Regulation of the epithelial Na+ channel by endothelin-1 in rat collecting duct

Abstract

We used patch-clamp electrophysiology to investigate regulation of the epithelial Na+ channel (ENaC) by endothelin-1 (ET-1) in isolated, split-open rat collecting ducts. ET-1 significantly decreases ENaC open probability by about threefold within 5 min. ET-1 decreases ENaC activity through basolateral membrane ETB but not ETA receptors. In rat collecting duct, we find no role for phospholipase C or protein kinase C in the rapid response of ENaC to ET-1. ET-1, although, does activate src family tyrosine kinases and their downstream MAPK1/2 effector cascade in renal principal cells. Both src kinases and MAPK1/2 signaling are necessary for ET-1-dependent decreases in ENaC open probability in the split-open collecting duct. We conclude that ET-1 in a physiologically relevant manner rapidly suppresses ENaC activity in native, mammalian principal cells. These findings may provide a potential mechanism for the natriuresis observed in vivo in response to ET-1, as well as a potential cause for the salt-sensitive hypertension found in animals with impaired endothelin signaling.

Fig. 1.

Endothelin-1 (ET-1) rapidly decreases epithelial Na⁺ channel (ENaC) open probability in rat collecting ducts. A: representative continuous current trace from a cell-attached patch containing at least 3 ENaC before and after application of 20 nM ET-1 to the bath solution. This patch was formed on the apical membrane of a principal cell within a freshly isolated, split-open rat collecting duct. The seal was voltage clamped to −V_p = −60 mV. At this holding potential and with our recording solutions, inward Li⁺ current is downward. Areas under the gray bars over the continuous trace (top) are shown below at an expanded time scale. Dashed lines indicate the respective current levels shown to the right. B: summary graph of ENaC open probability changes in response to ET-1 from paired patch-clamp experiments performed on isolated, split-open rat collecting duct. Circles represent data from individual experiments with means shown as bars. *Significant decrease compared with before addition of ET-1.

Fig. 2.

ET-1 decreases ENaC open probability in collecting duct through the ET_B receptor. Representative gap-free current traces showing the effects of ET-1 in the presence of ET_A (BQ-123, A) and ET_B (BQ-788, C) receptor inhibitors. These traces are from cell-attached patches containing at least 2 ENaC formed on the apical plasma membranes of principal cells in isolated, split-open rat collecting ducts. Recording conditions and data display identical to Fig. 1A. Summary graphs of ENaC open probability changes in response to ET-1 in the presence of ET_A (B) and ET_B (D) receptor inhibitors from paired patch-clamp experiments performed on isolated, split-open rat collecting duct. Data displayed as in Fig. 1B. *Significant decrease compared with before addition of ET-1.

Fig. 3.

Src family tyrosine kinases signal ET-1-dependent decreases in ENaC open probability in native rat collecting duct. Summary graphs of ENaC open probability changes in response to ET-1 in the presence of PLC (U73122, A), PKC (Ro 31-8220, B), and src family tyrosine kinase (PP2, C) inhibitors. Paired patch-clamp results from experiments performed on isolated, split-open rat collecting ducts similar to those described in Fig. 1A. *Significant decrease in open probability compared with before addition of ET-1.

Fig. 4.

MAPK1/2 signaling plays a role in transducing ET-1-dependent decreases in ENaC open probability in native rat collecting duct. Summary graphs of ENaC open probability changes in response to ET-1 in the presence of 2 chemically distinct MEK1/2 inhibitors, PD98059 (A) and U0126 (B). Paired patch-clamp results from experiments performed on isolated, split-open rat collecting ducts as in Fig. 1A. Summary results displayed as in Fig. 1B.

Fig. 5.

ET-1 activates c-src and MAPK1/2 signaling through basolateral ET_B receptors in principal cells. A: representative continuous current trace from a cell-attached patch containing at least 3 ENaC before and after application of 20 nM ET-1. This patch was formed on the apical membrane of a mpkCCD_c14 principal cell clamped to −V_p = −60 mV. All other conditions the same as Fig. 1A. B: summary graph of ENaC open probability changes in response to ET-1 from paired patch-clamp experiments performed on mpkCCD_c14 principal cells. Results displayed as in Fig. 1B. *Significant decrease compared with before addition of ET-1. C: representative Western blots containing mpkCCD_c14 lysate from cells treated with vehicle (control) and ET-1 applied to the apical or basolateral membrane in the absence and presence of PP2. PMA was used as a positive control. Blots probed with anti-phospho-MAPK1/2 (top) and anti-MAPK1/2 (bottom) antibodies. D: summary graph of relative MAPK activation under control conditions and in response to PMA and ET-1 applied to the apical and basolateral membranes in the absence and presence of PP2. MAPK activation measured as the ratio of phospho-MAPK to total MAPK in Western blots similar to that shown in C. Values were normalized to control, which was set to 1, within each experiment. *Significant increase.