molecular mechanisms for the ACE2 upregulation by ARB treatment. ANG II significantly reduced ACE2 activity and downregulated ACE2 mRNA in cardiac myocytes, effects blocked by the ARB losartan, indicating that ANG II regulates ACE2. ANG II also reduced ACE2 mRNA in cardiac fibroblasts; however, no enzyme activity was detected, reflecting the limited expression of ACE2 in these cells. Endothelin-1 (ET-1) also significantly reduced myocyte ACE2 mRNA. The reduction in ACE2 mRNA by ANG II or ET-1 was blocked by inhibitors of mitogen-activated protein kinase kinase 1, suggesting that ANG II or ET-1 activates extracellular signal-regulated kinase (ERK) 1/ERK2 to reduce ACE2. Although ACE2 mRNA was not affected by ANG-(1-7), both the ANG II- and ET-1-mediated reductions in ACE2 mRNA were blocked by the heptapeptide. The ANG-(1-7) modulatory effect was prevented by the ANG-(1-7) receptor antagonist [D-Ala7]-ANG-(1-7), indicating that the ANG-(1-7) response was mediated by a specific AT(1-7) receptor. Myocyte treatment with atrial natriuretic peptide (ANP) also reversed the ACE2 mRNA downregulation by ANG II or ET-1, whereas treatment with ANP alone was ineffective. These results indicate that multiple hypertrophic and anti-hypertropic peptides regulate ACE2 production in myocytes, suggesting that ACE2 expression in the heart is dependent upon the compliment and concentration of regulatory molecules.     

Differential autocrine modulation of atrial and ventricular potassium currents and of oxidative stress in diabetic rats

The autocrine modulation of cardiac K(+) currents was compared in ventricular and atrial cells (V and A cells, respectively) from Type 1 diabetic rats. K(+) currents were measured by using whole cell voltage clamp. ANG II was measured by ELISA and immunofluorescent labeling. Oxidative stress was assessed by immunofluorescent labeling with dihydroethidium, a measure of superoxide ions. In V cells, K(+) currents are attenuated after activation of the renin-angiotensin system (RAS) and the resulting ANG II-mediated oxidative stress. In striking contrast, these currents are not attenuated in A cells. Inhibition of the angiotensin-converting enzyme (ACE) also has no effect, in contrast to current augmentation in V cells. ANG II levels are enhanced in V, but not in A, cells. However, the high basal ANG II levels in A cells suggest that in these cells, ANG II-mediated pathways are suppressed, rather than ANG II formation. Concordantly, superoxide ion levels are lower in diabetic A than in V cells. Several findings indicate that high atrial natriuretic peptide (ANP) levels in A cells inhibit RAS activation. In male diabetic V cells, in vitro ANP (300 nM-1 muM, >5 h) decreases oxidative stress and augments K(+) currents, but not when excess ANG II is present. ANP has no effect on ventricular K(+) currents when the RAS is not activated, as in control males, in diabetic males treated with ACE inhibitor and in diabetic females. In conclusion, the modulation of K(+) currents and oxidative stress is significantly different in A and V cells in diabetic rat hearts. The evidence suggests that this is largely due to inhibition of RAS activation and/or action by ANP in A cells. These results may underlie chamber-specific arrhythmogenic mechanisms.     

Enhanced intrarenal receptor-mediated prorenin activation in chronic progressive anti-thymocyte serum nephritis rats on high salt intake

Despite suppression of the circulating renin-angiotensin system (RAS), high salt intake (HSI) aggravates kidney injury in chronic kidney disease. To elucidate the effect of HSI on intrarenal RAS, we investigated the levels of intrarenal prorenin, renin, (pro)renin receptor (PRR), receptor-mediated prorenin activation, and ANG II in chronic anti-thymocyte serum (ATS) nephritic rats on HSI. Kidney fibrosis grew more severe in the nephritic rats on HSI than normal salt intake. Despite suppression of plasma renin and ANG II, marked increases in tubular prorenin and renin proteins without concomitant rises in renin mRNA, non-proteolytically activated prorenin, and ANG II were noted in the nephritic rats on HSI. Redistribution of PRR from the cytoplasm to the apical membrane, along with elevated non-proteolytically activated prorenin and ANG II, was observed in the collecting ducts and connecting tubules in the nephritic rats on HSI. Olmesartan decreased cortical prorenin, non-proteolytically activated prorenin and ANG II, and apical membranous PRR in the collecting ducts and connecting tubules, and attenuated the renal lesions. Cell surface trafficking of PRR was enhanced by ANG II and was suppressed by olmesartan in Madin-Darby canine kidney cells. These data suggest the involvement of the ANG II-dependent increase in apical membrane PRR in the augmentation of intrarenal binding of prorenin and renin, followed by nonproteolytic activation of prorenin, enhancement of renin catalytic activity, ANG II generation, and progression of kidney fibrosis in the nephritic rat kidneys on HSI. The origin of the increased tubular prorenin and renin remains to be clarified. Further studies measuring the urinary prorenin and renin are needed.     

Differential response of cardiac fibroblasts from young adult and senescent rats to ANG II

The intracardiac ANG II-forming pathway is activated in the senescent myocardium, raising the possibility of enhanced ANG II effects on cardiac fibroblasts. This study established an in vitro model of cultured cardiac fibroblasts from aged rats to examine if the response of these cells to ANG II is modified in the aged heart. Levels of mRNA encoding renin, angiotensinogen, and the AT(1) receptor subtype in cardiac fibroblasts from young adult and senescent rats were quantified by RT-PCR, net collagen production by a hydroxyproline-based assay, and transforming growth factor (TGF)-beta levels using a commercial kit. In cardiac fibroblasts from young adult rats, ANG II significantly enhanced AT(1) mRNA levels, net collagen production, and TGF-beta production. In fibroblasts from the aged myocardium, ANG II downregulated AT(1) mRNA expression, had a less pronounced effect on net collagen production, and had no effect on TGF-beta production. Such age-related modification of the response of cardiac fibroblasts to ANG II may counteract the effects of augmented intracardiac ANG II production in the senescent heart, limiting fibrogenesis.     

Novel roles of nuclear angiotensin receptors and signaling mechanisms

The renin-angiotensin system (RAS) constitutes an important hormonal system in the physiological regulation of blood pressure. The dysregulation of the RAS is considered a major influence in the development and progression of cardiovascular disease and other pathologies. Indeed, experimental and clinical evidence indicates that blockade of this system with angiotensin-converting enzyme (ACE) inhibitors or angiotensin type 1 receptor (AT1R) antagonists is an effective therapy to attenuate hypertension and diabetic renal injury, and to improve heart failure. Originally defined as a circulating system, multiple tissues express a complete RAS, and compelling evidence now favors an intracellular system involved in cell signaling and function. Within the kidney, intracellular expression of the three predominant ANG receptor subtypes is evident in the nuclear compartment. The ANG type 1 receptor (AT1R) is coupled to the generation of reactive oxygen species (ROS) through the activation of phosphoinositol-3 kinase (PI3K) and PKC. In contrast, both ANG type 2 (AT2R) and ANG-(1-7) (AT7R) receptors stimulate nitric oxide (NO) formation, which may involve nuclear endothelial NO synthase (eNOS). Moreover, blockade of either ACE2-the enzyme that converts ANG II to ANG-(1-7)-or the AT7 receptor exacerbates the ANG II-ROS response on renal nuclei. Finally, in a model of fetal programmed hypertension, the nuclear ROS response to ANG II is enhanced, while both AT2 and AT7 stimulation of NO is attenuated, suggesting that an imbalance in the intracellular RAS may contribute to the development of programming events. We conclude that a functional intracellular or nuclear RAS may have important implications in the therapeutic approaches to cardiovascular disease.     

Effects of spironolactone and eprosartan on cardiac remodeling and angiotensin-converting enzyme isoforms in rats with experimental heart failure

Angiotensin-converting enzyme (ACE)-2 is a newly described enzyme with antagonistic effects to those of the classical ACE (ACE-1). Both ANG II and aldosterone play an important role in the pathophysiology of congestive heart failure (CHF) and in the adverse cardiac remodeling during its development. In this study, we examined the effects of experimental CHF induced by an aortocaval fistula (ACF) and of its treatment with ANG II and aldosterone inhibitors on the relative levels of ACE-1 and ACE-2. We also compared the effects of spironolactone, an aldosterone antagonist, and eprosartan, an ANG II receptor antagonist, on heart hypertrophy and fibrosis in rats with ACF. Spironolactone (15 mg x kg(-1) x day(-1) ip, via minipump) or eprosartan (5 mg x kg(-1) x day(-1) ip, via minipump) was administered into rats with ACF for 14 and 28 days. Specific antibodies were used to determine the protein levels of myocardial ACE-1 and ACE-2. ACF increased the cardiac levels of ACE-1 and decreased those of ACE-2. Heart-to-body weight ratio significantly increased from 0.30 +/- 0.004% in sham-operated controls to 0.50 +/- 0.018% and 0.56 +/- 0.044% (P < 0.001) in rats with ACF, 2 and 4 wk after surgery, respectively, in association with increased plasma levels of aldosterone. The area occupied by collagen increased from 2.33 +/- 0.27% to 6.85 +/- 0.65% and 8.03 +/- 0.93% (P < 0.01), 2 and 4 wk after ACF, respectively. Both spironolactone and eprosartan decreased cardiac mass and collagen content and reversed the shift in ACE isoforms. ACF alters the ratio between ACE isoforms in a manner that increases local ANG II and aldosterone levels. Early treatment with both ANG II and aldosterone antagonists is effective in reducing this effect. Thus ACE isoform shift may represent an important component of the development of cardiac remodeling in response to hemodynamic overload, and its correction may contribute to the beneficial therapeutic effects of renin-angiotensin-aldosterone system inhibitors.     

Divergent mechanism regulating fluid intake and metabolism by the brain renin-angiotensin system

The purpose of this review is two-fold. First, I will highlight recent advances in our understanding of the mechanisms regulating angiotensin II (ANG II) synthesis in the brain, focusing on evidence that renin is expressed in the brain and is expressed in two forms: a secreted form, which may catalyze extracellular ANG I generation from glial or neuronal angiotensinogen (AGT), and an intracellular form, which may generate intracellular ANG in neurons that may act as a neurotransmitter. Second, I will discuss recent studies that advance the concept that the renin-angiotensin system (RAS) in the brain not only is a potent regulator of blood pressure and fluid intake but may also regulate metabolism. The efferent pathways regulating the blood pressure/dipsogenic effects and the metabolic effects of elevated central RAS activity appear different, with the former being dependent upon the hypothalamic-pituitary-adrenal axis, and the latter being dependent upon an interaction between the brain and the systemic (or adipose) RAS.     

Effect of angiotensin II blockade on a new congenic model of hypertension derived from transgenic Ren-2 rats

The generation of the Lew.Tg(mRen2) congenic hypertensive rat strain, developed through a backcross of the hypertensive (mRen2)27 transgenic rat with normotensive Lewis rats, provides a new model by which primary hypertension can be studied without the genetic variability found in the original strain. The purpose of this study was to characterize the Lew.Tg(mRen2) rats by dually investigating the effects of type 1 angiotensin II (ANG II) receptor (AT(1)) blockade and angiotensin-converting enzyme (ACE) activity inhibition on the ANG-(1-7)/ACE2 axis of the renin-angiotensin system in this new hypertensive model. The control of blood pressure elicited by 12-day administration of either lisinopril (mean difference change = 92 +/- 2, P < 0.05) or losartan (mean difference change = 69 +/- 2, P < 0.05) was associated with 54% and 33% increases in cardiac ACE2 mRNA and 54% and 43% increases in cardiac ACE mRNA, respectively. Lisinopril induced a 3.1-fold (P < 0.05) increase in renal cortical expression of ACE2, whereas losartan increased ACE2 mRNA 3.5-fold (P < 0.05). Both treatment regimens increased renal ACE mRNA 2.6-fold (P < 0.05). The two therapies augmented ACE2 protein activity, as well as increased cardiac and renal AT(1) receptor mRNAs. ACE inhibition reduced plasma ANG II levels (81%, P < 0.05) and increased plasma ANG-(1-7) (265%, P < 0.05), whereas losartan had no effect on the peptides. In contrast with what had been shown in normotensive rats, ACE inhibition decreased renal ANG II excretion and transiently decreased ANG-(1-7) excretion, whereas losartan treatment was associated with a consistent decrease in ANG-(1-7) urinary excretion rates. In response to the treatments, the expression of both renal cortical renin and angiotensinogen mRNAs was significantly augmented. The paradoxical effects of blockade of ANG II synthesis and activity on urinary excretion rates of the peptides and plasma angiotensins levels suggest that, in Lew.Tg(mRen2) congenic rats, a failure of compensatory ACE2 and ANG-(1-7)-dependent vasodepressor mechanisms may contribute both to the development and progression of hypertension driven by increased formation of endogenous ANG II.