Abstract
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.
Highlights
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]
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 epithelial Na؉ channel (ENaC) inhibition
We have identified Src family kinases as intermediate signaling proteins in a negative regulatory path for ENaC
Summary
Cell Culture—Experiments were performed using NIH 3T3 cells infected with retrovirus encoding cDNAs for rat ␣␥ ENaC subunits [39]. Pipette (intracellular) solutions for whole cell patch clamp experiments contained, in mM: 120 Tris aspartate, 3 Mg-ATP, 0.3 ADP, 0.1 CaCl2, 7 MgCl2, and 1 EGTA. Data acquisition and subsequent analyses for both whole cell and single channel experiments were performed using the pClamp 8.0 software package (Axon Instruments). The pipette solution for single channel studies contained, in mM: 280 lithium aspartate, 2 MgCl2, 5 TES, and 0.1 CaCl2. 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 MgCl2, 1 mM CaCl2, 1 mM orthovanadate, 4 Ci of [␥-32P]ATP) with an exogenous Src family kinase substrate, rabbit muscle enolase (10 g, Sigma). Samples were subjected to SDSPAGE and visualized by phosphorimage analysis
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