Distribution of transcellular calcium and sodium transport pathways along mouse distal nephron.

The organization of Na(+) and Ca(2+) transport pathways along the mouse distal nephron is incompletely known. We revealed by immunohistochemistry a set of Ca(2+) and Na(+) transport proteins along the mouse distal convolution. The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) characterized the distal convoluted tubule (DCT). The amiloride-sensitive epithelial Na(+) channel (ENaC) colocalized with NCC in late DCT (DCT2) and extended to the downstream connecting tubule (CNT) and collecting duct (CD). In early DCT (DCT1), the basolateral Ca(2+)-extruding proteins [Na(+)/Ca(2+) exchanger (NCX), plasma membrane Ca(2+)-ATPase (PCMA)] and the cytoplasmic Ca(2+)-binding protein calbindin D(28K) (CB) were found at very low levels, whereas the cytoplasmic Ca(2+)/Mg(2+)-binding protein parvalbumin was highly abundant. NCX, PMCA, and CB prevailed in DCT2 and CNT, where we located the apical epithelial Ca(2+) channel (ECaC1). Its subcellular localization changed from apical in DCT2 to exclusively cytoplasmic at the end of CNT. NCX and PMCA decreased in parallel with the fading of ECaC1 in the apical membrane. All three of them were undetectable in CD. These findings disclose DCT2 and CNT as major sites for transcellular Ca(2+) transport in the mouse distal nephron. Cellular colocalization of Ca(2+) and Na(+) transport pathways suggests their mutual interactions in transport regulation.

Localization of epithelial sodium channel and aquaporin-2 in rabbit kidney cortex.

The amiloride-sensitive epithelial sodium channel (ENaC) and the vasopressin-dependent water channel aquaporin-2 (AQP2) mediate mineralocorticoid-regulated sodium- and vasopressin-regulated water reabsorption, respectively. Distributions of ENaC and AQP2 have been shown by immunohistochemistry in rats. Functional data from rabbits suggest a different distribution pattern of these channels than in rats. We studied, by immunohistochemistry in the rabbit kidney cortex, the distributions of ENaC and AQP2, in conjunction with marker proteins for distal segments. In rabbit cortex ENaC is restricted to the connecting tubule (CNT) cells and cortical collecting duct (CCD) cells. The intracellular distribution of ENaC shifts from the apical membrane in the most upstream CNT cells to a cytoplasmic location further downstream in the CNT and in the CCD cells. AQP2 is detected in the CCD cells exclusively. The anatomic subdivisions in the rabbit distal nephron coincide exactly with distributions of apical transport systems. The differences between rabbits and rats in the distribution patterns of ENaC and AQP2 may explain functional differences in renal salt and water handling between these species.

Corticosteroid-dependent sodium transport in a novel immortalized mouse collecting duct principal cell line.

The final control of sodium balance takes place in the cortical collecting duct (CCD) of the nephron, where corticosteroid hormones regulate sodium reabsorption by acting through mineralocorticoid (MR) and/or glucocorticoid (GR) receptors. A clone of principal CCD cells (mpkCCDc14) has been established that is derived from a transgenic mouse (SV40 large T antigen under the control of the SV40 enhancer/L-type pyruvate kinase promoter). Cells grown on filters form polarized monolayers with high electrical transepithelial resistance (R(T) approximately 4700 ohm x cm2) and potential difference (P(D) approximately -50 mV) and have an amiloride-sensitive electrogenic sodium transport, as assessed by the short-circuit current method (Isc approximately 11 microA/cm2). Reverse transcription-PCR experiments using rat MR primers, [3H]aldosterone, and [3H]dexamethasone binding and competition studies indicated that the mpkCCDc14 cells exhibit specific MR and GR. Aldosterone increased Isc in a dose- (10(-10) to 10(-6) M) and time-dependent (2 to 72 h) manner, whereas corticosterone only transiently increased Isc (2 to 6 h). Consistent with the expression of 11beta-hydroxysteroid dehydrogenase type 2, which metabolizes glucocorticoids to inactive 11-dehydroderivates, carbenoxolone potentiated the corticosterone-stimulated Isc. Aldosterone (5x10(-7) M)-induced Isc (fourfold) was associated with a three- to fivefold increase in alpha-ENaC mRNA (but not in those for beta- or gamma-ENaC) and three- to 10-fold increases in alpha-ENaC protein synthesis. In conclusion, this new immortalized mammalian CCD clonal cell line has retained a high level of epithelial differentiation and sodium transport stimulated by aldosterone and therefore represents a useful mammalian cell system for identifying the genes controlled by aldosterone.

Epithelial Na+ channel subunits in rat taste cells: localization and regulation by aldosterone.

Amiloride-sensitive Na+ channels play an important role in transducing Na+ salt taste. Previous studies revealed that in rodent taste cells, the channel shares electrophysiological and pharmacological properties with the epithelial Na+ channel, ENaC. Using subunit-specific antibodies directed against alpha, beta, and gamma subunits of rat ENaC (rENaC), we observed cytoplasmic immunoreactivity for all three subunits in nearly all taste cells of fungiform papillae, and in about half of the taste cells in foliate and vallate papillae. The intensity of labeling in cells of vallate papillae was significantly lower than that of fungiform papillae, especially for beta and gamma subunits. Dual localization experiments showed that immunoreactivity for the taste cell-specific G protein, gustducin, occurs in a subset ofrENaC positive taste cells. Aldosterone is known to increase the amiloride sensitivity of the NaCl taste response. In our study, increases in blood aldosterone levels enhanced the intensity of apical immunoreactivity for beta and gamma rENaC in taste cells of all papillae. In addition, whole cell recordings from isolated taste cells showed that in fungiform papillae, aldosterone increased the number of amiloride-sensitive taste cells and enhanced the current amplitude. In vallate taste cells, which are normally unresponsive to amiloride, aldosterone treatment induced an amiloride sensitive current in about half of the cells. Immunoreactivity for rENaC subunits also was present in nonsensory epithelial cells, especially in the anterior portion of the tongue. In addition, immunoreactivity for all subunits, but especially beta and gamma, was associated with some nerve fibers innervating taste papillae. These extragustatory sites of rENaC expression may indicate a role for this channel in paracellular transduction of sodium ions.

Mutational analysis of cysteine-rich domains of the epithelium sodium channel (ENaC). Identification of cysteines essential for channel expression at the cell surface.

One of the characteristic features of the structure of the epithelial sodium channel family (ENaC) is the presence of two highly conserved cysteine-rich domains (CRD1 and CRD2) in the large extracellular loops of the proteins. We have studied the role of CRDs in the functional expression of rat alphabetagamma ENaC subunits by systematically mutating cysteine residues (singly or in combinations) into either serine or alanine. In the Xenopus oocyte expression system, mutations of two cysteines in CRD1 of alpha, beta, or gamma ENaC subunits led to a temperature-dependent inactivation of the channel. In CRD1, one of the cysteines of the rat alphaENaC subunit (Cys158) is homologous to Cys133 of the corresponding human subunit causing, when mutated to tyrosine (C133Y), pseudohypoaldosteronism type 1, a severe salt-loosing syndrome in neonates. In CRD2, mutation of two cysteines in alpha and beta but not in the gamma subunit also produced a temperature-dependent inactivation of the channel. The main features of the mutant cysteine channels are: (i) a decrease in cell surface expression of channel molecules that parallels the decrease in channel activity and (ii) a normal assembly or rate of degradation as assessed by nondenaturing co-immunoprecipitation of [35S]methionine-labeled channel protein. These data indicate that the two cysteines in CRD1 and CRD2 are not a prerequisite for subunit assembly and/or intrinsic channel activity. We propose that they play an essential role in the efficient transport of assembled channels to the plasma membrane.

Epithelial sodium channel regulatory proteins identified by functional expression cloning.

We describe here our current strategy for identifying and cloning proteins involved in the regulation of the epithelial sodium channel (ENaC). We have set up a complementation functional assay in the Xenopus laevis oocyte expression system. Using this assay, we have been able to identify a channel-activating protease (CAP-1) that can increase ENaC activity threefold. We propose a novel extracellular signal transduction pathway controlling ionic channels of the ENaC gene family that include genes involved in mechanotransduction (degenerins), in peptide-gated channels involved in neurotransmission (FaNaCh), in proton-gated channels involved in pH sensing (ASIC) or pain sensation (DRASIC).

Unusual degradation of alpha-beta complexes in Xenopus oocytes by beta-subunits of Xenopus gastric H-K-ATPase.

The catalytic alpha-subunit of oligomeric P-type ATPases such as Na-K-ATPase and H-K-ATPase requires association with a beta-subunit after synthesis in the endoplasmic reticulum (ER) to become stably expressed and functionally active. In this study, we have expressed the beta-subunit of Xenopus gastric H-K-ATPase (betaHK) in Xenopus oocytes together with alpha-subunits of H-K-ATPase (alphaHK) or Na-K-ATPase (alphaNK) and have followed the biosynthesis, assembly, and cell surface expression of functional pumps. Immunoprecipitations of Xenopus betaHK from metabolically labeled oocytes show that it is well expressed and, when synthesized without alpha-subunits, can leave the ER and become fully glycosylated. Xenopus betaHK can associate with both coexpressed alphaHK and alphaNK, but the alpha-beta complexes formed are degraded rapidly in or close to the ER and do not produce functional pumps at the cell surface as assessed by 86Rb uptake. A possible explanation of these results is that Xenopus betaHK may contain a tissue-specific signal that is important in the formation or correct targeting of functional alpha-beta complexes in the stomach but that cannot be recognized in Xenopus oocytes and in consequence leads to cellular degradation of the alpha-beta complexes in this experimental system.

Mutations causing Liddle syndrome reduce sodium-dependent downregulation of the epithelial sodium channel in the Xenopus oocyte expression system.

Liddle syndrome is an autosomal dominant form of hypertension resulting from deletion or missense mutations of a PPPxY motif in the cytoplasmic COOH terminus of either the beta or gamma subunit of the epithelial Na channel (ENaC). These mutations lead to increased channel activity. In this study we show that wild-type ENaC is downregulated by intracellular Na+, and that Liddle mutants decrease the channel sensitivity to inhibition by intracellular Na+. This event results at high intracellular Na+ activity in 1.2-2.4-fold higher cell surface expression, and 2.8-3.5-fold higher average current per channel in Liddle mutants compared with the wild type. In addition, we show that a rapid increase in the intracellular Na+ activity induced downregulation of the activity of wild-type ENaC, but not Liddle mutants, on a time scale of minutes, which was directly correlated to the magnitude of the Na+ influx into the oocytes. Feedback inhibition of ENaC by intracellular Na+ likely represents an important cellular mechanism for controlling Na+ reabsorption in the distal nephron that has important implications for the pathogenesis of hypertension.

Neonatal injection of native proton pump antigens induces autoimmune gastritis in mice.

BACKGROUND & AIMS: Autoimmune gastritis is associated with gastric H+, K(+)-adenosine triphosphatase (ATPase)-specific autoantibodies (HKAb). The (auto) antigen that triggers disease and the pathogenic role of the autoantibodies are unknown. The aim of this study was to analyze when these autoantibodies are produced during autoimmune gastritis in neonatally thymectomized mice and whether a native H+, K(+)-ATPase antigen preparation can induce disease in mice. METHODS: Autoantibodies were characterized by a novel assay based on immunoprecipitation of a functional H+, K(+)-ATPase expressed in Xenopus oocytes. Normal mice were injected intraperitoneally with H+, K(+)-ATPase-enriched gastric membranes in the absence of adjuvant. RESULTS: Conformational autoantibodies recognizing both H+, K(+)-ATPase subunits appeared simultaneously with the gastric lesions 1 month after thymectomy. Immunization of neonates, but not adults, induced a persistent autoimmune gastritis in the body mucosa, characterized by lymphocytic infiltrations, loss of parietal and chief cells, metaplasia, and H+, K(+)-ATPase-specific autoantibodies. The histopathological lesions of this new model are similar to those in humans and thymectomized mice. CONCLUSIONS: The onset of gastritis and autoantibody production parallels the expression of the H+, K(+)-ATPase during ontogeny. Exposure of the neonatal immune system to organ-specific antigens expressed late after birth induces autoimmune gastritis in adult mice.

Cystic fibrosis transmembrane conductance regulator inverts protein kinase A-mediated regulation of epithelial sodium channel single channel kinetics.

Abnormal regulation of ion channels by members of the ABC transport protein superfamily has been implicated in hyperinsulinemic hypoglycemia and in excessive Na+ absorption by airway epithelia in cystic fibrosis (CF). How ABC proteins regulate ion conductances is unknown, but must generally involve either the number or activity of specific ion channels. Here we report that the cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in CF, reverses the regulation of the activity of single epithelial sodium channels (ENaC) by cAMP. ENaC expressed alone in fibroblasts responded to activation of cAMP-dependent protein kinase with increased open probability (Po) and mean open time, whereas ENaC co-expressed with CFTR exhibited decreased Po and mean open time under conditions optimal for PKA-mediated protein phosphorylation. Thus, CFTR regulates ENaC at the level of single channel gating, by switching the response of single channel Po to cAMP from an increase to a decrease.

A reappraisal of aldosterone effects on the kidney: new insights provided by epithelial sodium channel cloning.

Sodium homeostasis is crucial for the control of extra-cellular volume and blood pressure. Regulation of sodium reabsorption is mainly achieved in the distal nephron by the mineralocorticoid aldosterone, but the molecular pathway of aldosterone action has largely remained unclear. Molecular genetic analysis of inherited diseases disturbing sodium homeostasis has now demonstrated that the amiloride-sensitive epithelial sodium channel is a major effector of aldosterone action. Mechanisms by which aldosterone regulates the epithelial sodium channel activity are beginning to emerge and will be of great importance for a better understanding of salt-sensitive hypertension.

Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach.

The epithelial amiloride-sensitive sodium channel (ENaC) controls transepithelial Na+ movement in Na(+)-transporting epithelia and is associated with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the functional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative detection of cell-surface-expressed ENaC. We have developed a quantitative assay based on the binding of 125I-labeled M2 anti-FLAG monoclonal antibody (M2Ab*) directed against a FLAG reporter epitope introduced in the extracellular loop of each of the alpha, beta, and gamma ENaC subunits. Insertion of the FLAG epitope into ENaC sequences did not change its functional and pharmacological properties. The binding specificity and affinity (Kd = 3 nM) allowed us to correlate in individual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (INa) with the number of ENaC wild-type and mutant subunits expressed at the cell surface. These experiments demonstrate that: (i) only heteromultimeric channels made of alpha, beta, and gamma ENaC subunits are maximally and efficiently expressed at the cell surface; (ii) the overall ENaC open probability is one order of magnitude lower than previously observed in single-channel recordings; (iii) the mutation causing Liddle syndrome (beta R564stop) enhances channel activity by two mechanisms, i.e., by increasing ENaC cell surface expression and by changing channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sensitive hypertension.

The rat distal colon P-ATPase alpha subunit encodes a ouabain-sensitive H+, K+-ATPase.

The functional properties and the pharmacological profile of the recently cloned cDNA colonic P-ATPase alpha subunit (Crowson, M.S., and Shull, G.E. (1992) J. Biol. Chem. 267, 13740-13748) were investigated by using the Xenopus oocyte expression system. Xenopus oocytes were injected with alpha subunit cRNAs from Bufo marinus bladder or rat distal colon and/or with beta subunit cRNA from B. marinus bladder. Two days after injection, K+ uptake was measured by using 86 Rb+ as a K+ surrogate, and pH measurements were performed by means of ion-selective microelectrodes. Co-injection of alpha and beta subunit cRNAs led to a large increase in 86Rb+ uptake, an intracellular alkalinization, and an extracellular medium acidification, as compared to alpha or beta injection alone. These results indicate that the colonic P-ATPase alpha subunit, like the bladder alpha subunit, acts as a functional H+,K+-ATPase, and that co-expression of alpha and beta subunits is required for the function. External K+ activation of the 86Rb+ uptake had a K1/2 of approximately 440 microM for the bladder isoform (consistent with the previously reported value (Jaisser, F., Horisberger, J.D., Geering, K., and Rossier, B.C. (1993) J. Cell. Biol. 123, 1421-1431) and a K1/2 of approximately 730 microM for the colonic isoform. Sch28080 was ineffective to reduce 86Rb+ uptake whereas ouabain inhibited the activity expressed from rat colon alpha subunit with a Ki of 970 microM when measured at the Vmax of the enzyme. We conclude that, when expressed in Xenopus oocytes, the rat colon P-ATPase alpha subunit encodes a ouabain-sensitive H+,K+-ATPase.

Human ATP1AL1 gene encodes a ouabain-sensitive H-K-ATPase.

The cDNA for ATP1AL1, the fifth member of the human Na-K-adenosinetriphosphatase (ATPase)/H-K-ATPase gene family, was recently cloned (A. V. Grishin, V. E. Sverdlov, M. B. Kostina, and N. N. Modyanov. FEBS Lett. 349: 144-150, 1994). The encoded protein (ATP1AL1) has all the primary structural features common to the catalytic alpha-subunit of ion-transporting P-type ATPases and is similar (63-64% identity) to the Na-K-ATPase alpha-subunit isoforms and the gastric H-K-ATPase alpha-subunit. In this study, ATP1AL1 was expressed in Xenopus laevis oocytes in combination with the beta-subunit of rabbit gastric H-K-ATPase. The functional properties of the stable alpha/beta-complex were studied by 86Rb+ uptake and demonstrated that ATP1AL1 is a novel human K(+)-dependent ATPase [apparent half-constant activation/(K1/2) for K+ approximately 375 microM)]. ATP1AL1-mediated inward K+ transport was inhibited by ouabain (inhibition constant approximately 13 microM) and was found to be inhibited by high concentrations of SCH-28080 (approximately 70% at 500 microM). ATP1AL1 expression resulted in the alkalinization of the oocytes' cytoplasm and ouabain-sensitive proton extrusion, as measured with pH-sensitive microelectrodes. These data argue that ATP1AL1 is the catalytic alpha-subunit of a human nongastric P-type ATPase capable of exchanging extracellular potassium for intracellular protons.

Cloning of a bovine renal epithelial Na+ channel subunit.

A bovine homologue of the rat and human epithelial Na+ channel subunits, alpha-rENaC and alpha-hENaC, was cloned. The cDNA clone, termed alpha-bENaC, was isolated from a bovine renal papillary collecting duct cDNA expression library. The bovine cDNA is 3,584 base pairs (bp) long, has an open reading frame of 2,094 bp encoding a 697-amino acid protein, and is 75-85% homologous to its rat and human counterparts. In vitro translation of the transcribed cRNA yields an 80-kDa polypeptide and one at 92 kDa in the presence of pancreatic microsomes. The clone exhibits consensus sequences for N-linked glycosylation and for phosphorylation by protein kinase C, but not for protein kinase A. After expression in Xenopus laevis oocytes, a small amiloride-sensitive Na+ conductance that exhibited inward rectification and a reversal potential greater than +30 mV, consistent with the predicted equilibrium potential for Na+, was identified. The expressed alpha-bENaC-associated Na+ current was not responsive to elevations in adenosine 3',5'-cyclic monophosphate but could be stimulated by phorbol 12-myristate 13-acetate, an activator of protein kinase C. alpha-bENaC also formed amiloride-sensitive chimeric channels when coexpressed with the rat beta- and gamma-ENaC subunits in Xenopus oocytes. alpha-bENaC therefore represents a novel isoform of a growing family of epithelial Na+ channels.

Lung epithelial Na channel subunits are differentially regulated during development and by steroids.

Because the alpha-subunit of the rat lung epithelial Na channel (rENaC) is not expressed until late fetal gestation, the developmental immaturity of alpha-rENaC may be involved in the premature fetal lung's inability to mount a Na-absorptive response to appropriate agonists. As previous work has shown that the beta- and gamma-rENaC subunits of the Na channel are required for maximal alpha-rENaC activity, we determined their developmental expression in the fetal lung. In addition, because thyroid and corticosteroid therapy can mature the in vivo fetal lamb lung's ability to transport Na, we wished to determine whether such treatment increased the expression of alpha-, beta-, and gamma-rENaC. Lungs were harvested from normal rat fetuses of 17 through 22 days gestation (term = 22 days), normal rat pups during the first week of life, and adult rats. Initial expression of alpha-rENaC was detected at 19 days gestation and progressively increased in utero. beta- and gamma-rENaC mRNA were not detected until 21 and 22 days gestation, and then only at very low levels. During the first week after birth, the levels of alpha-rENaC declined, whereas beta- and gamma-rENaC mRNA levels increased. This pre- and postnatal pattern of alpha-rENaC expression correlates with the endogenous glucocorticosteroid levels in the fetus and the rat pup's early postnatal corticosteroid resistance. Combined or separate treatment of pregnant rats (16 through 22 days gestational age) with thyroid-releasing hormone (TRH) and/or dexamethasone (Dex) for 48 h showed that Dex, but not TRH, could increase fetal lung alpha-rENaC mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Relative expression of the human epithelial Na+ channel subunits in normal and cystic fibrosis airways.

The availability of the newly cloned subunits (alpha, beta, gamma) of the epithelial Na+ channel (ENaC) permits molecular studies of the pathogenesis of the abnormal Na+ transport rates of cystic fibrosis (CF) airway epithelia. Northern analyses of airway epithelia showed that both normal and CF airway epithelia express ENaC subunit mRNAs in a ratio of alpha > beta > gamma. In situ hybridization studies revealed expression of all three ENaC subunits in the superficial epithelium and the alpha- and beta-subunits in the gland ductular and acinar epithelium of both normal and CF airways. Ribonuclease protection assays revealed that the steady-state levels of alpha-, beta-, and gamma-ENaC mRNAs were similar in CF and normal airway superficial epithelia. These findings indicate that 1) Na+ transport defects in CF airways disease may be expressed in glandular acinar and ductal epithelium as well as superficial epithelium, and 2) the molecular pathogenesis of Na+ hyperabsorption in CF airways does not reflect increased levels of Na+ channel mRNAs, and probably number, but reflects an absence of the normal inhibitory regulation of Na+ channels by CF transmembrane conductance regulator proteins.

CFTR as a cAMP-dependent regulator of sodium channels.

Cystic fibrosis transmembrane regulator (CFTR), the gene product that is mutated in cystic fibrosis (CF) patients, has a well-recognized function as a cyclic adenosine 3',5'-monophosphate (cAMP)-regulated chloride channel, but this property does not account for the abnormally high basal rate and cAMP sensitivity of sodium ion absorption in CF airway epithelia. Expression of complementary DNAs for rat epithelial Na+ channel (rENaC) alone in Madin Darby canine kidney (MDCK) epithelial cells generated large amiloride-sensitive sodium currents that were stimulated by cAMP, whereas coexpression of human CFTR with rENaC generated smaller basal sodium currents that were inhibited by cAMP. Parallel studies that measured regulation of sodium permeability in fibroblasts showed similar results. In CF airway epithelia, the absence of this second function of CFTR as a cAMP-dependent regulator likely accounts for abnormal sodium transport.

Labeling of a cysteine in the cardiotonic glycoside binding site by the steroid derivative HDMA.

The digoxigenin derivative N-hydroxysuccinimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate (HDMA) has been shown to covalently label the ouabain binding site of the Na,K-ATPase epsilon subunit [Antolovic et al. (1995) Eur. J. Biochem. 227, 61-67]. In the present study we observed both, labeling and inactivation of the activity, of wild type Na,K-ATPase overexpressed in Xenopus oocyte. In contrast, no significant inhibition and no labeling could be detected when a Cys-113 of the first transmembrane segment was mutated to serine, although the affinity of this mutant for digoxigenin or HDMA measured in acute inhibition experiments was similar to the wild type. This indicates that after docking of its genin moiety, HDMA can form a thioester bond with Cys-113.