Chronic activation of vasopressin-2 receptors induces hypertension in Liddle mice by promoting Na+ and water retention.

The renin-angiotensin-aldosterone and arginine vasopressin-V2 receptor-aquaporin-2 (AQP2) systems converge on the epithelial Na+ channel (ENaC) to regulate blood pressure and plasma tonicity. Although it is established that V2 receptors initiate renal water reabsorption through AQP2, whether V2 receptors can also induce renal Na+ retention through ENaC and raise blood pressure remains an open question. We hypothesized that a specific increase in V2 receptor-mediated ENaC activity can lead to high blood pressure. Our approach was to test effects of chronic activation of V2 receptors in Liddle mice, a genetic mouse model of high ENaC activity, and compare differences in ENaC activity, urine Na+ excretion, and blood pressure with control mice. We found that ENaC activity was elevated in Liddle mice and could not be stimulated further by administration of desmopressin (dDAVP), a V2 receptor-specific agonist. In contrast, Liddle mice showed higher levels of expression of AQP2 and aquaporin-3, but they could still respond to dDAVP infusion by increasing phospho-AQP2 expression. With dDAVP infusion, Liddle mice excreted smaller urine volume and less urine Na+ and developed higher blood pressure compared with control mice; this hypertension was attenuated with administration of the ENaC inhibitor benzamil. We conclude that V2 receptors contribute to hypertension in the Liddle mouse model by promoting primary Na+ and concomitant water retention.NEW & NOTEWORTHY Liddle syndrome is a classic model for hypertension from high epithelial Na+ channel (ENaC) activity. In the Liddle mouse model, vasopressin-2 receptors stimulate both ENaC and aquaporin-2, which increases Na+ and water retention to such an extent that hypertension ensues. Liddle mice will preserve plasma tonicity at the expense of a higher blood pressure; these data highlight the inherent limitation in which the kidney must use ENaC as a pathway to regulate both plasma tonicity and blood pressure.

Collecting Duct Nitric Oxide Synthase 1ß Activation Maintains Sodium Homeostasis During High Sodium Intake Through Suppression of Aldosterone and Renal Angiotensin II Pathways.

BACKGROUND: During high sodium intake, the renin-angiotensin-aldosterone system is downregulated and nitric oxide signaling is upregulated in order to remain in sodium balance. Recently, we showed that collecting duct nitric oxide synthase 1ß is critical for fluid-electrolyte balance and subsequently blood pressure regulation during high sodium feeding. The current study tested the hypothesis that high sodium activation of the collecting duct nitric oxide synthase 1ß pathway is critical for maintaining sodium homeostasis and for the downregulation of the renin-angiotensin-aldosterone system-epithelial sodium channel axis. METHODS AND RESULTS: Male control and collecting duct nitric oxide synthase 1ß knockout (CDNOS1KO) mice were placed on low, normal, and high sodium diets for 1 week. In response to the high sodium diet, plasma sodium was significantly increased in control mice and to a significantly greater level in CDNOS1KO mice. CDNOS1KO mice did not suppress plasma aldosterone in response to the high sodium diet, which may be partially explained by increased adrenal AT1R expression. Plasma renin concentration was appropriately suppressed in both genotypes. Furthermore, CDNOS1KO mice had significantly higher intrarenal angiotensin II with high sodium diet, although intrarenal angiotensinogen levels and angiotensin-converting enzyme activity were similar between knockout mice and controls. In agreement with inappropriate renin-angiotensin-aldosterone system activation in the CDNOS1KO mice on a high sodium diet, epithelial sodium channel activity and sodium transporter abundance were significantly higher compared with controls. CONCLUSIONS: These data demonstrate that high sodium activation of collecting duct nitric oxide synthase 1ß signaling induces suppression of systemic and intrarenal renin-angiotensin-aldosterone system, thereby modulating epithelial sodium channel and other sodium transporter abundance and activity to maintain sodium homeostasis.

Binding of human myeloperoxidase to red blood cells: Molecular targets and biophysical consequences at the plasma membrane level.

Myeloperoxidase (MPO) is an oxidant-producing enzyme that can also bind to cellular surface proteins. We found that band 3 protein and glycophorins A and B were the key MPO-binding targets of human red blood cells (RBCs). The interaction of MPO with RBC proteins was mostly electrostatic in nature because it was inhibited by desialation, exogenic sialic acid, high ionic strength, and extreme pH. In addition, MPO failed to interfere with the lectin-induced agglutination of RBCs, suggesting a minor role of glycan-recognizing mechanisms in MPO binding. Multiple biophysical properties of RBCs were altered in the presence of native (i.e., not hypochlorous acid-damaged) MPO. These changes included transmembrane potential, availability of intracellular Ca(2+), and lipid organization in the plasma membrane. MPO-treated erythrocytes became larger in size, structurally more rigid, and hypersensitive to acidic and osmotic hemolysis. Furthermore, we found a significant correlation between the plasma MPO concentration and RBC rigidity index in type-2 diabetes patients with coronary heart disease. These findings suggest that MPO functions as a mediator of novel regulatory mechanism in microcirculation, indicating the influence of MPO-induced abnormalities on RBC deformability under pathological stress conditions.

A fluorescence vital assay for the recognition and quantification of excitotoxic cell death by necrosis and apoptosis using confocal microscopy on neurons in culture.

An automated fluorescence method for the detection of neuronal cell death by necrosis and apoptosis with sequential acridine orange (AO) and ethidium bromide (EB) staining using confocal microscopy is described. Since cell nuclei during apoptosis become acidic, AO staining was utilized to distinguish live neurons from neurons undergoing apoptosis, using the AO property to shift its fluorescence from green at normal pH toward brilliant orange-red in the process of acidification. Further EB application labels nuclei of necrotic neurons in red. Sequential treatment by AO and EB can be employed as an express vitality test to count fractions of live and dead cell via apoptosis and necrosis, respectively. An algorithm of automatic quantification of cell types is based on the image correlation analysis. Our conclusion is validated by experiments with the vital dye trypan blue and the pharmacological study of receptor subtypes involved in the excitotoxicity. The approach described here, therefore, offers an express, easy, sensitive and reproducible method by which necrosis and apoptosis can be recognized and quantified in a population of living neurons. Because this assay does not require any preliminary tissue treatment, fixation or dissociation in a cell suspension its utility is likely to be extended for measuring cell viability and cytotoxicity on a variety of living preparations (tissues, brain slices and cell cultures).