ATP masks stretch activation of epithelial sodium channels in A6 distal nephron cells

He-Ping Ma1, Li Li1, Zhen-Hong Zhou1, Douglas C. Eaton2, and David G. Warnock1

1 Division of Nephrology, Department of Medicine, The University of Alabama at Birmingham, Birmingham, Alabama 35294; and 2 Center for Cell and Molecular Signaling, Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The mechanosensitivity of the epithelial sodium channel (ENaC) is controversial. Using cell-attached patch-clamp techniques, we found that mechanical stretch stimulated ENaC in A6 distal nephron cells in only three of nine cell-attached patches. However, stretch consistently activated ENaC after apical ATP was scavenged with apical hexokinase plus glucose or after P2 receptors in the patch were blocked. The mean open probability (Po) of ENaC was increased from 0.31 ± 0.04 to 0.61 ± 0.06 (P < 0.001; n = 9) when patch pipettes contained hexokinase and glucose, or from 0.24 ± 0.05 to 0.55 ± 0.11 (P < 0.01; n = 7) when patch pipettes contained suramin, respectively. A poorly hydrolyzable ATP analog, ATPgamma S, in the patch pipettes inhibited ENaC, reducing the Po from 0.41 ± 0.06 to 0.19 ± 0.05 (P < 0.01; n = 8). Pretreatment of A6 cells with the phospholipase C (PLC) inhibitor U-73122 abolished the effect of ATP on ENaC activity. These data together suggest that ATP, acting through a PLC-dependent purinergic pathway, masks stretch-induced ENaC activation.

patch-clamp techniques; stretch; autocrine regulation; purinergic receptors; phospholipase C; adenosine 5'-triphosphate


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AS THEIR NAME IMPLIES, VERTEBRATE epithelial sodium channels (ENaCs) are generally associated with sodium transport in epithelial tissues like kidney, colon, and lung. However, the homology of ENaCs with mechanosensation-associated genes identified in invertebrates like Caenorhabditis elegans and Drosophila melanogaster (5-7) has suggested to some investigators that ENaCs, besides transporting sodium, might also be mechanosensitive channels. Nonetheless, the mechanosensitivity of ENaC remains a matter of controversy despite a variety of studies attempting to address this issue. Examples of studies in which ENaC appears to be stretch sensitive include the observations that the purified renal ENaC in planar lipid bilayer membranes can be activated by hydrostatic pressure (1, 4, 8) and that expression of alpha -ENaC in mammalian cells results in stretch-activated channel activity (10). Recent studies have shown that osmotic stretch regulates rat alpha -, beta -, and gamma -ENaC expressed in Xenopus laevis oocytes (9). However, using the oocyte expression technique as well, other investigators demonstrated that osmotic swelling failed to activate ENaC (2). Furthermore, negative pressure applied to patch pipettes activated native ENaC in principal cells of the rat cortical collecting tubule in <30% of cell-attached patches (13).

Because mechanical stretch can induce cellular ATP release (14, 15), P2 receptors might be stimulated by stretch. Since, in preliminary work, we have shown that ATP inhibits ENaC activity in A6 distal nephron cells, we hypothesized that the inhibitory effect of stretch-induced ATP release on ENaC might obscure a stretch-induced ENaC activation. To support this hypothesis, we have shown that mechanical stretch consistently activates ENaC after we have eliminated apical ATP by hydrolysis with hexokinase plus glucose or after blocking of P2 receptors and that addition of exogenous ATP inhibits ENaC. These findings suggest that ATP masks stretch activation of ENaC via an autocrine mechanism in A6 cells. We argue that the variability of this purinergic regulation from patch to patch may account for the variable activation by stretch of ENaC activity observed in principal cells.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. A6 distal nephron cells were purchased from American Type Culture Collection (Rockville, MD) at passage 68. The cells were cultured in a plastic flask in modified NCTC-109 media (GIBCO BRL) with 10% fetal bovine serum (GIBCO BRL) at 26°C and 4% CO2. Cells from passages 72-82 were removed from the flasks and plated on permeable supports attached to Snapwell inserts (Corning Costar). The permeable supports were coated with rat tail collagen according to the manufacturer's protocol.

Patch-clamp technique. Immediately before use, a Snapwell insert was thoroughly washed with NaCl bath solution (see Chemicals and solutions) and transferred into the patch chamber mounted in the stage of a Leitz inverted microscope. Using patch-clamp techniques, cell-attached recordings were established on the apical membrane of A6 cells with polished micropipettes with tip resistance of 2.5-5 MOmega . Under the above culture conditions, a patch seal (seal resistance >20 GOmega ) was usually formed after positive pressure in the patch pipette was released or after a slightly negative pressure (<2 cmH2O) was applied. Approximately 10% of patches requiring a stronger negative pressure to form a seal >20 GOmega were excluded from testing the response of ENaC to mechanical stretch. Single-channel current filtered at 1 kHz was recorded on videotapes with a modified Sony PCM video converter (Vetter Instruments). Before digitization with pClamp 8 software (Axon Instruments), single-channel records were low-pass filtered at 100 Hz. Open probability (Po) was calculated by using at least 2 min of a single-channel record. Experiments were conducted at 22-23°C.

Chemicals and solutions. Most chemicals, including HEPES, hexokinase, and ATP, were obtained from Sigma. U-73122 and U-73343 were purchased from Calbiochem. Hexokinase plus glucose was used to trap free ATP in the patch pipette by catalyzing the hydrolysis of the gamma -phosphate of ATP to phosphorylate glucose. If negative pressure caused any ATP to be released from cytoplasm, the released ATP would be rapidly consumed to phosphorylate glucose in the presence of hexokinase. Suramin was purchased from Research Biochemicals International and used to block P2 receptors. The NaCl bath solution contained (in mM) 100 NaCl, 3.4 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES, at a pH of 7.4. All the concentrations throughout this study are shown as the final concentration.

Statistical analysis. A paired t-test or analysis of variance for multiple comparisons was used for statistical analysis, as we previously described (12). A P value <0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To examine the regulation of ENaC by mechanical perturbation, cell-attached experiments were performed in A6 distal nephron cells. We previously cultured the cells on permeable supports for 10-14 days before patch-clamp recordings were made (12). However, it seemed that ENaC completely lost the sensitivity to mechanical stretch if cells were cultured for more than 10 days. In the present study, we plated the cells with a density that allowed the cells to become confluent within 5 days for patching. Under this condition, we found that stretch did stimulate ENaC but inconsistently from patch to patch. Application of negative pressure to the patch pipette failed to activate ENaC in six of nine cell-attached patches. In the other three patches, negative pressure appeared to stimulate ENaC, but the mean Po of the entire group was not significantly changed (0.36 ± 0.07 vs. 0.42 ± 0.06; P > 0.05; n = 9) (Fig. 1, A and B). These results are very consistent with previous studies accomplished in rat cortical collecting tubule (13).


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Fig. 1.   Inconsistent activation of epithelial sodium channel (ENaC) by mechanical stretch in A6 cells. A: representative single-channel recordings from 9 cell-attached patches. Negative pressure (-3 cmH2O) had no effect in 6 patches (top trace) and activated ENaC in 3 patches (bottom trace). B: summary plots of open probability (Po) of ENaC before and after stretch under control conditions. A6 cells were cultured on Snapwell permeable supports. Upward events are channel openings. C, baseline when channels are closed.

Surprisingly, consistent activation was induced by negative pressure after trapping of ATP with hexokinase and glucose in the patch pipettes or after blocking of P2 receptors with 100 µM suramin in the patch pipettes (Fig. 2A). After these treatments, the mean Po of ENaC was increased from 0.31 ± 0.04 to 0.61 ± 0.06 (P < 0.001; n = 9) (Fig. 2B) and from 0.24 ± 0.05 to 0.55 ± 0.11 (P < 0.01; n = 7) (Fig. 2C), respectively. The Po in one patch after trapping of ATP was 0.89, and the Po values in two patches after blocking of P2 receptors were 0.85 and 0.93, respectively. Mechanical stretch did not further stimulate the channel activity in these three patches. Because the high basal activity seemed to saturate stretch activation, these patches were not included in the above statistical analysis. Interestingly, we never observed a channel with the Po higher than 0.8 in a total of nine cell-attached patches when patch pipettes contained neither hexokinase nor suramin, as shown in Fig. 1. Because mechanical stretch can cause the release of ATP at the luminal surface of cells (15), ATP release might mask stretch-induced ENaC activation via an autocrine mechanism mediated by apical purinergic receptors in the plasma membrane. If ATP does play an autocrine role in regulating ENaC activity, exogenous ATP in the patch pipette should inhibit ENaC activity. Because ENaC activity is highly variable from patch to patch, a protocol was designed to use the same patch as a control. The tip of the patch pipette was filled with an ATP-free solution; the main body of the pipette was back-filled with a solution containing 200 µM ATPgamma S (a poorly hydrolyzable ATP analog). Control channel activity was recorded before ATPgamma S diffused down the pipette to the patched membrane to bind its receptor. Approximately 3 min after patch formation, ENaC activity began to fall. A significant decrease was observed during the period from 6 to 9 min. The mean Po for the interval from 0 to 3 min after patch formation was decreased from 0.41 ± 0.06 (0-3 min) to 0.19 ± 0.05 (6-9 min) (P < 0.01, n = 8) (Fig. 3, A and B). To eliminate the possibility that the decrease is actually due to a channel rundown, channel activity under control conditions was continuously recorded. Although the Po obviously dropped 6 min later in one patch, the mean Po for the interval from 6 to 9 min (0.31 ± 0.04; n = 7) was not significantly lower than that for the interval from 0 to 3 min (0.37 ± 0.07; n = 7) (Fig. 3, C and D). The data are consistent with a previous report (12) that ENaC activity in cell-attached patches can be recorded for >10 min without a significant rundown. Furthermore, the effect of ATP on ENaC activity was also examined, using a protocol in which both the tip and the rear part of the patch pipette were filled with a solution containing 200 µM ATPgamma S. The inhibition of ENaC by intrapipette ATPgamma S occurred almost immediately after patch formation. Only a few short openings were observed in all of three cell-attached patches. The effect of mechanical stretch on ENaC activity was also tested after the inhibitory effect of ATP occurred. Under this condition, application of negative pressure to the patch pipette failed to stimulate ENaC in all three cell-attached patches we tested. The representative recording was shown in Fig. 3A (inset a).


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Fig. 2.   Consistent activation of ENaC by stretch after either trapping of ATP or blocking of purinergic receptors. A: representative responses to negative pressure when patch pipettes contained either 6 U/ml hexokinase plus 5 mM glucose (top trace) or 100 µM suramin (bottom trace). B and C: summary plots of Po values before and after stretch when patch pipettes contained hexokinase plus glucose or suramin in the pipettes, respectively.



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Fig. 3.   Inhibition of ENaC by a poorly hydrolyzable ATP analog in patch pipettes. A: representative single-channel activity of ENaC in a cell-attached patch before [0-2 min after patch formation (top trace)] and after [6-8 min after patch formation (bottom trace)]. ATPgamma S diffused to the apical receptor in the patch membrane. Inset a: negative pressure no longer stimulated ENaC activity when ENaC was already inhibited by 200 µM ATPgamma S in the patch pipette. B: summary plots of Po values before (0-3 min) and after ATPgamma S (6-9 min). C: representative single-channel activity of ENaC in a cell-attached patch under control conditions [0-2 min after patch formation (top trace); 6-8 min after patch formation (bottom trace)]. D: summary plots of Po values of control ENaC activity (0-3 min after patch formation) and continuous recordings (6-9 min after patch formation).

To further test the signaling pathways of ATP, both the tip and the rear part of the patch pipette were filled with a solution containing 50 µM ATP to allow a quick onset of the inhibition. In contrast to control channel activity (Po = 0.37 ± 0.07; n = 7) as shown in Fig. 3, significantly lower ENaC activity (Po = 0.16 ± 0.03; n = 5) was recorded when the patch pipette contained 50 µM ATP. However, after pretreatment of A6 cells with phospholipase C (PLC) inhibitor (5 µM U-73122), 50 µM ATP in the patch pipette no longer inhibited ENaC (Po = 0.35 ± 0.06; n = 7). However, ATP (50 µM) in the patch pipette still inhibited ENaC (Po = 0.19 ± 0.03; n = 5) after pretreatment with 5 µM U-73343, an inactive analog of U-73122 (Fig. 4, A-D). These results suggest that ATP in the patch pipette is able to inhibit ENaC activity via a P2 receptor coupled to a PLC-mediated pathway.


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Fig. 4.   Abolishment of ATP-induced ENaC inhibition by a PLC inhibitor. A: ENaC activity under control conditions. B: ENaC activity after pretreatment of A6 cells with U-73122 for 15 min. C: ENaC activity after pretreatment with U-73343 for 15 min. D: summary plots of Po values under each condition shown in A-C with 50 µM ATP in patch pipettes.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Consistent with previous studies in rat cortical collecting tubules (13), we show that stretch activation of the ENaC was not observed in nearly 70% of cell-attached patches established on the apical membrane of A6 distal nephron cells. Interestingly, membrane stretch stimulated ENaC in almost all the cell-attached patches we tested after hexokinase trapping of ATP or after blocking of P2 receptors with 100 µM suramin in the patch pipette. Furthermore, we also demonstrate that exogenous ATP in the patch pipette significantly inhibited ENaC activity. Because stretch failed to stimulate ENaC when the patch pipette contained exogenous ATP (insert a, Fig. 3), we argue that ATP signaling may also render the channels insensitive to stretch activation because the inhibitory effects are already in place. Recent studies show that mechanical perturbation can induce ATP release (15). Taken together, these results imply that mechanical perturbation produces a dual regulation of the renal ENaC. Both downregulation and upregulation of the ENaC are simultaneously induced by membrane tension. The downregulation is mediated by stretch-induced ATP release, whereas the upregulation is either a direct result of ENaC mechanosensitivity or an indirect effect of cytoskeleton perturbation. This investigation provides an explanation for the controversial findings on the mechanosensitivity of the ENaC. The inconsistent activation of the renal ENaC in cell-attached experiments of Palmer and Frindt (13) could be due to variability in the composition of patches. Stretch-induced activation would be recorded if only an ENaC channel is in the patch that does not also contain a P2 receptor or possibly the ATP release mechanism. To provide further evidence for the contradictory effects of ATP and stretch on ENaC activity, we have also tried to correlate these two effects with the age of the culture. Although the stimulatory effect of stretch is dependent on the age of the culture, the inhibitory effect of ATP could occur no matter how long the cells have been cultured (data not shown). Recent reports show that ATP mobilizes intracellular Ca2+ in A6 cells via the P2Y2 (also known as a P2U) receptor (3). It is well known that the P2Y family of receptors is coupled to PLC, and, therefore, it is not surprising that our data shows that U-73122, a specific PLC inhibitor, blocks the effect of ATP on ENaC activity (Fig. 4). Although intracellular Ca2+ and PKC regulate renal ENaC (11), it is unlikely that phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis resulting from the activation of PLC by the putative P2Y2 receptor in the isolated membrane patch generates sufficient inositol 1,4,5-trisphosphate to mobilize intracellular Ca2+ or enough diacylglycerol to stimulate PKC. Because it has recently been demonstrated that ENaC requires PIP2 for normal activity (16), we speculate that PLC-mediated loss or consumption of PIP2 itself is probably more important than the local increase in Ca2+ concentration at the cytosolic surface of the patched membrane in mediating the inhibition of ENaC by purinergic receptors. However, the physiological relevance of the observations presented here remains to be further investigated.


    ACKNOWLEDGEMENTS

This work was supported by a National Kidney Foundation Young Investigator Award (to H.-P. Ma), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant 1R01-DK-56305 (to D. C. Eaton), and National Institute of Diabetes and Digestive and Kidney Diseases Grant 1R01-DK-53161 (to D. G. Warnock).


    FOOTNOTES

Address for reprint requests and other correspondence: H.-P. Ma, Nephrology Division, Dept. of Medicine, The Univ. of Alabama at Birmingham, 1530 Third Ave. South, Sparks Bldg. 865, Birmingham, AL 35294-0017 (E-mail: hepingma{at}uab.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajprenal.00147.2001

Received 10 May 2001; accepted in final form 30 October 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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5.   Canessa, CM, Horisberger JD, and Rossier BC. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 361: 467-470, 1993[ISI][Medline].

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