Distinct regulation of Na+ reabsorption and Cl- secretion by arginine vasopressin in the amphibian cell line A6

Chalfant, Michael L.; Coupaye-Gerard, Brigitte; Kleyman, Thomas R.

doi:10.1152/ajpcell.1993.264.6.c1480

Cited by 57 publications

(46 citation statements)

References 3 publications

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“…The biphasic time course of the I sc response to cAMP (Fig. 2) was consistent with previous studies in MDCK cells (43,54) and in A6 cells (4,8,29,44,58) that showed a rapid increase in anion secretion followed by a more gradual increase in Na ϩ absorption. The secretory anion current, represented by NS-I sc , was partially inhibited by 200 M glibenclamide (Figs.…”

Section: Discussionsupporting

confidence: 90%

“…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.…”

mentioning

confidence: 75%

“…We also examined whether such a mechanism could account for the later decay in Na ϩ absorption after cAMP stimulation observed in previous studies with this and similar epithelia (8,29,34,43,44) and the effect of increases in [Cl Ϫ ] i on the density of ENaC subunits in the apical membrane of these FL-MDCK cells.…”

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confidence: 94%

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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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Section: Discussionsupporting

confidence: 90%

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confidence: 75%

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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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“…There is thus excellent agreement between the observed and predicted responses, and so the changes in G Cl Ϫ and G Na ϩ that we now report can explain the well-documented effects of isoprenaline upon these cells (23,30,(36)(37)(38). Moreover, in A6 cells, a Na ϩ -absorbing cell line derived from the amphibian kidney, cAMP-dependent agonists have been reported to elicit a biphasic response similar to that described above (7). This pattern of response may thus be a generalized feature of salt-absorbing epithelia.…”

Section: Discussionsupporting

confidence: 85%

β-Adrenoceptor-mediated control of apical membrane conductive properties in fetal distal lung epithelia

Collett

Ramminger

Olver

et al. 2002

American Journal of Physiology-Lung Cellular and Molecular Phys

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Distal lung epithelial cells isolated from fetal rats were cultured (48 h) on permeable supports so that transepithelial ion transport could be quantified electrometrically. Unstimulated cells generated a short-circuit current (Isc) that was inhibited (ϳ80%) by apical amiloride. The current is thus due, predominantly, to the absorption of Na ϩ from the apical solution. Isoprenaline increased the amiloride-sensitive Isc about twofold. Experiments in which apical membrane Na ϩ currents were monitored in basolaterally permeabilized cells showed that this was accompanied by a rise in apical Na ϩ conductance (GNaϩ). Isoprenaline also increased apical Cl Ϫ conductance (GClϪ) by activating an anion channel species sensitive to glibenclamide but unaffected by 4,4Ј-diisothiocyanatostilbene-2,2Ј-disulfonic acid (DIDS). The isoprenaline-evoked changes in G Na ϩ and GClϪ could account for the changes in Isc observed in intact cells. Glibenclamide had no effect upon the isoprenaline-evoked stimulation of I sc or GNaϩ demonstrating that the rise in GClϪ is not essential to the stimulation of Na ϩ transport. alveolar ion transport; Ussing chambers; permeabilized epithelia THROUGHOUT FETAL LIFE the distal lung epithelia secrete fluid into the developing air spaces (31) and establish a distending pressure that is crucial to lung morphogenesis (11). However, this liquid must be removed from the lungs if the newborn infant is to breathe at birth. The absorption of this liquid occurs during the final stages of gestation and is dependent upon the active withdrawal of Na ϩ from the lung lumen, a process that can be controlled via ␤-adrenoceptors (23,30,(36)(37)(38). The means by which this control is achieved are not fully understood, but there is evidence that the process involves a rise in apical Na ϩ conductance (G Na ϩ) (15,20). It is also known that ␤-adrenoceptor agonists can increase apical Cl Ϫ conductance (G Cl Ϫ) in these cells, and it has been suggested that this may facilitate Na ϩ transport by increasing the driving force for Na ϩ entry (16,29). It is, however, difficult to see how the ionic gradients that normally prevail in epithelial cells could allow Na ϩ transport to be controlled in this way (see Refs. 20,44). To clarify the means by which ␤-adrenoceptor agonists can control Na ϩ absorption in the distal lung epithelia, we now explore the effects of isoprenaline upon the conductive properties of the apical membrane. METHODS Isolation and culture of rat fetal distal lung epithelial cells.Fetuses removed from anesthetized (3% halothane), 20-day pregnant (term ϭ 22 days) rats were immediately decapitated, and their lung tissue was collected into ice-cold, Ca 2ϩ -and Mg 2ϩ -free Hanks' balanced salt solution. The anesthetized animals were then killed (cervical dislocation/exsanguination) before regaining consciousness. The fetal lung tissue was chopped into pieces (Ͻ0.5 mm) and disaggregated using 0.2% trypsin/0.012% DNase (2 ϫ 20 min, 37°C) followed by 0.1% collagenase/0.012% DNase [15 min, 37°C, both in Dulbecco's mo...

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“…Grown on permeable supports, they acquire polarity and express highly selective sodium channels (Kleyman et al 1989;Hamilton & Benos, 1990;Benos et al 1995). In addition, amiloride-sensitive Na¤ channels, cAMP-induced Na¤ channels (Verrey, 1994), highly conductive Cl¦ channels (Marunaka & Eaton, 1990) and non-selective cation channels (Hamilton & Benos, 1990) have been demonstrated in the same cells, together with the effect of factors like vasopressin Chalfont et al 1993;Smith et al 1995), aldosterone (Kemendy et al 1992;Willis et al 1992), insulin (Marunaka et al 1992;Record et al 1996) and cAMP (Hamilton & Eaton, 1991;Nakahari & Marunaka, 1995) on these channels. AÜ cells can be used as experimental analogues of frog skin with the advantage that they can be easily patched, while Cacoµ cells are, for similar reasons, equally convenient analogues of the mammalian large intestine.…”

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confidence: 99%

Activation of Ionic Channels by Deoxycholate in Frog and Human Cell Lines

Maurício

Ferreira

1999

Experimental Physiology

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summary Humans, after extensive ileal resection, frequently suffer from diarrhoea, which may be due to an increased delivery of deoxycholate (DOC) to the large intestine. In the frog skin the addition of DOC (0·5 mÒ) to the apical side induced the activation of amiloride-sensitive Na¤ channels and an increase in the unidirectional Cl¦ fluxes. Here we used two established cell lines (AÜ and Cacoµ) to study the effect of DOC on ion channels at cell and membrane level using the patch-clamp technique. In AÜ cells subcultured directly on Petri dishes and studied in the whole-cell configuration, DOC induced an increase in cell conductance of 110·3 ± 4 pS pF¢ (N = 8) which was reduced to 89 ± 14 pS pF¢ (N = 8) by the addition of DIDS (0·5 mÒ). The absolute values of these two effects were not statistically different (P < 0·2). In Cacoµ cells, the addition of DOC (0·5 mÒ) induced, after 1 min, an increase in cell conductance of 583 ± 16 pS pF¢ (N = 8) which was reduced to 560·4 ± 16 pS pF¢ (N = 8) by DIDS (0·5 mÒ) and N_phenylanthranilic acid (DPC; 0·5 mÒ). The two values were not statistically different (P < 0·4). In Cacoµ cells subcultured under the same conditions, DOC induced an increase in cell conductance of 1710 ± 64 pS pF¢ (N = 6). Subsequent addition of amiloride (0·1 mÒ) reduced the cell conductance to 1558 ± 33 pS pF¢ (N = 6). These two mean values were statistically different allowing for an error of the second kind < 0·05. In cells in which DOC produced a conductance increase of 1010 ± 10 pS pF¢, gadolinium (0·5 mÒ) induced a fall in cell conductance of 1800 ± 10 pS pF¢. In Cacoµ cells, addition of DOC (0·5 mÒ) to the bath reversibly induced the appearance of or an increase in channel activity in patches studied in cell-attached and excised inside-out configuration. In inside-out experiments (N = 13) DOC (0·5 mÒ) induced the appearance of channel activity with conductances and reversal potentials (Er) of 27·7 ± 1·9 pS and 0·8 ± 5·7 mV, respectively. In cell-attached patches (N = 13) these values were 24·9 ± 4·4 pS and −18·1 ± 6·4 mV. In excised inside-out patches from Cacoµ cells, subjected to electrochemical gradients for Na¤, K¤ and Cl¦, (+85, −85 and 0 mV, respectively), addition of DOC also induced an increase in the baseline conductance and a shift in the reversal potential from values around +25 mV to values around 0 mV. Bile salts activated both anionic and cationic channels and did not require the presence of intracellular factors for these effects. We suggest that they act at the membrane level.introduction The occurrence of diarrhoea in humans after extensive ileal resections (Hofmann, 1967;Hofmann & Poley, 1969) has been attributed to the action of products resulting from bacterial degradation of bile acids in the colon. Such products would inhibit absorption or even stimulate secretion in the large intestine. The addition of deoxycholate (DOC) to the apical side of frog skin stimulated transepithelial sodium transport (Lima et al. 1996) by activating amiloride-sensitive sodium channels. In the...

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Distinct regulation of Na+ reabsorption and Cl- secretion by arginine vasopressin in the amphibian cell line A6

Cited by 57 publications

References 3 publications

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

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

β-Adrenoceptor-mediated control of apical membrane conductive properties in fetal distal lung epithelia

Activation of Ionic Channels by Deoxycholate in Frog and Human Cell Lines

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Distinct regulation of Na+ reabsorption and Cl- secretion by arginine vasopressin in the amphibian cell line A6

Cited by 57 publications

References 3 publications

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

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

β-Adrenoceptor-mediated control of apical membrane conductive properties in fetal distal lung epithelia

Activation of Ionic Channels by Deoxycholate in Frog and Human Cell Lines

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

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