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Am J Physiol Cell Physiol 290: C404-C410, 2006. First published September 28, 2005; doi:10.1152/ajpcell.00210.2005
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NERVOUS SYSTEM CELL BIOLOGY

ENaC proteins are required for NGF-induced neurite growth

Heather A. Drummond, Marise M. Furtado, Samuel Myers, Samira Grifoni, Kimberly A. Parker, Angela Hoover, and David E. Stec

Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, Mississippi

Submitted 2 May 2005 ; accepted in final form 22 September 2005


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Neurite growth is required for nervous system development and repair. Multiple signals, including neurotrophic factors and intact mechanosensing mechanisms, interact to regulate neurite growth. Degenerin/epithelial Na+ channel (DEG/ENaC) proteins have been identified as putative mechanosensors in sensory neurons. Recently, others have shown that the neurotrophic factor NGF stimulates expression of acid-sensing ion channel molecules, which are members of the DEG/ENaC family. However, it is unknown whether NGF regulates ENaC expression or whether ENaC expression is required for neurite formation. Therefore, the aims of the present study were to determine whether ENaC expression is 1) regulated by NGF and 2) required for NGF-induced neurite growth in pheochromocytoma PC-12 cells. We found NGF-induced expression of {beta}- and {gamma}-subunits of ENaC, but not {alpha}-ENaC. Tyrosine kinase A (TrkA) receptor blockade abolished NGF-induced {beta}- and {gamma}-ENaC expression and neurite formation. NGF-induced neurite formation was inhibited by disruption of ENaC expression using 1) pharmacological blockade with benzamil, a specific ENaC inhibitor; 2) small interfering RNA; and 3) dominant-negative ENaC molecules. These data indicate NGF-TrkA regulation of ENaC expression may be required for neurite growth and may suggest a novel role for DEG/ENaC proteins in neuronal remodeling and differentiation.

mechanosensation; degenerins; neurotrophins; tyrosine kinase A; pheochromocytoma cells

NEURITE GROWTH IS REQUIRED for development of the nervous system, target organ innervation, neural control of organ systems, and nerve regeneration after neuronal injury. Neurotrophic factors are well-studied regulators of neurite growth and include NGF, brain-derived neurotrophic factor, neurotrophin-3, and IGF (5, 11, 26). NGF-stimulated neurite growth is mediated primarily by activation of high-affinity tyrosine kinase A (TrkA) receptors and, to a smaller extent, activation of low-affinity p75 receptors (3, 8). However, the downstream mechanisms by which neurotrophic factors stimulate neurite formation are not fully understood.

One potential mechanism by which neurotrophins stimulate neurite formation may be by inducing expression of putative mechanosensory elements, which permit interactions between neurons and their extracellular environment. Three lines of evidence support this possibility. First, mechanosensitive proteins are found at the site of growth in nerve endings and growth cones (4, 25, 27, 28, 43). Second, normal neurite formation and elongation require activation of mechanosensitive proteins (17, 32, 39). Third, neurotrophic factors upregulate putative mechanosensory proteins in sensory neurons (7, 29, 30). These data suggest that mechanosensitive elements contribute to neurite growth and may be regulated by neurotrophic factors.

Members of the degenerin/epithelial Na+ channel (DEG/ENaC) protein family have been identified as potential components of mechanosensitive ion channel complexes involved in baroreception, touch sensation, and proprioception in sensory neurons (1, 9, 13, 15, 33, 35). Despite the fact that acid-sensing ion channel (ASIC) transcripts, which are DEG/ENaC family members, are NGF targets (10, 29, 30), the role of mechanosensory DEG/ENaC proteins in neurite formation has never been addressed. Therefore, we wanted to address the hypothesis that NGF induces neurite formation by stimulating the expression of mechanosensory ENaC proteins. To address this hypothesis, we sought to determine whether 1) NGF increases the expression of ENaC message and protein, 2) NGF-induced ENaC expression is mediated by TrkA receptor activation, and 3) disruption of ENaC and ASIC1 expression prevents NGF-induced neurite growth in neuronal pheochromocytoma (PC)-12 cells. Our results suggest that ENaC expression is regulated by NGF-TrkA/p75 receptor interactions and is required for NGF-induced neurite growth.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
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PC-12 cell culture. Rat PC-12 cells (American Type Culture Collection, Manassas, VA), a neuronal cell model commonly used to study neurite formation, were maintained in 75-cm2 culture flasks in culture medium [Kaighn's modification of Ham's F-12 medium (F12K; Invitrogen, Carlsbad, CA) supplemented with 1.5 g/l sodium bicarbonate, 15% horse serum, 2.5% FBS, and 1% penicillin/streptomycin] at 37°C in a 5% CO2 atmosphere. For all experiments, PC-12 cells were plated at 2.5 x 105 cells/ml on 100-mm2 culture dishes or collagen-coated eight-well glass slides. To determine NGF regulation, culture medium was supplemented with NGF (10–100 ng/ml) for 24 h, followed by RT-PCR or immunostaining to detect ENaC message or protein as described below. To determine whether TrkA or p75 receptors mediated the response, anti-TrkA and anti-p75 receptor antibodies (2 ng/ml; Abcam, Cambridge, MA) were added to the NGF-supplemented medium. Both antibodies have been used previously to block TrkA and p75 receptor activation (30). Cultures were then immunostained to detect changes in {beta}- and {gamma}-ENaC protein or assayed for neurite growth as described below.

RT-PCR. We used RT-PCR to determine whether NGF induces {alpha}-, {beta}-, and {gamma}-ENaC transcript expression. Total RNA was isolated from harvested cells using RNA STAT-60 (Tel-Test, Friendswood, TX) that was DNase treated (Ambion, Austin, TX) and quantified using spectrophotometry. RNA (1 µg) was reverse transcribed using random hexamers and avian myeloblastosis virus RT (Promega, Madison, WI). cDNA was amplified using the following primer sequences: {alpha}-rENaC, 5'-AGTACCTCAGCTACCCAGTGAGC-3' and 5'-TTGACCGGGACATCGCTACCAT-3'; {beta}-rENaC, 5'-CAAGAAGAAGGCCATGTGGT-3' and 5'-GTACTGGAAGGGGCTGGAAT-3'; and {gamma}-rENaC, 5'-AATCCTTACAGATACACTG-3' and 5'-TTCCTTTCTCATACTGATG-3'. Samples were preheated on a Robocycler (Stratagene, La Jolla, CA) to 94°C for 3 min and then cycled 40 times at 94°C for 20 s, at 60°C ({alpha}-rENaC) or at 55°C ({beta}-rENaC and {gamma}-rENaC) for 20 s, and at 72°C for 1 min. As negative controls, RT was omitted from the reverse transcription reaction. PCR products were separated using electrophoresis, visualized using ethidium bromide, and sequenced to confirm identity. Expected PCR product sizes were as follows: {alpha}-rENaC, 900 bp; {beta}-rENaC, 177 bp; and {gamma}-rENaC, 376 bp.

Semiquantitative immunostaining. To study the effects of NGF and neurotrophin receptor blockade and small interfering RNA (siRNA) on protein expression, we used semiquantitative immunostaining. To use immunofluorescence in a semiquantitative manner, we took extreme care to maintain uniform experimental conditions. To minimize sampling variability, the following steps were taken. All samples (control and experimental) were 1) run in parallel and treated identically, 2) examined under identical conditions, and 3) repeated at least three times. Antigen affinity-purified rabbit anti-{beta}-ENaC and sheep anti-{gamma}-ENaC antibodies (1:100 dilution) have been used previously for immunolabeling (13, 14). Rabbit anti-ASIC1 was obtained from Chemicon International (Temecula, CA). Cells were plated onto collagen-coated eight-well glass slides as described above and incubated with NGF (10–100 ng/ml) for 24 h to induce neurite formation. Immediately afterward, all samples were washed in PBS, fixed in 4% paraformaldehyde for 10 min, and then rinsed with PBS. Samples were air dried on a 37°C block so that cells adhered to the slides. After being dried, samples were reequilibrated in PBS and blocked with 5% normal donkey serum (NDS) for 1 h. Cells were then coincubated with anti-ENaC antibodies in 5% NDS (rabbit anti-{beta}-ENaC and sheep anti-{gamma}-ENaC, 1:100 dilution) overnight at 4°C, washed with PBS, and incubated with fluorescence-labeled secondary antibodies for 1 h [Cy3-conjugated donkey anti-rabbit F(ab')2 (1:100 dilution; Jackson ImmunoResearch, West Grove, PA) and Alexa Fluor 488-conjugated donkey anti-sheep IgG (1:1,000 dilution; Molecular Probes, Eugene, OR)]. Samples were rinsed with PBS and coverslipped with Gel Mount (Biomeda, Foster City, CA). Immunolabeling was examined using a laser scanning confocal microscope (Leica Microsystems, Exton, PA). Fluorescence intensity was normalized for cell size and calculated as relative fluorescence units/cell area (µm2).

Neurite growth assay. Neurite growth was assessed using a standard neurite growth assay (23, 24). PC-12 cells were plated onto collagen-coated eight-well coverglass slides as described previously. Cells were examined using transmitted light confocal microscopy, and cells with neurites twice the length of the soma were considered to have neurites. Neurite growth was quantified as the percentage of cells with neurites. Clumped cells were eliminated from the analysis. Approximately 100 neurons per group were included in the analysis.

To confirm TrkA and p75 receptor involvement in NGF-stimulated neurite formation, cells plated onto eight-well slides were coincubated with NGF and anti-TrkA and/or p75 receptor antibodies as described earlier. To determine whether pharmacological ENaC inhibition blocks neurite formation, cells were treated with NGF (50 ng/ml) in the presence of benzamil (100 nM-10 µM; Biomol, Plymouth Meeting, PA), a specific ENaC inhibitor, for 24 h. To determine whether expression disruption of specific ENaC molecules blocks NGF-induced neurite formation, cultures were transfected with {beta}- and/or {gamma}-ENaC siRNA or dominant-negative molecules and maintained for 48–72 h. Neurite formation was induced with NGF (50 ng/ml, 24 h) and assessed using a standard neurite growth assay described previously.

siRNA and dominant-negative fusion molecules. To determine whether specific DEG/ENaC proteins are required for NGF-induced neurite formation, we knocked down message and protein using siRNA and dominant-negative fusion molecule constructs.

Validated siRNA molecules directed to {beta}-rENaC (Scnn_1b, no. 16804), {gamma}-rENaC (Scnn_1g, no. 16704), and rASIC1 (ACCN2, no. 52768) were purchased from Ambion. As a negative control, we used a nontargeting siRNA control molecule that activates the RNA-inducing silencing complex (RISC, no. D001210-02; Dharmacon, Chicago, IL). Quantitation of knockdown was performed using semiquantitative immunofluorescence as detailed previously.

Dominant-negative constructs for ENaC proteins ({beta}I41X and {gamma}L160X) were generated using standard PCR cloning techniques. The extreme NH2-terminal coding regions of {beta}- and {gamma}-rENaC cDNA were ligated into enhanced green fluorescent protein (EGFP) expression vector (pEGFP-C1; BD Biosciences Clontech, Palo Alto, CA) at the EcoRI and BamHI restriction enzyme sites. Gene fusion regions were sequenced to confirm that the NH2-terminal coding regions were in frame with EGFP. A diagram of the expression constructs is shown in Fig. 5A. Dominant-negative constructs encoding similar regions of ENaC and other DEG family members are known to silence the expression of these proteins effectively (1, 21). Dominant-negative and siRNA molecules were transfected into PC-12 cells using lipid-mediated transfection.



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  		Fig. 5. Disrupting {beta}-ENaC and {gamma}-ENaC protein inhibits NGF-induced neurite growth. A: diagram of dominant-negative constructs. A cytomegalovirus (CMV) promoter drives expression of enhanced green fluorescent protein (EGFP) fused to the NH2-terminal portion of {beta}-ENaC or {gamma}-ENaC. B: representative images show that lipid-mediated transfection with EGFP-labeled {beta}-ENaC (EGFP-{beta}I41X) and {gamma}-ENaC (EGFP-{gamma}L160X) dominant-negative molecules had a transfection efficiency of ~80% on the basis of EGFP-transfected cells. C: transfection with 1 µg/ml EGFP-{beta}I41X and EGFP-{gamma}L160X prevented NGF-induced neurite growth. Data are means ± SE. *P ≤ 0.05 vs. NGF control.

 
Lipid-mediated cell transfection. To study the effect of decreased ENaC expression on NGF-induced neurite formation, PC-12 cells were transfected with siRNA or dominant-negative EGFP fusion molecules using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were grown on collagen-coated, eight-well glass slides at 2.5 x 105 cells/ml. Cells were transfected upon reaching 70% confluence. Lipofectamine 2000 was complexed with 1 µg/ml cDNA or 100 ng/ml siRNA at a 1:2 oligonucleotide-to-Lipofectamine ratio in OptiMEM I medium for 4 h. After this procedure, cultures were supplemented with culture medium and maintained for 48–72 h before addition of NGF.

Statistical analyses. Statistical analyses were performed using SigmaStat software, version 3.0 (SPSS, Chicago, IL). Groups were compared using ANOVA, and Student-Newman-Keuls post hoc tests were performed to determine statistical differences among groups (P ≤ 0.05).


   RESULTS
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 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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NGF induces expression of {beta}- and {gamma}-ENaC in PC-12 cells. To determine whether NGF stimulated ENaC message in PC-12 cells, we used RT-PCR. As shown in Fig. 1A, in NGF unstimulated cells, we could not detect expression of {alpha}-, {beta}-, or {gamma}-ENaC transcripts, even after performing a second round of PCR with nested primers (data not shown). However, after stimulation with NGF (50 ng/ml, 24 h), we were able to detect expression of {beta}- and {gamma}-ENaC, but not {alpha}-ENaC message. These data indicate that NGF induced {beta}-ENaC and {gamma}-ENaC expression at the transcriptional level. We observed similar results using immunostaining as shown in Fig. 1, B and C. In unstimulated cells, {beta}- and {gamma}-ENaC relative immunofluorescence intensity was not different from the no-primary-antibody negative control; however, exposure to NGF increased {beta}- and {gamma}-ENaC immunofluorescence intensity remarkably (23, 24) (Fig. 1, B and C, respectively). These data show that NGF induced {beta}- and {gamma}-ENaC message and protein in PC-12 cells.



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  		Fig. 1. NGF induces {beta}-epithelial Na+ channel ({beta}-ENaC) and {gamma}-ENaC expression in pheochromocytoma (PC)-12 cells. A: RT-PCR analysis showing that NGF (50 ng/ml, 24 h) stimulates {beta}-ENaC and {gamma}-ENaC, but not {alpha}-ENaC, message in PC-12 cells. Arrows denote predicted product size. Immunostaining shows that NGF (50 ng/ml, 24 h) also profoundly increases {beta}-ENaC (B) and {gamma}-ENaC (C) protein expression in PC-12 cells. Data are means ± SE of relative immunofluorescence intensity (RFU)/cell area (µm2). Representative immunostaining images are shown. Experiments were repeated at least 3 times. *P ≤ 0.05 vs. no-NGF control.

 
TrkA receptors primarily mediate NGF-induced {beta}- and {gamma}-ENaC expression and neurite growth. To determine whether TrkA or p75 receptors mediate NGF-induced ENaC expression, {beta}- and {gamma}-ENaC immunolabeling were examined in PC-12 cells treated with NGF (10–100 ng/ml) in the presence or absence of specific TrkA or p75 antibodies. As shown in Fig. 2, A and B, NGF produced a concentration-dependent increase in {beta}- and {gamma}-ENaC immunofluorescence intensity compared with unstimulated cells. TrkA receptor blockade prevented NGF-induced increases in {beta}- and {gamma}-ENaC expression. To a lesser extent, p75 receptor blockade also inhibited {beta}-ENaC immunolabeling at all NGF concentrations used. Unexpectedly, p75 receptor blockade at the highest NGF concentration (100 ng/ml) significantly increased NGF-induced {gamma}-ENaC immunofluorescence intensity.



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  		Fig. 2. NGF-induced {beta}- and {gamma}-ENaC expression is primarily mediated via the tyrosine kinase A (TrkA) receptor. NGF increased {beta}-ENaC (A) and {gamma}-ENaC (B) immunofluorescence in a concentration-dependent manner (open bars). TrkA receptor blockade (gray bars) with TrkA antibody (2 ng/ml) prevented NGF-induced {beta}- and {gamma}-ENaC immunofluorescence. Blocking p75 receptor (filled bars) with anti-p75 antibody (2 ng/ml) produced a smaller decrease in {beta}-ENaC but not {gamma}-ENaC immunofluorescence. C: TrkA receptor blockade also profoundly inhibited NGF-induced neurite growth, whereas p75 receptor blockade decreased NGF-mediated neurite growth to a lesser but significant extent. Data are means ± SE. *P ≤ 0.05 vs. NGF control.

 
As shown by others, NGF (50 ng/ml, 24 h) induced neurite formation in ~40% of cells, which was prevented by TrkA and to a lesser extent by p75 receptor blockade (Fig. 2C).

Disruption of ENaC activity prevents NGF-induced neurite growth. To determine whether ENaC expression is required for neurite formation, we studied NGF-induced neurite growth after ENaC inhibition. We used three different approaches to disrupt ENaC expression: 1) pharmacological blockade of ENaC activity with benzamil (6, 20), 2) gene silencing using siRNA, and 3) protein silencing using expression of dominant-negative molecules.

ENaC inhibition with benzamil blocks neurite formation. As shown in Fig. 3, benzamil (100 nM-10 µM) prevented NGF-induced neurite formation in a concentration-dependent manner. These data suggest that ENaC activity is required for NGF-induced neurite formation.



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  		Fig. 3. Benzamil (Benz) prevents NGF-induced neurite growth. The specific ENaC blocker benzamil decreased NGF-induced neurite growth in PC-12 cells in a concentration-dependent manner. Representative images are shown for some groups. Data are means ± SE. *P ≤ 0.05 vs. NGF control.

 
{beta}- and {gamma}-ENaC, but not ASIC1, siRNA molecules block neurite formation. To determine whether {beta}-ENaC, {gamma}-ENaC, and ASIC1 molecules are required for neurite formation, we silenced {beta}- and/or {gamma}-ENaC gene expression using siRNA. As shown in Fig. 4, siRNA molecules to {beta}-ENaC, {gamma}-ENaC, and ASIC1 (Fig. 4, A and B) suppress expression of their respective proteins. Transfection of {beta}- and/or {gamma}-ENaC siRNA molecules inhibited neurite formation compared with negative controls [Lipofectamine alone and nontargeting, RISC-activating siRNA molecules (Fig. 4C)]. These data indicate expression of {beta}- and {gamma}-ENaC, but not ASIC1, transcripts is required for NGF-mediated neurite formation.



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  		Fig. 4. Silencing {beta}-ENaC and {gamma}-ENaC, but not acid-sensing ion channel (ASIC)1, message inhibits NGF-induced neurite growth. A: quantitation of small interfering RNA (siRNA) on {beta}-ENaC, {gamma}-ENaC, and ASIC1 protein expression. siRNA molecules silenced the respective protein expression to or near background signal levels as indicated by dashed line. RISC, RNA-inducing silencing complex. B: representative images of {beta}-ENaC, {gamma}-ENaC, and ASIC1 immunolabeling 48 h after siRNA transfection. C: transfection with 100 ng/ml {beta}- and/or {gamma}-ENaC siRNA prevented NGF-induced neurite growth compared with Lipofectamine (Lfx) and nontargeting siRNA controls. ASIC1 siRNA did not disrupt neurite formation. Data are means ± SE. *P ≤ 0.05 vs. NGF control.

 
Expression of dominant-negative constructs inhibits NGF-induced neurite formation. In our third approach to silencing ENaC expression, we used dominant-negative {beta}- and {gamma}-ENaC molecules. Transfected cells were identified and visualized using EGFP and fluorescence microscopy. Transfection efficiency was estimated at ~80% on the basis of EGFP fluorescence (Fig. 5B). Compared with EGFP-transfected PC-12 cells, expression of the {beta}- and/or {gamma}-ENaC dominant-negative molecules (EGFP-{beta}-ENaCI41X and EGFP-{gamma}-ENaCL160X, respectively) nearly abolished NGF-induced neurite formation (Fig. 5C). Combined with our findings regarding benzamil and {beta}- and/or {gamma}-ENaC siRNA molecules, these data suggest {beta}- and {gamma}-ENaC expression are required for NGF-mediated neurite growth.


   DISCUSSION
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GRANTS
 REFERENCES
 
Mechanosensory processes contribute to neurite formation. Neurotrophic factor initiation of neurogenesis is well established (5, 11, 26). Yet, the downstream signaling events mediating neurite formation and remodeling are poorly understood. Studies from other laboratories have indicated that mechanosensitive processes are required for normal neurite formation. For example, gadolinium (stretch-activated channel blocker) or integrin disruption blocks neurite formation (36, 42). The role of DEG/ENaC proteins as mechanosensors in neurite formation has never been addressed, although evidence suggests that 1) expression of ASIC transcripts, which are DEG/ENaC family members, are targets of NGF regulation (10, 29, 30); and 2) DEG/ENaC channels may function as mechanosensors in sensory neurons (1, 9, 13, 15, 33, 35). In the present study, we describe two major findings addressing the role of ENaC proteins in neurite formation. First, {beta}- and {gamma}-ENaC expression are regulated by NGF-TrkA/p75 receptor interactions. Second, ENaC expression is required for neurite formation in neuronal PC-12 cells. These data provide evidence that DEG/ENaC proteins may contribute to neurite formation.

DEG/ENaC cation channel family. DEG/ENaC proteins are expressed in a diverse range of tissues and species in which many are known to form cation-selective ion channels. In epithelial tissue, {alpha}-, {beta}-, and {gamma}-ENaC proteins form a non-voltage-gated Na+-selective channel that contributes to Na+ and water transport in the kidney, lung, and colon (18, 22, 38). A growing body of evidence suggests that DEG/ENaC proteins may also play a role in mechanosensation (1, 9, 13, 15, 33, 35). Some DEG/ENaC members are expressed at the site of mechanotransduction in mechanosensitive tissues such as sensory neurons and vascular smooth muscle, and disruption of DEG/ENaC activity using pharmacological inhibition or knockout animal models alters mechanosensitive responses in some tissues (13, 15, 34, 35).

NGF-TrkA/p75 receptor interactions stimulate ENaC expression in neurons. In unstimulated PC-12 cells, we were unable to detect {alpha}-, {beta}-, or {gamma}-ENaC transcript or protein expression. However, after stimulation of ENaC expression with NGF, we detected transcription and protein expression for {beta}- and {gamma}-ENaC, but not for {alpha}-ENaC. This expression pattern has also been observed in sensory neurons (13, 15). Because Mamet et al. (29, 30) showed that NGF-TrkA receptor interactions stimulate transcription of ASIC molecules, it is not surprising that the expression of ENaC molecules is also regulated by NGF.

NGF-induced neurite growth is mediated primarily by high-affinity TrkA receptors (2, 8, 31); however, activation of low-affinity p75 receptors can augment the TrkA-mediated response (16, 19). Herein, we present similar findings in that most of the effects of NGF were mediated by TrkA receptor activation. Unexpectedly, p75 receptor blockade augmented {gamma}-ENaC but not {beta}-ENaC immunolabeling, but only at the highest NGF concentration (100 ng/ml). This suggests that p75 receptor activation may differentially regulate {beta}- and {gamma}-ENaC proteins, at least at higher concentrations. Although the signal transduction pathway mediating NGF-induced ENaC expression is not known, it may involve the activation of phosphoinositide 3-kinase and serum and glucocorticoid-inducible kinase pathways, because the same pathways are activated by NGF-TrkA activation and are known to upregulate ENaC expression (12, 37, 40, 41, 44).

Disruption of ENaC expression blocks neurite formation. To determine whether ENaC molecules contribute to NGF-induced neurite formation, we evaluated neurite formation in NGF-stimulated cells after the blockade of ENaC expression using three approaches. In the first approach, we blocked channel activity with benzamil (100 nM–10 µM), a specific ENaC inhibitor, at low doses. In the second and third approaches, we blocked transcript and protein expression using {beta}- and {gamma}-ENaC siRNA and dominant-negative molecules, respectively. All approaches yielded similar results in that disruption of ENaC expression blunted neurite formation. Disruption of ASIC1 expression did not significantly inhibit neurite formation, which suggests that ASIC1 does not play a pivotal role in neurite formation in vitro. These data provide evidence that ENaC expression is required for NGF-mediated neurite growth.

Despite our findings that ENaC inhibition with siRNA and dominant-negative molecules has a profound effect on neurite formation in vitro, it is unlikely that DEG/ENaC molecules play a prominent role in the development of the nervous system, because ENaC-null mice display no gross neurodevelopmental defects. The presence of less obvious neurological defects in {beta}- and {gamma}-ENaC-null mice has not been evaluated. Alternatively, ENaC proteins may not play a role in neurogenesis; instead, they may participate in neuronal regeneration after injury or degenerative diseases such as multiple sclerosis.

In summary, our data demonstrate that NGF-TrkA/p75 receptor interactions induce {beta}- and {gamma}-ENaC expression, which are required for neurite formation. We speculate that {beta}- and {gamma}-ENaC proteins may be components of mechanosensitive ion channel complexes and transduce ECM-membrane interactions; that is, they may permit the neurite to "feel" its way through its environment. The results of these studies provide insight into the mechanisms underlying neurite formation and may contribute to the understanding of neuronal repair after injury and degenerative diseases.


   GRANTS
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REFERENCES
 
Financial support was provided by National Heart, Lung, and Blood Institute Grant R01 HL-071603-01A1.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. A. Drummond, Dept. of Physiology and Biophysics, Univ. of Mississippi Medical Center, 2500 North State St., N615, Jackson, MS 39216 (e-mail: hdrummond{at}physiology.umsmed.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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