SRC family kinases mediate epithelial Na+ channel inhibition by endothelin.

The epithelial Na(+) channel (ENaC) is implicated in the pathogenesis of salt-sensitive hypertension. Recent evidence from animal models suggests that the vasoactive peptide, endothelin (ET-1), may be an important negative regulator of ENaC in vivo. We investigated the signaling pathway involved in endothelin-mediated ENaC inhibition. Experiments were performed in NIH 3T3 cells stably expressing genes for the three (alpha, beta, and gamma) ENaC subunits. In whole cell patch clamp experiments, we found that ET-1 treatment induced a dose-dependent decrease in amiloride-sensitive currents. Using receptor-specific antagonists, we determined that the effects of ET-1 were attributed to activation of the ET(B) receptor. Moreover, the inhibitory effect of ET-1 on ENaC could be completely blocked when cells were pretreated with the selective Src family kinase inhibitor, PP2. Further studies revealed that basal Src family kinase activity strongly regulates ENaC whole cell currents and single channel gating. These results suggest that Src family kinases lie in a signaling pathway activated by ET-1 and are components of a novel negative regulatory cascade resulting in ENaC inhibition.

The epithelial Na ؉ channel (ENaC) is implicated in the pathogenesis of salt-sensitive hypertension. Recent evidence from animal models suggests that the vasoactive peptide, endothelin (ET-1), may be an important negative regulator of ENaC in vivo. We investigated the signaling pathway involved in endothelin-mediated ENaC inhibition. Experiments were performed in NIH 3T3 cells stably expressing genes for the three (␣, ␤, and ␥) ENaC subunits. In whole cell patch clamp experiments, we found that ET-1 treatment induced a dose-dependent decrease in amiloride-sensitive currents. Using receptor-specific antagonists, we determined that the effects of ET-1 were attributed to activation of the ET B receptor. Moreover, the inhibitory effect of ET-1 on ENaC could be completely blocked when cells were pretreated with the selective Src family kinase inhibitor, PP2. Further studies revealed that basal Src family kinase activity strongly regulates ENaC whole cell currents and single channel gating. These results suggest that Src family kinases lie in a signaling pathway activated by ET-1 and are components of a novel negative regulatory cascade resulting in ENaC inhibition.
Endothelin (ET) 1 1, a potent vasoactive peptide originally described as an endothelial cell-derived factor, is the founding member of a family of related 21-amino acid peptides (1,2). Each endothelin (ET-1, ET-2, and ET-3) is encoded by a distinct gene and is processed from an inactive precursor via two proteolytic cleavages to generate a biologically active peptide (3,4). Endothelins are synthesized in many cell types, including endothelial, epithelial, fibroblast, and cardiac muscle cells, and function as autocrine or paracrine factors to regulate different cellular processes (1,(5)(6)(7). The two endothelin receptors, ET A and ET B , are broadly expressed with overlapping, but distinct, distributions (8 -11). ET A and ET B are heterotrimeric G pro-tein-coupled receptors (GPCRs) that can couple with multiple G␣ subunits, depending on cell type (12)(13)(14). In addition, ET receptors stimulate the activity of nonreceptor tyrosine kinases in some cells (5,15,16). Although this activation is generally thought to mediate the mitogenic effects of endothelins, acute activation of NHE3 via ET-1 is blocked ϳ50% by tyrosine kinase inhibitors (17,18). The ET-mediated activation of NHE3 occurs specifically via ET B receptors (19).
Recent evidence indicates that renal ET B receptors may be important for sodium handling. The function of ET B has been examined in rats with naturally occurring mutation of the ET B gene. These studies indicate that ET B plays an essential role in development of enteric neurons because absence of functional ET B receptors causes perinatal lethality resulting from megacolon (20,21). Gariepy et al. (22) rescued this phenotype by specifically expressing ET B only in adrenergic neurons using the dopamine ␤-hydroxylase promoter (22). On high salt diets, these rats developed salt-sensitive hypertension, which was restored to normal when animals were treated with amiloride, a potent inhibitor of the epithelial Na ϩ channel (ENaC) (22,23). These data indicate that endothelin, acting on ET B receptors, mediates tonic inhibition of ENaC, and that this inhibition may be required for the maintenance of blood pressure.
ENaC, the product of three distinct genes (␣, ␤, and ␥), is a low conductance Na ϩ -selective channel that provides the ratelimiting step in electrogenic Na ϩ transport across the apical membrane of many epithelial tissues including the distal nephron (24 -26). Analysis of two human disorders, Liddle syndrome and pseudohypoaldosteronism type 1, indicates that ENaC in renal epithelia plays a critical role in salt homeostasis and the control of blood pressure (27)(28)(29). Although all pathways that regulate apical membrane Na ϩ transport via ENaC are not known, several hormones stimulate ENaC activity or expression (30,31). For example, aldosterone acts on cytosolic mineralocorticoid receptors to potently stimulate the absorption of Na ϩ across cells of the distal nephron and colon (32). Although this stimulation is accomplished in part via increased transcription of ENaC subunits, aldosterone also induces the transcription of the serum and glucocorticoid-regulated kinase (SGK), which activates ENaC (33,34). In addition, vasopressin and insulin are known to stimulate the activity or expression of ENaC in some model systems (35)(36)(37). In contrast, few hormonal pathways that potently inhibit ENaC activity have been described.
Evidence that rats and mice lacking functional ET B receptors develop salt-sensitive hypertension supports a role for ET-1 inhibition of ENaC activity. In A6 cells, a Xenopus model of the distal nephron, ET-1 dramatically down-regulates ENaC activity, although the pathway linking ET B receptors to ENaC was not identified (38). Therefore, we sought to identify the signaling pathway linking activation of ET receptors to inhibition of ENaC in mammalian cells. We found that ET B receptors act via Src family kinases to potently inhibit ENaC open probability. This provides a novel mechanism of ENaC regulation and explains the inhibitory influence of ET B on ENaC activity. The identification of signaling proteins that potently inhibit Na ϩ absorption in the distal nephron could potentially provide additional therapeutic targets for the treatment of human hypertension.

EXPERIMENTAL PROCEDURES
Cell Culture-Experiments were performed using NIH 3T3 cells infected with retrovirus encoding cDNAs for rat ␣␤␥ ENaC subunits (39). Clones stably expressing ENaC subunits were maintained in a humidified incubator with 5% CO 2 , in Dulbecco's modified Eagle's medium (DMEM) with 10% bovine calf serum, 10 M amiloride, 100 units/ml penicillin, 0.1 mg/ml streptomycin, 300 g/ml G418, and 1 g/ml puromycin. Cells for patch clamp experiments were grown on 35-mm culture dishes in standard media and were treated with 2 mM sodium butyrate and 1 M dexamethasone 24 h prior to recording to induce ENaC expression (39). Cells used for biochemical experiments were maintained at subconfluent states and were moved into serumfree media at least 24 h prior to study.
Electrophysiology-NIH 3T3 cells expressing ␣␤␥ rat ENaC subunits were used in patch clamp analyses. Patch pipettes were pulled from glass capillary tubes using a microelectrode puller and fire-polished to achieve electrode resistances of 5-8 megohms. Pipette (intracellular) solutions for whole cell patch clamp experiments contained, in mM: 120 Tris aspartate, 3 Mg-ATP, 0.3 ADP, 0.1 CaCl 2 , 7 MgCl 2 , and 1 EGTA. Additionally, phosphorylated (activating) or nonphosphorylated peptides (control; Calbiochem) modeled on the amino acid sequence of the Src kinase carboxyl terminus (amino acid residues 521-533) were used where indicated, to modulate the activity of endogenous Src family kinases (40). The peptides were dissolved in the whole cell pipette solution at a final concentration of 10 M. The standard bath solution for whole cell experiments was composed of, in mM: 150 lithium aspartate, 5 TES, 2 MgCl 2 , and 1 CaCl 2. As indicated, amiloride (final concentration of 10 M) was added to the bath solution during whole cell recordings.
Whole cell and single channel currents were recorded with an Axopatch 1C amplifier (Axon Instruments). Data was acquired at 2 kHz and filtered at 200 Hz during whole cell recordings (50 Hz for single channel recordings) using a low pass four-pole Bessel filter. Data acquisition and subsequent analyses for both whole cell and single channel experiments were performed using the pClamp 8.0 software package (Axon Instruments).
For whole cell patch clamp experiments, cells were voltage-clamped at 0 mV and pulsed to Ϫ40 mV every 100 ms for 400-ms intervals. This sequence was repeated for 180 s. Next, cells were voltage clamped at 0 mV and pulsed for 600 ms from Ϫ160 mV to ϩ20 mV, in 20-mV increments. Current-voltage (IV) curves were generated from average current measurements starting 200 ms after each voltage pulse. Whole cell amiloride-sensitive currents (I amil ) were calculated from the difference in current in the presence and absence of 10 M amiloride.
In Vitro Kinase Assays-Kinases were immunoprecipitated from NIH 3T3 cell lysates using an antibody directed against the conserved COOH terminus of the Src family kinase sequence that recognizes all Src kinase family members (rabbit polyclonal antibody; Santa Cruz), or using antibodies to specific Src family kinases (␣-Src kinase, Oncogene; ␣-Yes kinase, Wako). Lysates were incubated with antibody at 4°C for 3-4 h before a 1:1 mixture of Protein A/G-agarose was added. After 1 h of incubation with Protein A/G-agarose at 4°C, immunoprecipitates were washed in binding buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% Triton X-100, pH 7.4), and resuspended in a modified pipette solution/kinase assay buffer (150 mM Tris aspartate, 2 mM MgCl 2 , 1 mM CaCl 2 , 1 mM orthovanadate, 4 Ci of [␥-32 P]ATP) with an exogenous Src family kinase substrate, rabbit muscle enolase (10 g, Sigma). Additionally, we tested whether ENaC cytosolic domains were substrates for Src family kinases. GST-ENaC fusion proteins were generated by subcloning the cDNAs encoding the NH 2 -and COOHterminal subunits of human ␣, ␤, and ␥ ENaC into the pGEX-2TK (Amersham Pharmacia Biotech) expression vector, which contains an engineered PKA phosphorylation site. Purified fusion proteins were immobilized on glutathione-Sepharose in the standard kinase assay buffer described above and incubated with purified PKA catalytic subunit (Promega) or purified Src kinase (Upstate Biotechnology). Samples were incubated at 30°C for 45 min, and reactions were stopped by the addition of Laemmli sample buffer. Samples were subjected to SDS-PAGE and visualized by phosphorimage analysis.

RESULTS
ET-1 Inhibition of ENaC-We used a previously described NIH 3T3 fibroblast cell model system that stably expresses ␣␤␥ ENaC subunits (39). Similar to those reports, we observed ENaC current density that ranged from 10 to 30 pA/pF (at Ϫ160 mV) under whole cell patch clamp conditions. Specific ENaC currents were detected as the difference current (I amil ) in the presence and absence of 10 M amiloride, a selective ENaC channel blocker (Fig. 1A). Generally, treatment with amiloride decreased whole cell currents by 50 -70%, reflecting the expression level and activity of ENaC in these cells. ET-1 treatment markedly inhibited amiloride-sensitive current. Full inhibition of I amil was observed in cells treated with 10 nM ET-1 (Fig. 1B). Concentrations of ET-1 in the range of 1 pM to 10 nM produced a dose-dependent inhibition of I amil (Fig. 1C).
ET-1 Activation of ET B Inhibits ENaC-The ET-1 concentration that resulted in 50% inhibition of I amil was ϳ50 pM, in a range consistent with ET-1 activation of ET B receptors. To test this possibility, cells were pretreated with 1 M BQ-788, a selective ET B receptor antagonist. Under these conditions, BQ-788 prevented the inhibition of ENaC currents by 100 pM ET-1 ( Fig. 2A). In contrast, 100 pM ET-1 was fully inhibitory in the presence of 1 M BQ-123, a selective ET A receptor antagonist (Fig. 2B). These results demonstrate that, in NIH 3T3 cells, ET B receptor signaling is responsible for ET-1 inhibition of ENaC.
Src Inhibitor Prevents ET-1 Inhibition of ENaC-We have demonstrated previously that Src family kinases potently inhibit ENaC when coexpressed in Xenopus oocytes. 2 Therefore, we investigated whether Src family kinases were involved in mediating the effects of ET-1 on ENaC. We used a cell-permeable specific Src family kinase inhibitor, PP2, in whole cell patch clamp experiments. PP2 competes with ATP for binding to Src family kinases (41). In in vitro kinase assays, purified Src kinase did not phosphorylate a known substrate, enolase, in the presence of 1 M PP2 (Fig. 3A, lane 2). The negative control compound PP3 shares a similar structure but does not affect Src kinase activity and did not inhibit the phosphorylation of enolase (Fig. 3A, lane 3). Cells were incubated in bath solutions containing 1 M either PP2 or PP3 and subsequently treated with 100 pM ET-1. In cells incubated in PP3, ET-1 inhibited I amil as observed previously (Fig. 3B). In contrast, in cells pretreated with PP2, I amil was not inhibited by 100 pM ET-1. This observation implicates Src family kinase activity in the inhibition of ENaC by ET-1.
To demonstrate biochemically that Src family kinases are involved in ET-1 signaling, we examined changes in phosphotyrosine levels in response to ET-1 stimulation. Cells were treated with 10 nM ET-1 for 10 min, lysed, subjected to SDS-PAGE, and transferred to membranes. Total cell lysates were blotted with anti-phosphotyrosine antibody (monoclonal antibody clone 4G10; Fig. 3C). We observed a dramatic increase in tyrosine phosphorylation in response to ET-1. In particular, tyrosine phosphorylation of ϳ85-, 65-, 48-, and 43-kDa proteins were enhanced by ET-1 treatment. Additionally, in vitro kinase assays measuring the activity of Src family kinases show a significant increase in kinase activity in cells treated with 10 nM ET-1 (72 Ϯ 20% increase over control; p Ͻ 0.05; Fig. 3D).
These results indicate that ET-1 signaling activates tyrosine kinase pathways that include Src family kinases.
Activation of Endogenous Src Family Kinases Inhibit ENaC-To further show that activation of Src family kinases mediates inhibition of ENaC, we used peptides modeled on the conserved Src family kinase COOH terminus to activate the endogenous kinases. c-Src is inactivated by phosphorylation of tyrosine 527, by COOH-terminal Src kinase (CSK; Ref. 43). Intramolecular interactions between the Src homology 2 domain and this phosphotyrosine residue (Tyr 527 ) maintain c-Src kinase in an inactive state. Introduction of phosphorylated peptides containing the sequence of the Src carboxyl terminus disrupts the intramolecular interactions that hold the kinase in the folded, inactive state. Addition of activating phosphorylated peptide to c-Src and c-Yes kinase-specific immunoprecipitates significantly increased phosphorylation of enolase in in vitro kinase assays (Fig. 4A). As a control, immunoprecipitates incubated with the nonphosphorylated peptide, which does not compete for Src homology 2 binding, show a low level of phosphorylation. These assays demonstrate that c-Src and c-Yes kinase can be activated by the introduction of phosphorylated carboxyl-terminal tail peptides. In whole cell patch clamp experiments, activation of endogenous Src family kinases resulted in a significant decrease in amiloride-sensitive currents (Fig. 4B). Dialysis of cells with the nonphosphorylated, control peptide did not alter ENaC activity. These experiments reveal that the cell's endogenous Src family kinases are poised to potently inhibit ENaC when activated by appropriate signals.
Src Family Kinase Inhibitor, PP2, Increases Whole Cell ENaC Currents and Alters Single Channel Gating-We have shown that activation of Src family kinases can dramatically reduce I amil (Fig. 4B). However, it is evident that Src family kinase activity exists at a basal level in unstimulated cells (Fig.  3D). To examine whether this basal level of kinase activity influences ENaC currents, we measured I amil in the presence of PP2 and PP3. We found that treatment of cells with 1 M PP2 significantly increased I amil over control (PP3) (Fig. 5). These results suggest that basal Src family kinase activity mediates tonic inhibition of ENaC in this model system.
Previous work in A6 cell lines describes the inhibition of ENaC by ET-1 (38). In that study, ET-1 inhibited ENaC P o and enhanced mean closed time. Therefore, to test whether Src family kinases could mediate similar effects on ENaC in mammalian cells, we studied ENaC in cell-attached single channel patches in the presence of PP2 or PP3. A representative experiment is shown in Fig. 6A. Channel openings, represented by upward deflections in the trace, reveal classical ENaC characteristics, e.g. low amplitude and slow gating channel transitions (44). Patches from PP2-treated cells contained ENaC that were open for extended periods (up to tens of seconds), compared with patches from PP3-treated cells. The all-points histograms in Fig. 6A   between channel opening events.
Src Family Kinases Do Not Regulate ENaC by Direct Phosphorylation of the Channel Subunits-In several instances, Src family kinases regulate ion transport by direct phosphorylation of the ion channel or transporter (53,54). To investigate this possibility as a mechanism for the regulation of ENaC by Src family kinases, we incubated purified GST fusion proteins encoding the ENaC subunit cytosolic domains with purified Src kinase (Fig. 7). The fusion proteins were generated in-frame with GST from the pGEX-2TK expression vector, which contains an engineered PKA phosphorylation site. As expected, each of the immobilized NH 2 -and COOH-terminal ENaC fusion proteins were phosphorylated by exogenous PKA. However, purified, active Src kinase failed to phosphorylate any of the ENaC cytosolic domain fusion proteins. These results demonstrate that ENaC subunit cytosolic domains do not serve as Src family kinase substrates in vitro. DISCUSSION We have identified Src family kinases as intermediate signaling proteins in a negative regulatory path for ENaC. Previous reports linked ET-1 signaling to inhibition of ENaC, but no information existed concerning the signaling pathway involved (22,38). Our studies confirmed previous findings that Src fam-ily kinases are activated by ET-1 signaling (5,16). We report the novel finding that Src family kinases potently inhibit ENaC and that this action mediates the inhibitory effect of ET B on ENaC function. ET-1 is a well known regulator of systemic blood pressure; as such, its action on vascular targets has been intensely studied. Additional physiological roles of this hormone are suggested by ET A and ET B receptor localization in nonvascular tissues, such as the nephron (11). Analysis of the ET receptor distribution in the nephron shows that ET B is expressed at 10-fold higher levels than ET A , suggesting a dominant role for ET B in renal tubule epithelial cells (45,46). Importantly, adult rats lacking functional ET B receptor activity display enhanced Na ϩ reabsorption in the distal nephron, implicating ET B in regulation of tubular function (22). The elevated blood pressure in these animals was reversed to normal when treated with amiloride, a potent inhibitor of ENaC. Our finding that Src family kinases robustly down-regulate ENaC activity may be critical to understanding how ET B receptors regulate blood pressure.
Although the phenotype of the ET B receptor-deficient rat is consistent with an inhibitory effect of ET B on ENaC activity, the in vivo model does not exclude the role of indirect effects from ET B in the vasculature, the possibility that other hormones may be involved, or that the effects were mediated by changes in ENaC gene expression. Studies in cell culture models provide systems to examine ET-1 regulation of ENaC in isolation, separate from the influences of circulating hormones and complex multisystem regulatory feedback mechanisms. In A6 cells, a Xenopus model of the distal nephron, ENaC single channel activity was strongly inhibited by ET-1 (38). This effect was attributed to the activation of ET B and resulted in decreases in ENaC P o and increases in channel mean closed time. Our studies in NIH 3T3 cells both confirmed and extended the previously published results. We found that ET-1 potently inhibited ENaC in mammalian cells and that this effect was totally dependent on the activation of Src family kinases (Figs.  1 and 3). Examination of ENaC at the single channel level showed that Src family kinase activity decreases ENaC P o and mean open time, whereas increasing the channel mean closed time (Fig. 6). Because the regulation of ENaC by ET-1 is robust in cell culture and heterologous expression systems, it is unlikely that the regulation of ENaC by ET-1 observed in vivo is a secondary consequence of the actions of other hormones or signals. Instead, the effects of ET-1 on ENaC regulation are most likely caused by direct activation of intracellular signal transduction pathways involving Src kinases that link the receptor to ENaC.
ET A and ET B are GPCRs, but several published reports show that ET-1 treatment causes a significant increase in cellular tyrosine phosphorylation and Src kinase activity (5,47). There are many examples of cross-talk between GPCRs and tyrosine kinases, for instance Src and the calcium-sensitive tyrosine kinase, Pyk2, couple lysophosphatidic acid (G␣ i -coupled) and bradykinin (G␣ q -coupled) receptors with the mitogen-activated protein kinase pathway (48). Additionally, inhibitors of tyrosine kinases (herbimycin A) and Src kinases (PP1), block activation of mitogen-activated protein kinase cascades by thrombin receptors (G␣ q -coupled) (49,50). The mechanism responsible for linking these signaling cascades is not fully understood, but could involve the activation of Ca 2ϩ -sensitive kinases or direct actions of G-protein subunits on tyrosine kinase effectors (48,51). Here, we showed that treatment of NIH 3T3 cells with 10 nM ET-1 markedly increased total cellular tyrosine phosphorylation and Src family kinase activity, in particular (Fig. 3, C and D). Further, the inhibitory effects of ET-1 on ENaC activity could be completely blocked if cells were pretreated with Src family kinase inhibitors (Fig. 3B). Together, these data strongly argue that Src family kinases contribute to the ET-1 signaling cascade. Src family kinases are potent mitogenic agents, and ET-1-mediated gene expression of atrial natriuretic peptide in primary cardiomyocytes is Src kinase-dependent (5). However, the time frame of our observations eliminates the possibility that changes in expression of ENaC regulatory proteins accounts for the acute effects of Src family kinases on ENaC. Here, we describe a paradigm in which ET-1 rapidly alters ion transport via a transcriptionindependent mechanism. This effect is similar to previous reports of ET-1 activation of the Na ϩ /H ϩ exchanger 3 (NHE3), in which ET-1 effects (via ET B ) were blocked ϳ50% by tyrosine kinase inhibitors (17). Additionally, regulation of Na-HCO 3 co-transport by carbachol through activation of muscarinic receptors was completely inhibited by the Src family kinase inhibitor, PP1 (52). These examples provide important evidence that cross-talk between GPCRs and tyrosine kinases can have immediate effects on ion transport, in addition to long term mitogenic effects on gene transcription. Moreover, our in vitro studies suggest that the phenotype of the ET B -deficient rat and mouse may not be explained solely by changes in the expression of ENaC subunit genes or by the involvement of other hormonal systems. The mechanism by which Src family kinases inhibit ENaC activity remains to be determined. Our single channel data clearly show changes in channel open probability consistent with inhibition of whole cell currents by Src family kinases (Fig. 4). We found that inhibition of Src family kinase activity caused ENaC channels to display short average closed times (Fig. 6). This suggests that active Src family kinases tonically inhibit ENaC by altering the channel gating. The low ENaC P o associated with Src activation is consistent with the strong inhibition of whole cell currents. Our experiments do not exclude the possibility that Src kinase activity also regulates the number of ENaC channels on the cell surface. However, our single channel data showed a strong inhibition of channel open probability that is sufficient to alone account for the inhibition of whole cell currents by Src family kinases.
We find that Src family kinase activity is required for the inhibition of ENaC by ET-1. Therefore, it will be crucial to identify the relevant proteins that are downstream of Src family kinases to further define the molecular mechanism of this inhibition. Previous reports describing ion transport regulation by Src family kinases include direct phosphorylation of channels or transporters (53,54). This seems unlikely in the case of ENaC. The in vitro phosphorylation of ENaC subunits has been investigated; however, only phosphoserine/threonine, but not phosphotyrosine residues were observed (55,56). Moreover, the ENaC subunit cytosolic domains do not contain consensus sites for Src kinase phosphorylation. Our data (Fig. 7) support these findings and demonstrate that ENaC cytosolic domains are not substrates for Src kinase in vitro. Therefore, it is likely that Src family kinases inhibit ENaC by an indirect mechanism. These findings are similar to the regulation of ENaC by the serumglucocorticoid-regulated kinase (SGK), where SGK activation increases ENaC activity by enhancing cell-surface expression (34). When coexpressed in Xenopus laevis oocytes, SGK increases ENaC activity by stimulating the translocation of ENaC channels to the cell surface in a kinase-dependent manner (34). The mechanism that accounts for enhanced cell-surface expression is unknown, but similar to Src, SGK does not directly phosphorylate ENaC and the target of its kinase activity is unidentified (56). Our results, taken together with these findings on SGK regulation of ENaC, make it clear that unidentified molecular mechanisms exist for control of Na ϩ conductance. These mechanisms may vary importantly with cell type and with the upstream hormonal signal involved. Clearly there are intermediary proteins that are required for the regulation of ENaC by SGK and Src family kinases, identification of these proteins will be the subject of future studies.
Several general mechanisms can be envisioned by which Src family kinases may act to indirectly inhibit ENaC activity. First, Src family kinase activation may modulate the activity of a serine/threonine kinase. Insulin stimulates receptor tyrosine kinase activity, increases serine/threonine phosphorylation of ␤ and ␥ ENaC, and increases ENaC activity (55). Therefore, it is possible that Src kinases may enhance or suppress the activity of a serine/threonine kinase, which in turn, modulates ENaC activity. Second, Src family kinase activation may alter the activity of a phosphatase that subsequently dephosphorylates the channel or an associated regulator. Third, Src family kinases may induce cytoskeletal rearrangements that affect the association of stimulatory or inhibitory proteins with ENaC. Finally, the target of Src family kinase activity could be an ENaC-associated cytosolic protein, whose phosphorylation state influences its association with the channel. Binding or release from the channel may potentially alter channel gating. Given the complexity of ENaC regulation, any of these models could account for the effects of Src family kinases.
The involvement of ET-1 and ET B in the genesis of saltsensitive hypertension may provide new insights into the molecular pathophysiology of renal disease. Our novel finding that Src family kinases are required for endothelin-mediated inhibition of ENaC provides further understanding of Na ϩ transport regulation, with potential implications for elucidating causes of human hypertension. It will be intriguing to examine the hypertensive population for variations in ET-1, ET B , and Src family kinase activities in the nephron. Such alterations may help to understand the mechanisms of salt-sensitive hypertension and/or essential hypertension. In additional, identifying the Src family kinase members (Src, Yes, Fyn, etc.) that mediate these effects in vivo could provide insights into understanding the functional specificity of Src family kinase signaling. Our identification of Src family kinases as intermediaries of ET-1 modulation of ENaC provides another potential therapeutic target for the treatment of human hypertension.