Bradykinin acutely inhibits activity of the epithelial Na+ channel in mammalian aldosterone-sensitive distal nephron

Activation of the renal kallikrein-kinin system results in natriuresis and diuresis, suggesting its possible role in renal tubular sodium transport regulation. Here, we used patch-clamp electrophysiology to directly assess the effects of bradykinin (BK) on the epithelial Na(+) channel (ENaC) activity in freshly isolated split-opened murine aldosterone-sensitive distal nephrons (ASDNs). BK acutely inhibits ENaC activity by reducing channel open probability (P(o)) in a dose-dependent and reversible manner. Inhibition of B2 receptors with icatibant (HOE-140) abolished BK actions on ENaC. In contrast, activation of B1 receptors with the selective agonist Lys-des-Arg(9)-BK failed to reproduce BK actions on ENaC. This is consistent with B2 receptors playing a critical role in mediating BK signaling to ENaC. BK has little effect on ENaC P(o) when G(q/11) was inhibited with Gp antagonist 2A. Moreover, inhibition of phospholipase C (PLC) with U73122, but not saturation of cellular cAMP levels with the membrane-permeable nonhydrolysable cAMP analog 8-cpt-cAMP, prevents BK actions on ENaC activity. This argues that BK stimulates B2 receptors with subsequent activation of G(q/11)-PLC signaling cascade to acutely inhibit ENaC activity. Activation of BK signaling acutely depletes apical PI(4,5)P(2) levels. However, inhibition of Ca(2+) pump SERCA of the endoplasmic reticulum with thapsigargin does not prevent BK signaling to ENaC. Furthermore, caffeine, while producing a similar rise in [Ca(2+)](i) as in response to BK stimulation, fails to recapitulate BK actions on ENaC. Therefore, we concluded that BK acutely inhibits ENaC P(o) in mammalian ASDN via stimulation of B2 receptors and following depletion of PI(4,5)P(2), but not increases in [Ca(2+)](i).     

Oxidation of CaMKII determines the cardiotoxic effects of aldosterone

Excessive activation of the β-adrenergic, angiotensin II (Ang II) and aldosterone signaling pathways promotes mortality after myocardial infarction, and antagonists targeting these pathways are core therapies for treating this condition. Catecholamines and Ang II activate the multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), the inhibition of which prevents isoproterenol-mediated and Ang II-mediated cardiomyopathy. Here we show that aldosterone exerts direct toxic actions on myocardium by oxidative activation of CaMKII, causing cardiac rupture and increased mortality in mice after myocardial infarction. Aldosterone induces CaMKII oxidation by recruiting NADPH oxidase, and this oxidized and activated CaMKII promotes matrix metalloproteinase 9 (MMP9) expression in cardiomyocytes. Myocardial CaMKII inhibition, overexpression of methionine sulfoxide reductase A (an enzyme that reduces oxidized CaMKII) or NADPH oxidase deficiency prevented aldosterone-enhanced cardiac rupture after myocardial infarction. These findings show that oxidized myocardial CaMKII mediates the cardiotoxic effects of aldosterone on the cardiac matrix and establish CaMKII as a nodal signal for the neurohumoral pathways associated with poor outcomes after myocardial infarction.     

The angiotensin Ⅱ type 1 receptor and receptor-associated proteins

INTRODUCTIONThe renin-angiotensin system (RAS) is consid-ered to be the major regulator of blood pressure)electrolyte balance and renal, neuronal as well as en-docrine functions related to cardiovascular control.The RAS is the key factor in most cases essential hy-pertension, as indicated by successes in treatment ofhypertensive patients with various angiotensin I con-verting enzyme (ACE) inhibitors and receptor block-ers. Renin was a central subject of intense investigation because of…     

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.     

Angiotensin II type 1 receptor-modulated signaling pathways in neurons

Mammalian brain contains high densities of angiotensin II (Ang II) type 1 (AT1) receptors, localized mainly to specific nuclei within the hypothalamus and brainstem regions. Neuronal AT1 receptors within these areas mediate the stimulatory actions of central Ang II on blood pressure, water and sodium intake, and vasopressin secretion, effects that involve the modulation of brain noradrenergic pathways. This review focuses on the intracellular events that mediate the functional effects of Ang II in neurons, via AT1 receptors. The signaling pathways involved in short-term changes in neuronal activity, membrane ionic currents, norepinephrine (NE) release, and longer-term neuromodulatory actions of Ang II are discussed. It will be apparent from this discussion that the signaling pathways involved in these events are often distinct.     

Pleiotropic AT1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin II

Angiotensin II (Ang II) activates a wide spectrum of signaling responses via the AT1 receptor (AT1R) that mediate its physiological control of blood pressure, thirst, and sodium balance and its diverse pathological actions in cardiovascular, renal, and other cell types. Ang II-induced AT1R activation via Gq/11 stimulates phospholipases A2, C, and D, and activates inositol trisphosphate/Ca2+ signaling, protein kinase C isoforms, and MAPKs, as well as several tyrosine kinases (Pyk2, Src, Tyk2, FAK), scaffold proteins (G protein-coupled receptor kinase-interacting protein 1, p130Cas, paxillin, vinculin), receptor tyrosine kinases, and the nuclear factor-kappaB pathway. The AT1R also signals via Gi/o and G11/12 and stimulates G protein-independent signaling pathways, such as beta-arrestin-mediated MAPK activation and the Jak/STAT. Alterations in homo- or heterodimerization of the AT1R may also contribute to its pathophysiological roles. Many of the deleterious actions of AT1R activation are initiated by locally generated, rather than circulating, Ang II and are concomitant with the harmful effects of aldosterone in the cardiovascular system. AT1R-mediated overproduction of reactive oxygen species has potent growth-promoting, proinflammatory, and profibrotic actions by exerting positive feedback effects that amplify its signaling in cardiovascular cells, leukocytes, and monocytes. In addition to its roles in cardiovascular and renal disease, agonist-induced activation of the AT1R also participates in the development of metabolic diseases and promotes tumor progression and metastasis through its growth-promoting and proangiogenic activities. The recognition of Ang II's pathogenic actions is leading to novel clinical applications of angiotensin-converting enzyme inhibitors and AT1R antagonists, in addition to their established therapeutic actions in essential hypertension.     

Cross-talk between aldosterone and angiotensin signaling in vascular smooth muscle cells

In hypertension or other forms of cardiovascular disease, the chronic activation of the renin-angiotensin-aldosterone system (RAAS) leads to dysfunction of the vasculature, including, increased vascular tone, inflammation, fibrosis and thrombosis. Cross-talk between the main mediators of the RAAS, aldosterone and angiotensin (Ang) II, participates in the development of this vascular dysfunction. Recent studies have highlighted the molecular mechanisms supporting this cross-talk in vascular smooth muscle cells (VSMCs). Some of the signaling pathways activated by the Ang II type 1 receptor (AT₁R) are dependent on the mineralocorticoid receptor (MR) and vice versa. VSMC signaling pathways involved in migration and growth are under the control of cross-talk between aldosterone and Ang II. A synergistic mechanism leads to potentiation of signaling pathways activated by each agent. The genomic and non-genomic mechanisms activated by aldosterone cooperate with Ang II to regulate vascular tone and gene expression of pro-inflammatory and pro-fibrotic molecules. This cross-talk is dependent on the non-receptor tyrosine kinase c-Src, and on receptor tyrosine kinases, EGFR and PDGFR, and leads to activation of MAP kinases and growth, migration and inflammatory effects. These new findings will contribute to development of better treatments for conditions in which the RAAS is excessively activated.     

The angiotensin II-AT1 receptor stimulates reactive oxygen species within the cell nucleus

We and others have reported significant expression of the Ang II Type 1 receptor (AT1R) on renal nuclei; thus, the present study assessed the functional pathways and distribution of the intracellular AT1R on isolated nuclei. Ang II (1 nM) stimulated DCF fluorescence, an intranuclear indicator of reactive oxygen species (ROS), while the AT1R antagonist losartan or the NADPH oxidase (NOX) inhibitor DPI abolished the increase in ROS. Dual labeling of nuclei with antibodies against nucleoporin 62 (Nup62) and AT1R or the NADPH oxidase isoform NOX4 revealed complete overlap of the Nup62 and AT1R (99%) by flow cytometry, while NOX4 was present on 65% of nuclei. Treatment of nuclei with a PKC agonist increased ROS while the PKC inhibitor GF109203X or PI3 kinase inhibitor LY294002 abolished Ang II stimulation of ROS. We conclude that the Ang II-AT1R-PKC axis may directly influence nuclear function within the kidney through a redox sensitive pathway.     

The epithelial sodium channel and the control of sodium balance

Studies aiming at the elucidation of the genetic basis of rare monogenic forms of hypertension have identified mutations in genes coding for the epithelial sodium channel ENaC, for the mineralocorticoid receptor, or for enzymes crucial for the synthesis of aldosterone. These genetic studies clearly demonstrate the importance of the regulation of Na⁺ absorption in the aldosterone-sensitive distal nephron (ASDN), for the maintenance of the extracellular fluid volume and blood pressure.Recent studies aiming at a better understanding of the cellular and molecular basis of ENaC-mediated Na⁺ absorption in the distal part of nephron, have essentially focused on the regulation ENaC activity and on the aldosterone-signaling cascade. ENaC is a constitutively open channel, and factors controlling the number of active channels at the cell surface are likely to have profound effects on Na⁺ absorption in the ASDN, and in the amount of Na⁺ that is excreted in the final urine.A number of membrane-bound proteases, kinases, have recently been identified that increase ENaC activity at the cell surface in heterologous expressions systems. Ubiquitylation is a general process that regulates the stability of a variety of target proteins that include ENaC. Recently, deubiquitylating enzymes have been shown to increase ENaC activity in heterologous expressions systems.These regulatory mechanisms are likely to be nephron specific, since in vivo studies indicate that the adaptation of the renal excretion of Na⁺ in response to Na⁺ diet occurs predominantly in the early part (the connecting tubule) of the ASDN.An important work is presently done to determine in vivo the physiological relevance of these cellular and molecular mechanisms in regulation of ENaC activity. The contribution of the protease-dependent ENaC regulation in mediating Na⁺ absorption in the ASDN is still not clearly understood. The signaling pathway that involves ubiquitylation of ENaC does not seem to be absolutely required for the aldosterone-mediated control of ENaC. These in vivo physiological studies presently constitute a major challenge for our understanding of the regulation of ENaC to maintain the Na⁺ balance.     

Angiotensin II stimulates the Na+/H+ exchanger in human umbilical vein endothelial cells via AT1 receptor

Angiotensin II (Ang II) has an important role in cardiovascular regulation and in the control of electrolyte balance, and its role in the regulation of Na+ transcellular movements through its actions on the activity of Na+/K+ ATPase is well documented. We showed previously that human umbilical vein endothelial cells (HUVEC) express the Ang II type 1 (AT1) receptor, which mediates Ang II modulation of Na+/K+ ATPase activity (1). We here investigate the effects of Ang II on the activity of the Na+/H+ exchanger in HUVEC. When compared with controls, incubation of HUVEC for 20 min with different concentrations of Ang II provoked significant increases in Na+/H+ activity. The stimulation was dose dependent between 1 and 10 nM Ang II and varied with time of incubation up to 20 min. The maximal response, obtained with 10 nM Ang II after 20 min treatment, resulted in a 65% increment in Na+/H+ activity. Preincubation of HUVEC with 10 microM DuP753 blocked Na+/H+ activation by Ang II. These results suggest that the effects of Ang II on both the Na+/K+ ATPase and the Na+/H+ exchanger may increase the transendothelial flux of Na+ and are mediated by the AT1 receptor.     

E Prostanoid-1 receptor regulates renal medullary αENaC in rats infused with angiotensin II

E Prostanoid (EP) receptors play an important role in urinary Na⁺ excretion. In the kidney, the epithelial sodium channel (ENaC) is the rate-limiting-step for Na⁺ reabsorption. We hypothesized that activation of EP1/EP3 regulates the expression of ENaC in the face of renin-angiotensin-aldosterone-system (RAAS) activation. In primary cultures of inner medullary collecting duct (IMCD) cells, sulprostone (EP1 > EP3 agonist, 1 μM) and 17 Phenyl trinor (17 Pt, EP1 agonist, 10 μM) prevented the up-regulation of αENaC mRNA induced by aldosterone (10 nM). In Sprague-Dawley rats infused with angiotensin II (0.4 μg/kg/min), αENaC expression was up-regulated in renal cortex and medulla coincidently with high plasma aldosterone levels. Sulprostone and/or 17 Pt prevented this effect in renal medulla but not in cortex. Immunocytochemistry demonstrated that IMCD cells express EP1. Our results suggest that specific activation of EP1 receptor during RAAS activation antagonizes the action of aldosterone on αENaC expression in the renal medulla.     

The epithelial sodium channel: from molecule to disease

Genetic analysis has demonstrated that Na absorption in the aldosterone-sensitive distal nephron (ASDN) critically determines extracellular blood volume and blood pressure variations. The epithelial sodium channel (ENaC) represents the main transport pathway for Na⁺ absorption in the ASDN, in particular in the connecting tubule (CNT), which shows the highest capacity for ENaC-mediated Na⁺ absorption. Gain-of-function mutations of ENaC causing hypertension target an intracellular proline-rich sequence involved in the control of ENaC activity at the cell surface. In animal models, these ENaC mutations exacerbate Na⁺ transport in response to aldosterone, an effect that likely plays an important role in the development of volume expansion and hypertension. Recent studies of the functional consequences of mutations in genes controlling Na⁺ absorption in the ASDN provide a new understanding of the molecular and cellular mechanisms underlying the pathogenesis of salt-sensitive hypertension.