Subsets of retinal neurons and glia express P2Y1 receptors

Recent evidence suggests that extracellular ATP modulates retinal processing and could play a role in modulating glial cells during retinal diseases. Here, we evaluated the localization of P2Y₁ receptors in the rat retina using indirect immunofluorescence immunocytochemistry. We observed labeling within defined populations of inner retinal neurons and Müller cell processes and end feet. Double labeling of P2Y₁ receptor with choline acetyltransferase revealed extensive colocalization indicating the expression of this receptor by cholinergic amacrine cells. Ganglion cell labeling for P2Y₁ receptors was also observed. Having established the normal pattern of immunolabeling within the retina, we next examined whether immunolabeling was altered by retinal disease. P2Y₁ receptor immunolabeling of Müller cells was of greater intensity following light-induced retinal degeneration, suggesting that Müller cell gliosis is accompanied by changes in P2Y₁ receptor expression. Overall, these data provide further evidence for a role of extracellular ATP in retinal signaling within subsets of retinal neurons as well as glia.     

AT2 receptors: beneficial counter-regulatory role in cardiovascular and renal function

The renin–angiotensin system (RAS) is a coordinated hormonal cascade intimately involved in cardiovascular and renal control and blood pressure regulation. Angiotensin II (Ang II), the major RAS effector peptide, binds two distinct receptors, the angiotensin type-1 receptor (AT₁R) and the angiotensin type-2 (AT₂R) receptor. The vast majority of the physiological actions of Ang II, almost all of them detrimental, are mediated by AT₁Rs. In contrast, AT₂Rs negatively modulate the actions of AT₁Rs under the majority of circumstances and generally possess beneficial effects. AT₂Rs induce vasodilation in both resistance and capacitance vessels, mediating natriuresis directly and via interactions with dopamine D1 receptors in the renal proximal tubule. AT₂Rs inhibit renin biosynthesis and secretion and protect the kidneys from inflammation and ischemic injury. Our understanding of the exact role of AT₂Rs in physiology and pathophysiology continues to expand; the purpose of this review is to provide an up-to-date summary of the functional role of AT₂Rs at the organ, tissue, cellular, and subcellular levels with emphasis on the vascular and renal actions that bear on blood pressure regulation and hypertension.     

Microinjection of angiotensin II in the caudal ventrolateral medulla induces hyperalgesia

Nociceptive transmission from the spinal cord is controlled by supraspinal pain modulating systems that include the caudal ventrolateral medulla (CVLM). The neuropeptide angiotensin II (Ang II) has multiple effects in the CNS and at the medulla oblongata. Here we evaluated the expression of angiotensin type 1 (AT₁) receptors in spinally-projecting CVLM neurons, and tested the effect of direct application of exogenous Ang II in the CVLM on nociceptive behaviors. Although AT₁-immunoreactive neurons occurred in the CVLM, only 3% of AT₁-positive neurons were found to project to the dorsal horn, using double-immunodetection of the retrograde tracer cholera toxin subunit B. In behavioral studies, administration of Ang II (100 pmol) in the CVLM gave rise to hyperalgesia in both the tail-flick and formalin tests. This hyperalgesia was significantly attenuated by local administration of the AT₁ antagonist losartan. The present study demonstrates that Ang II can act on AT₁ receptors in the CVLM to modulate nociception. The effect on spinal nociceptive processing is likely indirect, since few AT₁-expressing CVLM neurons were found to project to the spinal cord. The renin-angiotensin system may also play a role in other supraspinal areas implicated in pain modulation.     

Nongenomic action of aldosterone on colocalization of angiotensin II type 1 and type 2 receptors in rat kidney

Previous in vitro studies have demonstrated that angiotensin II type 1 and type 2 receptors (AT₁R and AT₂R) are co-localized and can form AT₁R/AT₂R dimerization in rat proximal tubular cells. Aldosterone non-genomically enhances angiotensin II receptor dimerization. We found no other in vivo studies in the literature regarding the effect of aldosterone on colocalization of AT₁R and AT₂R in whole kidney. Male Wistar rats were intraperitoneally injected with either normal saline solution (sham group) or aldosterone (experimental group). Colocalization of renal AT₁R and AT₂R proteins was examined by double immunohistochemical staining. The colocalization of AT₁R and AT₂R proteins was more prominent in the glomerulus, distal convoluted tubules, and cortical collecting ducts while colocalization was weak and diffused in the proximal convoluted tubules and peritubular capillaries in both groups. Our in vivo study showed aldosterone did not alter a constitutive colocalization of AT₁R and AT₂R proteins in the renal cortex and medulla. However, these proteins were colocalized more prominently in the renal cortex.     

Distribution of angiotensin IV binding sites (AT4 receptor) in the human forebrain, midbrain and pons as visualised by in vitro receptor autoradiography

Angiotensin IV and other AT₄ receptor agonists, improve memory retention and retrieval in the passive avoidance and swim maze learning paradigms. Angiotensin IV binding sites (also known as the AT₄ receptors) are widely distributed in guinea pig and monkey (Macaca fascicularis) brains where high densities of the binding sites have been detected in the hippocampus, neocortex and motor nuclei. However, the distribution of the binding sites in the human brain is not known. We have recently localised the angiotensin IV binding sites (AT₄ receptors) in post-mortem human brain using iodinated Nle-angiotensin IV, a higher affinity and more stable analogue of angiotensin IV. This radioligand bound with relatively high affinity and specificity to angiotensin IV binding sites. In competition studies on consecutive sections through the prefrontal cortex and claustrum, angiotensin IV, Nle-angiotensin IV and LVV-hemorphin 7 competed for the binding of ¹²⁵I[Nle]-angiotensin IV with nanomolar affinities. Angiotensin II and the AT₁ and AT₂ receptor antagonists were ineffective in competing for the binding at concentrations of up to 10 μM. We found high densities of ¹²⁵I[Nle]-angiotensin IV binding sites throughout the cerebral cortex including the insular, entorhinal, prefrontal and cingulate cortices. Very high densities of the binding sites were observed in the claustrum, choroid plexus, hippocampus and pontine nucleus. Some thalamic nuclei displayed high densities of binding including the anteroprincipal, ventroanterior, anteromedial, medial dorsal and ventrolateral nuclei. The caudate nucleus, putamen, many amygdaloid nuclei and the red nucleus all displayed moderate densities of binding with a higher level detected in the substantia nigra pars compacta. In the hypothalamus, high densities binding sites were found in the ventromedial nucleus with lower levels in the dorsomedial and paraventricular nuclei. The distribution of ¹²⁵I[Nle]-angiotensin IV binding sites in the human brain is similar to that found in other species and supports multiple roles for the binding sites in the central nervous system, including facilitation of memory retention and retrieval.     

Angiotensin III modulates the nociceptive control mediated by the periaqueductal gray matter

Endogenous angiotensin (Ang) II and/or an Ang II-derived peptide, acting on Ang type 1 (AT₁) and Ang type 2 (AT₂) receptors, can carry out part of the nociceptive control modulated by periaqueductal gray matter (PAG). However, neither the identity of this putative Ang-peptide, nor its relationship to Ang II antinociceptive activity was clarified. Therefore, we have used tail-flick and incision allodynia models combined with an HPLC time course of Ang metabolism, to study the Ang III antinociceptive effect in the rat ventrolateral (vl) PAG using peptidase inhibitors and receptor antagonists. Ang III injection into the vlPAG increased tail-flick latency, which was fully blocked by Losartan and CGP 42,112A, but not by divalinal-Ang IV, indicating that Ang III effect was mediated by AT₁ and AT₂ receptors, but not by the AT₄ receptor. Ang III injected into the vlPAG reduced incision allodynia. Incubation of Ang II with punches of vlPAG homogenate formed Ang III, Ang (1–7) and Ang IV. Amastatin (AM) inhibited the formation of Ang III from Ang II by homogenate, and blocked the antinociceptive activity of Ang II injection into vlPAG, suggesting that aminopeptidase A (APA) formed Ang III from Ang II. Ang III can also be formed from Ang I by a vlPAG alternative pathway. Therefore, the present work shows, for the first time, that: (i) Ang III, acting on AT₁ and AT₂ receptors, can elicit vlPAG-mediated antinociception, (ii) the conversion of Ang II to Ang III in the vlPAG is required to elicit antinociception, and (iii) the antinociceptive activity of endogenous Ang II in vlPAG can be ascribed preponderantly to Ang III.     

AT1 receptor-mediated angiotensin II activation and chemotaxis of T lymphocytes

Angiotensin II (Ang II), a central renin–angiotensin system (RAS) effector molecule, and its receptors, AT₁ and AT₂, have been shown to be involved in the inflammatory aspects of different diseases, however the cellular mechanisms underlying the regulation of immunity are not fully understood. In this work, using spleen-derived CD4⁺ and CD8⁺ T lymphocytes activated in vitro, we tested the influence of Ang II on different aspects of the T cell function, such as activation and adhesion/transmigration through endothelial basal membrane proteins. The addition of 10⁻⁸ M Ang II did not change any of the parameters evaluated. However, 10⁻⁶ M losartan, an AT₁ receptor antagonist: (i) reduced the percentage of CD25⁺ and CD69⁺ cells of both subsets; (ii) inhibited adhesion of these cells to fibronectin or laminin by 53% or 76%, respectively and (iii) significantly reduced transmigration through fibronectin or laminin by 57% or 43%, respectively. In addition, 10⁻⁶ M captopril, an angiotensin-converting enzyme inhibitor had similar effects to Ang II, however its effects were reverted by exogenous Ang II (10⁻⁸ M). None of these responses was modified by 10⁻⁷ M PD123319, an AT₂ antagonist. These data reinforce the notion of endogenous production of Ang II by T cells, which is important for T cell activation, and adhesion/transmigration induced on interaction with basal membrane proteins, possibly involving AT₁ receptor activation. Moreover, AT₁ receptor expression is 10-fold higher in activated T lymphocytes compared with naive cells, but AT₂ receptor expression did not change after T cell receptor triggering.     

Angiotensin II type 2 receptor regulates the development of pancreatic endocrine cells in mouse embryos

Background : We previously identified a local renin‐angiotensin system (RAS) regulating the differentiation of an isolated population of human pancreatic progenitor cells. Major RAS components that regulate organogenesis have been also described in embryos; however, it is not known whether a local RAS is present in the fetal pancreas. We now hypothesize that angiotensin II type 1 (AT₁) and type 2 (AT₂) receptors are expressed in mouse embryonic pancreas and involved in regulating endocrine cell development. Results: Differential expression of AT₁ and AT₂ receptors was observed in the mouse pancreata in late embryogenesis. Systemic AT₂, but not AT₁, receptor blockade during the second transition in pancreatic development (from embryonic day 12.0 onward) reduced the β‐cell to α‐cell ratio of the neonate islets, impaired their insulin secretory function and the glucose tolerance of the pups. Studies with pancreas explants ex vivo revealed regulation by AT₂ receptors of the differentiation of pancreatic progenitors into insulin‐producing cells and of the proliferation of the differentiated cell, actions that did not result from reduced angiogenesis as a secondary effect of AT₂ receptor antagonism. Conclusions: These data revealed an AT₂ receptor‐mediated mechanism regulating pancreatic endocrine cell development in vivo. Developmental Dynamics 243:415–427, 2014. © 2013 Wiley Periodicals, Inc.     

Expression of angiotensin II and its receptors in the normal and hypoxic amoeboid microglial cells and murine BV-2 cells

Involvement of the local angiotensin receptor system in the central nervous system is well documented, yet its cellular localization and role in the glial cells have remained elusive. This study reports expression of angiotensin II and its receptors namely, angiotensin II receptor type 1 (AT₁) and angiotensin II receptor type 2 (AT₂) in the amoeboid microglial cells in the neonatal rat brain. In rats subjected to hypoxia, the amount of angiotensin II released in the corpus callosal tissue was reduced as revealed by enzyme immunoassay. Expression of AT₁ mRNA and protein was down-regulated after hypoxic exposure, but AT₂ was up-regulated. In BV-2 cells exposed to hypoxia for 4 h, expression of AT₁ mRNA was reduced but AT₂ was increased. These changes were further intensified respectively in LPS-stimulated microglia. Edaravone enhanced AT₁ expression but suppressed AT₂ expression significantly in lipopolysaccharide-stimulated cells. Neutralization of AT₂ with its antiserum significantly increased mRNA expression of tumor necrosis factor-α and interleukin-1β but decreased that of transforming growth factor-beta1. In conclusion, the present results suggest that AT₁ may be linked to regulation of vasodilation for increase of blood flow in hypoxic conditions, while up-regulated expression of AT₂ may reduce inflammatory responses through suppression of proinflammatory cytokines and elimination of free radicals.     

Evolving concepts on regulation and function of renin in distal nephron

Sustained stimulation of the intrarenal/intratubular renin–angiotensin system in a setting of elevated arterial pressure elicits renal vasoconstriction, increased sodium reabsorption, proliferation, fibrosis, and eventual renal injury. Activation of luminal AT₁ receptors in proximal and distal nephron segments by local Ang II formation stimulates various transport systems. Augmented angiotensinogen (AGT) production by proximal tubule cells increases AGT secretion contributing to increased proximal Ang II levels and leading to spillover of AGT into the distal nephron segments, as reflected by increased urinary AGT excretion. The increased distal delivery of AGT provides substrate for renin, which is expressed in principal cells of the collecting tubule and collecting ducts, and is also stimulated by AT₁ receptor activation. Renin and prorenin are secreted into the tubular lumen and act on the AGT delivered from the proximal tubule to form more Ang I. The catalytic actions of renin and or prorenin may be enhanced by binding to prorenin receptors on the intercalated cells or soluble prorenin receptor secreted into the tubular fluid. There is also increased luminal angiotensin converting enzyme in collecting ducts facilitating Ang II formation leading to stimulation of sodium reabsorption via sodium channel and sodium/chloride co-transporter. Thus, increased collecting duct renin contributes to Ang II-dependent hypertension by augmenting distal nephron intratubular Ang II formation leading to sustained stimulation of sodium reabsorption and progression of hypertension.     

Immunohistochemical localization of angiotensin AT1 receptors in the rat carotid body

The carotid body (CB) is a major peripheral arterial chemoreceptor that initiates respiratory and cardiovascular adjustments to maintain homeostasis. Recent evidence suggests that circulating or locally produced hormones like angiotensin II acting via AT₁ receptors modulate its activity in a paracrine-autocrine manner. The aim of this study was to examine the immunohistochemical localization of AT₁ receptor in the CB of adult rats and to compare its expression in vehicle-treated animals, and after the long-term application of its selective blocker losartan. Immunohistochemistry revealed that a subset of CB glomeruli and the vast majority of neurons in the adjacent superior cervical ganglion (SCG) were strongly AT₁ receptor-immunoreactive. In the CB immunostaining was observed in the chemosensory glomus cells typically aggregated in cell clusters while the nerve fibers in-between and large capillaries around them were immunonegative. Exogenous administration of losartan for a prolonged time significantly reduces the intensity of AT₁ receptor immunostaining in the CB glomus cells and SCG neurons. Our results show that AT₁ receptors are largely expressed in the rat CB under physiological conditions, and their expression is down-regulated by losartan treatment.     

Genetic deletion and overexpression of angiotensin II receptors

Angiotensin II receptors are essential components of the renin-angiotensin system transducing angiotensin II mediated signals across the plasma membrane of many cell types in the cardiovascular system. To date, three subtypes of angiotensin II receptors have been identified by molecular cloning, termed angiotensin II type 1 (AT_1A, AT_1B) and type 2 (AT₂) receptors. This review focuses on recent transgenic animal models which have been generated to study the in vivo significance of angiotensin receptor diversity. AT_1A receptors are the major blood pressure regulators and have a potent growth-stimulatory effect on cardiac myocytes in vivo. The AT_1B receptor subtype may participate in the control of vascular tone if AT_1A receptors are absent. AT₂ receptors are abundantly expressed during embryonic development and may also play a role in blood pressure regulation by influencing vascular development and differentiation. Received: 16 February 1998 / Accepted: 10 August 1998     

Stable transfection of rat angiotensin II receptor subtypes in mouse adrenocortical Y1 cells

Summary A procedure is described for stably transfecting mouse adrenocortical Y1 cells with AT_1a and AT_1b angiotensin II (AII) receptor subtypes using the lipofectin reagent, DOTAP. The transfected cells are screened by radioreceptor assay using¹²⁵I-[Sar¹,Ile⁸]AII to select those cells that express high levels of the AII receptors. The establishment of individual transfected Y1 cell lines expressing AT_1a and AT_1b receptors provides a valuable model system in which to compare the function and signal transduction mechanisms of these two highly homologous AII receptor subtypes.     

Angiotensin II induces hyperresponsiveness of bronchial smooth muscle via an activation of p42/44 ERK in rats

Angiotensin II (Ang II) might be an important mediator in the pathogenesis of bronchial asthma, although the mechanisms of airway hyperresponsiveness caused by Ang II are not yet clear. Whether p42/44 ERK contributes to the Ang II-elicited bronchial smooth muscle (BSM) hyperresponsiveness in rats was presently examined. The RT-PCR analyses revealed that Ang II AT_1A, AT_1B, and AT₂ receptors, angiotensinogen, angiotensin-converting enzyme, but not renin, were expressed in the lungs, trachea, and main bronchi of rats. Only a small and transient contraction was induced by the application of Ang II in the main bronchial smooth muscle; the contraction was inhibited by losartan, an AT₁ receptor antagonist. The contractions induced by carbachol (CCh), high K⁺ depolarization, and sodium fluoride (NaF; a G protein activator) were augmented by pretreatment with Ang II. The BSM hyperresponsiveness induced by Ang II was abolished by losartan. Furthermore, the Ang II-induced BSM hyperresponsiveness to CCh was attenuated by pretreatment with U-0126, a p42/44 ERK kinase (MEK-1/2) inhibitor. In conclusion, Ang II-induced BSM hyperresponsiveness through the activation of p42/44 ERK may play an important role in the pathophysiology of bronchial asthma, although Ang II itself caused a small force development in the bronchial smooth muscle.     

Angiotensin-(1–7) and angiotension II in the rostral ventrolateral medulla modulate the cardiac sympathetic afferent reflex and sympathetic activity in rats

The rostral ventrolateral medulla (RVLM) plays a pivotal role in regulating sympathetic vasomotor activity. The cardiac sympathetic afferent reflex (CSAR) contributes to the enhanced sympathetic outflow in chronic heart failure and hypertension. The aim of the present study was to determine whether angiotensin (Ang) II and Ang-(1–7) in the RVLM modulate the CSAR and sympathetic activity. Bilateral sinoaortic denervation and vagotomy were carried out in anesthetized rats. The CSAR was evaluated as the renal sympathetic nerve activity (RSNA) response to epicardial application of capsaicin. The effects of bilateral microinjection of Ang II, Ang-(1–7), the AT₁ receptor antagonist losartan or the Mas receptor antagonist d-alanine-Ang-(1–7) (A-779) into the RVLM were determined. Either Ang II or Ang-(1–7) enhanced the CSAR as well as increased RSNA and mean arterial pressure (MAP) in a dose-dependent manner. Pretreatment with losartan but not the A-779 abolished the effects of Ang II, while A-779 but not the losartan eliminated the effects of Ang-(1–7). The RVLM microinjection of losartan alone had no direct effect on the CSAR, RSNA, and MAP, but A-779 alone attenuated the CSAR and decreased RSNA and MAP. These results indicate that Ang-(1–7) is as effective as Ang II in sensitizing the CSAR and increasing sympathetic outflow. In contrast to Ang II, the effects of Ang-(1–7) are not mediated by AT₁ receptors but by Mas receptors. Mas receptors, but not the AT₁ receptors, in the RVLM are involved in the tonic control of the CSAR.     

Sex differences in the subcellular distribution of angiotensin type 1 receptors and NADPH oxidase subunits in the dendrites of C1 neurons in the rat rostral ventrolateral medulla

The rostral ventrolateral medulla (RVLM), a region critical for the tonic and reflex control of arterial pressure, contains a group of adrenergic (C1) neurons that project to the spinal cord and directly modulate pre-ganglionic sympathetic neurons. Epidemiological data suggest that there are gender differences in the regulation of blood pressure. One factor that could be involved is angiotensin II signaling and the associated production of reactive oxygen species (ROS) by NADPH oxidase, which is emerging as an important molecular substrate for central autonomic regulation and dysregulation. In this study dual electron microscopic immunolabeling was used to examine the subcellular distribution of the angiotensin type 1 (AT₁) receptor and two NADPH oxidase subunits (p47 and p22) in C1 dendritic processes, in tissue from male, proestrus (high estrogen) and diestrus (low estrogen) female rats. Female dendrites displayed significantly more AT₁ labeling and significantly less p47 labeling than males. While elevations in AT₁ labeling primarily resulted from higher levels of receptor on the plasma membrane, p47 labeling was reduced both on the plasma membrane and in the cytoplasm. Across the estrous cycle, proestrus females displayed significantly higher levels of AT₁ labeling than diestrus females, which resulted exclusively from plasma membrane density differences. In contrast, p47 labeling did not change across the estrous cycle, indicating that ROS production might reflect AT₁ receptor membrane density. No significant differences in p22 labeling were observed. These findings demonstrate that both sex and hormonal levels can selectively affect the expression and subcellular distribution of components of the angiotensin II signaling pathway within C1 RVLM neurons. Such effects could reflect differences in the capacity for ROS production, potentially influencing short term excitability and long term gene expression in a cell group which is critically involved in blood pressure regulation, potentially contributing to gender differences in the risk of cardiovascular disease.     

Extracellular signal regulated kinase and SMAD signaling both mediate the angiotensin II driven progression towards overt heart failure in homozygous TGR(mRen2)27

Angiotensin (Ang) II is a key player in left ventricular (LV) remodeling and cardiac fibrosis. Its effects are thought to be transferred at least in part by mitogen-activated protein kinases (MAPK), transforming growth factor (TGF) ₁, and the Smad pathway. In this study we sought to elucidate whether Ang II related effects on LV dysfunction and fibrosis in vivo are mediated via MAPK or rather via Smad stimulation. We treated homozygous REN2 rats (7–11 weeks) with placebo, Ang II type 1 (AT₁) receptor blocker or tyrphostin A46 (TYR), an inhibitor of epidermal growth factor receptor tyrosine kinase that blocks extracellular signal-regulated kinase (ERK) activity. REN2 rats had LV hypertrophy (LVH) and LV dysfunction that progressed to heart failure between 10 and 13 weeks. Blood pressure normalized over time. Renin, N-terminal atrial natriuretic peptide (N-ANP), and ERK were activated while p38 MAPK was not. Treatment with AT₁ receptor blockade prevented LVH and right ventricular hypertrophy, normalized systolic and diastolic dP/dt, N-ANP levels, and reduced collagen apposition. Similarly, TYR reduced LVH, N-ANP levels, and collagen apposition. Myocardial ERK activation did not depend on AT₁ receptor signaling as it was not affected by AT₁ receptor blockade. TYR abolished myocardial ERK activity. Smad2 activation was inhibited by AT₁ receptor blockade but was unaltered by TYR. Ang II induced LV remodeling and fibrosis are dependent on both ERK and Smad2 activation. This process is prevented by both AT₁ receptor blockade and TYR, and therefore inhibition of either pathway is equally efficacious in restoring LV function and architecture.R.A.D.B. and S.P. contributed equally to this work