Evidence for a Physiological Mitochondrial Angiotensin II System in the Kidney Proximal Tubules: Novel Roles of Mitochondrial Ang II/AT1a/O2- and Ang II/AT2/NO Signaling.

The present study tested the hypotheses that overexpression of an intracellular Ang II (angiotensin II) fusion protein, mito-ECFP/Ang II, selectively in the mitochondria of mouse proximal tubule cells induces mitochondrial oxidative and glycolytic responses and elevates blood pressure via the Ang II/AT1a receptor/superoxide/NHE3 (the Na+/H+ exchanger 3)-dependent mechanisms. A PT-selective, mitochondria-targeting adenoviral construct encoding Ad-sglt2-mito-ECFP/Ang II was used to test the hypotheses. The expression of mito-ECFP/Ang II was colocalized primarily with Mito-Tracker Red FM in mouse PT cells or with TMRM in kidney PTs. Mito-ECFP/Ang II markedly increased oxygen consumption rate as an index of mitochondrial oxidative response (69.5%; P<0.01) and extracellular acidification rate as an index of mitochondrial glycolytic response (34%; P<0.01). The mito-ECFP/Ang II-induced oxygen consumption rate and extracellular acidification rate responses were blocked by AT1 blocker losartan (P<0.01) and a mitochondria-targeting superoxide scavenger mito-TEMPO (P<0.01). By contrast, the nonselective NO inhibitor L-NAME alone increased, whereas the mitochondria-targeting expression of AT2 receptors (mito-AT2/GFP) attenuated the effects of mito-ECFP/Ang II (P<0.01). In the kidney, overexpression of mito-ECFP/Ang II in the mitochondria of the PTs increased systolic blood pressure 12±3 mm Hg (P<0.01), and the response was attenuated in PT-specific PT-Agtr1a-/- and PT-Nhe3-/- mice (P<0.01). Conversely, overexpression of AT2 receptors selectively in the mitochondria of the PTs induced natriuretic responses in PT-Agtr1a-/- and PT-Nhe3-/- mice (P<0.01). Taken together, these results provide new evidence for a physiological role of PT mitochondrial Ang II/AT1a/superoxide/NHE3 and Ang II/AT2/NO/NHE3 signaling pathways in maintaining blood pressure homeostasis.

Recent Research Advances in Renin-Angiotensin-Aldosterone System Receptors.

PURPOSE OF REVIEW: The renin-angiotensin-aldosterone system (RAAS) plays important roles in regulating blood pressure and body fluid, which contributes to the pathophysiology of hypertension and cardiovascular/renal diseases. However, accumulating evidence has further revealed the complexity of this signal transduction system, including direct interactions with other receptors and proteins. This review focuses on recent research advances in RAAS with an emphasis on its receptors. RECENT FINDINGS: Both systemically and locally produced angiotensin II (Ang II) bind to Ang II type 1 receptor (AT1R) and elicit strong biological functions. Recent studies have shown that Ang II-induced activation of Ang II type 2 receptor (AT2R) elicits the opposite functions to those of AT1R. However, accumulating evidence has now expanded the components of RAAS, including (pro)renin receptor, angiotensin-converting enzyme 2, angiotensin 1-7, and Mas receptor. In addition, the signal transductions of AT1R and AT2R are regulated by not only Ang II but also its receptor-associated proteins such as AT1R-associated protein and AT2R-interacting protein. Recent studies have indicated that inappropriate activation of local mineralocorticoid receptor contributes to cardiovascular and renal tissue injuries through aldosterone-dependent and -independent mechanisms. Since the mechanisms of RAAS signal transduction still remain to be elucidated, further investigations are necessary to explore novel molecular mechanisms of the RAAS, which will provide alternative therapeutic agents other than existing RAAS blockers.

Reversal of Aortic Enlargement Induced by Increased Biomechanical Forces Requires AT1R Inhibition in Conjunction With AT2R Activation.

Objective- Pharmacological inhibition of the AT1R (angiotensin II type 1 receptor) with losartan can attenuate ascending aortic remodeling induced by transverse aortic constriction (TAC). In this study, we investigated the role of the AT2R (angiotensin II type 2 receptor) and MasR (Mas receptor) in TAC-induced ascending aortic dilation and remodeling. Approach and Results- Wild-type C57BL/6J mice were subjected to sham or TAC surgeries in the presence and absence of various drugs. Aortic diameters were assessed by echocardiography, central blood pressure was measured in the ascending aorta 2 weeks post-operation, and histology and gene expression analyses completed. An angiotensin-converting enzyme inhibitor, captopril, decreased systolic blood pressure to the same level as losartan but did not attenuate aortic dilation, adventitial inflammation, medial collagen deposition, elastin breakage, or Mmp9 (matrix metalloproteinase-9) expression when compared with TAC mice. In contrast, co-administration of captopril with an AT2R agonist, compound 21, attenuated aortic dilation, medial collagen content, elastin breaks, and Mmp9 expression, whereas co-administration of captopril with a MasR agonist (AVE0991) did not reverse aortic dilation and led to aberrant aortic remodeling. An AT2R antagonist, PD123319, reversed the protective effects of losartan in TAC mice. Treatment with compound 21 alone showed no effect on TAC-induced aortic enlargement, blood pressure, elastin breakage, or Mmp9 expression. Conclusions- Our data indicate that when AT1R signaling is blocked, AT2R activation is a key modulator to prevent aortic dilation that occurs with TAC. These data suggest that angiotensin-converting enzyme inhibitor may not be as effective as losartan for slowing aneurysm growth because losartan requires intact AT2R signaling to prevent aortic enlargement.

The angiotensin II type 2 receptors protect renal tubule mitochondria in early stages of diabetes mellitus.

Diabetic nephropathy correlates more closely to defective mitochondria and increased oxidative stress in the kidney than to hyperglycemia. A key driving factor of diabetic nephropathy is angiotensin II acting via the G-protein-coupled cell membrane type 1 receptor. The present study aimed to investigate the role of the angiotensin II type 2 receptor (AT2R) at the early stages of diabetic nephropathy. Using receptor binding studies and immunohistochemistry we found that the mitochondria in renal tubules contain high-affinity AT2Rs. Increased renal mitochondrial AT2R density by transgenic overexpression was associated with reduced superoxide production of isolated mitochondria from non-diabetic rats. Streptozotocin-induced diabetes (28 days) caused a drop in the ATP/oxygen ratio and an increase in the superoxide production of isolated renal mitochondria from wild-type diabetic rats. This correlated with changes in the renal expression profile and increased tubular epithelial cell proliferation. AT2R overexpression in tubular epithelial cells inhibited all diabetes-induced renal changes including a drop in mitochondrial bioenergetics efficiency, a rise in mitochondrial superoxide production, metabolic reprogramming, and increased proliferation. Thus, AT2Rs translocate to mitochondria and can contribute to reno-protective effects at early stages of diabetes. Hence, targeted AT2R overexpression in renal cells may open new avenues to develop novel types of drugs preventing diabetic nephropathy.

Chronic administration of the angiotensin type 2 receptor agonist C21 improves insulin sensitivity in C57BL/6 mice.

The renin-angiotensin system modulates insulin action. Angiotensin type 1 receptor exerts a deleterious effect, whereas the angiotensin type 2 receptor (AT2R) appears to have beneficial effects providing protection against insulin resistance and type 2 diabetes. To further explore the role of the AT2R on insulin action and glucose homeostasis, in this study we administered C57Bl/6 mice with the synthetic agonist of the AT2R C21 for 12 weeks (1 mg/kg per day; ip). Vehicle-treated animals were used as control. Metabolic parameters, glucose, and insulin tolerance, in vivo insulin signaling in main insulin-target tissues as well as adipose tissue levels of adiponectin, and TNF-α were assessed. C21-treated animals displayed decreased glycemia together with unaltered insulinemia, increased insulin sensitivity, and increased glucose tolerance compared to nontreated controls. This was accompanied by a significant decrease in adipocytes size in epididymal adipose tissue and significant increases in both adiponectin and UCP-1 expression in this tissue. C21-treated mice showed an increase in both basal Akt and ERK1/2 phosphorylation levels in the liver, and increased insulin-stimulated Akt activation in adipose tissue. This positive modulation of insulin action induced by C21 appeared not to involve the insulin receptor. In C21-treated mice, adipose tissue and skeletal muscle became unresponsive to insulin in terms of ERK1/2 phosphorylation levels. Present data show that chronic pharmacological activation of AT2R with C21 increases insulin sensitivity in mice and indicate that the AT2R has a physiological role in the conservation of insulin action.

Angiotensin II Triggers Peripheral Macrophage-to-Sensory Neuron Redox Crosstalk to Elicit Pain.

Injury, inflammation, and nerve damage initiate a wide variety of cellular and molecular processes that culminate in hyperexcitation of sensory nerves, which underlies chronic inflammatory and neuropathic pain. Using behavioral readouts of pain hypersensitivity induced by angiotensin II (Ang II) injection into mouse hindpaws, our study shows that activation of the type 2 Ang II receptor (AT2R) and the cell-damage-sensing ion channel TRPA1 are required for peripheral mechanical pain sensitization induced by Ang II in male and female mice. However, we show that AT2R is not expressed in mouse and human dorsal root ganglia (DRG) sensory neurons. Instead, expression/activation of AT2R on peripheral/skin macrophages (MΦs) constitutes a critical trigger of mouse and human DRG sensory neuron excitation. Ang II-induced peripheral mechanical pain hypersensitivity can be attenuated by chemogenetic depletion of peripheral MΦs. Furthermore, AT2R activation in MΦs triggers production of reactive oxygen/nitrogen species, which trans-activate TRPA1 on mouse and human DRG sensory neurons via cysteine modification of the channel. Our study thus identifies a translatable immune cell-to-sensory neuron signaling crosstalk underlying peripheral nociceptor sensitization. This form of cell-to-cell signaling represents a critical peripheral mechanism for chronic pain and thus identifies multiple druggable analgesic targets.SIGNIFICANCE STATEMENT Pain is a widespread health problem that is undermanaged by currently available analgesics. Findings from a recent clinical trial on a type II angiotensin II receptor (AT2R) antagonist showed effective analgesia for neuropathic pain. AT2R antagonists have been shown to reduce neuropathy-, inflammation- and bone cancer-associated pain in rodents. We report that activation of AT2R in macrophages (MΦs) that infiltrate the site of injury, but not in sensory neurons, triggers an intercellular redox communication with sensory neurons via activation of the cell damage/pain-sensing ion channel TRPA1. This MΦ-to-sensory neuron crosstalk results in peripheral pain sensitization. Our findings provide an evidence-based mechanism underlying the analgesic action of AT2R antagonists, which could accelerate the development of efficacious non-opioid analgesic drugs for multiple pain conditions.

Dimerization of AT2 and Mas Receptors in Control of Blood Pressure.

PURPOSE OF REVIEW: Angiotensin type 2 receptor (AT2R) and receptor Mas (MasR) are part of the "protective arm" of the renin angiotensin system. Gene and pharmacological manipulation studies reveal that AT2R and MasR are involved in natriuretic, vasodilatory, and anti-inflammatory responses and in lowering blood pressure in various animal models under normal and pathological conditions such as salt-sensitive hypertension, obesity, and diabetes. The scope of this review is to discuss co-localization and heterodimerization as potential molecular mechanisms of AT2R- and MasR-mediated functions including antihypertensive activities. RECENT FINDINGS: Accumulating evidences show that AT2R and MasR are co-localized, make a heterodimer, and are functionally interdependent in producing their physiological responses. Moreover, ang-(1-7) preferably may be an AT1R-biased agonist while acting as a MasR agonist. The physical interactions of AT2R and MasR appear to be an important mechanism by which these receptors are involved in blood pressure regulation and antihypertensive activity. Whether heteromers of these receptors influence affinity or efficacy of endogenous or synthetic agonists remains a question to be considered.

Immunologic Effects of the Renin-Angiotensin System.

Inappropriate activation of the renin-angiotensin system (RAS) exacerbates renal and vascular injury. Accordingly, treatment with global RAS antagonists attenuates cardiovascular risk and slows the progression of proteinuric kidney disease. By reducing BP, RAS inhibitors limit secondary immune activation responding to hemodynamic injury in the target organ. However, RAS activation in hematopoietic cells has immunologic effects that diverge from those of RAS stimulation in the kidney and vasculature. In preclinical studies, activating type 1 angiotensin (AT1) receptors in T lymphocytes and myeloid cells blunts the polarization of these cells toward proinflammatory phenotypes, protecting the kidney from hypertensive injury and fibrosis. These endogenous functions of immune AT1 receptors temper the pathogenic actions of renal and vascular AT1 receptors during hypertension. By counteracting the effects of AT1 receptor stimulation in the target organ, exogenous administration of AT2 receptor agonists or angiotensin 1-7 analogs may similarly limit inflammatory injury to the heart and kidney. Moreover, although angiotensin II is the classic effector molecule of the RAS, several RAS enzymes affect immune homeostasis independently of canonic angiotensin II generation. Thus, as reviewed here, multiple components of the RAS signaling cascade influence inflammatory cell phenotype and function with unpredictable and context-specific effects on innate and adaptive immunity.

Novel players in cardioprotection: Insulin like growth factor-1, angiotensin-(1-7) and angiotensin-(1-9).

Insulin-like growth factor-1, angiotensin-(1-7) and angiotensin-(1-9) have been proposed to be important mediators in cardioprotection. A large body of evidence indicates that insulin like growth factor-1 has pleotropic actions in the heart (i.e., contractility, metabolism, hypertrophy, autophagy, senescence and cell death) and, conversely, its deficiency is associated with impaired cardiac function. Recently, we reported that insulin like growth factor-1 receptor is also located in plasma membrane invaginations with perinuclear localization, highlighting the role of nuclear Ca(2+) signaling in the heart. In parallel, angiotensin-(1-7) and angiotensin (1-9) acting through Mas receptor and angiotensin type 2 receptor have emerged as a novel anti-hypertensive molecules promoting vasodilatation and preventing heart hypertrophy. In this review we discuss the scientific evidence available regarding insulin-like growth factor-1, angiotensin-(1-7) and angiotensin-(1-9) in cardioprotection and its potential application as novel therapeutic targets for treating cardiac diseases.

Angiotensin II type 2 receptor- and acetylcholine-mediated relaxation: essential contribution of female sex hormones and chromosomes.

Angiotensin-induced vasodilation, involving type 2 receptor (AT2R)-induced generation of nitric oxide (NO; by endothelial NO synthase) and endothelium-derived hyperpolarizing factors, may be limited to women. To distinguish the contribution of female sex hormones and chromosomes to AT2R function and endothelium-derived hyperpolarizing factor-mediated vasodilation, we made use of the four-core genotype model, where the testis-determining Sry gene has been deleted (Y(-)) from the Y chromosome, allowing XY(-) mice to develop a female gonadal phenotype. Simultaneously, by incorporating the Sry gene onto an autosome, XY(-)Sry and XXSry transgenic mice develop into gonadal male mice. Four-core genotype mice underwent a sham or gonadectomy (GDX) operation, and after 8 weeks, iliac arteries were collected to assess vascular function. XY(-)Sry male mice responded more strongly to angiotensin than XX female mice, and the AT2R antagonist PD123319 revealed that this was because of a dilator AT2R-mediated effect occurring exclusively in XX female mice. The latter could not be demonstrated in XXSry male and XY(-) female mice nor in XX female mice after GDX, suggesting that it depends on both sex hormones and chromosomes. Indeed, treating C57bl/6 GDX male mice with estrogen could not restore angiotensin-mediated, AT2R-dependent relaxation. To block acetylcholine-induced relaxation of iliac arteries obtained from four-core genotype XX mice, both endothelial NO synthase and endothelium-derived hyperpolarizing factor inhibition were required, whereas in four-core genotype XY animals, endothelial NO synthase inhibition alone was sufficient. These findings were independent of gonadal sex and unaltered after GDX. In conclusion, AT2R-induced relaxation requires both estrogen and the XX chromosome sex complement, whereas only the latter is required for endothelium-derived hyperpolarizing factors.

Sex- and age-related differences in the chronic pressure-natriuresis relationship: role of the angiotensin type 2 receptor.

Sex hormones regulate the renin-angiotensin system. For example, estrogen enhances expression of the angiotensin type 2 receptor. We hypothesized that activation of the angiotensin type 2 receptor shifts the chronic pressure-natriuresis relationship leftward in females compared with males and that this effect is lost with age. Mean arterial pressure was measured by radiotelemetry in adult (4 mo old) and aged (14 mo old) wild-type and angiotensin type 2 receptor knockout male and female mice. Chronic pressure-natriuresis curves were constructed while mice were maintained on a normal-salt (0.26%) diet and following 6 days of high salt (5.0%) diet. Mean arterial pressure was lower in adult wild-type females than males (88 ± 1 and 97 ± 1 mmHg, respectively), a difference that was maintained with age, but was absent in adult knockout mice. In wild-type females, the chronic pressure-natriuresis relationship was shifted leftward compared with knockout females, an effect that was lost with age. In males, the chronic pressure-natriuresis relationship was not influenced by angiotensin type 2 receptor deficiency. Compared with age-matched females, the chronic pressure-natriuresis relationships in male mice were shifted rightward. Renal expression of the angiotensin type 2 receptor was fourfold greater in adult wild-type females than males. With age, the angiotensin type 2 receptor-to-angiotensin type 1 receptor balance was reduced in females. Conversely, in males, angiotensin receptor expression did not vary significantly with age. In conclusion, the angiotensin type 2 receptor modulates the chronic pressure-natriuresis relationship in an age- and sex-dependent manner.

ANG-(3-4) inhibits renal Na+-ATPase in hypertensive rats through a mechanism that involves dissociation of ANG II receptors, heterodimers, and PKA.

The physiological roles of ANG-(3-4) (Val-Tyr), a potent ANG II-derived peptide, remain largely unknown. The present study 1)investigates whether ANG-(3-4) modulates ouabain-resistant Na(+)-ATPase resident in proximal tubule cells and 2) verifies whether its possible action on pumping activity, considered the fine tuner of Na(+) reabsorption in this nephron segment, depends on blood pressure. ANG-(3-4) inhibited Na(+)-ATPase activity in membranes of spontaneously hypertensive rats (SHR) at nanomolar concentrations, with no effect in Wistar-Kyoto (WKY) rats or on Na(+)-K(+)-ATPase. PD123319 (10(-7) M) and PKA(5-24) (10(-6) M), an AT2 receptor (AT2R) antagonist and a specific PKA inhibitor, respectively, abrogated this inhibition, indicating that AT2R and PKA are central in this pathway. Despite the lack of effect of ANG-(3-4) when assayed alone in WKY rats, the peptide (10(-8) M) completely blocked stimulation of Na(+)-ATPase induced by 10(-10) M ANG II in normotensive rats through a mechanism that also involves AT2R and PKA. Tubular membranes from WKY rats had higher levels of AT2R/AT1R heterodimers, which remain associated in 10(-10) M ANG II and dissociate to a very low dimerization state upon addition of 10(-8) M ANG-(3-4). This lower level of heterodimers was that found in SHR, and heterodimers did not dissociate when the same concentration of ANG-(3-4) was present. Oral administration of ANG-(3-4) (50 mg/kg body mass) increased urinary Na(+) concentration and urinary Na(+) excretion with a simultaneous decrease in systolic arterial pressure in SHR, but not in WKY rats. Thus the influence of ANG-(3-4) on Na(+) transport and its hypotensive action depend on receptor association and on blood pressure.

Analgesic efficacy and mode of action of a selective small molecule angiotensin II type 2 receptor antagonist in a rat model of prostate cancer-induced bone pain.

OBJECTIVE: The pathobiology of prostate cancer (PCa)-induced bone pain (PCIBP) has both inflammatory and neuropathic components. Previously, we showed that small molecule angiotensin II type 2 receptor (AT2 R) antagonists with >1,000-fold selectivity over the angiotensin II type 1 receptor produced dose-dependent analgesia in a rat model of neuropathic pain. Here, we assessed the analgesic efficacy and mode of action of the AT2 R antagonist, EMA200, in a rat model of PCIBP. METHODS: At 14-21 days after unilateral intratibial injection of AT3B PCa cells, rats exhibiting hindpaw hypersensitivity received single intravenous bolus doses of EMA200 (0.3-10 mg/kg) or vehicle, and analgesic efficacy was assessed. The mode of action was investigated using immunohistochemical, Western blot, and/or molecular biological methods in lumbar dorsal root ganglia (DRGs) removed from drug-naïve and EMA200-treated PCIBP rats relative to sham-control rats. RESULTS: Intravenous bolus doses of EMA200 produced dose-dependent analgesia in PCIBP rats. Lumbar DRG levels of angiotensin II, nerve growth factor (NGF), tyrosine kinase A (TrkA), phospho-p38 mitogen-activated protein kinase (MAPK), and phospho-p44/p42 MAPK, but not the AT2 R, were increased significantly (P < 0.05) in PCIBP rats, c.f. the corresponding levels for sham controls. EMA200 produced analgesia in PCIBP rats by reducing elevated angiotensin II levels in the lumbar DRGs to attenuate augmented angiotensin II/AT2 R signaling. This in turn reduced augmented NGF/TrkA signaling in the lumbar DRGs. The net result was inhibition of p38 MAPK and p44/p42 MAPK activation. CONCLUSION: Small molecule AT2 R antagonists are worthy of further investigation as novel analgesics for relief of intractable PCIBP and other pain types where hyperalgesia worsens symptoms.

Characterization of ion currents of murine CD117(pos) stem cells in vitro and their modulation under AT2 R stimulation.

AIM: Hematopoietic stem cells, especially CD117(pos) cells, have been found to possess a regenerative potential in various tissues, in particular cardiac muscle. However, the characterization of the relevant ion currents of stem cells prior to implantation lacks documentation. Activation of angiotensin II type 2 receptor (AT2 R) can lead to further cell differentiation and receptor auto-expression and might thus influence electrophysiological properties of CD117(pos) stem cells. This study was designed to functionally characterize membrane currents of CD117(pos) cells under normal and AT2 R-stimulated conditions. METHODS: CD117(pos) murine bone marrow stem cells were isolated with MACS technique and stimulated for the AT2 R with angiotensin II and losartan for 3-5 days prior to patch-clamp measurements. RT-PCR was used to determine channel expression. Endothelial properties were analysed with immunocytochemistry and acLDL uptake assay. RESULTS: A well-expressed inward rectifying current (IKir ) was identified in cultured CD117(pos) cells. Furthermore, a ZD 7288 (HCN channel blocker)-sensitive current component was isolated. Voltage-dependent potassium currents and chloride currents were less expressed. A small fraction of cells demonstrated voltage- and time-dependent inward currents. In AT2 R-stimulated cells inward rectifying the hyperpolarization-induced inward currents were slightly attenuated on the translational level but showed increased mRNA expression. Cultured CD117(pos) cells express CD31 and VEGFR-2 and significantly increased the uptake of acLDL. CONCLUSIONS: CD117(pos) cells do not have properties of action potential-generating cells and moderately change their excitability during AT2 R stimulation. Electrophysiological and molecular properties of control and AT2 R-stimulated cells point to a differentiation to vascular endothelial cells. This could increase beneficial vascularization in injured tissues.

Effect of angiotensin II type 2 receptor on stroke, cognitive impairment and neurodegenerative diseases.

Here, we briefly review the role of the renin-angiotensin system (RAS) in cognitive impairment and neurodegenerative disease, mainly discussing our experimental studies on the angiotensin II type 2 (AT(2)) receptor. Ischemic brain damage is enhanced in mice with overexpression of angiotensin II, with reduced cerebral blood flow in the penumbra and an increase in oxidative stress in the ischemic area. Angiotensin II binds two types of receptors, type 1 (AT(1)) and type 2 (AT(2)). Our previous experiments showed that AT(1) receptor signaling has a harmful effect, and AT(2) receptor signaling has a protective effect on the brain after stroke. AT(2) receptor signaling in bone marrow stromal cells or hematopoietic cells was shown to prevent ischemic brain damage after middle cerebral artery occlusion. In contrast, AT(2) receptor signaling also affects cognitive function. We showed that direct stimulation of the AT(2) receptor by a newly generated direct AT(2) receptor agonist, Compound 21 (C21), enhanced cognitive function in wild-type (C57BL6) mice and an Alzheimer's disease mouse model with intracerebroventricular injection of amyloid ß (1-40). Finally, we carried out clinical research by investigating the levels of RAS components in patients with neurodegenerative diseases. We observed a reduction of angiotensin II and angiotensin converting enzyme (ACE) 2 levels, and an increase in ACE level in cerebrospinal fluid from patients with multiple sclerosis. These results suggest that RAS is also involved in neurodegenerative disease. Therefore, regulation of RAS might be a new therapeutic target to protect neurons from neural diseases.

Angiotensin AT2 receptor activates the cyclic-AMP signaling pathway in eel.

A unique angiotensin type 2 receptor (AT2) that induces a cAMP signaling pathway was cloned and characterized for the first time in fish, Anguilla japonica. Phylogeny and synteny results showed that the AT2s among fishes and tetrapods share the same origin despite a sub-cluster formation among eel, salmon, and zebrafish. The eel AT2 was expressed abundantly in the spleen and localized at straight arterioles and ellipsoid regions prior to the sinusoid, suggesting a role in the regulation of microcirculation and/or immune response. Various angiotensin (Ang) peptides, including Ang II, Ang III, and Ang IV, were detected in the spleen by a radioimmunoassay coupled with HPLC separation, and these endogenous peptides stimulated a cAMP signaling, which has no crosstalk with cGMP pathway. The common and contrasting features of AT2 between fishes and mammals imply some ancestral characters of AT2, which are important information for receptor binding and evolutionary studies.

Advanced glycation end-products activate the renin-angiotensin system through the RAGE/PI3-K signaling pathway in podocytes.

PURPOSE: The purpose of this study was to investigate the effects of advanced glycation end-products (AGEs) on the components of the renin-angiotensin system (RAS) in podocytes and to understand the mechanism of these effects. METHODS: Immortalized mouse podocytes were exposed to various concentrations of AGEs for different time intervals. The expression levels of angiotensinogen (AGT), angiotensin II type 1 and 2 receptors (AT1R and AT2R) and renin were examined by real-time PCR and western blot; the receptor for AGEs (RAGE) and both Akt and phosphorylated Akt were examined by western blot; levels of angiotensin II (Ang II) were assayed by ELISA, and the activity of angiotensin-converting enzyme (ACE) was evaluated by measuring the production of hippuric acid in vitro. RESULTS: Treatment with AGEs resulted in significant increases in the expression of AGT (62%, P=0.002) and AT1R (59%, P=0.01). Moreover, Ang II levels increased significantly in both cell lysates (70%, P=0.018) and conditioned media (65%, P=0.01). ACE activity was also significantly higher in cell lysates (68% , P= 0.035) and conditioned media (65%, P=0.023). There were no changes in renin or AT2R expression (P > 0.05). AGEs did increase the expression of RAGE by 50% (P=0.012) and the phosphorylation of Akt by 100% (P=0.001). When podocytes were pretreated with anti-RAGE antibody (50 µg/ml) or the phosphoinositide 3-kinase (PI3-K) inhibitor, LY294002 (10 µM), the AGEs-induced increases in AGT and AT1R expression were reduced. Likewise, Ang II levels and ACE activity decreased significantly. CONCLUSION: AGEs activate the RAS in podocytes through the RAGE-PI3-K/Akt-dependent pathway and lead to an increase in podocyte apoptosis.

Dopamine and angiotensin type 2 receptors cooperatively inhibit sodium transport in human renal proximal tubule cells.

Little is known regarding how the kidney shifts from a sodium and water reclaiming state (antinatriuresis) to a state where sodium and water are eliminated (natriuresis). In human renal proximal tubule cells, sodium reabsorption is decreased by the dopamine D(1)-like receptors (D(1)R/D(5)R) and the angiotensin type 2 receptor (AT(2)R), whereas the angiotensin type 1 receptor increases sodium reabsorption. Aberrant control of these opposing systems is thought to lead to sodium retention and, subsequently, hypertension. We show that D(1)R/D(5)R stimulation increased plasma membrane AT(2)R 4-fold via a D(1)R-mediated, cAMP-coupled, and protein phosphatase 2A-dependent specific signaling pathway. D(1)R/D(5)R stimulation also reduced the ability of angiotensin II to stimulate phospho-extracellular signal-regulated kinase, an effect that was partially reversed by an AT(2)R antagonist. Fenoldopam did not increase AT(2)R recruitment in renal proximal tubule cells with D(1)Rs uncoupled from adenylyl cyclase, suggesting a role of cAMP in mediating these events. D(1)Rs and AT(2)Rs heterodimerized and cooperatively increased cAMP and cGMP production, protein phosphatase 2A activation, sodium-potassium-ATPase internalization, and sodium transport inhibition. These studies shed new light on the regulation of renal sodium transport by the dopaminergic and angiotensin systems and potential new therapeutic targets for selectively treating hypertension.

The involvement of angiotensin type 1 and type 2 receptors in estrogen-induced cell proliferation and vascular endothelial growth factor expression in the rat anterior pituitary.

The aim of our study was to examine the involvement of renin-angiotensin system (RAS) in estrogen-induced lactotropes proliferation and vascular endothelial growth factor (VEGF) expression in rat pituitary. The study was performed on Fisher 344 rats underwent 8-day treatment with diethylstilboestrol (DES). The proliferation index (PCNA) and VEGF expression in pituitary sections were estimated using immunohistochemical methods. Treatment with DES increased the number of PCNA-positive cells, VEGF-positive cells, and VEGF-positive blood vessels in pituitary. Stimulatory effect of estrogen on cell proliferation and VEGF expression in blood vessels was attenuated by losartan, PD123319, and captopril. VEGF immunoreactivity in pituitary cells of DES-treated rats was decreased by AT1 antagonist and not changed by AT2 blocker and ACE inhibitor. Our findings suggest the involvement of RAS in DES-induced cell proliferation and VEGF expression in pituitary. Both the AT1 and AT2 receptors appear to mediate the estrogen-dependent mitogenic and proangiogenic effects in rat pituitary.