Alpha(1)-adrenergic-mediated eNOS phosphorylation in intact arteries

Activation of arterial smooth muscle alpha(1)-adrenergic receptors results in vasoconstriction, as well as a secondary release of nitric oxide and slow vasodilation, presumably through gap junction communication from smooth muscle to endothelium. We hypothesized that this slow vasodilation is due to activation of eNOS through phosphorylation at Ser1179 and dephosphorylation at Thr495. Phosphorylation was measured by western blot using mouse mesenteric arteries that were cannulated and pressurized (75 mm Hg) and treated either by 1) 5 mm of phenylephrine superfusion (10(-5) M) (PE5), 2) 15 min of phenylephrine (PE15), 3) 15 min phenylephrine followed by acetylcholine (10(-4) M) (PE + ACh), or 4) 20 min time control with no treatment (NT) [4-5 arteries pooled per treatment per blot; 5 blots performed]. These treatments allowed correlation between vasomotor changes, namely maximal constriction (PE5), slow vasodilation (PE15), and maximal dilation (PE + ACh), and relative phosphorylation changes. Phosphorylation of eNOS at Ser1179 was increased relative to NT by more than 2-fold at PE5 and remained similarly increased at PE15 and PE + ACh. Phosphotylation of eNOS at Thr495 was less in all treatments relative to NT, but not significantly. Treatment with L-NAME (10(-4) M) or endothelial denudation indicated that the slow dilation in response to phenylephrine was completely due to nitric oxide synthase and was endothelial dependent. These results indicate that eNOS phosphorylation at Ser1179 occurs before the slow dilation and is not actively involved in this vasodilation or dilation to acetylcholine, but may play a permissive role in eNOS activation by other mechanisms. It is not yet known what mechanism is responsible for Ser1179 phosphorylation with phenylephrine stimulation. (C) 2012 Elsevier Inc. All rights reserved

The Effects of Salt and Aldosterone on Vascular and Renal Function

Hypertension is a global epidemic that increases risk for stroke, myocardial infarction, vascular disease and chronic kidney disease, making early detection and treatment imperative. Genetic factors and lifestyle choices such as diet can influence an individual’s risk of developing hypertension. The purpose of this study was to identify consequences of two risk factors, high salt diet (HSD) and increased aldosterone levels, on vascular and renal function with an emphasis on the role of the epithelial sodium channel (ENaC). Previous cell culture studies demonstrate that an increase in extracellular [Na+] coupled with increased aldosterone increases ENaC expression in endothelial cells, leading to decreased nitric oxide production. Therefore, we hypothesized that ENaC contributes to vascular dysfunction with HSD and aldosterone in intact vasculature. Inhibition of ENaC with amiloride in isolated vessels from combination treated animals increased acetylcholine sensitivity, however, the most robust changes we observed in vascular reactivity occurred with HSD alone. We discovered a dynamic response of the vasculature to various HSD treatment lengths, in part mediated by ENaC. We examined the effects of treatment alone or in combination on renal transporter expression given their importance to ion regulation and BP. Interestingly, HSD and aldosterone led to an increase in transcript and protein expression of all ENaC subunits in the kidney. Furthermore, the combined treatments led to an increase in renal inflammation, oxidative stress, and signs of fibrosis. Our full factorial treatments allowed us to assess the contribution of diet and aldosterone alone as well as in combination. Finally, we found that chronic HSD surprisingly kept mice from gaining weight with age, despite the fact food intake was comparable to that in control animals. These animals may have shifts in metabolic processes that explain their weight differences, and these changes occur differentially in various organs. Overall, our results have added to our understanding of how HSD and aldosterone alone and in combination increase risk for hypertension by affecting vascular and renal function. We have shown that ENaC plays a role in mediating the negative effects of this pathological state, and that it may be an attractive pharmacological target beyond the kidney

Deletion of the Gamma Subunit of ENaC in Endothelial Cells Does Not Protect against Renal Ischemia Reperfusion Injury

Acute kidney injury due to renal ischemia-reperfusion injury (IRI) may lead to chronic or end stage kidney disease. A greater understanding of the cellular mechanisms underlying IRI are required to develop therapeutic options aimed at limiting or reversing damage from IRI. Prior work has shown that deletion of the α subunit of the epithelial Na+ channel (ENaC) in endothelial cells protects from IRI by increasing the availability of nitric oxide. While canonical ENaCs consist of an α, β, and γ subunit, there is evidence of non-canonical ENaC expression in endothelial cells involving the α subunit. We therefore tested whether the deletion of the γ subunit of ENaC also protects mice from IRI to differentiate between these channel configurations. Mice with endothelial-specific deletion of the γ subunit and control littermates were subjected to unilateral renal artery occlusion followed by 48 h of reperfusion. No significant difference was noted in injury between the two groups as assessed by serum creatinine and blood urea nitrogen, levels of specific kidney injury markers, and histological examination. While deletion of the γ subunit did not alter infiltration of immune cells or cytokine message, it was associated with an increase in levels of total and phosphorylated endothelial nitric oxide synthase (eNOS) in the injured kidneys. Our studies demonstrate that even though deletion of the γ subunit of ENaC may allow for greater activation of eNOS, this is not sufficient to prevent IRI, suggesting the protective effects of α subunit deletion may be due, in part, to other mechanisms

Effects of amiloride on acetylcholine‐dependent arterial vasodilation evolve over time in mice on a high salt diet

Abstract The maintenance of endothelial health is required for normal vascular function and blood pressure regulation. The epithelial Na+ channel (ENaC) in endothelial cells has emerged as a new molecular player in the regulation of endothelial nitric oxide production and vascular stiffness. While ENaC expression in the kidney is negatively regulated by high [Na+], ENaC expression in isolated endothelial cells has been shown to increase in response to a high extracellular [Na+]. In culture, this increased expression leads to cellular stiffening and decreased nitric oxide release. In vivo, the effects of high salt diet on endothelial ENaC expression and activity have varied depending on the animal model utilized. Our aim in the present study was to examine the role of endothelial ENaC in mediating vasorelaxation in the C57Bl/6 mouse strain. We utilized pressure myography to test the responsiveness of thoracodorsal arteries to acetylcholine in mice with increased sodium consumption both in the presence and absence of increased aldosterone. ENaC’s contribution was assessed with the use of the specific inhibitor amiloride. We found that while aldosterone had very little effect on ENaC's contribution to acetylcholine sensitivity, a high salt diet led to an amiloride‐dependent shift in the acetylcholine response of vessels. However, the direction of this shift was dependent on the length of high salt diet administration. Overall, our studies reveal that ENaC's role in the endothelium may be more complicated than previously thought. The channel does not simply inhibit nitric oxide generation, but instead helps preserve a homeostatic response

Validation of commercially available antibodies directed against subunits of the epithelial Na+ channel

Abstract The epithelial Na+ channel (ENaC) is traditionally composed of three subunits, although non‐canonical expression has been found in various tissues including the vasculature, brain, lung, and dendritic cells of the immune system. Studies of ENaC structure and function have largely relied on heterologous expression systems, often with epitope‐tagged channel subunits. Relevant in vivo physiological studies have used ENaC inhibitors, mice with global or tissue specific knockout of subunits, and anti‐ENaC subunit antibodies generated by investigators or by commercial sources. Availability of well‐characterized, specific antibodies is imperative as we move forward in understanding the role of ENaC in non‐epithelial tissues where expression, subunit organization, and electrophysiological characteristics may differ from epithelial tissues. We report that a commonly used commercial anti‐α subunit antibody recognizes an intense non‐specific band on mouse whole kidney and lung immunoblots, which migrates adjacent to a less intense, aldosterone‐induced full length α‐subunit. This antibody localizes to the basolateral membrane of aquaporin 2 negative cells in kidney medulla. We validated antibodies against the β‐ and γ‐subunits from the same commercial source. Our work illustrates the importance of validation studies when using popular, commercially available anti‐ENaC antibodies

Regulation of cellular communication by signalling microdomains in the blood vessel wall

It has become increasingly clear that the accumulation of proteins in specific regions of the plasma membrane can facilitate cellular communication. These regions, termed signaling microdomains, are found throughout the blood vessel wall where cellular communication, both within and between cell types, must be tightly regulated to maintain proper vascular function. We will define a cellular signaling microdomain and apply this definition to the plethora of means by which cellular communication has been hypothesized to occur in the blood vessel wall. To that end, we make a case for three broad areas of cellular communication where signaling microdomains could play an important role: 1) paracrine release of free radicals and gaseous molecules such as nitric oxide and reactive oxygen species; 2) role of ion channels including gap junctions and potassium channels, especially those associated with the endotheliumderived hyperpolarization mediated signaling, and lastly, 3) mechanism of exocytosis that has considerable oversight by signaling microdomains, especially those associated with the release of von Willebrand factor. When summed, we believe that it is clear that the organization and regulation of signaling microdomains is an essential component to vessel wall function

Direct soluble guanylate cyclase activation improves vascular function in a mouse model of sickle cell disease

Sickle cell disease (SCD) is caused by a point mutation in the hemoglobin (Hb) β gene, which generates hemoglobin S (HbS). A less soluble tetramer that undergoes polymerization. Endothelial dysfunction caused by chronic intravascular hemolysis is a hallmark of SCD affecting multiple organs, including the lung. A common complication of SCD is pulmonary arterial hypertension (PAH) which is associated with early mortality. In the present study, we use an established animal model of SCD to investigate novel soluble guanylate cyclase (sGC) targeting in the pathobiology of global vascular dysfunction. \ud The transgenic BERK sickle mouse develops significant vascular dysfunction with age. Novel targeting of smooth muscle cell sGC represents a novel therapeutic approach for global vascular dysfunction

Intercalated Cell BKα Subunit is Required for Flow-Induced K+ Secretion

BK channels are expressed in intercalated cells (ICs) and principal cells (PCs) in the cortical collecting duct (CCD) of the mammalian kidney and have been proposed to be responsible for flow-induced K+ secretion (FIKS) and K+ adaptation. To examine the IC-specific role of BK channels, we generated a mouse with targeted disruption of the pore-forming BK α subunit (BKα) in ICs (IC-BKα-KO). Whole cell charybdotoxin-sensitive (ChTX-sensitive) K+ currents were readily detected in control ICs but largely absent in ICs of IC-BKα-KO mice. When placed on a high K+ (HK) diet for 13 days, blood [K+] was significantly greater in IC-BKα-KO mice versus controls in males only, although urinary K+ excretion rates following isotonic volume expansion were similar in males and females. FIKS was present in microperfused CCDs isolated from controls but was absent in IC-BKα-KO CCDs of both sexes. Also, flow-stimulated epithelial Na+ channel-mediated (ENaC-mediated) Na+ absorption was greater in CCDs from female IC-BKα-KO mice than in CCDs from males. Our results confirm a critical role of IC BK channels in FIKS. Sex contributes to the capacity for adaptation to a HK diet in IC-BKα-KO mice