This study examines whether serine proteases can activate the amiloride-sensitive sodium channel (ENaC) in mammalian kidney epithelial cells. The transepithelial sodium transport assessed by amiloride-sensitive short-circuit current appears to be sensitive to aprotinin, a protease inhibitor in a mouse cortical collecting duct cell line (mpkCCD(c14)). This result indicated that serine proteases may be implicated in the regulation of ENaC-mediated sodium transport. Using degenerated oligonucleotides to a previously isolated serine protease from Xenopus, xCAP1 (channel activating protease), a novel full-length serine protease (mCAP1), has been isolated and characterized. RNA analysis showed a broad pattern of expression in tissues (kidney, lung, colon, and salivary glands) expressing ENaC. Reverse transcription-PCR experiments also showed that mCAP1 was abundantly expressed in proximal tubule cells and was also expressed in intact and cultured collecting duct cells. Coexpression of the Xenopus, rat, or human alpha-, beta-, and gamma-ENaC subunits in Xenopus oocytes also showed that mCAP1 induces a significant increase in ENaC-mediated current accompanied by a decrease of channel molecules at the cell surface. It is proposed that this novel mouse channel activating protease may act as a regulator of ENaC within the kidney.

During the past several years, sites of expression of ion transport proteins in tubules from adult kidneys have been described and correlated with functional properties. Less information is available concerning sites of expression during tubule morphogenesis, although such expression patterns may be crucial to renal development. In the current studies, patterns of renal axial differentiation were defined by mapping the expression of sodium transport pathways during nephrogenesis in the rat. Combined in situ hybridization and immunohistochemistry were used to localize the Na-Pi cotransporter type 2 (NaPi2), the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), the thiazide-sensitive Na-Cl cotransporter (NCC), the Na/Ca exchanger (NaCa), the epithelial sodium channel (rENaC), and 11β-hydroxysteroid dehydrogenase (11HSD). The onset of expression of these proteins began in post-S-shape stages. NKCC2 was initially expressed at the macula densa region and later extended into the nascent ascending limb of the loop of Henle (TAL), whereas differentiation of the proximal tubular part of the loop of Henle showed a comparatively retarded onset when probed for NaPi2. The NCC was initially found at the distal end of the nascent distal convoluted tubule (DCT) and later extended toward the junction with the TAL. After a period of changing proportions, subsegmentation of the DCT into a proximal part expressing NCC alone and a distal part expressing NCC together with NaCa was evident. Strong coexpression of rENaC and 11HSD was observed in early nascent connecting tubule (CNT) and collecting ducts and later also in the distal portion of the DCT. Ontogeny of the expression of NCC, NaCa, 11HSD, and rENaC in the late distal convolutions indicates a heterogenous origin of the CNT. These data present a detailed analysis of the relations between the anatomic differentiation of the developing renal tubule and the expression of tubular transport proteins.

Na+ reabsorption from the epithelial surface of the respiratory tract plays a fundamental role in respiratory physiology. As in the epithelia of the renal collecting tubule and distal colon, Na+ enters across the luminal surface of respiratory epithelial cells via a recently cloned amiloride-sensitive multisubunit (alpha, beta, gamma) epithelial Na+ channel. We have examined the cellular expression at the mRNA level of the alpha-, beta-, and gamma-subunits of rat epithelial Na+ channel (rENaC) in the rat lung and upper airway epithelial cells using in situ hybridization. A large prevalence of alpha- and gamma-rENaC subunit expression (over beta) was found in tracheal epithelium, in a subpopulation of alveolar cells, presumably type II pneumocytes, and in nasal and tracheal gland acini. In contrast, equivalent levels of expression of all three subunits were detected in bronchiolar epithelium and in rat nasal gland ducts. This diversity of expression may reflect cell-specific functions of the amiloride-sensitive Na+ channel along the respiratory tract.

Sodium transport across the apical membrane, via amiloride sensitive sodium channels, is the limiting step of sodium absorption in transporting epithelia with high intercellular electrical resistance, such as the distal parts of the colon and of the renal tubule. Several types of amiloride sensitive sodium channels have been functionally characterized: one of them (type I) with high selectivity and low conductance for sodium is under the control of aldosterone and antidiuretic hormone. This channel has been cloned (2): it is formed of three subunits, alpha, beta and gamma. The distribution of these subunits has been examined in several epitheliums at the mRNA (in situ hybridization) and protein (immunocytochemistry) levels. All three subunits are expressed in the most superficial cells of the distal colon, in principal cells of the renal distal tubule and cortical collecting duct, in striated ducts of serous acini of salivary glands, and in excretory ducts of sweat glands. Immunocytochemistry established the apical localization of the channel subunit proteins. No expression was detected in other cell types of these tissues. These results highlight the crucial role of the type I amiloride sensitive sodium channel in the control of sodium homeostasis at the level of tight, aldosterone-sensitive epitheliums. Furthermore, novel questions are opened, in view of the sodium channel being a member of a highly conserved family of mechanoreceptors, and of its implication in some human genetic diseases.

A highly selective, amiloride-sensitive, epithelial sodium channel from rat colon (rENaC), composed of three homologous subunits termed alpha, beta, and gamma rENaC, has been cloned by functional expression and was proposed to mediate electrogenic sodium reabsorption in aldosterone-responsive epithelia. To determine whether rENaC could account for sodium absorption in vivo, we studied the cellular localization of the sodium channel messenger RNA subunits by in situ hybridization and their cellular and subcellular distribution by immunocytochemistry in the kidney, colon, salivary, and sweat glands of the rat. In the kidney, we show that the three subunit mRNAs are specifically co-expressed in the renal distal convoluted tubules (DCT), connecting tubules (CNT), cortical collecting ducts (CCD), and outer medullary collecting ducts (OMCD), but not in the inner medullary collecting ducts (IMCD). We demonstrate co-localization of alpha, beta, and gamma subunit proteins in the apical membrane of a majority of cells of CCD and OMCD. Our data indicate that alpha, beta, and gamma subunit mRNAs and proteins are co-expressed in the distal nephron (excepting IMCD), a localization that correlates with the previously described physiological expression of amiloride-sensitive electrogenic sodium transport. Our data, however, suggest that another sodium transport protein mediates electrogenic amiloride-sensitive sodium reabsorption in IMCD. We also localized rENaC to the surface epithelial cells of the distal colon and to the secretory ducts of the salivary gland and sweat gland, providing further evidence consistent with the hypothesis that the highly selective, amiloride-sensitive sodium channel is physiologically expressed in aldosterone-responsive cells.

In the brain, corticosteroids bind to intracellular glucocorticoid (GR) and mineralocorticoid (MR) receptors, affecting target gene transcription, and thereby altering neuronal function, including electrophysiological activity. The hippocampus very highly expresses both MR and GR; however, MR-regulated hippocampal transcripts have not yet been described. We investigated the effects of adrenalectomy +/- glucocorticoid or mineralocorticoid replacement on expression of mRNAs encoding alpha-subunit isoforms of Na(+)-K(+)-adenosinetriphosphatase, a critical transmembrane ion gradient-regulating enzyme. Aldosterone significantly increased alpha 3-subunit mRNA expression in dentate gyrus granule cells (62% increase compared with adrenalectomy) and in CA1 and CA4 hippocampal neurons (37 and 38%), but not in CA2, CA3, parietal cortex neurons, or glia. This effect was not reproduced by dexamethasone, and none of the corticosteroid manipulations altered alpha 1- or alpha 2-subunit mRNA expression at any site examined. Aldosterone-mediated upregulation of hippocampal alpha 3-subunit mRNA expression may underlie, at least in part, the specific actions of MR ligands on hippocampal function. The observation that aldosterone differentially affects alpha 3-isoform mRNA expression in distinct neuronal populations, associated with the established aldosterone modulations of alpha 1-isoform mRNA in epithelial cells, supports the presence of cell-specific factors that regulate MR-mediated transcriptional activity.

The mineralocorticoid receptor displays equal affinity for aldosterone and corticosterone. It has been proposed that aldosterone selectivity in vivo is achieved by the conversion of corticosterone into its inactive metabolite 11-dehydrocorticosterone by 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). To test this hypothesis, we transfected rat liver 11 beta HSD cDNA into TBM cells, a sodium-transporting cell line. These cells respond equally well to aldosterone and corticosterone, indicating that endogenous 11 beta HSD is expressed at low levels in TBM cells. Although exogenous rat liver 11 beta HSD was expressed at high levels in transfected cells, mineralocorticoid selectivity was not observed. By contrast, the biologically inactive 11-dehydrocorticosterone was readily converted into corticosterone, a potent agonist for sodium transport. Our results indicate that rat liver 11 beta HSD behaves predominantly as a reductase in TBM cells. Another 11 beta HSD isoform is likely to be responsible for the dehydrogenase reaction in aldosterone-responsive cells.

Recently, a putative distal colon H(+)-K(+)-ATPase alpha-subunit has been identified and characterized (M. S. Crowson and G. E. Shull. J. Biol. Chem. 267:13740-13748, 1992). In the present study, we report the tissue and cell expression of this putative H(+)-K(+)-ATPase. The results indicate that, first, in the gut, the putative H(+)-K(+)-ATPase alpha-subunit is restricted to the distal part of the colon and is predominantly expressed in surface epithelial cells, in marked contrast to the alpha 1-subunit of Na(+)-K(+)-ATPase that is also expressed in glands. These data suggest that the H(+)-K(+)-ATPase alpha-subunit is a potential marker for terminal differentiation of distal colon. Second, in the uterus, the putative H(+)-K(+)-ATPase is restricted to the region of the myometrium between the inner and midmuscular zone that is very rich in vascular supply and nerve cells. This striking expression suggests that the H(+)-K(+)-ATPase may not be involved in the control of pH and potassium concentration of the uterine fluid but rather in distinct functions of vascular and/or nerve cells. Third, with the use of three independent and different approaches (Northern blot analysis, ribonuclease protection assay, and in situ hybridization), we were unable to detect any significant amount of H(+)-K(+)-ATPase transcripts in kidney tissue. Our data suggest that the putative distal colon H(+)-K(+)-ATPase is probably distinct from the kidney isoform. Finally, we report the sequence of a set of degenerate oligonucleotides that are useful to clone alpha-subunits of the Na(+)-K(+)-/H(+)-K(+)-ATPase gene family in different tissues and different species.

The expression of the three alpha-isoforms of Na(+)-K(+)-adenosine triphosphatase (ATPase) was examined in rat brain and rat kidney by Northern blot analysis. All three isoforms were detected in brain tissue while alpha 1-isoform was observed in whole kidney, suggesting that either this isoform was solely expressed in this organ or that alpha 2- and/or alpha 3-isoforms were not detected only because of their restricted distribution among a minority of specialized tubular cells. To distinguish between these two possibilities, in situ hybridization with rat alpha 1-, alpha 2-, and alpha 3-ATPase cRNA was performed on rat kidney sections. Results show that alpha 1-isoform expression largely predominates in the loop of Henle, distal tubule, and cortical collecting tubule. The labeling was drastically reduced by preincubation of sections with RNase. A sense cRNA probe, used as control, did not hybridize. With alpha 2- and alpha 3-probes, the labeling was low and uniformly distributed. In contrast, these two isoforms were clearly expressed in the brain, together with alpha 1. We conclude that only alpha 1-isoform of the Na(+)-K(+)-ATPase is detectable along the rat nephron. Its expression predominates in the tubular segments known to have a high Na(+)-K(+)-ATPase activity.