Xi-Juan Chen

Nitric oxide inhibits lung sodium transport through a cGMP-mediated inhibition of epithelial cation channels

We used the patch-clamp technique to study the effect of nitric oxide (NO) on a cation channel in rat type II pneumocytes [alveolar type II (AT II) cells]. Single-channel recordings from the apical surface of AT II cells in primary culture showed a predominant cation channel with a conductance of 20.6 +/- 1.1 (SE) pS (n = 9 cell-attached patches) and Na(+)-to-K+ selectivity of 0.97 +/- 0.07 (n = 7 cell-attached patches). An NO donor, S-nitrosoglutathione (GSNO; 100 microM), inhibited the basal cation-channel activity by 43% [open probability (Po), control 0.28 +/- 0.05 vs. GSNO 0.16 +/- 0.03; P < 0.001; n = 16 cell-attached patches], with no significant change in the conductance. GSNO reduced the Po by reducing channel mean open and increasing mean closed times. GSNO inhibition was reversed by washout. The inhibitory effect of NO was confirmed by using a second donor of NO, S-nitroso-N-acetylpenicillamine (100 microM; Po, control 0.53 +/- 0.05 vs. S-nitroso-N-acetylpenicillamine 0.31 +/- 0.04; -42%; P < 0.05; n = 5 cell-attached patches). The GSNO effect was blocked by methylene blue (a blocker of guanylyl cyclase; 100 microM), suggesting a role for cGMP. The permeable analog of cGMP, 8-bromo-cGMP (8-BrcGMP; 1 mM), inhibited the cation channel in a manner similar to GSNO (Po, control 0.38 +/- 0.06 vs. 8-BrcGMP 0.09 +/- 0.02; P < 0.05; n = 7 cell-attached patches). Pretreatment of cells with 1 microM KT-5823 (a blocker of protein kinase G) abolished the inhibitory effect of GSNO. The NO inhibition of channels was not due to changes in cell viability. Intracellular cGMP was found to be elevated in AT II cells treated with NO (control 13.4 +/- 3.6 vs. GSNO 25.4 +/- 4.1 fmol/ml; P < 0.05; n = 6 cell-attached patches). We conclude that NO suppresses the activity of an Na(+)-permeant cation channel on the apical surface of AT II cells. This action appears to be mediated by a cGMP-dependent protein kinase.We used the patch-clamp technique to study the effect of nitric oxide (NO) on a cation channel in rat type II pneumocytes [alveolar type II (AT II) cells]. Single-channel recordings from the apical surface of AT II cells in primary culture showed a predominant cation channel with a conductance of 20.6 ± 1.1 (SE) pS ( n = 9 cell-attached patches) and Na+-to-K+selectivity of 0.97 ± 0.07 ( n = 7 cell-attached patches). An NO donor, S-nitrosoglutathione (GSNO; 100 μM), inhibited the basal cation-channel activity by 43% [open probability ( P o), control 0.28 ± 0.05 vs. GSNO 0.16 ± 0.03; P < 0.001; n = 16 cell-attached patches], with no significant change in the conductance. GSNO reduced the P o by reducing channel mean open and increasing mean closed times. GSNO inhibition was reversed by washout. The inhibitory effect of NO was confirmed by using a second donor of NO, S-nitroso- N-acetylpenicillamine (100 μM; P o, control 0.53 ± 0.05 vs. S-nitroso- N-acetylpenicillamine 0.31 ± 0.04; -42%; P < 0.05; n = 5 cell-attached patches). The GSNO effect was blocked by methylene blue (a blocker of guanylyl cyclase; 100 μM), suggesting a role for cGMP. The permeable analog of cGMP, 8-bromo-cGMP (8-BrcGMP; 1 mM), inhibited the cation channel in a manner similar to GSNO ( P o, control 0.38 ± 0.06 vs. 8-BrcGMP 0.09 ± 0.02; P < 0.05; n = 7 cell-attached patches). Pretreatment of cells with 1 μM KT-5823 (a blocker of protein kinase G) abolished the inhibitory effect of GSNO. The NO inhibition of channels was not due to changes in cell viability. Intracellular cGMP was found to be elevated in AT II cells treated with NO (control 13.4 ± 3.6 vs. GSNO 25.4 ± 4.1 fmol/ml; P < 0.05; n = 6 cell-attached patches). We conclude that NO suppresses the activity of an Na+-permeant cation channel on the apical surface of AT II cells. This action appears to be mediated by a cGMP-dependent protein kinase.

Antisense oligonucleotides against the α-subunit of ENaC decrease lung epithelial cation-channel activity

Amiloride-sensitive Na+ transport by lung epithelia plays a critical role in maintaining alveolar Na+ and water balance. It has been generally assumed that Na+transport is mediated by the amiloride-sensitive epithelial Na+ channel (ENaC) because molecular biology studies have confirmed the presence of ENaC subunits α, β, and γ in lung epithelia. However, the predominant Na+-transporting channel reported from electrophysiological studies by most laboratories is a nonselective, high-conductance channel that is very different from the highly selective, low-conductance ENaC reported in other tissues. In our laboratory, single-channel recordings from apical membrane patches from rat alveolar type II (ATII) cells in primary culture reveal a nonselective cation channel with a conductance of 20.6 ± 1.1 pS and an Na+-to-K+selectivity of 0.97 ± 0.07. This channel is inhibited by submicromolar concentrations of amiloride. Thus there is some question about the relationship between the gene product observed with single-channel methods and the cloned ENaC subunits. We have employed antisense oligonucleotide methods to block the synthesis of individual ENaC subunit proteins (α, β, and γ) and determined the effect of a reduction in the subunit expression on the density of the nonselective cation channel observed in apical membrane patches on ATII cells. Treatment of ATII cells with antisense oligonucleotides inhibited the production of each subunit protein; however, single-channel recordings showed that only the antisense oligonucleotide targeting the α-subunit resulted in a significant decrease in the density of nonselective cation channels. Inhibition of the β- and γ-subunit proteins alone or together did not cause any changes in the observed channel density. There were no changes in open probability or other channel characteristics. These results support the hypothesis that the α-subunit of ENaC alone or in combination with some protein other than the β- or γ-subunit protein is the major component of lung alveolar epithelial cation channels.

Ascorbate Deficiency Impairs Sodium Transport by Distal Lung Epithelia ♦ 1954

Deficiency of antioxidants has been shown to increase the incidence and severity of ARDS and the propensity for acute edematous lung injury. This study examines the effect of ascorbate (Asc) deficiency on lung epithelial sodium transport. Adult guinea pigs were fed Asc-deficient diet for 7, 14, and 21 days to create mild, moderate, and severe Asc deficiency. The patch-clamp technique was used to study single-channel activity in alveolar type II (AT II) cells. Asc deficiency was confirmed by the significantly lower Asc levels in plasma, lung tissue, and AT-II cells, and by decreased hydroxylation of proline and lysine in Asc-deficient animals. Table 1 shows the effect of Asc deficiency on the probability of finding open sodium channels. There was a two-fold increase in the probability of finding no channel activity in cell-attached apical patches from 14 and 21 days (not 7 days) Asc- deficient animals (Table 1). Addition of Asc to the culture medium and culture of AT-II cells on type IV collagen resulted in partial restoration of channel activity. Table 2 shows patches with no activity in control and 21-day deficient animals. The level of Asc deficiency seen in these animals has been reported in chronic asthmatics, premature infants, and in chronic smokers. We speculate that clinially-relevant levels of Asc deficiency may impair sodium transport mechanisms in the distal lung epithelia resulting in an increased propensity for pulmonary edema.

Beta-Adrenergic Agonists Stimulate Lung Epithelial Sodium Transport Via a cAMP-Mediated Activation of Amiloride-Sensitive Cation Channels † 1682

Beta-Adrenergic Agonists Stimulate Lung Epithelial Sodium Transport Via a cAMP-Mediated Activation of Amiloride-Sensitive Cation Channels † 1682

Nitric oxide inhibits sodium transport by distal lung epithelia via a cGMP-mediated inhibition of sodium channels. |[dagger]| 1520

Active sodium (Na+) transport by distal lung epithelium plays a critical role in maintaining normal alveolar fluid balance and in preventing pulmonary edema. The effect of inhaled nitric oxide (NO) on lung epithelial Na+ transport is largely unknown. We have used the patch-clamp technique to study the effect of NO on a Na+ permeant cation channel in rat type-II pneumocytes (ATII cells). Single-channel recordings from the apical surface of ATII cells in primary culture showed a predominant cation channel with unitary conductance of 17.6 pS and Na+/K+ selectivity of 0.9. Nitric oxide donor GSNO (100μM) inhibited the basal cation channel activity by 56% (nPo control vs. GSNO, x±SEM, 0.29±.07 vs. 0.13±.04, n=6, p <0.05). The effect was rapid with onset of less than 2 min and sustained through up to 30 min of recording. GSNO inhibition was reversed by washout, and the unitary conductance remained unchanged. The inhibitory effect of NO was confirmed by using a second donor of NO, SNAP (100μM) (0.45±.09 vs. 0.26±.06, n=6, p <0.05). The GSNO effect was blocked by methylene blue (100μM) suggesting a role for cGMP. The soluble analog of cGMP, 8Br-cGMP (100μM) inhibited the cation channel similar to that seen with GSNO (nPo control vs. Br-cGMP, 0.35±.08 vs. 0.17±.06, -52%, n=6, p <0.05). To determine if the activation of cGMP-dependent protein kinase was necessary for NO to inhibit Na+ channels, cells were treated with KT 5823 (a blocker of protein kinase G) prior to treatment with GSNO. KT 5823 abolished the inhibitory effect of GSNO. The NO inhibition of channels was not due to changes in cell viability. We conclude that NO suppresses the activity of a Na+ permeant cation channel on the apical surface of ATII cells. This action appears to be mediated by cGMP-induced activation of a protein kinase. The modulation of Na+ channels by NO may have important clinical implications since it can adversely effect alveolar fluid balance resulting in the development of pulmonary edema.