Increased expression of cyclooxygenase-2 in the renal cortex of human prorenin receptor gene-transgenic rats.

Increased macula densa cyclooxygenase-2 (COX-2) is observed in diabetic rats and may contribute to hyperfiltration states. However, the signals mediating increased COX-2 expression in diabetic rats remain undetermined. We recently found that non-proteolytic activation of prorenin by site-specific binding proteins, such as prorenin receptor, plays a pivotal role in the development of diabetic nephropathy. The present study was designed to determine the contribution of prorenin receptor to renal cortical COX-2 expression. The COX-2 mRNA and protein levels of six 4-week-old male wild-type rats and six human prorenin receptor gene-transgenic (hProRenRcTg) rats were measured by real-time polymerase chain reaction methods, Western blotting, and immunohistochemistry, and compared. There were no differences between the two groups in arterial pressure measured by telemetry, urinary sodium excretion, or renal levels of rat prorenin receptor mRNA. The renal cortical COX-2 mRNA levels of the hProRenRcTg rats were significantly higher than those of the wild-type rats, and the renal cortical COX-2 protein levels were also higher in hProRenRcTg rats than in the wild-type rats. Immunohistochemical analysis revealed that COX-2 immunostaining was predominantly present in the macula densa cells, and significantly more COX-2-positive cells were present in the hProRenRcTg rats than in the wild-type rats. In addition, COX-2 inhibition with NS398 significantly decreased renal cortical blood flow in the hProRenRcTg rats but not in the wild-type rats. These results strongly suggest that human prorenin receptor directly or indirectly contributes to the regulation of renal cortical COX-2 expression.

Metalloprotease-dependent ErbB ligand shedding in mediating EGFR transactivation and vascular remodelling.

AngII (angiotensin II) and its G-protein-coupled AT(1) receptor play critical roles in mediating cardiovascular diseases such as hypertension, atherosclerosis and restenosis after vascular injury. It is widely believed that AngII promotes these diseases by inducing vascular remodelling that involves hypertrophy, hyperplasia and migration of VSMCs (vascular smooth muscle cells). We have shown that transactivation of an ErbB family receptor, EGFR (epidermal growth factor receptor; ErbB1), is essential for VSMC hypertrophy and migration induced by AngII. However, the precise signal transduction mechanism by which AngII transactivates EGFR/ErbB1 and whether other ErbBs are also required for AngII function remains unclear. Recent studies suggest an involvement of a metalloprotease-dependent ErbB family ligand production in the transactivation. Here, we will discuss the roles and mechanisms of AngII/AT(1) receptor in promoting ErbB receptors transactivation in VSMCs. Further elucidation of this ErbB activation machinery not only will give us a better understanding of the critical molecular mechanism underlying vascular remodelling stimulated by AngII, but will also contribute to development of novel treatment strategies for cardiovascular diseases.

Dual effects of angiotensin II type 2 receptor on cardiovascular hypertrophy.

Roles of angiotensin II (Ang II) in the regulation of cardiovascular system under normal and pathological condition have been well documented. Although two major subtypes of Ang II receptors, AT(1) and AT(2), are found in various proportions, the role and signaling mechanisms of AT(2) in the control of hypertrophic responses of cardiac ventricle and vasculature are not clear. Although earlier reports indicated that AT(2)'s functions are essentially growth suppression, an increasing number of recent reports indicate that AT(2) in cardiovascular tissues are often growth promoting. In some tissues AT(1) and AT(2) seem to share a common signaling pathways, at least in part. This review focuses on the accumulating evidence for the AT(2) function in the cardiovascular system.

Overexpression of the human angiotensin II type 1 receptor in the rat heart augments load induced cardiac hypertrophy.

Angiotensin II is known to stimulate cardiac hypertrophy and contractility. Most angiotensin II effects are mediated via membrane bound AT1 receptors. However, the role of myocardial AT1 receptors in cardiac hypertrophy and contractility is still rarely defined. To address the hypothesis that increased myocardial AT1 receptor density causes cardiac hypertrophy apart from high blood pressure we developed a transgenic rat model which expresses the human AT1 receptor under the control of the alpha-myosin heavy-chain promoter specifically in the myocardium. Expression was identified and quantified by northern blot analysis and radioligand binding assays, demonstrating overexpression of angiotensin II receptors in the transgenic rats up to 46 times the amount seen in nontransgenic rats. Coupling of the human AT1 receptor to rat G proteins and signal transduction cascade was verified by sensitivity to GTP-gamma-S and increased sensitivity of intracellular Ca2+ [Ca2+]i to angiotensin II in fluo-3 loaded transgenic cardiomyocytes. Transgenic rats exhibited normal cardiac growth and function under baseline conditions. Pronounced hypertrophic growth and contractile responses to angiotensin II, however, were noted in transgenic rats challenged by volume and pressure overload. In summary, we generated a new transgenic rat model that exhibits an upregulated myocardial AT1 receptor density and demonstrates augmented cardiac hypertrophy and contractile response to angiotensin II after volume and pressure overload, but not under baseline conditions.

The angiotensin II type 1 receptor and receptor-associated proteins.

The mechanisms of regulation, activation and signal transduction of the angiotensin II (Ang II) type 1 (AT1) receptor have been studied extensively in the decade after its cloning. The AT1 receptor is a major component of the renin-angiotensin system (RAS). It mediates the classical biological actions of Ang II. Among the structures required for regulation and activation of the receptor, its carboxyl-terminal region plays crucial roles in receptor internalization, desensitization and phosphorylation. The mechanisms involved in heterotrimeric G-protein coupling to the receptor, activation of the downstream signaling pathway by G proteins and the Ang II signal transduction pathways leading to specific cellular responses are discussed. In addition, recent work on the identification and characterization of novel proteins associated with carboxyl-terminus of the AT1 receptor is presented. These novel proteins will advance our understanding of how the receptor is internalized and recycled as they provide molecular mechanisms for the activation and regulation of G-protein-coupled receptors.

Increased AT(1) receptors in adrenal gland of AT(2) receptor gene-disrupted mice.

Angiotensin II (Ang II) AT(2) receptor-gene disrupted mice have increased systemic blood pressure and response to exogenous Angiotensin II. To clarify the mechanism of these changes, we studied adrenal AT(1) receptor expression and mRNA by receptor autoradiography and in situ hybridization in female AT(2) receptor-gene disrupted mice (agtr 2-/-) and wild-type controls (agtr 2+/+). We found high expression of AT(1) receptor binding and mRNA in adrenal zona glomerulosa of female wild-type mice. AT(2) receptors and mRNA were highly expressed in adrenal medulla of wild-type mice, but were not detected in zona glomerulosa. There was no AT(2) receptor binding or mRNA in adrenal glands of AT(2) receptor-gene disrupted mice. In these animals, AT(1) receptor binding and mRNA were increased in adrenal zona glomerulosa and AT(1) receptor mRNA was increased in the adrenal medulla when compared with wild-type animals.The present data support the hypothesis of an interaction or cross talk between AT(2) and AT(1) receptors in adrenal gland. The significant increase in AT(1) receptor expression in the absence of AT(2) receptor transcription may be partially responsible for the increased blood pressure and for the enhanced response to exogenously administered Angiotensin II in this model.

Unique regulation of c-Jun N-terminal kinase by PYK2/CAK-beta in angiotensin II-stimulated vascular smooth muscle cells.

Activation of tyrosine kinases is believed to play a central role in angiotensin II (AngII) signaling. Here, we have investigated whether a tyrosine kinase, PYK2, is functionally involved in AngII-induced c-Jun N-terminal kinase (JNK) activation in vascular smooth muscle cells (VSMCs). Adenovirus expressing PYK2 kinase-inactive mutant K457A or a tyrosine phosphorylation site mutant Y402F was transfected in VSMCs. AngII-induced JNK phosphorylation was markedly enhanced by K457A, whereas it was suppressed by Y402F. Protein synthesis induced by AngII was also enhanced by K457A and inhibited by Y402F. In this regard, K457A suppressed PYK2 kinase activation by AngII, whereas it enhanced AngII-induced PYK2 Tyr(402) phosphorylation. By contrast, Y402F inhibited PYK2 Tyr(402) phosphorylation, whereas it markedly enhanced AngII-induced PYK2 kinase activation. Thus, we conclude that PYK2 kinase activity negatively regulates JNK activation and protein synthesis, whereas Tyr(402) phosphorylation positively regulates these events in AngII-stimulated VSMCs, suggesting a unique role of PYK2 in mediating vascular remodeling.

Angiotensin II type 2 receptor is essential for left ventricular hypertrophy and cardiac fibrosis in chronic angiotensin II-induced hypertension.

BACKGROUND: The roles of angiotensin II (Ang II) in the regulation of heart function under normal and pathological conditions have been well documented. Although 2 types of Ang II receptor (AT(1) and AT(2)) are found in various proportions, most studies have focused on AT(1)-coupled events. In the present study, we examined the hypothesis that signaling by AT(2) is important to the development of left ventricular hypertrophy and cardiac fibrosis by Ang II infusion in mice lacking the AT(2) gene (Agtr2-/Y). METHODS AND RESULTS: Male Agtr2-/Y and age-matched wild-type (WT) mice were treated long-term with Ang II, infused at a rate of 4.2 ng. kg(-1). min(-1) for 3 weeks. Ang II elevated systolic blood pressure to comparable levels in Agtr2-/Y and WT mice. WT mice developed prominent concentric cardiac hypertrophy, prominent fibrosis, and impaired diastolic relaxation after Ang II infusion. In contrast, there was no cardiac hypertrophy in Agtr2-/Y mice. Agtr2-/Y mice, however, did not show signs of heart failure or impairment of ventricular relaxation and only negligible fibrosis after Ang II infusion. The absence of fibrosis may be a clue to the absence of impairment in ventricular relaxation and account for the normal left ventricular systolic and diastolic performances in Agtr2-/Y mice. CONCLUSIONS: Chronic loss of AT(2) by gene targeting abolished left ventricular hypertrophy and cardiac fibrosis in mice with Ang II-induced hypertension.

Calcitonin receptor gene polymorphism in japanese women: correlation with body mass and bone mineral density.

We have identified a polymorphism at position 1377 of the calcitonin receptor (CTR) gene which generates CC, CT, or TT genotype. In this study, the genotypes of the CTR and their relationship with the body height, the body weight, the bone mineral density (BMD), and osteocalcin levels were examined in 152 healthy Japanese women aged 16-43 years. The CTR genotypic frequencies in the Japanese population were 77.0% for CC, 20.4% for CT, and 2.6% for TT. The height, BMD, and osteocalcin levels were not significantly different among these three genotypes. The body weight adjusted for height in these three groups was significantly different in the order of TT, CT, and CC (P = 0.0454 by analysis of covariance). In combined analysis of the VDR genotype (B,b) and the CTR genotype (C,T), the body height was found to be significantly different between CCB and others (P = 0.0236). In addition, analysis of the CTR genotypic frequency using 64 blood samples from Japanese and 47 blood samples from Caucasians indicated that there was a significant difference between the two races (P < 0.0001). We found that C allele was predominant in the Japanese population, however, Caucasians have an almost equal ratio of the C and T. In conclusion, the CTR allele is one of the genetic factors regulating body weight in Japanese women.

Changes of AT(2) receptor levels in the rat adrenal cortex and medulla induced by bilateral nephrectomy and its modulation by circulating ANG II.

We studied regulation of the AT(2) receptor by investigating the effect of bilateral nephrectomy (bNX) in Sprague-Dawley rats. The expression of aldosterone synthase (CYP11B2) and AT(2) receptor mRNA was detected by nonradioactive in situ hybridization. AT(2) receptor mRNA was detected in cells of the first two or three subcapsular cell layers of the zona glomerulosa (ZG) and in the medulla of sham-operated animals. After bNX, the number and area of distribution of AT(2) receptor-positive cells increased in the ZG. This was associated with an enlargement of the steroidogenic active ZG and with reduced proliferation rate (sham 5.9 +/- 0.9%; bNX 2.4 +/- 0.2%; p<0.02). Infusion of angiotensin II (ANG II; 200 ng/kg/min SC for 56 hr) to bNX rats did not reverse the effect of nephrectomy on the distribution of AT(2) receptor expression, although mRNA levels per cell were reduced compared to NX alone. ANG II infusion decreased proliferation rate further (0.4 +/- 0.07%; p<0.001). In the adrenal medulla after bNX, decreased expression of the AT(2) receptor was associated with increased proliferation (2.6 +/- 0.2% vs 6.6 +/- 0.5%). These results demonstrate differential regulation of the AT(2) receptor in the adrenal gland and suggest that expression of the AT(2) receptor is involved in regulating proliferation and differentiation in the ZG and medulla. (J Histochem Cytochem 49:649-656, 2001)

3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors block calcium-dependent tyrosine kinase Pyk2 activation by angiotensin II in vascular endothelial cells. involvement of geranylgeranylation of small G protein Rap1.

We recently reported the calcium-dependent activation of tyrosine kinase Pyk2 by angiotensin II (Ang II) in pulmonary vein endothelial cells (PVEC). Since Pyk2 has no calcium binding domain, and neither Ca(2+) nor Ca(2+)/calmodulin directly activates Pyk2, it is not clear how Ca(2+) transduces the signal to activate Pyk2, a key tyrosine kinase, in the early events of Ang II signaling. In the present study, we investigated the mechanism of the calcium-dependent activation of Pyk2 in response to Ang II by using 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors and isoprenoid intermediates in PVEC. We have obtained substantial evidence indicating that Ang II activates Pyk2 through calcium-mediated activation of the geranylgeranylated small G protein Rap1 and the Rap1 association with Pyk2. Thus, the small G protein Rap1 is an intermediary signaling molecule linking Ang II-induced calcium signal to Pyk2 activation in PVEC. In addition, our results indicate that 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, a class of cholesterol-lowering drugs, could interrupt Ang II signaling independent of cholesterol lowering in endothelial cells.

Localization and characterization of a short isoform of the corticotropin-releasing factor receptor type 2alpha (CRF(2)alpha-tr) in the rat brain.

We have recently isolated a cDNA encoding a short isoform of the corticotropin-releasing factor (CRF) receptor subtype, referred to as CRF(2)alpha-tr, from the rat amygdala. The present study determined the localization of the truncated receptor mRNA in the rat brain by in situ hybridization histochemistry. The results showed significant levels of hybridization in the lateral septum, central nucleus of the amygdala, cortico-amygdaloid nucleus, ventromedial nucleus of the hypothalamus (VMH), and frontal cortex. In the physiological study, antidepressive drugs increased the expression of CRF(2)alpha-tr mRNA and the total binding activity to CRF in the rat amygdala. These findings suggest that CRF(2)alpha-tr may regulate endogeneous CRF release in the amygdala.

N-acetylcysteine inhibits angiotensin ii-mediated activation of extracellular signal-regulated kinase and epidermal growth factor receptor.

Angiotensin II (Ang II) is known to stimulate reactive oxygen species (ROS) generation and epidermal growth factor (EGF) receptor transactivation to mediate growth-promoting signals such as extracellular signal-regulated kinase (ERK) in vascular smooth muscle cells (VSMCs). However, how ROS and EGF receptor interact to orchestrate these signals in VSMCs remains unclear. Here we found that an antioxidant, N-acetylcysteine, inhibited ERK activation and EGF receptor tyrosine phosphorylation induced by Ang II. Moreover, H(2)O(2) stimulates EGF receptor tyrosine phosphorylation and EGF receptor inhibitors attenuated H(2)O(2)-induced ERK activation. These data indicate that ROS mediate Ang II-induced EGF receptor transactivation, a critical mechanism for ERK-dependent growth in VSMCs.

Increased AT(1) receptor expression and mRNA in kidney glomeruli of AT(2) receptor gene-disrupted mice.

The proposed feedback between angiotensin II AT(2) and AT(1) receptors prompted us to study AT(1) receptor expression in kidneys of male AT(2) receptor-gene disrupted mice (agtr2 -/y). In wild-type (agtr2 +/y) mice, AT(1) receptor binding and mRNA is abundant in glomeruli, and AT(1) receptor binding is also high in the inner stripe of the outer medulla. AT(2) receptors are scarce, primarily associated to cortical vascular structures. In agtr2 -/y mice, AT(1) receptor binding and mRNA were increased in the kidney glomeruli, and AT(1) receptor binding was higher in the rest of the cortex and outer stripe of the outer medulla, but not in its inner stripe, indicating different cellular regulation. Although AT(2) receptor expression is very low in male agtr 2 +/y mice, their gene disruption alters AT(1) receptor expression. AT(1) upregulation alone may explain the AT(2) gene-disrupted mice phenotype such as increased blood pressure, higher sensitivity to angiotensin II, and altered renal function. The indirect AT(1)/AT(2) receptor feedback could have clinical significance because AT(1) antagonists are widely used in medical practice.

Activation of MAPKs by angiotensin II in vascular smooth muscle cells. Metalloprotease-dependent EGF receptor activation is required for activation of ERK and p38 MAPK but not for JNK.

In cultured vascular smooth muscle cells (VSMC), the vasculotrophic factor, angiotensin II (AngII) activates three major MAPKs via the G(q)-coupled AT1 receptor. Extracellular signal-regulated kinase (ERK) activation by AngII requires Ca(2+)-dependent "transactivation" of the EGF receptor that may involve a metalloprotease to stimulate processing of an EGF receptor ligand from its precursor. Whether EGF receptor transactivation also contributes to activation of other members of MAPKs such as p38MAPK and c-Jun N-terminal kinase (JNK) by AngII remains unclear. In the present study, we have examined the effects of a synthetic metalloprotease inhibitor BB2116, and the EGF receptor kinase inhibitor AG1478 on AngII-induced activation of MAPKs in cultured VSMC. BB2116 markedly inhibited ERK activation induced by AngII or the Ca(2+) ionophore without affecting the activation by EGF or PDGF. BB2116 as well as HB-EGF neutralizing antibody inhibited the EGF receptor transactivation by AngII, suggesting a critical role of HB-EGF in the metalloprotease-dependent EGF receptor transactivation. In addition to the ERK activation, activation of p38MAPK and JNK by AngII was inhibited by an AT1 receptor antagonist, RNH6270. and EGF markedly activate p38MAPK, whereas but not EGF markedly activates JNK, indicating the possible contribution of the EGF receptor transactivation to the p38MAPK activation. The findings that both BB2116 and AG1478 specifically inhibited activation of p38MAPK but not JNK by AngII support this hypothesis. From these data, we conclude that ERK and p38MAPK activation by AngII requires the metalloprotease-dependent EGF receptor transactivation, whereas the JNK activation is regulated without involvement of EGF receptor transactivation.

Pain threshold, learning and formation of brain edema in mice lacking the angiotensin II type 2 receptor.

The main biological role of angiotensin II type 2 receptor (AT2) has not been established. We made use of targeted disruption of the mouse AT2 gene to examine the functional role of the AT2 receptor in the central nervous system (CNS). We have previously shown that AT2-deficient mice displayed anxiety-like behavior in comparisons with wild-type mice. In the present study, we analyzed the pain threshold, learning behavior and brain edema formation using the tail-flick test, the tail-pinch test, the passive avoidance task and cold injury, respectively. In the passive avoidance task and cold injury, no differences were found between wild-type mice and AT2-deficient mice. In contrast, the pain threshold was significantly lower in AT2-deficient mice, compared with findings in wild-type mice. The immunohistochemical distribution of beta-endorphin in the brain was analyzed quantitatively in AT2-deficient mice and wild-type mice, using microphotometry. The fluorescence intensity of beta-endorphin in the arcuate nucleus of the medial basal hypothalamus (ARC) was significantly lower in AT2-deficient mice, compared with findings in wild-type mice. We found that the AT2 receptor does not influence learning behavior and brain edema formation. As AT2-deficient mice have increased sensitivity to pain and decreased levels of brain beta-endorphin, AT2 receptors may perhaps mediate regulation of the pain threshold.

Salutary role for angiotensin in partial urinary tract obstruction.

BACKGROUND: In the neonatal period, angiotensin II (Ang II) is up-regulated and induces a timely development of the renal pelvis and ureteral peristalsis, thereby protecting the kidney from hydronephrosis. We tested the possibility that in adulthood, Ang II may act salutarily on the kidney structure during partial urinary tract obstruction by inducing adaptive changes in the peristaltic machinery. METHODS: Adult male Sprague-Dawley rats were subjected to partial unilateral ureteral obstruction (UUO) and divided into two groups, that is, those treated with (group L, N = 21) and those without (group C, N = 21) an angiotensin type 1 (AT1) receptor antagonist (losartan). Control animals were sham operated (N = 10). Rats were sacrificed either at day 7 or day 14. RESULTS: The degree of hydronephrosis determined morphometrically was significantly more severe in group L than group C at both day 7 and day 14, indicating that Ang II inhibition accentuated hydronephrosis. The measurement of upstream pressure within the partially ligated ureter in vivo revealed that losartan significantly attenuates the frequency of ureteral peristaltic activities. In in vitro studies using ureteral strips harvested from normal adult Sprague-Dawley rats (N = 10), Ang II (10(-8) mol/L) was shown to augment contraction, which was completely inhibited by losartan (10(-6) mol/L). CONCLUSIONS: Ang II has a salutary effect of protecting kidneys from hydronephrosis during partial ureteral obstruction through its ability to augment ureteral peristalsis.

Increased gene expression of components of the renin-angiotensin system in glomeruli of genetically hypertensive rats.

OBJECTIVE: The renin-angiotensin system (RAS) is implicated in the development of hypertensive glomerulosclerosis. However, no experimental evidence exists that clearly demonstrates activation of glomerular RAS in hypertensive nephropathy. We used stroke-prone spontaneously hypertensive rats (SHRSP) to examine whether RAS components are increased in glomeruli of SHRSP and whether this increase leads to an increase in mRNA levels for transforming growth factor-beta1 (TGF-beta1). METHODS: We examined the sequential changes of urinary albumin excretion (UAE), morphology, and glomerular mRNA expression for TGF-beta1 and fibronectin (FN) in relation to glomerular mRNA expression for angiotensinogen (ATN), angiotensin converting enzyme (ACE), angiotensin II type 1a (AT1a), and type 1b (AT1b) receptors, and intervention with angiotensin II type 1 receptor antagonist candesartan and equihypotensive hydralazine. RESULTS: In SHRSP, UAE was normal at 9 weeks of age, but became higher, beginning at 12 weeks of age, than that in the age-matched Wistar-Kyoto (WKY) rats, while SHRSP showed no glomerulosclerosis until 14 weeks of age; it was marked at 24 weeks. Plasma renin activity and plasma angiotensin II level was equivalent in the 9- and 12-week-old SHRSP and the WKY rats; both parameters, however, were elevated in 24-week-old SHRSP as compared with age-matched control. RNase protection assays showed that glomerular levels of ATN, ACE, and AT1a and AT1b receptors mRNA were significantly increased in 9-, 12-, and 14-week-old, but not in 24-week-old SHRSP, compared with age-matched WKY rats. Northern blot analysis showed that glomerular levels of TGF-beta1 and FN mRNA were higher in SHRSP than in WKY rats at all time points. Candesartan reduced UAE to control levels, whereas hydralazine reduced UAE but not to control levels. Candesartan administration for 12 weeks virtually prevented the progression of glomerulosclerosis. While candesartan reduced mRNA levels for RAS components, TGF-beta1, and FN to control levels, hydralazine was not effective in this respect. Conclusion Results suggest that increases in glomerular RAS components that occur independently of circulating RAS alter glomerular permselectivity and increase the glomerular expression of TGF-beta1 and FN in young SHRSP. Findings in old SHRSP suggest that altered glomerular permselectivity and an increased glomerular expression of TGF-beta1 and FN may be associated with the activation of systemic RAS.