Activation of the epithelial Na+ channel in the collecting duct by vasopressin contributes to water reabsorption

Abstract

We used patch-clamp electrophysiology on isolated, split-open murine collecting ducts (CD) to test the hypothesis that regulation of epithelial sodium channel (ENaC) activity is a physiologically important effect of vasopressin. Surprisingly, this has not been tested directly before. We ask whether vasopressin affects ENaC activity distinguishing between acute and chronic effects, as well as, parsing the cellular signaling pathway and molecular mechanism of regulation. In addition, we quantified possible synergistic regulation of ENaC by vasopressin and aldosterone associating this with a requirement for distal nephron Na+ reabsorption during water conservation vs. maintenance of Na+ balance. We find that vasopressin significantly increases ENaC activity within 2-3 min by increasing open probability (P(o)). This activation was dependent on adenylyl cyclase (AC) and PKA. Water restriction (18-24 h) and pretreatment of isolated CD with vasopressin (approximately 30 min) resulted in a similar increase in P(o). In addition, this also increased the number (N) of active ENaC in the apical membrane. Similar to P(o), increases in N were sensitive to inhibitors of AC. Stressing animals with water and salt restriction separately and jointly revealed an important effect of vasopressin: conservation of water and Na+ each independently increased ENaC activity and jointly had a synergistic effect on channel activity. These results demonstrate a quantitatively important action of vasopressin on ENaC suggesting that distal nephron Na+ reabsorption mediated by this channel contributes to maintenance of water reabsorption. In addition, our results support that the combined actions of vasopressin and aldosterone are required to achieve maximally activated ENaC.

Fig. 1.

Arginine vasopressin (AVP) rapidly increases epithelial sodium channel (ENaC) activity by increasing open probability (P_o). A: typical current trace from a cell-attached patch formed on the apical membrane of a principal cell in a freshly isolated, split-open murine collecting duct before and after treatment with AVP. This patch contains at least 4 ENaC and is from a collecting duct isolated from an animal maintained on standard chow. The full trace is shown at top with areas of the trace below the gray bars marked 1 and 2 before and after AVP, respectively, shown with an expanded time scale below. Dashed lines note current levels with C demarking the closed level. This patch was clamped to a holding potential of −V_p = −60 mV with the collecting duct bathed by physiological saline and Li⁺ the permeant cation in the pipette solution. With this holding potential and solutions, inward current through ENaC is downward. B: summary graph of 16 paired experiments testing AVP action on ENaC similar to the representative experiment shown in A. *Significant increase compared with before treatment with AVP.

Fig. 2.

Adenylyl cylase (AC) and PKA are required for AVP actions on ENaC P_o. Summary graphs of paired experiments testing the acute effects of AVP on ENaC P_o in the presence of inhibitors of AC (A) and PKA (B and C). Results are from experiments similar to those in Fig. 1A but with the addition that collecting ducts were pretreated with inhibitor for ≥30 min before use. D: summary graph of resting ENaC P_o in principal cells from control collecting ducts (isolated from animals fed standard chow) and those pretreated with inhibitors of AC (MDL) and PKA (Rp and H-89). *Significantly lower P_o compared with control.

Fig. 3.

Water restriction and exogenous AVP activate ENaC in a similar manner. A: representative current traces of ENaC in cell-attached patches formed on the apical membranes of principal cells in collecting ducts isolated from mice maintained with standard chow in the absence (1. control) and presence (2. AVP) of pretreating with AVP at least 30 min before seal formation. In addition, a representative trace for ENaC in a principal cell from a collecting duct isolated from a mouse maintained on standard chow but water restricted for 24 h before CD isolation is also shown (3. H₂O restriction). All other conditions identical to Fig. 1A. Summary graphs of resting ENaC P_o (B) and N (C) in control collecting ducts and those pretreated with AVP and isolated from water-restricted animals. *Significantly greater compared with the control group.

Fig. 4.

Need to conserve water and sodium works in synergy to set ENaC activity in the collecting duct. Summary graphs of ENaC P_o (A), N (B), frequency (f; C), and fNP_o (D) in principal cells from collecting ducts isolated from animals fed a high-Na⁺ or nominally Na⁺-free diet with free and restricted access to water. Results from cell-attached patches. *Significant increase compared with free access to water. **Significant increase compared with high [Na⁺].

Fig. 5.

ENaC is most active when AVP and aldosterone are both elevated. This figure contains a representation of the effects on ENaC P_o, N, and f of restricting dietary Na⁺ and access to water. The 4 large circles represent typical apical membranes of principal cells in collecting ducts isolated from the 4 scenarios tested in Fig. 4: high Na⁺ + free access to water, high Na⁺ + water restriction, low Na⁺ + free access to water, and high Na⁺ + water restriction. The smallest circles represent the possibility of having active ENaC in the membrane. The P_o of these potentially active channels is noted by a gray scale with channels having the highest P_o being black and channels with a lower P_o being light gray and those that are currently not present in the membrane white. Dashed circles represent channel clustering.