bodies directed against subunits of the epithelial Na channel (ENaC) were used together with electrophysiological measurements in the cortical collecting duct to investigate the processing of the proteins in rat kidney with changes in Na or K intake. When animals were maintained on a low-Na diet for 7-9 days, the abundance of two forms of the ␣-subunit, with apparent masses of 85 and 30 kDa, increased. Salt restriction also increased the abundance of the ␤-subunit and produced an endoglycosidase H (Endo H)-resistant pool of this subunit. The abundance of the 90-kDa form of the ␥-subunit decreased, whereas that of a 70-kDa form increased and this peptide also exhibited Endo H-resistant glycosylation. These changes in ␣-and ␥-subunits were correlated with increases in Na conductance elicited by a 4-h infusion with aldosterone. Changes in all three subunits were correlated with decreases in Na conductance when Na-deprived animals drank saline for 5 h. We conclude that ENaC subunits are mainly in an immature form in salt-replete rats. With Na depletion, the subunits mature in a process that involves proteolytic cleavage and further glycosylation. Similar changes occurred in ␣-and ␥-but not ␤-subunits when animals were treated with exogenous aldosterone, and in ␤-and ␥-but not ␣-subunits when animals were fed a high-K diet. Changes in the processing and maturation of the channels occur rapidly enough to be involved in the daily regulation of ENaC activity and Na reabsorption by the kidney. Na channels; Na depletion; Na repletion; aldosterone; potassium intake; proteolytic cleavage REGULATION OF EPITHELIAL NA transport by salt depletion and aldosterone entails the stimulation of the activity of apical Na channels in epithelia such as the renal connecting tubule and collecting duct (8,20). However, the mechanisms underlying this regulation are incompletely understood. The three subunits (␣, ␤, and ␥) of the epithelial Na channel (ENaC) comprise the apical membrane the Na channel in absorptive epithelia (4). In the mammalian colon, aldosterone enhances the transcription and translation of ␤-and ␥-subunits (2). In A6 cells derived from Xenopus laevis kidney, the hormone has little effect on mRNA abundance but increases rates of translation of subunit protein (1, 17). In the mammalian kidney, all three subunits are constitutively expressed. Modest increases in the expression of ␣-ENaC have been observed in response to elevated aldosterone levels in vivo at both the mRNA and protein levels (2, 15), but these changes do not appear to account for the stimulation of Na channel activity. Immunocytochemical evidence suggests that Na channel protein moves from intracellular sites to the plasma membrane of collecting duct and connecting tubule cells during Na depletion (13-15), suggesting that channel trafficking may play a dominant role in the response to the hormone in the kidney. These intracellular locations have not been precisely identified.Immunoblots indicate that one subunit of the channel, ␥-ENaC, is cleaved during the ...

Expression of epithelial Na channel (ENaC) protein in the apical membrane of rat kidney tubules was assessed by biotinylation of the extracellular surfaces of renal cells and by membrane fractionation. Rat kidneys were perfused in situ with solutions containing NHS-biotin, a cell-impermeant biotin derivative that attaches covalently to free amino groups on lysines. Membranes were solubilized and labeled proteins were isolated using neutravidin beads, and surface ␤ and ␥ ENaC subunits were assayed by immunoblot. Surface ␣ ENaC was assessed by membrane fractionation. Most of the ␥ ENaC at the surface was smaller in molecular mass than the full-length subunit, consistent with cleavage of this subunit in the extracellular moiety close to the fi rst transmembrane domains. Insensitivity of the channels to trypsin, measured in principal cells of the cortical collecting duct by whole-cell patch-clamp recording, corroborated this fi nding. ENaC subunits could be detected at the surface under all physiological conditions. However increasing the levels of aldosterone in the animals by feeding a low-Na diet or infusing them directly with hormone via osmotic minipumps for 1 wk before surface labeling increased the expression of the subunits at the surface by two-to fi vefold. Salt repletion of Na-deprived animals for 5 h decreased surface expression. Changes in the surface density of ENaC subunits contribute signifi cantly to the regulation of Na transport in renal cells by mineralocorticoid hormone, but do not fully account for increased channel activity.on May 9, 2018 jgp.rupress.org Downloaded from

, which is important for transduction processes. We have recently shown that glucocorticoid receptors (GR) stimulate absorption of Na ϩ by semicircular canal duct (SCCD) epithelia. In the present study, we sought to determine the presence of genes involved in the control of the amiloride-sensitive Na ϩ transport pathway in rat SCCD epithelia and whether their level of expression was regulated by glucocorticoids using quantitative real-time RT-PCR. Transcripts were present for ␣-, ␤-, and ␥-subunits of the epithelial sodium channel (ENaC); the ␣1-, ␣3-, ␤1-, and ␤3-isoforms of Na ϩ -K ϩ -ATPase; inwardly rectifying potassium channels [IC50 of short circuit current (Isc) for Ba 2ϩ : 210 M] Kir2

Mortality in postdiarrheal hemolytic-uremic syndrome (HUS) is associated with brain injury. Normally, brain cells are resistant to Shiga toxin (Stx), the putative pathogenic toxin in HUS. However, exposure of human brain endothelial cells (HBECs) to tumor necrosis factor (TNF) and/or interleukin (IL)-1 markedly up-regulates Stx receptor (globotriaosylceramide; Gb3) expression and cytotoxicity. To investigate how Gb3 is augmented, ceramide glucosyltransferase (CGT), lactosylceramide synthase (GalT2), Gb3 synthase (GalT6), and alpha-galactosidase were studied in HBECs exposed to TNF and IL-1. TNF, both alone and in combination with IL-1, increased Stx-1 toxicity, Gb3 content, and Stx-1 binding. TNF in combination with IL-1 increased CGT, GalT2, and GalT6 but did not change alpha-galactosidase activities or mRNA levels. Cytokine treatment did not change CGT, GalT2, or GalT6 mRNA half-lives. Thus, inflammatory cytokine up-regulation of the sensitivity of HBECs to Stx-1 is the result of up-regulation, most likely via transcription, of the activities of 3 enzymes involved in Gb3 synthesis.

ABSTRACT. Cellular injury in post-diarrheal hemolytic-uremic syndrome (D+HUS) is related to shigatoxin (Stx) binding to globotriaosylceramide (Gb3). High renal Gb3 expression may determine renal susceptibility in D+HUS; however, the molecular mechanism(s) responsible for such relatively abundant Gb3 levels are unknown. Consequently, kidney cells expressing high Gb3 (cultured human proximal tubule cells [HPT]) were compared with non-kidney cells with low Gb3 content (cultured human brain microvascular endothelial cells [HBEC]). HPT were much more sensitive to the cytotoxic and protein synthesis inhibitory effects of Stx-1; this correlated with Gb3 content and 125I-Stx-1 binding. HPT had greater Gb3 synthase (GalT6) and lower α-galactosidase activities than HBEC, whereas lactosylceramide synthase (GalT2) activity was higher in HBEC. Ceramide glucosyltransferase (CGT) activity was similar between the two cell types. The higher HPT GalT6 activity was associated with increased GalT6 mRNA steady-state levels, but no difference in GalT6 mRNA half-life. The lower HPT α-galactosidase activity was associated with reduced α-galactosidase mRNA steady-state levels but no difference in α-galactosidase mRNA half-life. Higher HBEC GalT2 activity was associated with increased steady-state GalT2 mRNA levels. These studies suggest that high renal Gb3 expression is due to enhanced GalT6 gene transcription and reduced α-galactosidase gene transcription and occur despite relatively low GalT2 activity.