Alpha(1)-antitrypsin inhibits epithelial Na+ transport in vitro and in vivo - PubMed (original) (raw)

Alpha(1)-antitrypsin inhibits epithelial Na+ transport in vitro and in vivo

Ahmed Lazrak et al. Am J Respir Cell Mol Biol. 2009 Sep.

Abstract

A variety of studies have shown that Na(+) reabsorption across epithelial cells depends on the protease-antiprotease balance. Herein, we investigate the mechanisms by which alpha(1)-antitrypsin (A1AT), a major anti-serine protease in human plasma and lung epithelial fluid and lacking a Kunitz domain, regulates amiloride-sensitive epithelial Na(+) channel (ENaC) function in vitro and in vivo. A1AT (0.05 mg/ml = 1 microM) decreased ENaC currents across Xenopus laevis oocytes injected with human alpha,beta,gamma-ENaC (hENaC) cRNAs, and human lung Clara-like (H441) cells expressing native ENaC, in a partially irreversible fashion. A1AT also decreased ENaC single-channel activity when added in the pipette but not in the bath solutions of ENaC-expressing oocytes patched in the cell-attached mode. Incubation of A1AT with peroxynitrite (ONOO(-)), an oxidizing and nitrating agent, abolished its antiprotease activity and significantly decreased its ability to inhibit ENaC. Intratracheal instillation of normal but not ONOO(-)-treated A1AT (1 microM) in C57BL/6 mice also decreased Na(+)-dependent alveolar fluid clearance to the same level as amiloride. Incubation of either H441 cells or ENaC-expressing oocytes with normal but not ONOO(-)-treated A1AT decreased their ability to cleave a substrate of serine proteases. A1AT had no effect on amiloride-sensitive currents of oocytes injected with hENaC bearing Liddle mutations, presumably because these channels remain at the surface longer than the wild-type channels. These data indicate that A1AT may be an important modulator of ENaC activity and of Na(+)-dependent fluid clearance across the distal lung epithelium in vivo by decreasing endogenous protease activity needed to activate silent ENaC.

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Figures

**Figure 1.

Time course of inward Na+ current (INa) and amiloride-sensitive current (Iamil) inhibition across epithelial Na+ channel (ENaC)–expressing oocytes by native or peroxynitrite (ONOO−)-treated α1-antitrypsin (A1AT). Xenopus oocytes were injected with human α, β and γ-hENaC cRNAs (8.4 ng each). (A) INa values were recorded 48 hours later while the membrane potential (Vm) was altered from −40 to −140 mV for 600 milliseconds, before and after addition of either normal (closed circles) or ONOO−-treated (open circles) A1AT (0.05 mg/ml = 1 μM) into the perfusing solution (ND96). Amiloride (Amil.; 10 μM) was added into the bath as indicated. Results are displayed of a typical experiment, which was repeated 15 times using three different batches of oocytes with identical results (see C for mean values). (B) The same as in (A), except that oocytes were perfused with an ND96 containing trypsin (1 μM) and no amiloride at the indicated times. Results are shown of a typical experiment that was repeated at least 10 times. (C) Iamil–voltage relationships for oocytes incubated with either vehicle or A1AT (1 μM) for 45 minutes and then placed in ND96 for the indicated times. Iamil values were calculated by subtracting currents remaining after addition of amiloride (10 μM) from INa. Values are means (±1 SEM); n = 15 oocytes for each condition. Closed squares, vehicle; immediately after perfusion; open circles, 2 hours after A1AT perfusion; closed triangles, 4 hours after A1AT perfusion; open inverted triangles, 6 hours after A1AT perfusion. All values of A1AT-perfused oocytes are significantly different from control (ANOVA followed by the Bonferroni t test; P < 0.01).

**Figure 2.

SDS-PAGE of normal and ONOO−-treated A1AT. A1AT (2 μg) was incubated with vehicle (lanes 1 and 2) or ONOO− (1 mM; lanes 3 and 4) for 15 minutes. Proteins were then separated by SDS-PAGE and stained with GelCode. Molecular weights (kD) of standards are shown. A typical blot is shown that was repeated at least three different times.

**Figure 3.

INa at −140 mV after exposure of ENaC-expressing oocytes to native and ONOO−-treated A1AT (1 μM) for 2 hours. Box–whisker graph showing individual data points, boxes (25th to 75th percentiles of the data), whisker bars (5th to 95th percentiles), and means (±1 SEM) of INa just before and after perfusion with amiloride (Amil.; 10 μM) for the indicated conditions. P < 0.01 compared with its corresponding control using the t test.

**Figure 4.

Single-channel currents recorded from cell-attached patches of ENaC-expressing oocytes. Oocytes were patched in the cell-attached mode, as described in the text, and currents were measured at a pipette holding potential of −100 mV (_V_holding = _V_apical − _P_pipette); amplitude distribution histograms were generated as described in M

aterials and

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. Each trace represents 1-minute recordings for the indicated time after formation of the Giga seal. The close states are indicated by the small bar to the left of each record. (A and B) Control; (C and D) addition of A1AT (1 μM) in the bath at time zero; (E and F) A1AT (1 μM) was included in the pipette solution at the beginning of the patch. Results are shown from typical experiments reproduced at least 13 times with different oocytes from different batches.

**Figure 5.

A1AT decreases inward INa of human cells. H441 cells were patched in the whole-cell mode and held at −40 mV. A1AT (0.05 mg/ml; 1 μM) or vehicle was then added in the recording chamber and INa values were then recorded while pulsing the Vm from −40 to −140 mV every 5 seconds for a 50-minute period. Amiloride (2 μM) was added when steady-state INa values were obtained after addition of A1AT. (A) Typical time courses of INa while pulsing the Vm from −40 to −140 mV. (B) The same protocol as in (A) showing that addition of 1 μM trypsin increased INa. (C) Current–voltage curves. Values are means (±1 SEM; n = 6). Solid circles, vehicle; open inverted triangles, A1AT; solid triangles, amiloride (2 μM) after vehicle. INa values of A1AT-treated oocytes were significantly different from the corresponding controls (P < 0.01).

**Figure 6.

Measurements of oocyte and H441 cell protease activity after addition of A1AT. A total of 20 oocytes (A) or 105 H441 cells (B) were incubated with vehicle (solid squares), native (solid circles), or ONOO−-treated (1 mM; open squares) A1AT (1 μM) for 45 minutes and then added to a cuvette containing Boc-Gln-Ala-Arg-AMC (50 μM). Vertical arrow in A indicates the time that oocytes were ruptured by vigorous pipetting. Fluorescence was measured continuously for the next 60 minutes at 460 nm after excitation at 380 nm. Results are shown of typical experiments that were repeated three times.

**Figure 7.

(A) A1AT does not decrease currents in oocytes injected with Liddle ENaC. Continuous recording of inward INa by pulsing the Vm from −40 to −140 mV. In contrast to results obtained with wild-type ENaC, incubation of Liddle ENaC–injected oocytes with A1AT (0.05 mg/ml; 1 μM) had no effect on INa. On the other hand, amiloride (10 μM) decreased INa to zero. Results are shown of a typical experiment that was repeated nine times with identical results. (B) Inhibition of the proteasome system does not prevent the A1AT inhibition of ENaC. Mean values are displayed of INa of oocytes injected with wild-type ENaC and incubated with either MG-132 (red line, 0.1 μM for 24 hours; orange line, 4 μM for 2 hours) or vehicle (black line) before being perfused with A1AT (1 μM). For purposes of clarity, SEs were not included.

**Figure 8.

Incubation of ENaC-expressing oocytes with A1AT decreases surface but not total γ-ENaC levels. (A) Total γ-ENaC. (B) Surface γ-ENaC. Lane 1, water injected oocytes; lane 2, α,β,γ-hENaC–injected oocytes; lanes 3 and 4, α,β,γ-hENaC–injected oocytes exposed to 0.05 mg/ml A1AT for 20 and 40 minutes, respectively, before lysis. Results are shown of a typical experiment that was repeated three different times using three different batches of oocytes. For each lane, proteins from 5 (total) or 25 (surface) different oocytes were pooled. (C) Density values of cleaved (lightly shaded bars) and not-cleaved (darkly shaded bars) γ-ENaC bands shown in (B) using AlphaEaseFC 4.0 software (Alpha Innotech Corp., San Leandro, CA). Values shown are X = mean (±1 SEM); n = 3 (three distinct sets of oocytes were used).

**Figure 9.

A1AT decreases alveolar fluid clearance (AFC) in vivo. AFC was measured across the distal lung spaces of anesthetized and ventilated C57BL/6 mice 30 minutes after instillation of a solution containing 5% albumin and vehicle, A1AT (1 μM), ONOO−-treated A1AT (1 μM), or 1.5 mM amiloride. Values are means (±SEM); numbers in parentheses are numbers of mice. *P < 0.05 and **P < 0.01 compared with control.

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Alpha(1)-antitrypsin inhibits epithelial Na+ transport in vitro and in vivo - PubMed (original) (raw)