Arginine vasopressin and forskolin regulate apical cell surface expression of epithelial Na+ channels in A6 cells

Kleyman, Thomas R.; Ernst, Stephen A.; Coupaye-Gerard, Brigitte

doi:10.1152/ajprenal.1994.266.3.f506

Cited by 66 publications

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“…This will increase the apical Ca2+ influx and, therefore, stim ulate transcellular transport of Ca2+. A similar m echanism has been postulated for th e stim ulatory effect of PTH on Ca2+ influx in the distal nephron and for the effect of vasopressin on epithelial Na+ channels [26,29,30].…”

Section: Discussionmentioning

confidence: 67%

Co-ordinated control of apical calcium influx and basolateral calcium efflux in rabbit cortical collecting system

et al. 1997

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Summary Transcellular Ca2+ transport in the distal nephron involves passive Ca2+ influx at the apical membrane, diffusion through the cytosol and active extrusion across the opposing basolateral membrane. The molecular identity of the apical Ca2+ entry step is still elusive, but its regulatory aspects have been analyzed in the present study. To this end, rabbit connecting and cortical collecting tubular cells were cultured on permeable and transparent supports and the apical Ca2+ influx was deduced from Mn2+ quenching of Ca2+ independent Fura-2 fluorescence, while the intracellular Ca2+ concentration ([Ca2+],) was measured simultaneously. In parallel experiments, transcellular Ca2+ transport was determined isotopically as 45Ca2+ flux from the apical to basolateral compartment. Decreasing the apical pH from 7.4 to 5.9 inhibited transcellular Ca2+ transport by 53 ± 1%, whereas apical Ca2+ influx was reduced by 39 ± 7% and [Ca2+], decreased by 18 ± 3%. Reversal of the Na+/Ca2+ exchanger by iso-osmotic replacement of Na+ by N-methyl-Dglucamine in the basolateral compartment resulted in 50 ± 5% inhibition of Ca2* transport, 46 ± 3% reduction of apical Ca2+ influx and 60 ± 3% increase in [Ca2+]r In the absence of basolateral Ca2+, however, this manoeuvre decreased [Ca2*], by 21 ± 8%, while Ca2+ transport and apical Ca2+ influx were reduced by the same magnitude as in the presence of Ca2+, that is by 53 ± 6% and 45 ± 4%, respectively. Stimulation of adenylyl cyclase with forskolin (10-5 M) increased transcellular Ca2+ transport by 108 ± 40%, stimulated apical Cas+ influx by 120 ± 17% and increased [Ca2+]i by 110 ± 2%. In conclusion, the apical Ca2+ influx is regulated by apical pH, intracellular cAMP and basolateral Na+/Ca2+ exchanger activity, and is coupled in an 1:1 fashion to the rate of transepithelial Ca2+ transport.

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Section: Discussionmentioning

confidence: 67%

Co-ordinated control of apical calcium influx and basolateral calcium efflux in rabbit cortical collecting system

et al. 1997

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“…The FLAG epitope allows the number of ENaC subunits residing in the apical membrane of the transfected MDCK cells to be quantified by the surface binding of 125 I-labeled anti-FLAG antibodies. Our initial studies with intact FL-MDCK monolayers in DMEM medium showed they had a high shortcircuit current (I sc ) and that cAMP treatment produced a rapid transient peak within 5 min followed by a broad peak that decayed over 20 min (43), as also described in A6 and M-1 cultures (8,29,34,44). The biphasic response to cAMP in all of these epithelia has been attributed to rapid Cl Ϫ secretion by CFTR activation followed by a slower cAMP-dependent activation of ENaC that is blunted by the inhibitory effect of CFTR.…”

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confidence: 77%

Inhibition of ENaC by intracellular Cl⁻in an MDCK clone with high ENaC expression

Xie

Schafer

2004

American Journal of Physiology-Renal Physiology

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We examined the effects of intracellular Cl− concentration ([Cl−]i) on the epithelial Na channel (ENaC) in a line of Madin-Darby canine kidney (MDCK) cells (FL-MDCK) with a high rate of Na+ transport produced by stable retroviral transfection with rENaC subunits (Morris RG and Schafer JA. J Gen Physiol 120: 71–85, 2002). Treatment with cAMP (100 μM 8-cpt-cAMP plus 100 μM IBMX) stimulated ENaC-mediated Na+ absorption as well as Cl− secretion via cystic fibrosis transmembrane conductance regulator, which was characterized in α-toxin-permeabilized monolayers to have the anion selectivity sequence NO3− > Br− > Cl− > I−. With the use of FL-MDCK monolayers in which the basolateral membrane was permeabilized by nystatin, the ENaC conductance of the apical membrane [determined from the amiloride-sensitive short-circuit current (AS- Isc) driven by an apical-to-basolateral Na+ concentration gradient] was progressively inhibited by increasing the [Cl−] in the basolateral solution (and hence in the cytosol), but it was insensitive to the [Cl−] in the apical solution. This inhibitory effect of [Cl−]i occurred regardless of the presence or absence of net Cl− transport. However, from fluorometric measurements using the Cl−-sensitive dye 6-methoxy- N-(3-sulfopropyl)-quinolinium in intact FL-MDCK monolayers on permeable supports, cAMP, which activates both Na+ absorption and Cl− secretion, produced a decrease of [Cl−]i from 76 ± 14 to 36 ± 8 mM ( P = 0.03). Thus it might be expected that activation of Cl− secretion by cAMP would lead to stimulation rather than inhibition of ENaC. In the nystatin-treated monolayers, an increase in [Cl−]i from 15 to 145 mM decreased AS- Isc from 24.5 ± 1.0 to 10.2 ± 1.6 μA/cm2. This inhibition of ENaC could be attributed to nearly proportional decreases in the density of ENaC in the apical membrane from 1.91 ± 0.16 to 1.32 ± 0.17 fmol/cm2 and in the intrinsic channel activity (the average current per ENaC subunit) from 13.3 ± 1.2 to 8.2 ± 1.4 μA/fmol.

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“…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 in sodium transport is most dramatic) (10,(17)(18)(19).Since the action of aldosterone appears to involve a mechanism that increases the P o of sodium channels, an examination of post-translational mechanisms that alter P o may offer some insight into the mechanism of aldosterone action, but identifying signal transduction pathways that can increase sodium channel P o in A6 cells has been difficult. There have been many suggestions about potential aldosterone-induced post-translational modifications, but in the context of our previous results (20 -22), one is particularly interesting.…”

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confidence: 82%

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

Becchetti¹,

Kemendy²,

Stockand³

et al. 2000

Journal of Biological Chemistry

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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 ...

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Arginine vasopressin and forskolin regulate apical cell surface expression of epithelial Na+ channels in A6 cells

Cited by 66 publications

References 0 publications

Co-ordinated control of apical calcium influx and basolateral calcium efflux in rabbit cortical collecting system

Co-ordinated control of apical calcium influx and basolateral calcium efflux in rabbit cortical collecting system

Inhibition of ENaC by intracellular Cl⁻in an MDCK clone with high ENaC expression

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

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Arginine vasopressin and forskolin regulate apical cell surface expression of epithelial Na+ channels in A6 cells

Cited by 66 publications

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Co-ordinated control of apical calcium influx and basolateral calcium efflux in rabbit cortical collecting system

Co-ordinated control of apical calcium influx and basolateral calcium efflux in rabbit cortical collecting system

Inhibition of ENaC by intracellular Cl−in an MDCK clone with high ENaC expression

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

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Inhibition of ENaC by intracellular Cl⁻in an MDCK clone with high ENaC expression