High salt intake down-regulates colonic mineralocorticoid receptors, epithelial sodium channels and 11β-hydroxysteroid dehydrogenase type 2

Besides the kidneys, the gastrointestinal tract is the principal organ responsible for sodium homeostasis. For sodium transport across the cell membranes the epithelial sodium channel (ENaC) is of pivotal relevance. The ENaC is mainly regulated by mineralocorticoid receptor mediated actions. The MR activation by endogenous 11β-hydroxy-glucocorticoids is modulated by the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). Here we present evidence for intestinal segment specific 11β-HSD2 expression and hypothesize that a high salt intake and/or uninephrectomy (UNX) affects colonic 11β-HSD2, MR and ENaC expression. The 11β-HSD2 activity was measured by means of 3H-corticosterone conversion into 3H-11-dehydrocorticosterone in Sprague Dawley rats on a normal and high salt diet. The activity increased steadily from the ileum to the distal colon by a factor of about 3, an observation in line with the relevance of the distal colon for sodium handling. High salt intake diminished mRNA and protein of 11β-HSD2 by about 50% (p<0.001) and reduced the expression of the MR (p<0.01). The functionally relevant ENaC-β and ENaC-γ expression, a measure of mineralocorticoid action, diminished by more than 50% by high salt intake (p<0.001). The observed changes were present in rats with and without UNX. Thus, colonic epithelial cells appear to contribute to the protective armamentarium of the mammalian body against salt overload, a mechanism not modulated by UNX.

Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain

Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Navs) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Na(v)s remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Navs, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury-induced neuropathic pain was used, and an Na(v)1.7-specific inhibitor, ProTxII, allowed the isolation of Na(v)1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Na(v)1.7 and Na(v)1.8 currents. The redistribution of Na(v)1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L(-/-)). SNS-Nedd4L(-/-) mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Na(v)1.7 and Na(v)1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Na(v)s and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain.

Mice carrying ubiquitin-specific protease 2 (Usp2) gene inactivation maintain normal sodium balance and blood pressure

Ubiquitylation plays an important role in the control of Na⁺ homeostasis by the kidney. It is well established that the epithelial Na⁺ channel ENaC is regulated by the ubiquitin-protein ligase NEDD4-2, limiting ENaC cell surface expression and activity. Ubiquitylation can be reversed by the action of deubiquitylating enzymes (DUBs). One such DUB, USP2-45, was identified previously as an aldosterone-induced protein in the kidney and is also a circadian output gene. In heterologous expression systems, USP2-45 binds to ENaC, deubiquitylates it, and enhances channel density and activity at the cell surface. Because the role of USP2-45 in renal Na⁺ transport had not been studied in vivo, we investigated here the effect of Usp2 gene inactivation in this process. We demonstrate first that USP2-45 protein has a rhythmic expression with a peak at ZT12. Usp2-KO mice did not show any differences from wild-type littermates with respect to the diurnal control of Na⁺ or K⁺ urinary excretion and plasma levels either on a standard diet or after acute and chronic changes to low- and high-Na⁺ diets, respectively. Moreover, they had similar aldosterone levels on either a low- or high-Na⁺ diet. Blood pressure measurements using telemetry did not reveal variations compared with control mice. Usp2-KO mice did not display alterations in expression of genes involved in sodium homeostasis or the ubiquitin system, as evidenced by transcriptome analysis in the kidney. Our data suggest that USP2 does not play a primary role in the control of Na⁺ balance or blood pressure.

Sodium/hydrogen exchanger NHA2 in osteoclasts: subcellular localization and role in vitro and in vivo

NHA2 was recently identified as a novel sodium/hydrogen exchanger which is strongly upregulated during RANKL-induced osteoclast differentiation. Previous in vitro studies suggested that NHA2 is a mitochondrial transporter required for osteoclast differentiation and bone resorption. Due to the lack of suitable antibodies, NHA2 was studied only on RNA level thus far. To define the protein's role in osteoclasts in vitro and in vivo, we generated NHA2-deficient mice and raised several specific NHA2 antibodies. By confocal microscopy and subcellular fractionation studies, NHA2 was found to co-localize with the late endosomal and lysosomal marker LAMP1 and the V-ATPase a3 subunit, but not with mitochondrial markers. Immunofluorescence studies and surface biotinylation experiments further revealed that NHA2 was highly enriched in the plasma membrane of osteoclasts, localizing to the basolateral membrane of polarized osteoclasts. Despite strong upregulation of NHA2 during RANKL-induced osteoclast differentiation, however, structural parameters of bone, quantified by high-resolution microcomputed tomography, were not different in NHA2-deficient mice compared to wild-type littermates. In addition, in vitro RANKL stimulation of bone marrow cells isolated from wild-type and NHA2-deficient mice yielded no differences in osteoclast development and activity. Taken together, we show that NHA2 is a RANKL-induced plasmalemmal sodium/hydrogen exchanger in osteoclasts. However, our data from NHA2-deficient mice suggest that NHA2 is dispensable for osteoclast differentiation and bone resorption both in vitro and in vivo.

Cardiac sodium channel Na(v)1.5 and interacting proteins: Physiology and pathophysiology

The cardiac voltage-gated Na(+) channel Na(v)1.5 generates the cardiac Na(+) current (INa). Mutations in SCN5A, the gene encoding Na(v)1.5, have been linked to many cardiac phenotypes, including the congenital and acquired long QT syndrome, Brugada syndrome, conduction slowing, sick sinus syndrome, atrial fibrillation, and dilated cardiomyopathy. The mutations in SCN5A define a sub-group of Na(v)1.5/SCN5A-related phenotypes among cardiac genetic channelopathies. Several research groups have proposed that Na(v)1.5 may be part of multi-protein complexes composed of Na(v)1.5-interacting proteins which regulate channel expression and function. The genes encoding these regulatory proteins have also been found to be mutated in patients with inherited forms of cardiac arrhythmias. The proteins that associate with Na(v)1.5 may be classified as (1) anchoring/adaptor proteins, (2) enzymes interacting with and modifying the channel, and (3) proteins modulating the biophysical properties of Na(v)1.5 upon binding. The aim of this article is to review these Na(v)1.5 partner proteins and to discuss how they may regulate the channel's biology and function. These recent investigations have revealed that the expression level, cellular localization, and activity of Na(v)1.5 are finely regulated by complex molecular and cellular mechanisms that we are only beginning to understand.

Deubiquitylating enzyme USP2 counteracts Nedd4-2-mediated downregulation of KCNQ1 potassium channels

KCNQ1 (Kv7.1), together with its KCNE β subunits, plays a pivotal role both in the repolarization of cardiac tissue and in water and salt transport across epithelial membranes. Nedd4/Nedd4-like (neuronal precursor cell-expressed developmentally downregulated 4) ubiquitin-protein ligases interact with the KCNQ1 potassium channel through a PY motif located in the C terminus of KCNQ1. This interaction induces ubiquitylation of KCNQ1, resulting in a reduced surface density of the channel. It was reported recently that the epithelial sodium channel is regulated by the reverse process-deubiquitylation-mediated by USP2 (ubiquitin-specific protease 2).

Neuronal precursor cell-expressed developmentally down-regulated 4-1 (NEDD4-1) controls the sorting of newly synthesized Ca(V)1.2 calcium channels

Neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) proteins are ubiquitin ligases, which attach ubiquitin moieties to their target proteins, a post-translational modification that is most commonly associated with protein degradation. Nedd4 ubiquitin ligases have been shown to down-regulate both potassium and sodium channels. In this study, we investigated whether Nedd4 ubiquitin ligases also regulate Ca(v) calcium channels. We expressed three Nedd4 family members, Nedd4-1, Nedd4-2, and WWP2, together with Ca(v)1.2 channels in tsA-201 cells. We found that Nedd4-1 dramatically decreased Ca(v) whole-cell currents, whereas Nedd4-2 and WWP2 failed to regulate the current. Surface biotinylation assays revealed that Nedd4-1 decreased the number of channels inserted at the plasma membrane. Western blots also showed a concomitant decrease in the total expression of the channels. Surprisingly, however, neither the Ca(v) pore-forming α1 subunit nor the associated Ca(v)β and Ca(v)α(2)δ subunits were ubiquitylated by Nedd4-1. The proteasome inhibitor MG132 prevented the degradation of Ca(v) channels, whereas monodansylcadaverine and chloroquine partially antagonized the Nedd4-1-induced regulation of Ca(v) currents. Remarkably, the effect of Nedd4-1 was fully prevented by brefeldin A. These data suggest that Nedd4-1 promotes the sorting of newly synthesized Ca(v) channels for degradation by both the proteasome and the lysosome. Most importantly, Nedd4-1-induced regulation required the co-expression of Ca(v)β subunits, known to antagonize the retention of the channels in the endoplasmic reticulum. Altogether, our results suggest that Nedd4-1 interferes with the chaperon role of Ca(v)β at the endoplasmic reticulum/Golgi level to prevent the delivery of Ca(v) channels at the plasma membrane.

Regulation of the cardiac sodium channel Nav1.5 by utrophin in dystrophin-deficient mice

Duchenne muscular dystrophy (DMD) is a severe striated muscle disease due to the absence of dystrophin. Dystrophin deficiency results in dysfunctional sodium channels and conduction abnormalities in hearts of mdx mice. Disease progression in the mdx mouse only modestly reflects that of DMD patients, possibly due to utrophin up-regulation. Here, we investigated mice deficient in both dystrophin and utrophin [double knockout (DKO)] to assess the role of utrophin in the regulation of the cardiac sodium channel (Na(v)1.5) in mdx mice.

Cardiac Sodium Channel Nav1.5 Mechanosensitivity is Inhibited by Ranolazine

The cardiac action potential (AP) is initiated by the depolarizing inward sodium current (I(Na)). The pore-forming subunit of the cardiac sodium channel, Na(v)1.5, is the main ion channel that conducts I(Na) in cardiac cells. Despite the large number of studies investigating Na(v)1.5, year after year, we are still learning new aspects regarding its roles in normal cardiac function and in diseased states. The clinical relevance of this channel cannot be understated. The cardiac I(Na) is the target of the class 1 anti-arrhythmic drugs(1), which are nowadays less frequently prescribed because of their well-documented pro-arrhythmic properties(2). In addition, since the first description in 1995 by Keating's group(3) of mutations in patients suffering from congenital long QT syndrome (LQTS) type 3, several hundred genetic variants in SCN5A, the gene coding for Na(v)1.5, have been reported and investigated(4). Interestingly, many of these genetic variants have been found in patients with diverse cardiac manifestations(5) such as congenital LQTS type 3, Brugada syndrome, conduction disorders, and more recently, atrial fibrillation and dilated cardiomyopathy. This impressive list underlines the importance of Na(v)1.5 in cardiac pathologies and raises the question about possible unknown roles and regulatory mechanisms of this channel in cardiac cells. Recent studies have provided experimental evidence that the function of Na(v)1.5, among many other described regulatory mechanisms(6), is also modulated by the mechanical stretch of the membrane in which it is embedded(7), thus suggesting that Na(v)1.5, like other ion channels, is "mechanosensitive". What does this mean? (SELECT FULL TEXT TO CONTINUE).

Neuronal expression of the ubiquitin ligase Nedd4-2 in rat dorsal root ganglia: modulation in the spared nerve injury model of neuropathic pain

Neuronal hyperexcitability following peripheral nerve lesions may stem from altered activity of voltage-gated sodium channels (VGSCs), which gives rise to allodynia or hyperalgesia. In vitro, the ubiquitin ligase Nedd4-2 is a negative regulator of VGSC α-subunits (Na(v)), in particular Na(v)1.7, a key actor in nociceptor excitability. We therefore studied Nedd4-2 in rat nociceptors, its co-expression with Na(v)1.7 and Na(v)1.8, and its regulation in pathology. Adult rats were submitted to the spared nerve injury (SNI) model of neuropathic pain or injected with complete Freund's adjuvant (CFA), a model of inflammatory pain. L4 dorsal root ganglia (DRG) were analyzed in sham-operated animals, seven days after SNI and 48 h after CFA with immunofluorescence and Western blot. We observed Nedd4-2 expression in almost 50% of DRG neurons, mostly small and medium-sized. A preponderant localization is found in the non-peptidergic sub-population. Additionally, 55.7 ± 2.7% and 55.0 ± 3.6% of Nedd4-2-positive cells are co-labeled with Na(v)1.7 and Na(v)1.8 respectively. SNI significantly decreases the proportion of Nedd4-2-positive neurons from 45.9 ± 1.9% to 33.5 ± 0.7% (p<0.01) and the total Nedd4-2 protein to 44% ± 0.13% of its basal level (p<0.01, n=4 animals in each group, mean ± SEM). In contrast, no change in Nedd4-2 was found after peripheral inflammation induced by CFA. These results indicate that Nedd4-2 is present in nociceptive neurons, is downregulated after peripheral nerve injury, and might therefore contribute to the dysregulation of Na(v)s involved in the hyperexcitability associated with peripheral nerve injuries.

Rufinamide attenuates mechanical allodynia in a model of neuropathic pain in the mouse and stabilizes voltage-gated sodium channel inactivated state

Background: Voltage-gated sodium channels dysregulation is important for hyperexcitability leading to pain persistence. Sodium channel blockers currently used to treat neuropathic pain are poorly tolerated. Getting new molecules to clinical use is laborious. We here propose a drug already marketed as anticonvulsant, rufinamide. Methods: We compared the behavioral effect of rufinamide to amitriptyline using the Spared Nerve Injury neuropathic pain model in mice. We compared the effect of rufinamide on sodium currents using in vitro patch clamp in cells expressing the voltage-gated sodium channel Nav1.7 isoform and on dissociated dorsal root ganglion neurons to amitriptyline and mexiletine. Results: In naive mice, amitriptyline (20 mg/kg) increased withdrawal threshold to mechanical stimulation from 1.3 (0.6–1.9) (median [95% CI]) to 2.3 g (2.2–2.5) and latency of withdrawal to heat stimulation from 13.1 (10.4–15.5) to 30.0 s (21.8–31.9), whereas rufinamide had no effect. Rufinamide and amitriptyline alleviated injury-induced mechanical allodynia for 4 h (maximal effect: 0.10 ± 0.03 g (mean ± SD) to 1.99 ± 0.26 g for rufinamide and 0.25 ± 0.22 g to 1.92 ± 0.85 g for amitriptyline). All drugs reduced peak current and stabilized the inactivated state of voltage-gated sodium channel Nav1.7, with similar effects in dorsal root ganglion neurons. Conclusions: At doses alleviating neuropathic pain, amitriptyline showed alteration of behavioral response possibly related to either alteration of basal pain sensitivity or sedative effect or both. Side-effects and drug tolerance/compliance are major problems with drugs such as amitriptyline. Rufinamide seems to have a better tolerability profile and could be a new alternative to explore for the treatment of neuropathic pain.

Sodium nitroprusside induces cell death and cytoskeleton degradation in adult rat cardiomyocytes in vitro: implications for anthracycline-induced cardiotoxicity

Sodium nitroprusside (SNP) is used clinically as a rapid-acting vasodilator and in experimental models as donor of nitric oxide (NO). High concentrations of NO have been reported to induce cardiotoxic effects including apoptosis by the formation of reactive oxygen species. We have therefore investigated effects of SNP on the myofibrillar cytoskeleton, contractility and cell death in long-term cultured adult rat cardiomyocytes at different time points after treatment. Our results show, that SNP treatment at first results in a gradual increase of cytoskeleton degradation marked by the loss of actin labeling and fragmentation of sarcomeric structure, followed by the appearance of TUNEL-positive nuclei. Already lower doses of SNP decreased contractility of cardiomyocytes paced at 2 Hz without changes of intracellular calcium concentration. Ultrastructural analysis of the cultured cells demonstrated mitochondrial changes and disintegration of sarcomeric alignment. These adverse effects of SNP in cardiomyocytes were reminiscent of anthracycline-induced cardiotoxicity, which also involves a dysregulation of NO with the consequence of myofibrillar degradation and ultimately cell death. An inhibition of the pathways leading to the generation of reactive NO products, or their neutralization, may be of significant therapeutic benefit for both SNP and anthracycline-induced cardiotoxicity.

Inhibition of the human epithelial calcium channel TRPV6 by 2-aminoethoxydiphenyl borate (2-APB)

TRPV6, a highly calcium-selective member of the transient receptor potential (TRP) channel superfamily, is a major pathway for calcium absorption in the fetal and adult body. It is expressed abundantly in the duodenum, the placenta and exocrine tissues. TRVP6 was postulated to contribute to store-operated calcium channel (SOC) activity in certain cell types such as exocrine cells. In this study, we tested 2-APB, a widely used SOC inhibitor on human TRPV6 (hTRPV6) activity using fluorescence imaging, patch clamp and radioactive tracer techniques in transiently and stably transfected HEK293 cells. We found that the basal calcium and cadmium influx was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells. 2-APB inhibited hTRPV6 activity in both transient and stably transfected cells. This effect was slightly sensitive toward extracellular calcium. The extracellular sodium concentration did not affect the inhibition of hTRPV6 by 2-APB. However, N-methyl-d-glucamine significantly diminished the inhibitory effect of 2-APB presumably through direct interaction with this compound. Furthermore, 2-APB inhibited the activity of TRPV6 orthologs but not human TRPV5. 2-APB may serve as a parental compound for the development of therapeutic strategies specifically targeting the hTRPV6 calcium channel.

Sodium/hydrogen exchanger NHA2 is critical for insulin secretion in β-cells

NHA2 is a sodium/hydrogen exchanger with unknown physiological function. Here we show that NHA2 is present in rodent and human β-cells, as well as β-cell lines. In vivo, two different strains of NHA2-deficient mice displayed a pathological glucose tolerance with impaired insulin secretion but normal peripheral insulin sensitivity. In vitro, islets of NHA2-deficient and heterozygous mice, NHA2-depleted Min6 cells, or islets treated with an NHA2 inhibitor exhibited reduced sulfonylurea- and secretagogue-induced insulin secretion. The secretory deficit could be rescued by overexpression of a wild-type, but not a functionally dead, NHA2 transporter. NHA2 deficiency did not affect insulin synthesis or maturation and had no impact on basal or glucose-induced intracellular Ca(2+) homeostasis in islets. Subcellular fractionation and imaging studies demonstrated that NHA2 resides in transferrin-positive endosomes and synaptic-like microvesicles but not in insulin-containing large dense core vesicles in β-cells. Loss of NHA2 inhibited clathrin-dependent, but not clathrin-independent, endocytosis in Min6 and primary β-cells, suggesting defective endo-exocytosis coupling as the underlying mechanism for the secretory deficit. Collectively, our in vitro and in vivo studies reveal the sodium/proton exchanger NHA2 as a critical player for insulin secretion in the β-cell. In addition, our study sheds light on the biological function of a member of this recently cloned family of transporters.