Expression of the Cystic Fibrosis Phenotype in a Renal Amphibian Epithelial Cell Line

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We used single channel methods on A6 renal cells to study the regulation by methylation reactions of epithelial sodium channels. 3-Deazaadenosine (3-DZA), a methyltransferase blocker, produced a 5-fold decrease in sodium transport and a 6-fold decrease in apical sodium channel activity by decreasing channel open probability (P o ). 3-Deazaadenosine also blocked the increase in channel open probability associated with addition of aldosterone. Sodium channel activity in excised "insideout" patches usually decreased within 1-2 min; in the presence of S-adenosyl-L-methionine (AdoMet), activity persisted for 5-8 min. Sodium channel mean time open (t open ) before and after patch excision was higher in the presence of AdoMet than in untreated excised patches but less than t open in cell-attached patches. Sodium channel activity in excised patches exposed to both AdoMet and GTP usually remained stable for more than 10 min, and P o and the number of active channels per patch were close to values in cell-attached patches from untreated cells. These findings suggest that a methylation reaction contributes to the activity of epithelial sodium channels in A6 cells and is directed to some regulatory element closely connected with the channel, whose activity also depends on the presence of intracellular GTP.The amiloride-blockable, highly selective, epithelial sodium channel (ENaC) 1 present on the apical surface of principal cells in mammalian renal cortical collecting tubules is the primary site for the regulation of total body sodium balance and blood pressure. The cell line, A6, derived from distal tubules of Xenopus laevis nephrons, is a good experimental model for the study of these sodium channels. When grown on permeable supports in the presence of aldosterone, A6 cells express an apical sodium channel with properties identical to those of channels in mammalian tissues (1).Despite many studies, the mechanism by which aldosterone stimulates apical sodium transport is still poorly understood. It is known that the complex between aldosterone and its intracellular receptor activates gene expression and induces the synthesis of proteins (2-9); however, little is known about the cellular functions of the induced proteins except that the final result is an increase in sodium transport (2, 9 -11). Originally, because of the observation that protein synthesis was required for aldosterone to increase sodium transport, it was postulated that aldosterone induced sodium channel synthesis and insertion. However, earlier studies in A6 cells showed that aldosterone increases Na ϩ entry at the apical membrane by changing the activity of channels that are already present in the apical membrane and not by increasing the number of channels (13). Although interpretation of other electrophysiological data remains controversial (14 -16), biochemical methods support the original observation that ENaC mRNA and ENaC protein in the apical membrane does not increase in the presence of aldosterone (at least in the first 2-4 h when the increase ...

Section: Exogenous Methyltransferase Does Not Increase the Effects Ofsupporting

confidence: 77%

Methylation Increases the Open Probability of the Epithelial Sodium Channel in A6 Epithelia

Becchetti¹,

Kemendy²,

Stockand³

et al. 2000

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“…In X. laevis, the only sequence with identity to these oligonucleotides as described by a blastn (National Center for Biotechnology Information) search was SAHHase. Addition of exogenous single-stranded oligonucleotides to A6 cells followed a protocol similar to that previously described by this laboratory (13). Briefly, competent A6 cell monolayers washed with phosphatebuffered saline were treated for 24 h prior to experimentation with 5-10 M oligonucleotide dissolved in basic media.…”

Section: Molecular Biological Methodsmentioning

confidence: 99%

S-Adenosyl-l-homocysteine Hydrolase Regulates Aldosterone-induced Na+ Transport

Stockand

Al-Baldawi

Al‐Khalili

et al. 1999

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Aldosterone-induced Na؉ reabsorption, in part, is regulated by a critical methyl esterification; however, the signal transduction pathway regulating this methylation remains unclear. The A6 cell line was used as a model epithelia to investigate regulation of aldosteroneinduced Na ؉ transport by S-adenosyl-L-homocysteine hydrolase (SAHHase), the only enzyme in vertebrates known to catabolize S-adenosyl-L-homocysteine (SAH), an end product inhibitor of methyl esterification. Sodium reabsorption was decreased within 2 h by 3-deazaadenosine, a competitive inhibitor of SAHHase, with a half inhibitory concentration between 40 and 50 M. Aldosterone increased SAH catabolism by activating SAHHase. Increased SAH catabolism was associated with a concomitant increase in S-adenosylmethionine catabolism. Moreover, SAH decreased substrate methylation. Antisense oligonucleotide complementary to SAHHase mRNA decreased SAHHase activity and Na ؉ current by approximately 50%. Overexpression of SAHHase increased SAHHase activity and dependent substrate methyl esterification. Whereas basal Na ؉ current was not affected by overexpression of SAHHase, aldosterone-induced current in SAHHase-overexpressing cells was significantly potentiated. These results demonstrate that aldosterone induction of SAHHase activity is necessary for a concomitant relief of the methylation reaction from end product inhibition by SAH and the subsequent increase in Na ؉ reabsorption. Thus, regulation of SAHHase activity is a control point for aldosterone signal transduction, but SAHHase is not an aldosterone-induced protein.

“…Functional expression of WT-CFTR, but not ⌬F508-CFTR, is associated with an inhibition of func- tional ENaC expression in oocytes (31). Such CFTR-mediated inhibition of ENaC is associated with a decrease in Na ϩ channel open probability, not with changes in levels of ENaC mRNA or protein expression (8,42,43). The forskolin/IBMX-regulated inhibition of ENaC by CFTR does not require expression of the full-length CFTR protein.…”

Section: Cftr Is a CLmentioning

confidence: 99%

“…In addition to functioning as a cAMPactivated, ATP-dependent Cl Ϫ channel, CFTR influences the transepithelial transport of other solutes, including Na ϩ via the epithelial sodium channel (ENaC), Cl Ϫ via an outwardly rectifying Cl Ϫ channel, HCO3 Ϫ , glutathione, and ATP (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Functional interactions between CFTR and ENaC have been observed in both epithelial and non-epithelial cells (2)(3)(4)(5)(6)(7)(8). The activation of CFTR is generally associated with an inhibition of ENaC, although activation of CFTR leads to activation of ENaC in the sweat duct (9), suggesting that the regulatory interactions between these two transporters are complex.…”

mentioning

confidence: 99%

Genistein Restores Functional Interactions between ΔF508-CFTR and ENaC in Xenopus Oocytes

Suaud¹,

Li²,

Jiang³

et al. 2002

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The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its Cl ؊ channel properties, has regulatory interactions with other epithelial ion channels including the epithelial Na ؉ channel (ENaC). Both the open probability and surface expression of wild type CFTR Cl ؊ channels are increased significantly when CFTR is co-expressed in Xenopus oocytes with ␣␤␥-ENaC, and conversely, the activity of ENaC is inhibited following wild type CFTR activation. Using the Xenopus oocyte expression system, a lack of functional regulatory interactions between ⌬F508-CFTR and ENaC was observed following activation of ⌬F508-CFTR by forskolin and isobutylmethylxanthine (IBMX). Whole cell currents in oocytes expressing ENaC alone decreased in response to genistein but increased in response to a combination of forskolin and IBMX followed by genistein. In contrast, ENaC currents in oocytes coexpressing ENaC and ⌬F508-CFTR remained stable following stimulation with forskolin/IBMX/genistein. Furthermore, co-expression of ⌬F508-CFTR with ENaC enhanced the forskolin/IBMX/genistein-mediated activation of ⌬F508-CFTR. Our data suggest that genistein restores regulatory interactions between ⌬F508-CFTR and ENaC and that combinations of protein repair agents, such as 4-phenylbutyrate and genistein, may be necessary to restore ⌬F508-CFTR function in vivo.