Apical release of ATP and UTP can activate P2Y(2) receptors in the aldosterone-sensitive distal nephron (ASDN) and inhibit the open probability (P(o)) 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 P(o), 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 P2Y(2) receptors in mice (P2Y(2)(-/-); littermates to wild-type mice) or inhibition of apical P2Y-receptor activation in wild-type mice prevents dietary Na(+)-induced lowering of ENaC P(o). Although they lack suppression of ENaC P(o) by dietary NaCl, P2Y(2)(-/-) 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 P2Y(2)(-/-) mice. The studies indicate a key role of the apical ATP/UTP-P2Y(2)-receptor system in the inhibition of ENaC P(o) in the ASDN in response to an increase in Na(+) intake, thereby contributing to NaCl homeostasis and blood pressure regulation.

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 P2Y 2 receptors. ENaC in collecting ducts isolated from mice lacking this receptor have blunted responses to ATP. P2Y 2 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 P2Y 2 ؊/؊ mice. Loss of collecting duct P2Y 2 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 P2Y 2 ؊/؊ 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 P2Y 2 receptor ؊/؊ mice. P2Y 2 receptor activation by nucleotides thus provides physiologically important regulation of ENaC and electrolyte handling in mammalian kidney. Systemic Naϩ balance influences blood pressure. Consequently, body Na ϩ content is under tight negative-feedback control by the renin-angiotensin-aldosterone system. Discretionary Na ϩ reabsorption in the aldosterone-sensitive distal renal nephron, including the collecting duct, fine-tunes plasma Na ϩ levels. Here, the activity of the luminal epithelial Na ϩ channel (ENaC) 2 is limiting for Na ϩ transport (1-3). ENaC is an end-effector of the renin-angiotensin-aldosterone system with aldosterone increasing ENaC activity. The importance of ENaC and its proper regulation to control of blood pressure is highlighted by several diseases associated with gain and loss of ENaC function (3, 4). For instance, gain of ENaC function results in inappropriate Na ϩ conservation and hypertension (e.g. Liddle syndrome). Conversely, loss of ENaC function results in renal salt wasting associated with hypotension (e.g. pseudohypoaldosteronism type 1).Although extrinsic regulation of ENaC in the distal nephron by hormones originating outside the kidney is considered pivotal to blood pressure control, complementary regulation of ENaC by autocrine/paracrine factors originating from intrarenal sources is just now becoming appreciated. ATP has been identified as a candidate signaling molecule possibly mediating intrinsic control of distal nephron Na ϩ reabsorption (5-14). ATP and other...

The mechanisms underlying "aldosterone escape," which refers to the excretion of sodium (Na ϩ ) during high Na ϩ intake despite inappropriately increased levels of mineralocorticoids, are incompletely understood. Because local purinergic tone in the aldosterone-sensitive distal nephron downregulates epithelial Na ϩ channel (ENaC) activity, we tested whether this mechanism mediates aldosterone escape. Here, urinary ATP concentration increased with dietary Na ϩ intake in mice. Physiologic concentrations of ATP decreased ENaC activity in a dosage-dependent manner. P2Y 2 Ϫ/Ϫ mice, which lack the purinergic receptor, had significantly less increased Na ϩ excretion than wild-type mice in response to high-Na ϩ intake. Exogenous deoxycorticosterone acetate and deletion of the P2Y 2 receptor each modestly increased the resistance of ENaC to changes in Na ϩ intake; together, they markedly increased resistance. Under the latter condition, ENaC could not respond to changes in Na ϩ intake. In contrast, as a result of aldosterone escape, wild-type mice had increased Na ϩ excretion in response to high-Na ϩ intake regardless of the presence of high deoxycorticosterone acetate. These data suggest that control of ENaC by purinergic signaling is necessary for aldosterone escape.

We used patch-clamp electrophysiology to investigate regulation of the epithelial Na ϩ 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.salt-sensitive hypertension; systemic blood pressure ENDOTHELIN-1 (ET-1) is a powerful vasoconstricting peptide hormone that is an important regulator of systemic blood pressure (53). Independent of its vascular effects, ET-1 also affects renal Na ϩ and water handling favoring natriuresis and diuresis. While circulating ET-1 arises from endothelial cells, local ET-1 systems also exist. For instance in the kidney, the collecting duct produces significant amounts of 25,38,51). ET-1 targets cells through two distinct receptor subtypes, ET A and ET B (32, 41). Renal collecting duct cells have both types of receptors and are able to bind 49,50). Thus, collecting duct-derived ET-1, acting in a paracrine/ autocrine manner, is an important regulator of renal Na ϩ handling (2,20,26,42).Regulated Na ϩ reabsorption in the renal collecting duct, in part, controls blood pressure. Here, activity of the aldosteronesensitive epithelial Na ϩ channel (ENaC) is limiting for Na ϩ transport (reviewed in Refs. 19,30,31). Dysfunction and inappropriate regulation of ENaC consequently result in improper renal Na ϩ handling and thus, blood pressure disorders. For instance, gain of ENaC function in rodents and humans is causative for hypertension associated with the hallmarks of low plasma renin activity and aldosterone levels (1,22,23,45,46). Amiloride, an ENaC blocker, ameliorates this hypertension.Spotting lethal (sl) rats have a naturally occurring null mutation of ET B (17). These rats, when rescued from lethal intestinal aganglionosis by directed ET B transgene expression in the enteric nervous system, are particularly sensitive to DOCA and salt-induced hypertension (18,33,34). Similarly, mice with collecting duct-specific knockout of the ET B receptor have elevated blood pressure that further increases with high salt feeding (20). Collecting duct-specific ET-1 knockout, moreover, leads to hypertension exacerbated by high salt (2, 42). Plasma renin activity and aldoste...

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) are physiologically important second messengers. These molecules bind effector proteins to modulate activity. Several types of ion channels, including the epithelial Na+ channel (ENaC), are phosphoinositide effectors capable of directly interacting with these signaling molecules. Little, however, is known of the regions within ENaC and other ion channels important to phosphoinositide binding and modulation. Moreover, the molecular mechanism of this regulation, in many instances, remains obscure. Here, we investigate modulation of ENaC by PI(3,4,5)P3 and PI(4,5)P2 to begin identifying the molecular determinants of this regulation. We identify intracellular regions near the inner membrane interface just following the second transmembrane domains in β- and γ- but not α-ENaC as necessary for PI(3,4,5)P2 but not PI(4,5)P2 modulation. Charge neutralization of conserved basic amino acids within these regions demonstrated that these polar residues are critical to phosphoinositide regulation. Single channel analysis, moreover, reveals that the regions just following the second transmembrane domains in β- and γ-ENaC are critical to PI(3,4,5)P3 augmentation of ENaC open probability, thus, defining mechanism. Unexpectedly, intracellular domains within the extreme N terminus of β- and γ-ENaC were identified as being critical to down-regulation of ENaC activity and Po in response to depletion of membrane PI(4,5)P2. These regions of the channel played no identifiable role in a PI(3,4,5)P3 response. Again, conserved positive-charged residues within these domains were particularly important, being necessary for exogenous PI(4,5)P2 to increase open probability. We conclude that β and γ subunits bestow phosphoinositide sensitivity to ENaC with distinct regions of the channel being critical to regulation by PI(3,4,5)P3 and PI(4,5)P2. This argues that these phosphoinositides occupy distinct ligand-binding sites within ENaC to modulate open probability.