Collecting duct-specific endothelin B receptor knockout increases ENaC activity

Abstract

Collecting duct (CD)-derived endothelin-1 (ET-1) acting via endothelin B (ETB) receptors promotes Na(+) excretion. Compromise of ET-1 signaling or ETB receptors in the CD cause sodium retention and increase blood pressure. Activity of the epithelial Na(+) channel (ENaC) is limiting for Na(+) reabsorption in the CD. To test for ETB receptor regulation of ENaC, we combined patch-clamp electrophysiology with CD-specific knockout (KO) of endothelin receptors. We also tested how ET-1 signaling via specific endothelin receptors influences ENaC activity under differing dietary Na(+) regimens. ET-1 significantly decreased ENaC open probability in CD isolated from wild-type (WT) and CD ETA KO mice but not CD ETB KO and CD ETA/B KO mice. ENaC activity in WT and CD ETA but not CD ETB and CD ETA/B KO mice was inversely related to dietary Na(+) intake. ENaC activity in CD ETB and CD ETA/B KO mice tended to be elevated under all dietary Na(+) regimens compared with WT and CD ETA KO mice, reaching significance with high (2%) Na(+) feeding. These results show that the bulk of ET-1 inhibition of ENaC activity is mediated by the ETB receptor. In addition, they could explain the Na(+) retention and elevated blood pressure observed in CD ET-1 KO, CD ETB KO, and CD ETA/B KO mice consistent with ENaC regulation by ET-1 via ETB receptors contributing to the antihypertensive and natriuretic effects of the local endothelin system in the mammalian CD.

Fig. 1.

Endothelin-1 (ET-1) decreases epithelial Na⁺ channel (ENaC) open probability (P_o) in wild-type (WT) and collecting duct (CD) endothlin A (ETA) knockout (KO) but not CD ETB and CD ETA/B KO mice. Representative gap-free current traces of ENaC in cell-attached patches made on the apical membranes of principal cells in split-open ASDN from WT (A) and CD ETA (B), ETB (C), and ETA/B KO (D) mice. The activity of ENaC before and after addition of 20 nM ET-1 is shown. Dashed lines show the respective current levels with C denoting the closed state. Areas under the bars labeled 1 (before) and 2 (after) are shown below at an expanded time scale. Summary graphs documenting the effects of ET-1 on ENaC P_o from paired experiments similar to those in A–D are shown to the right: WT (E) and CD ETA (F), ETB (G), and ETA/B KO (H). *Significant decrease (P < 0.05) compared with before addition of ET-1.

Fig. 2.

ENaC P_o in the presence of exogenous ET-1 is greater in CD ETB and CD ETA/B KO mice but not CD ETA KO mice compared with that in WT mice. Summary graph showing the relative P_o of ENaC after ET-1 treatment in aldosterone-sensitive distal nepfron (ASDN) from WT mice compared with that in CD ETA, ETB, and ETA/B KO mice. Number of experiments in each group indicated. *Significantly greater compared with WT.

Fig. 3.

ENaC in CD ETB and ETA/B KO mice is less responsive to changes in dietary Na⁺ intake. Summary graphs of the activity of ENaC in ASDN isolated from WT (A) and CD ETA (B), ETB (C), and ETA/B (D) KO mice maintained with different dietary Na⁺ regimens. Number of experiments in each group indicated. *Significantly less compared with sodium-free (<0.01% [Na⁺]) condition.

Fig. 4.

ENaC activity in the presence of high dietary Na⁺ is elevated in CD ETB and ETA/B KO mice. Summary graphs of the activity of ENaC in the ASDN isolated from WT and CD ETA, ETB. and ETA/B KO mice maintained with low- (A), regular- (B). and high-[Na⁺] diets (C). *Significantly greater compared with WT.

Fig. 5.

The ability of ENaC to respond to changes in dietary Na⁺ is compromised in CD ETB and ETA/B KO mice. Fractional ENaC activity (activity observed with a high-Na⁺ diet divided by that with a Na⁺-free diet) for WT and CD ETA, ETB, and ETA/B KO mice.