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

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

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