Serum- and glucocorticoid-regulated kinase 1 (SGK1) is an aldosterone-regulated early response gene product that regulates the activity of several ion transport proteins, most notably that of the epithelial sodium channel (ENaC). Recent evidence has established that SGK1 phosphorylates and inhibits Nedd4-2 (neural precursor cell-expressed, developmentally down-regulated protein 4-2), a ubiquitin ligase that decreases cell surface expression of the channel and possibly stimulates its degradation. The mechanistic basis for this SGK1-induced Nedd4-2 inhibition is currently unknown. In this study we show that SGK1-mediated phosphorylation of Nedd4-2 induces its interaction with members of the 14-3-3 family of regulatory proteins. Through functional characterization of Nedd4-2-mutant proteins, we demonstrate that this interaction is required for SGK1-mediated inhibition of Nedd4-2. The concerted action of SGK1 and 14-3-3 appears to disrupt Nedd4-2-mediated ubiquitination of ENaC, thus providing a mechanism by which SGK1 modulates the ENaC-mediated Na(+) current. Finally, the expression pattern of 14-3-3 is also consistent with a functional role in distal nephron Na(+) transport. These results demonstrate a novel, physiologically significant role for 14-3-3 proteins in modulating ubiquitin ligase-dependent pathways in the control of epithelial ion transport.

Role of small conductance calcium-activated potassium channels expressed in PVN in regulating sympathetic nerve activity and arterial blood pressure in rats. Am J Physiol Regul Integr Comp Physiol 303: R301-R310, 2012. First published May 30, 2012 doi:10.1152/ajpregu.00114.2012.-Small conductance Ca 2ϩ -activated K ϩ (SK) channels regulate membrane properties of rostral ventrolateral medulla (RVLM) projecting hypothalamic paraventricular nucleus (PVN) neurons and inhibition of SK channels increases in vitro excitability. Here, we determined in vivo the role of PVN SK channels in regulating sympathetic nerve activity (SNA) and mean arterial pressure (MAP). In anesthetized rats, bilateral PVN microinjection of SK channel blocker with peptide apamin (0, 0.125, 1.25, 3.75, 12.5, and 25 pmol) increased splanchnic SNA (SSNA), renal SNA (RSNA), MAP, and heart rate (HR) in a dose-dependent manner. Maximum increases in SSNA, RSNA, MAP, and HR elicited by apamin (12.5 pmol, n ϭ 7) were 330 Ϯ 40% (P Ͻ 0.01), 271 Ϯ 40% (P Ͻ 0.01), 29 Ϯ 4 mmHg (P Ͻ 0.01), and 34 Ϯ 9 beats/min (P Ͻ 0.01), respectively. PVN injection of the nonpeptide SK channel blocker UCL1684 (250 pmol, n ϭ 7) significantly increased SSNA (P Ͻ 0.05), RSNA (P Ͻ 0.05), MAP (P Ͻ 0.05), and HR (P Ͻ 0.05). Neither apamin injected outside the PVN (12.5 pmol, n ϭ 6) nor peripheral administration of the same dose of apamin (12.5 pmol, n ϭ 5) evoked any significant changes in the recorded variables. PVNinjected SK channel enhancer 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (DCEBIO, 5 nmol, n ϭ 4) or N-cyclohexyl-N-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidin]amine (CyPPA, 5 nmol, n ϭ 6) did not significantly alter the SSNA, RSNA, MAP, and HR. Western blot and RT-PCR analysis of punched PVN tissue showed abundant expression of SK1-3 channels. We conclude that SK channels expressed in the PVN play an important role in the regulation of sympathetic outflow and cardiovascular function. paraventricular nucleus; heart rate; sympathetic nervous system; cardiovascular function ACTIVITY of presympathetic hypothalamic paraventricular nucleus (PVN) neurons regulate the sympathetic nervous system (11,12,47,51) response to physiological challenges. These neurons also play a well-described role in activating the sympathetic nervous system in cardiovascular diseases (17,28,36,39,47,48,51,53). Even with the significant literature support for the role of presympathetic PVN neurons in controlling sympathetic nerve activity (SNA) and arterial blood pressure (ABP), not much is known about the intrinsic membrane properties of these neurons and their effect on cardiovascular function.The excitability of presympathetic PVN neurons is regulated by the integration of synaptic inputs with intrinsic membrane properties. Evidence for this is shown in PVN neurotransmitter systems that include GABA (30, 38), glutamate (9, 28, 31), nitric oxide (4, 20), and ANG II (7, 13, 29), which modulate the activity of presympathetic PVN neurons in vitro and SNA in vivo. One class of cha...

The epithelial Na ؉ channel (ENaC) plays a central role in control of epithelial surface hydration and vascular volume. Similar to other ion channels, ENaC activity is set, in part, by its membrane levels. The small G protein RhoA increases ENaC activity by increasing the membrane levels of this channel. We hypothesize that RhoA increases ENaC activity by promoting channel trafficking to the plasma membrane. Few experimental methods are available to directly visualize trafficking of ion channels to the plasma membrane. Here we combine electrophysiology with two complementary imaging methods, total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching, to study the mechanistic basis of RhoA actions on ENaC. Patch clamp results demonstrate that RhoA increases ENaC activity in an additive manner with dominant-negative dynamin. This is consistent with a mechanism of increased ENaC trafficking to the membrane. Direct visualization of ENaC movement near the plasma membrane with total internal reflection fluorescence-fluorescence recovery after photobleaching revealed that RhoA accelerates ENaC trafficking toward the membrane. RhoA-facilitated movement of the channel was sensitive to disrupting the endomembrane system. Moreover, facilitating retrieval decreased ENaC activity but not trafficking toward the membrane. ENaC at the plasma membrane clustered and was laterally immobile suggesting that the cytoskeleton tethers or corrals membrane resident channels or membranedirected vesicles containing ENaC. Disrupting microtubules but not microfilaments led to reorganization of ENaC clusters and slowed trafficking toward the membrane. The cytoskeleton is an established target for RhoA signaling. We conclude that RhoA, likely through effects on the cytoskeleton, promotes ENaC trafficking to the plasma membrane to increase channel membrane levels and activity.Ion channels are integral membrane proteins. Regulated trafficking of these proteins to and from the plasma membrane in part controls their activity. This is true for ENaC, 3 which is a nonvoltage-gated, Na ϩ -selective ion channel. ENaC is localized to the apical plasma membrane of epithelia particularly that lining the distal renal nephron and colon and alveolar spaces (1-3). ENaC activity in this epithelia is limiting for Na ϩ (re)absorption. Thus, ENaC plays an important physiological role in negative feedback control of blood pressure, as well as epithelial surface hydration in the lungs. Indeed, improper regulation and dysfunction of ENaC are causative for some pulmonary and blood pressure disorders (4 -6).Corticosteroids are the primary endocrine modulators of ENaC activity. One mechanism by which these steroids increase ENaC activity is by increasing the levels of this channel in the apical plasma membrane (1-3). We recently reported that the small G protein RhoA similarly increases ENaC activity by elevating membrane levels of the channel (7, 8). Regulation of ion channels, including several types of K ϩ channels (e.g. KCNA, K...

Abstract-The hypothalamic paraventricular nucleus (PVN) plays an important role in the sympathoexcitatory response to elevated plasma angiotensin II (Ang II). However, the mechanism by which Ang II influences sympathetic activity is not fully understood.In this study, we tested the hypothesis that GABA(␥-aminobutyric acid)-ergic function in the PVN is reduced by peripheral infusion of Ang II. To accomplish this, rats received either intravenous Ang II (12 ng/kg per minute) or vehicle (D5W) for 7 days, and renal sympathetic nerve activity (SNA), mean arterial pressure (MAP), and heart rate (HR) responses were recorded after unilateral PVN microinjection of the GABA-A receptor antagonist bicuculline methiodide (BMI, 0.1 nmol). Results indicate that in contrast to a significant increase in renal SNA, MAP, and HR observed in vehicle-infused rats (PϽ0.05), BMI injection into the PVN of Ang II-infused animals was without effect on all recorded variables. In a separate groups of animals, ganglionic blockade produced a significantly greater fall in MAP (PϽ0.01) in Ang II-infused rats than in vehicle-infused control rats, indicating that the contribution of SNA to the maintenance of blood pressure was elevated in the Ang II-infused group. Overall, these data indicate that cardiovascular and sympathoexcitatory responses to acute GABA-A receptor antagonism in the PVN are significantly blunted in rats after 7 days of intravenous infusion of Ang II. We conclude that an Ang II-induced reduction in GABAergic inhibition within the PVN may contribute to elevated SNA observed in this study. Key Words: angiotensin II Ⅲ hypothalamus Ⅲ sympathetic nervous system Ⅲ hypertension, arterial A s the dominant inhibitory neurotransmitter in the mammalian brain, ␥-aminobutyric acid (GABA) 1 plays an important role in regulating cardiovascular function. 2,3 In brain regions such as the nucleus tractus solitarius 2 and rostral ventrolateral medulla 2,3 that control sympathetic nerve activity (SNA), GABA tonically suppresses neuronal activity and excitability. In the hypothalamic paraventricular nucleus (PVN), neuronal activity is also regulated by GABA 4 -6 and by a number of excitatory neurotransmitters as well. 7,8 Among the latter is the peptide angiotensin II (Ang II). Ang II-containing fibers arise from Ang II-sensitive regions of the forebrain known as circumventricular organs (CVO), which lack a complete blood-brain barrier. 9,10 Neurons in two forebrain CVOs, the subfornical organ and organum vasculosum of the lamina terminalis, express Ang II AT 1 receptors in high density 11 and appear to sense and respond to circulating Ang II. 9,12-15 Although Ang II increases SNA through these and other central actions, 16,17 the mechanism by which circulating Ang II enhances PVN neuronal excitability is not fully understood.On the basis of literature evidence, it is clear that GABA 4 -6 and Ang II 13-15 can each act individually within the PVN to influence cardiovascular function. More recently, it has been shown that local GABA-Ang II interacti...

Hypertension (HTN) resulting from subcutaneous infusion of ANG II and dietary high salt (HS) intake involves sympathoexcitation. Recently, we reported reduced small-conductance Ca(2+)-activated K(+) (SK) current and increased excitability of presympathetic neurons in the paraventricular nucleus (PVN) in ANG II-salt HTN. Here, we hypothesized that ANG II-salt HTN would be accompanied by altered PVN SK channel activity, which may contribute to sympathoexcitation in vivo. In anesthetized rats with normal salt (NS) intake, bilateral PVN microinjection of apamin (12.5 pmol/50 nl each), the SK channel blocker, remarkably elevated splanchnic sympathetic nerve activity (SSNA), renal sympathetic nerve activity (RSNA), and mean arterial pressure (MAP). In contrast, rats with ANG II-salt HTN demonstrated significantly attenuated SSNA, RSNA, and MAP (P < 0.05) responses to PVN-injected apamin compared with NS control rats. Next, we sought to examine the individual contributions of HS and subcutaneous infusion of ANG II on PVN SK channel function. SSNA, RSNA, and MAP responses to PVN-injected apamin in rats with HS alone were significantly attenuated compared with NS-fed rats. In contrast, sympathetic nerve activity responses to PVN-injected apamin in ANG II-treated rats were slightly attenuated with SSNA, demonstrating no statistical difference compared with NS-fed rats, whereas MAP responses to PVN-injected apamin were similar to NS-fed rats. Finally, Western blot analysis showed no statistical difference in SK1-SK3 expression in the PVN between NS and ANG II-salt HTN. We conclude that reduced SK channel function in the PVN is involved in the sympathoexcitation associated with ANG II-salt HTN. Dietary HS may play a dominant role in reducing SK channel function, thus contributing to sympathoexcitation in ANG II-salt HTN.