James D. Stockand

Paracrine Regulation of the Epithelial Na+ Channel in the Mammalian Collecting Duct by Purinergic P2Y2 Receptor Tone

Growing evidence implicates a key role for extracellular nucleotides in cellular regulation, including of ion channels and renal function, but the mechanisms for such actions are inadequately defined. We investigated purinergic regulation of the epithelial Na+ channel (ENaC) in mammalian collecting duct. We find that ATP decreases ENaC activity in both mouse and rat collecting duct principal cells. ATP and other nucleotides, including UTP, decrease ENaC activity via apical P2Y2 receptors. ENaC in collecting ducts isolated from mice lacking this receptor have blunted responses to ATP. P2Y2 couples to ENaC via PLC; direct activation of PLC mimics ATP action. Tonic regulation of ENaC in the collecting duct occurs via locally released ATP; scavenging endogenous ATP and inhibiting P2 receptors, in the absence of other stimuli, rapidly increases ENaC activity. Moreover, ENaC has greater resting activity in collecting ducts from P2Y2-/- mice. Loss of collecting duct P2Y2 receptors in the knock-out mouse is the primary defect leading to increased ENaC activity based on the ability of direct PLC stimulation to decrease ENaC activity in collecting ducts from P2Y2-/- mice in a manner similar to ATP in collecting ducts from wild-type mice. These findings demonstrate that locally released ATP acts in an autocrine/paracrine manner to tonically regulate ENaC in mammalian collecting duct. Loss of this intrinsic regulation leads to ENaC hyperactivity and contributes to hypertension that occurs in P2Y2 receptor-/- mice. P2Y2 receptor activation by nucleotides thus provides physiologically important regulation of ENaC and electrolyte handling in mammalian kidney.

Direct Activation of the Epithelial Na+ Channel by Phosphatidylinositol 3,4,5-Trisphosphate and Phosphatidylinositol 3,4-Bisphosphate Produced by Phosphoinositide 3-OH Kinase

The phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is accepted to be a direct modulator of ion channel activity. The products of phosphoinositide 3-OH kinase (PI3K), PtdIns(3,4)P2 and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), in contrast, are not. We report here activation of the epithelial Na+ channel (ENaC) reconstituted in Chinese hamster ovary cells by PI3K. Insulin-like growth factor-I also activated reconstituted ENaC and increased Na+ reabsorption across renal A6 epithelial cell monolayers via PI3K. Neither IGF-I nor PI3K affected the levels of ENaC in the plasma membrane. The effects of PI3K and IGF-I on ENaC activity paralleled changes in the plasma membrane levels of the PI3K product phospholipids, PtdIns(3,4)P2/PtdIns(3,4,5)P3, as measured by evanescent field fluorescence microscopy. Both PtdIns(3,4)P2 and PtdIns(3,4,5)P3 activated ENaC in excised patches. Activation of ENaC by PI3K and its phospholipid products corresponded to changes in channel open probability. We conclude that PI3K directly modulates ENaC activity via PtdIns(3,4)P2 and PtdIns(3,4,5)P3. This represents a novel transduction pathway whereby growth factors, such as IGF-I, rapidly modulate target proteins independent of signaling elicited by kinases downstream of PI3K.

Regulation of the epithelial Na+ channel by endothelin-1 in rat collecting duct

We used patch-clamp electrophysiology to investigate regulation of the epithelial Na+ channel (ENaC) by endothelin-1 (ET-1) in isolated, split-open rat collecting ducts. ET-1 significantly decreases ENaC open probability by about threefold within 5 min. ET-1 decreases ENaC activity through basolateral membrane ETB but not ETA receptors. In rat collecting duct, we find no role for phospholipase C or protein kinase C in the rapid response of ENaC to ET-1. ET-1, although, does activate src family tyrosine kinases and their downstream MAPK1/2 effector cascade in renal principal cells. Both src kinases and MAPK1/2 signaling are necessary for ET-1-dependent decreases in ENaC open probability in the split-open collecting duct. We conclude that ET-1 in a physiologically relevant manner rapidly suppresses ENaC activity in native, mammalian principal cells. These findings may provide a potential mechanism for the natriuresis observed in vivo in response to ET-1, as well as a potential cause for the salt-sensitive hypertension found in animals with impaired endothelin signaling.

Ras Activates the Epithelial Na+ Channel through Phosphoinositide 3-OH Kinase Signaling

Aldosterone induces expression and activation of the GTP-dependent signaling switch K-Ras. This small monomeric G protein is both necessary and sufficient for activation of the epithelial Na+ channel (ENaC). The mechanism by which K-Ras enhances ENaC activity, however, is uncertain. We demonstrate here that K-Ras activates human ENaC reconstituted in Chinese hamster ovary cells in a GTP-dependent manner. K-Ras influences ENaC activity most likely by affecting open probability. Inhibition of phosphoinositide 3-OH kinase (PI3K) abolished K-Ras actions on ENaC. In contrast, inhibition of other K-Ras effector cascades, including the MAPK and Ral/Rac/Rho cascades, did not affect K-Ras actions on ENaC. Activation of ENaC by K-Ras, moreover, was sensitive to co-expression of dominant negative p85PI3K. The G12:C40 effector-specific double mutant of Ras, which preferentially activates PI3K, enhanced ENaC activity in a manner sensitive to inhibition of PI3K. Other effector-specific mutants preferentially activating MAPK and RalGDS signaling had no effect. Constitutively active PI3K activated ENaC independent of K-Ras with the effects of PI3K and K-Ras on ENaC not being additive. We conclude that K-Ras activates ENaC via the PI3K cascade.

Regulation of Na+ Reabsorption by the Aldosterone-induced Small G Protein K-Ras2A

Xenopus laevis A6 cells were used as model epithelia to test the hypothesis that K-Ras2A is an aldosterone-induced protein necessary for steroid-regulated Na+ transport. The possibility that increased K-Ras2A alone is sufficient to mimic aldosterone action on Na+ transport also was tested. Aldosterone treatment increased K-Ras2A protein expression 2.8-fold within 4 h. Active Ras is membrane associated. After aldosterone treatment, 75% of K-Ras was localized to the plasma membrane compared with 25% in the absence of steroid. Aldosterone also increased the amount of active (phosphorylated) mitogen-activated protein kinase kinase likely through K-Ras2A signaling. Steroid-induced K-Ras2A protein levels and Na+ transport were decreased with antisense K-ras2A oligonucleotides, showing that K-Ras2A is necessary for the natriferic actions of aldosterone. Aldosterone-induced Na+ channel activity, was decreased from 0.40 to 0.09 by pretreatment with antisense rasoligonucleotide, implicating the luminal Na+ channel as one final effector of Ras signaling. Overexpression of K-Ras2A increased Na+ transport approximately 2.2-fold in the absence of aldosterone. These results suggest that aldosterone signals to the luminal Na+ channel via multiple pathways and that K-Ras2A levels are limiting for a portion of the aldosterone-sensitive Na+ transport.

Insight toward epithelial Na+ channel mechanism revealed by the acid-sensing ion channel 1 structure

The epithelial Na+ channel/degenerin (ENaC/DEG) protein family includes a diverse group of ion channels, including nonvoltage‐gated Na+ channels of epithelia and neurons, and the acid‐sensing ion channel 1 (ASIC1). In mammalian epithelia, ENaC helps regulate Na+ and associated water transport, making it a critical determinant of systemic blood pressure and pulmonary mucosal fluidity. In the nervous system, ENaC/DEG proteins are related to sensory transduction. While the importance and physiological function of these ion channels are established, less is known about their structure. One hallmark of the ENaC/DEG channel family is that each channel subunit has only two transmembrane domains connected by an exceedingly large extracellular loop. This subunit structure was recently confirmed when Jasti and colleagues determined the crystal structure of chicken ASIC1, a neuronal acid‐sensing ENaC/DEG channel. By mapping ENaC to the structural coordinates of cASIC1, as we do here, we hope to provide insight toward ENaC structure. ENaC, like ASIC1, appears to be a trimeric channel containing 1α, 1β, and 1γ subunit. Heterotrimeric ENaC and monomeric ENaC subunits within the trimer possibly contain many of the major secondary, tertiary, and quaternary features identified in cASIC1 with a few subtle but critical differences. These differences are expected to have profound effects on channel behavior. In particular, they may contribute to ENaC insensitivity to acid and to its constitutive activity in the absence of time‐ and ligand‐dependent inactivation. Experiments resulting from this comparison of cASIC1 and ENaC may help clarify unresolved issues related to ENaC architecture, and may help identify secondary structures and residues critical to ENaC function.

Differential Effects of Protein Kinase C on the Levels of Epithelial Na+ Channel Subunit Proteins

Regulation of epithelial Na+channel (ENaC) subunit levels by protein kinase C (PKC) was investigated in A6 cells. PKC activation altered ENaC subunit levels, differentially decreasing the levels of both β and γ, but not αENaC. Temporal regulation of β and γENaC by PKC differed; γENaC decreased with a time constant of 3.7 ± 1.0 h, whereas βENaC decreased in 13.9 ± 3.0 h. Activation of PKC also resulted in a decrease in trans-epithelial Na+reabsorption for up to 48 h. PMA activation of PKC resulted in negative feedback inhibition of PKC protein levels beginning within 4 h. Both β and γENaC levels, as well as transport tended toward pretreatment values after 48 h of PMA treatment. PKC inhibitors attenuated the effects of PMA on ENaC subunit levels and Na+ transport. These results directly show for the first time that PKC differentially regulates ENaC subunit levels by decreasing the levels of β and γ but not αENaC protein. These results imply a PKC-dependent, long term decrease in Na+ reabsorption.

Acute Regulation of the Epithelial Na+ Channel by Phosphatidylinositide 3-OH Kinase Signaling in Native Collecting Duct Principal Cells

Activity of the epithelial Na(+) channel (ENaC) is limiting for Na(+) reabsorption in the aldosterone-sensitive distal nephron. Hormones, including aldosterone and insulin, increase ENaC activity, in part by stimulating phosphatidylinositide 3-OH kinase (PI3-K) signaling. Recent studies in heterologous expression systems reveal a close spatiotemporal coupling between PI3-K signaling and ENaC activity with the phospholipid product of this kinase, PI(3,4,5)P(3), in some cases, directly binding the channel and increasing open probability (P(o)). This study tested whether this tight coupling plays a physiologic role in modulating ENaC activity in native tissue and polarized epithelial cells. IGF-I was found to increase Na(+) reabsorption across mpkCCD(c14) principal cell monolayers in a PI3-K-sensitive manner. Inhibition of PI3-K signaling, moreover, rapidly decreased Na(+) reabsorption and ENaC activity in mpkCCD(c14) cells that were treated with corticosteroids and IGF-I. These decreases paralleled changes in apical membrane PI(3,4,5)P(3) levels, demonstrating tight spatiotemporal coupling between ENaC activity and PI3-K/PI(3,4,5)P(3) signaling within this membrane. For further probing of the mechanism underpinning this coupling, cortical collecting ducts (CCD) were isolated from rat and split open to expose the apical membrane for patch-clamp analysis. Inhibition of PI3-K signaling with wortmannin and LY294002 but not its inactive analogue rapidly and markedly decreased the P(o) of ENaC. Moreover, IGF-I acutely increased P(o) of ENaC in CCD principal cells in a PI3-K-sensitive manner. Together, these observations stress the importance of tight spatiotemporal coupling between PI3-K signaling and ENaC within the apical membrane of principal cells to the physiologic control of this ion channel.

Dietary Na+ inhibits the open probability of the epithelial sodium channel in the kidney by enhancing apical P2Y2-receptor tone

Apical release of ATP and UTP can activate P2Y2 receptors in the aldosterone‐sensitive distal nephron (ASDN) and inhibit the open probability (Po) of the epithelial sodium channel (ENaC). Little is known, however, about the regulation and physiological relevance of this system. Patch‐clamp studies in freshly isolated ASDN provide evidence that increased dietary Na+ intake in wild‐type mice lowers ENaC Po, consistent with a contribution to Na+ homeostasis, and is associated with increased urinary concentrations of UTP and the ATP hydrolytic product, ADP. Genetic deletion of P2Y2 receptors in mice (P2Y2 −/−; litter‐mates to wild‐type mice) or inhibition of apical P2Y‐receptor activation in wild‐type mice prevents dietary Na+‐induced lowering of ENaC Po. Although they lack suppression of ENaC Po by dietary NaCl, P2Y2−/− mice do not exhibit NaCl‐sensitive blood pressure, perhaps as a consequence of compensatory down‐regulation of aldosterone levels. Consistent with this hypothesis, clamping mineralocorticoid activity at high levels unmasks greater ENaC activity and NaCl sensitivity of blood pressure in P2Y2−/− mice. The studies indicate a key role of the apical ATP/UTP‐P2Y2‐receptor system in the inhibition of ENaC Po in the ASDN in response to an increase in Na+ intake, thereby contributing to NaCl homeostasis and blood pressure regulation.—Pochynyuk, O., Rieg, T., Bugaj, V., Schroth, J., Fridman, A., Boss, G. R., Insel, P. A., Stockand, J. D., Vallon, V. Dietary Na+ inhibits the open probability of the epithelial sodium channel in the kidney by enhancing apical P2Y2‐receptor tone. FASEB J. 24, 2056–2065 (2010). www.fasebj.org

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

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.