Alveolar fluid clearance in the developing and mature lungs is believed to be mediated by some form of epithelial Na channels (ENaC). However, single-channel studies using isolated alveolar type II (ATII) cells have failed to demonstrate consistently the presence of highly selective Na+ channels that would be expected from ENaC expression. We postulated that in vitro culture conditions might be responsible for alterations in the biophysical properties of Na+ conductances observed in cultured ATII cells. When ATII cells were grown on glass plates submerged in media that lacked steroids, the predominant channel was a 21-pS nonselective cation channel (NSC) with a Na+-to-K+ selectivity of 1; however, when grown on permeable supports in the presence of steroids and air interface, the predominant channel was a low-conductance (6.6 +/- 3.4 pS, n = 94), highly Na+-selective channel (HSC) with a P(Na)/P(K) >80 that is inhibited by submicromolar concentrations of amiloride (K(0.5) = 37 nM) and is similar in biophysical properties to ENaC channels described in other epithelia. To establish the relationship of this HSC channel to the cloned ENaC, we employed antisense oligonucleotide methods to inhibit the individual subunit proteins of ENaC (alpha, beta, and gamma) and used patch-clamp techniques to determine the density of this channel in apical membrane patches of ATII cells. Overnight treatment of cells with antisense oligonucleotides to any of the three subunits of ENaC resulted in a significant decrease in the density of HSC channels in the apical membrane cell-attached patches. Taken together, these results show that when grown on permeable supports in the presence of steroids and air interface, the predominant channels expressed in ATII cells have single-channel characteristics resembling channels that are associated with the coexpression of the three cloned ENaC subunits alpha-, beta-, and gamma-ENaC.

Dopamine increases lung fluid clearance. This is partly due to activation of basolateral Na-K-ATPase. However, activation of Na-K-ATPase by itself is unlikely to produce large changes in transepithelial transport. Therefore, we examined apical and basolateral dopamine's effect on apical, highly selective sodium channels [epithelial sodium channels (ENaC)] in monolayers of an alveolar type 2 cell line (L2). Dopamine increased channel open probability (P(o)) without changing the unitary current. The D(1) receptor blocker SCH-23390 blocked the dopamine effect, but the D(2) receptor blocker sulpiride did not. The dopamine-mediated increase in ENaC activity was not a secondary effect of dopamine stimulation of Na-K-ATPase, since ouabain applied to the basolateral surface to block the activity of Na-K-ATPase did not alter dopamine-mediated ENaC activity. Protein kinase A (PKA) was not responsible for dopamine's effect since a PKA inhibitor, H89, did not reduce dopamine's effect. However, cpt-2-O-Me-cAMP, which selectively binds and activates EPAC (exchange protein activated by cAMP) but not PKA, increased ENaC P(o). An Src inhibitor, PP2, and the phosphatidylinositol-3-kinase inhibitor, LY-294002, blocked dopamine's effect on ENaC. In addition, an MEK blocker, U0126, an inhibitor of phospholipase A(2), and a protein phosphatase inhibitor also blocked the effect of dopamine on ENaC P(o). Finally, since the cAMP-EPAC-Rap1 pathway also activates DARPP32 (32-kDa dopamine response protein phosphatase), we confirmed that dopamine phosphorylates DARPP32, and okadaic acid, which blocks phosphatases (DARPP32), also blocks dopamine's effect. In summary, dopamine increases ENaC activity by a cAMP-mediated alternative signaling pathway involving EPAC and Rap1, signaling molecules usually associated with growth-factor-activated receptors.

Amiloride-sensitive sodium channels in the lung play an important role in lung fluid balance. Particularly in the alveoli, sodium transport is closely regulated to maintain an appropriate fluid layer on the surface of the alveoli. Alveolar type II cells appear to play an important role in this sodium transport. In alveolar type II cells, there are a variety of different amiloride-sensitive, sodium-permeable channels. This significant diversity appears to play a role in both normal lung physiology and pathologic states. In many epithelial tissues, amiloride-sensitive epithelial sodium channels (ENaC) are formed from three subunit proteins designated alpha-ENaC, beta-ENaC, and gamma-ENaC. At least part of the diversity of sodium-permeable channels in lung arises from assembling different combinations of these subunits to form channels with different biophysical properties and different mechanisms for regulation. This leads to epithelial tissue in the lung that has enormous flexibility to alter the magnitude and regulation of salt and water transport. In this article, we discuss the regulation of ENaCs composed of varying subunits and some of the implications of the regulation for normal pulmonary function.

Previous studies using whole-cell recording methods suggest that human B lymphocytes express an amiloride-sensitive, sodium-permeable channel. The present studies aim to determine whether this channel has biophysical properties and a molecular structure related to the ␣, ␤, and ␥ subunits of the epithelial sodium channel (ENaC). Reverse transcriptase polymerase chain reaction and Northern blots showed that human B lymphocytes express messages for both ␣-and ␤-but not ␥-ENaC. Western blots showed that both ␣-and ␤-but not ␥-ENaC proteins are expressed and strongly reduced by antisense oligonucleotides. Patch clamp experiments demonstrated that lymphocyte sodium channels are not active in cell-attached patches. However, membrane stretch can activate a 21-pS nonselective cation channel. The frequency of observance of this channel was significantly reduced by antisense oligonucleotide against ␣-ENaC but not by antisense oligonucleotide against ␤-ENaC, indicating that only the ␣ subunit of ENaC is necessary to form stretch-activated cation channels. Aldosterone (1.5 M) reduced the frequency of observance of 21-pS ␣-ENaC channels and simultaneously induced the appearance of spontaneously active 10-pS channels. Antisense oligonucleotide experiments showed that this 10-pS channel is formed from ␣-and ␤-ENaC. After expression of exogenous ␥-ENaC, aldosterone again reduced the frequency of observance of the 21-pS ␣-ENaC channel but induced the appearance of a 5-pS channel, presumably a ␣␤␥-ENaC channel. In the absence of aldosterone, the ␣ subunit forms an ␣-cryptic channel that is activated by stretch, and in the presence of aldosterone, ␤ and ␣ subunits together form an active channel that is modulated by aldosterone.The superfamily of proteins to which the epithelial sodium channel (ENaC) 1 belongs generally mediates cation transport across cell membranes. ENaC, itself, is usually associated with sodium transport across the apical membrane of a variety of epithelia including the colon, lung, and kidney. Since 1994, when ENaC was initially cloned from rat colon (1), the biophysical properties and molecular structure of ENaC have been studied extensively. Several lines of evidence suggest that ENaC, including human ENaC (hENaC), is typically composed of three subunits, ␣, ␤, and ␥, and that all three subunits are required to form a functional ␣␤␥-ENaC channel complex (1-6). In heterologous expression systems, maximal expression of the hENaC channel requires co-expression of all three subunits (7, 8), and in oocytes, expression of ␣-ENaC cRNA alone produces little expression of any amiloride-sensitive currents. However, in other cell types, expression of the exogenous ␣-ENaC subunit alone can form a stretch-activated nonselective cation channel (9), and a similar nonselective cation channel has also been described in native lung epithelial alveolar type II cells (10 -12). This lung cation channel appears to be formed from ␣-ENaC alone and is equally permeable to Na ϩ and K ϩ , is sensitive to steroid hormones, and has a h...

We and others have shown that epithelial Na ϩ channels (ENaC) in alveolar type 2 (AT2) cells are activated by ␤2 agonists, steroid hormones, elevated oxygen tension, and by dopamine. Although acetylcholine receptors (AChRs) have been previously described in the lung, there are few reports of whether cholinergic agonists alter sodium transport in the alveolar epithelium. Therefore, we investigated how cholinergic receptors regulate ENaC activity in primary cultures of rat AT2 cells using cell-attached patchclamp recordings to assess ENaC activity. We found that the muscarinic agonists, carbachol (CCh) and oxotremorine, activated ENaC in a dose-dependent manner but that nicotine did not. CCh-induced activation of ENaC was blocked by atropine. Western blotting and immunohistochemistry suggested that muscarinic M2 and M3 receptors (mAChRs) but not nicotinic receptors were present in AT2 cells. Endogenous RhoA and GTP-RhoA increased in response to CCh and the increase was reduced by pretreatment with atropine. We showed that Y-27632, an inhibitor of Rho-associated protein kinase (ROCK), abolished endogenous ENaC activity and inhibited the activation of ENaC by CCh. We also showed that ROCK signaling was necessary for ENaC stability in 2F3 cells, a model for AT2 cells. Our results showed that muscarinic agonists activated ENaC in rat AT2 cells through M2 and/or M3 mAChRs probably via a RhoA/ROCK signaling pathway.