Activity of the epithelial Na+ channel (ENaC) is the limiting step for discretionary Na+reabsorption in the cortical collecting duct. Xenopus laeviskidney A6 cells were used to investigate the effects of cytosolic phospholipase A2 (cPLA2) activity on Na+ transport. Application of aristolochic acid, a cPLA2 inhibitor, to the apical membrane of monolayers produced a decrease in apical [3H]arachidonic acid (AA) release and led to an approximate twofold increase in transepithelial Na+ current. Increased current was abolished by the nonmetabolized AA analog 5,8,11,14-eicosatetraynoic acid (ETYA), suggesting that AA, rather than one of its metabolic products, affected current. In single channel studies, ETYA produced a decrease in ENaC open probability. This suggests that cPLA2 is tonically active in A6 cells and that the end effect of liberated AA at the apical membrane is to reduce Na+ transport via actions on ENaC. In contrast, aristolochic acid applied basolaterally inhibited current, and the effect was not reversed by ETYA. Basolateral application of the cyclooxygenase inhibitor ibuprofen also inhibited current. Both effects were reversed by prostaglandin E2(PGE2). This suggests that cPLA2 activity and free AA, which is metabolized to PGE2, are necessary to support transport. This study supports the fine-tuning of Na+ transport and reabsorption through the regulation of free AA and AA metabolism.
- sodium reabsorption
- arachidonic acid
- A6 cells
- cytosolic phospholipase A2
transport of sodium across principal cells in the distal collecting duct of the kidney is via an apical amiloride-sensitive epithelial Na+channel (ENaC) and a coupled basolateral Na+-K+-ATPase. This system vectorially moves Na+ from the external environment to the organism's internal environment. The rate-limiting step for this process is by regulation of ENaC. The principal means of regulating Na+transport in the cortical collecting duct is hormonal via the mineralocorticoid, aldosterone, and the peptide, antidiuretic hormone (19, 21, 40). Both hormones increase the apical membrane conductance to Na+, thereby promoting Na+reabsorption. Much effort has been focused on understanding the cellular mechanism of these hormonal regulatory events; however, the intracellular mechanism(s) responsible for their effects are not well identified. Less well described are the mechanisms of fine-tuning and local, intratubular, or cellular control of Na+ uptake.
Phospholipase A2, arachidonic acid, and ion channels.
The phospholipases A2 (PLA2) are a family of enzymes that metabolize membrane phospholipids and are generally classified into three groups, secretory (sPLA2), Ca2+ independent, and cytosolic PLA2(cPLA2) (33). Although the latter was originally termed cytosolic because it is found in the cytosol, this term is somewhat misleading because the active form of cPLA2 is membrane associated. cPLA2 has long been recognized to play an important role in a number of cellular processes. cPLA2 hydrolyzes the sn-2 position of phospholipids, resulting in the release of free fatty acids, primarily arachidonic acid (AA) in eukaryotic cells, and lysophospholipids. Both the free fatty acids and the lysophospholipids then may themselves mediate a number of responses by both intracellular and extracellular signaling. It is also well documented that a variety of plasma membrane receptors act on PLA2 (5, 33). Upon release, AA may initiate signaling or can be metabolized into a wide range of signaling factors by cyclooxygenases, lipoxygenases, and cytochromeP-450 monooxygenases, producing a cascade of prostaglandins, leukotrienes, and monooxygenase products, respectively. Determined by the predominate active metabolic pathways, the release of AA produces a prodigiously diverse range of physiological and pathological effects. In particular, many ion channels are affected by either AA itself and/or the various metabolites of AA (31). Free unsaturated fatty acids, principally AA, have varying effects on ion channels, thus a priori, the effects of AA on a given ion channel cannot be guessed. The effects of AA can either be inhibitory (23) or stimulatory (11, 13, 39). In addition, many studies involving the addition of free AA, usually at concentrations exceeding normal physiological levels, may not adequately address the physiological role of AA or its metabolites on ion transport.
In the 1970s, it was suggested that Na+reabsorption in the toad bladder was associated with an increased turnover of phospholipids (22), most likely through the involvement of PLA (42). To determine the possible effects of phospholipid turnover, and, in particular, AA on amiloride-sensitive Na+ channels and transepithelial Na+reabsorption, we chose to manipulate free AA levels by altering cPLA2 activity, using aristolochic acid as a relatively selective inhibitor of cytosolic PLA2, a strategy that had proved effective in previous work involving the maxi-K+channel in GH3 cells (13, 14).
This paper demonstrates that cPLA2 is tonically active in transporting A6 cells and that this activity is necessary for the support of transepithelial transport. Interestingly, AA is shown to reduce Na+ transport, whereas the metabolic product of AA, prostaglandin E2 (PGE2), is found to be necessary to maintain normal transport. Furthermore, the sidedness of cPLA2 inhibition suggests there is a local apical membrane-associated pool of cPLA2 at or near the apical Na+ channel in addition to the perinuclear and endoplasmic reticular localization of cPLA2.
MATERIALS AND METHODS
The 2F3 clonal line of A6 cells or A6 cells were grown on permeable supports under conditions that have been demonstrated to produce transepithelial Na+ transport, as well as a predominance of the low-conductance highly selective amiloride-sensitive Na+ channel in the apical membrane (21). Open-circuit current measurements were made using the 2F3 clonal cell line (which, in general, produces more current than the A6 cell line). Short-circuit current and single channel measurements were made using the A6 cell line. It should be noted that A6 cells were found to respond identically to the 2F3 cell line in open-circuit current measurements, albeit with a lower magnitude of current. Cells were maintained at 26°C in 4% CO2 in a mixture of 7:3 Coon's F-12 and Leibovitz's L-15 with 25 mM NaHCO3 (pH 7.4), 10% fetal calf serum, 1% streptomycin, and 0.6% penicillin. Cells for transepithelial current measurements and assays involving free AA production or cPLA2 activity were grown on 25-mm Anapore supports (Nunc). Cells for patch-clamp analysis were plated on rat tail collagen-coated Millipore-CM filters attached to the bottom of custom-made Lucite rings. Cells on both types of permeable supports were grown to confluency in the above conditions in the presence of 1.5 μM aldosterone. Under these conditions, high-resistance monolayers formed within 7–14 days. Short-circuit current measurements were performed on A6 cells grown under the conditions reported by Blazer-Yost et al. (4).
Measurement of transepithelial current.
The quality of high-resistance monolayer formation was monitored using an epithelial voltohmmeter (EVOM; World Precision Instruments). This instrument consists of a pair of Ag-AgCl electrodes mounted in “chopstick” fashion attached to a customized voltohmmeter. Both transepithelial potential (in mV) and transepithelial resistance (in KΩ) were measured with this instrument. Transepithelial current was calculated by Ohm's law, expressed as μA/cm2, and is referred to as open-circuit current in the text to clearly distinguish it from the short-circuit currents measured in Ussing chambers. Although the open-circuit current technique tends to underestimate the amount of current that would be obtained in short-circuit current measurements, EVOM measurements proved consistent and reliable for detecting changes in transepithelial voltage, resistance, and current. Because cells used in EVOM measurements were at room temperature and not aerated with 95:5 gas, current tended to slowly decline with time (see Figs. 1 A and 7 A). Experiments were performed in serum-free A6 culture media. Upon replacement of serum- containing media with serum-free media, currents typically become relatively stable within 5 min. To corroborate open-circuit current measurements, conventional short-circuit current measurements were performed. A6 cells were grown to confluency on Transwell supports, and the supporting membrane was then placed in an Ussing chamber for measurement of short-circuit current as described by Blazer-Yost et al. (3). Current from individual monolayers was allowed to stabilize (∼1–2 h) before drug addition. Data were recorded on a strip chart recorder, and current magnitudes were subsequently measured for plotting. No qualitative difference was observed between the methods of measurement or the cell line used (2F3 vs. A6).
A commonly used indirect approach to assess the release of free AA involves monitoring the release of [3H]AA from cells (1). A6 cells on permeable supports were loaded with [3H]AA for 24 h. During this time, the [3H]AA was incorporated into the cellular lipids. Cells were then washed four times in PBS, and the efflux of labeled AA was monitored by a standard sample-and-replace method using fatty acid-free BSA in the efflux media to act as a trap for liberated free fatty acid. Samples were placed in scintillation cocktail (BioSafe II; Amersham), and radioactive decay was counted using a Packard 1900A scintillation counter. At the end of the sample period, cells were lysed in 0.1 N NaOH, and the cell lysate was counted to determine the counts remaining in the cells. Data were plotted as the percent counts remaining in the cells for each time point, and the slope of the relation was used to determine the relative rate of free AA release. This technique offers an advantage over PLA2 activity measurements in cell lysates in that efflux (AA release) can be monitored on the apical side of the monolayer, thus providing a more accurate assessment of the free AA at the apical membrane.
Single channel patch-clamp recording.
Cell-attached single channel patch data of ENaC were obtained as previously described by Kemendy et al. (25) and Ma and Ling (29). Briefly, A6 cells were cultured on custom Lucite rings and viewed under Hoffman modulation optics mounted on a Nikon Diaphot inverted microscope. Patch pipettes with a tip resistance of 7–9 MΩ were fabricated from TW150 glass on a Narishige PP-83 puller and fire-polished with a Narishige MF-83 polisher. Bath and pipette solutions contained (in mM): 96 NaCl, 3.4 KCl, 0.8 CaCl2, 0.8 MgCl2, and 10 HEPES at pH 7.4 (titrated with 1 N NaOH). Single channel currents from apical membrane patches were recorded with an Axon 1D amplifier and data digitized to a computer using a TL-1 data interface. Single channel data analysis was accomplished using pCLAMP software (Axon).
[3H]AA was obtained from Amersham. Aristolochic acid, ibuprofen, 5,8,11,14-eicosatetraynoic acid (ETYA), 7,7-dimethyleicosadienoic acid (DEDA), and PGE2were obtained from Biomol. All other reagents were obtained from either GIBCO BRL or Sigma.
Data are presented as means ± SE. Significance was estimated using the Student's t-test with P < 0.05 indicating a significant difference.
Inhibition of cPLA2 by application of the cPLA2 inhibitor, aristolochic acid, produced opposite effects depending on whether application was to the apical or basolateral side of the transporting epithelial monolayer. We show (below) that inhibition of cPLA2 from the apical side produces an increase in transepithelial current, whereas inhibition from the basolateral side produces an inhibition of transepithelial current. The apical effect is due to the downmodulation of apical Na+ channels (ENaC) by free AA produced by cPLA2 activity. Current inhibition after inhibition of cPLA2 from the basolateral side results predominately from an inhibition of PGE2 production within the cells. It is important to note that both results imply that cPLA2 is present and active in the aldosterone-stimulated monolayers and that disruption of cPLA2 activity has significant effects on the magnitude of transepithelial Na+ transport.
Inhibition of cPLA2 from the apical side.
The results of cPLA2 inhibition after apical application of aristolochic acid is shown in Fig.1. Open-circuit transepithelial current shows a marked increase on application of aristolochic acid to the apical surface. Current was 6.8 ± 0.2 μA/cm2 in the control monolayers and 7.5 ± 0.3 μA/cm2 in the treated group at time zero. With the addition of an apical media containing 200 μM aristolochic acid, current increased significantly within 1 min to 13.6 ± 0.6 μA/cm2(P < 0.001; Fig. 1 A). Addition of replacement apical media to the control group resulted in no significant change in current. Currents remained elevated approximately twofold for at least 5 min, with control and aristolochic acid-treated groups having currents of 5.6 ± 0.2 and 13.6 ± 0.5 μA/cm2, respectively. Apical aristolochic acid reduced transepithelial resistance from 1.22 ± 0.09 KΩ in control to 0.95 ± 0.06 KΩ. The increase in current with apical aristolochic acid was fully reversible, as shown in Fig. 1 B. Open-circuit transepithelial current was 5.9 ± 0.2 μA/cm2 before the apical addition of aristolochic acid. Apical aristolochic acid produced an increase in current to 9.6 ± 0.3 μA/cm2. Upon washout of apical aristolochic acid, currents were not significantly different from control, 5.9 ± 0.3 μA/cm2. All current was amiloride sensitive (data not shown).
In an effort to more fully describe the nature of current stimulation seen in the open-circuit current measurements with apical application of aristolochic acid, the short-circuit current method was applied. Identical results to those obtained in open-circuit conditions were observed upon apical application of 200 μM aristolochic acid under short-circuit conditions (Fig. 2). Starting current was 22.7 ± 1.4 μA/cm2 for the control group of monolayers and 20.3 ± 2 μA/cm2 for the treatment group. Current did not vary significantly in the control group over the course of the experiment, with the ending current being 22.7 ± 1.4 μA/cm2. Significant current stimulation was seen with the apical application of aristolochic acid within 20 s of addition (30.8 ± 1.6 μA/cm2,P < 0.001 vs. current at time 0). Current remained elevated during the 3-min time course with current at 40.2 ± 2.9 μA/cm2. The addition of 1 × 10−5 M amiloride to the apical bathing media reduced current in both the control and the aristolochic acid-treated groups, with current being 3.3 ± 1.1 and 3.5 ± 1.3 μA/cm2, respectively. There was no significant difference in the amiloride-insensitive current component between each group.
Because there have been some reports that aristolochic acid can affect the activity of sPLA2 (41), a specific inhibitor of sPLA2 was applied to the apical side of transporting monolayers. Figure 3 clearly demonstrates that 10 μM DEDA does not affect open-circuit transepithelial current. Current level began at 7.1 ± 0.2 μA/cm2, and addition of DEDA to the apical side did not significantly increase current; indeed, current was slightly decreased to 6.4 ± 0.3 μA/cm2. However, subsequent application of 100 μM apical aristolochic acid produced a significant increase in transepithelial current (12.5 ± 0.3 μA/cm2, P < 0.001). Thus the apical aristolochic acid effect is not due to actions on sPLA2.
Presumably, inhibition of cPLA2 by apical aristolochic acid application should reduce the amount of free AA present at the apical membrane. One means of accessing the liberation of free AA is to measure the release (efflux) of labeled AA from the monolayers. Figure4 A shows the efflux of [3H]AA from monolayers that had [3H]AA incorporated into the cellular lipid pool. Apical application of 200 μM aristolochic acid, as presumed, resulted in a decrease in the release (efflux) of free AA from the apical surface of the transporting monolayers. Although small, the efflux rate of [3H]AA from the apical surface was significantly reduced with apical aristolochic acid application (Fig. 4 B). Efflux rates were 0.045 ± 0.013 s−1 for the untreated group and 0.019 ± 0.004 s−1 for monolayers treated with apical aristolochic acid (P < 0.05). It should be mentioned that the actual release of free AA is underestimated by monitoring the efflux of AA, because free AA will partition into the cellular membrane and the cytosol as well as the external media. Nonetheless, a decrease in AA release was observed, thus indicating that available free AA at the apical membrane is reduced by the apical application of aristolochic acid. No appreciable change in [3H]AA efflux was detected with apical application of DEDA or with the basolateral application of aristolochic acid (data not shown).
The cPLA2 liberation of free AA from the sn-2 position of membrane lipids results in the production of a lysophospholipid product. Either free AA or the lysophospholipid product may act as a signaling molecule, and, in addition, free AA is the substrate for a number of enzymes and can result in a prodigious number of different signaling molecules. In an effort to ascertain which signaling pathways are involved, an analog of AA containing four triple bonds (ETYA) vs. the four double bonds of AA can be employed. ETYA acts as an unsaturated free fatty acid as well as an inhibitor of the enzymes that normally metabolize free AA. It has thus been used to distinguish between free fatty acid effects and AA metabolic product effects. As shown in Fig. 5, a 2-min apical application of 40 μM ETYA fully reverses the effect of current stimulation with apical application of aristolochic acid, thus implying that free AA is responsible for decreasing transepithelial current. Control currents were 5.7 ± 0.4 μA/cm2, which increased to 9.3 ± 0.7 μA/cm2 with apical application of 200 μM aristolochic acid. Subsequent apical addition of ETYA reduced currents to control values (5.6 ± 0.4 μA/cm2) and increased transepithelial resistance from 0.99 ± 0.05 KΩ to 1.16 ± 0.06 KΩ. Apical application of ETYA on control monolayers resulted in an ∼9% reduction of basal current, from 4.3 ± 0.2 to 3.9 ± 0.2 μA/cm2, which did not represent a significant difference (n = 12, data not shown). The lack of significance may arise from the presence of endogenous AA masking the effect of ETYA in the basal state.
To determine whether the stimulation of transepithelial current observed with apical application of aristolochic acid in monolayers was due to a free fatty acid-induced reduction in ENaC activity, the patch-clamp technique was employed. Figure6 shows the results of cell-attached patch-clamp recordings on A6 cells, specifically ENaC activity. Typical single channel records are shown in Fig. 6 A. After addition of 40 μM ETYA, the single channel open time decreases. With extensive washout, this decrease in channel open time can be partially reversed. A slight decrease in channel amplitude is also observed; however, this is not due to a change in the single channel conductance, as shown in Fig. 6 B, but rather represents a shift in the cell membrane potential, thus affecting the patch-clamp potential (∼8 mV). The voltage shift is not sufficient to produce the profound effects on channel open time observed in Fig. 6 A. Indeed, channel open time in the presence of ETYA is also decreased from control values if the patch-clamp potential is adjusted such that the channel amplitude remains constant (e.g., a constant patch potential, data not shown). Figure 6 C shows the mean results obtained from eight patches. ENaC open probability (P o) was 0.43 ± 0.05 before ETYA and was reduced 56 ± 9% to 0.18 ± 0.04 (P < 0.002) with the application of ETYA. Subsequent washout led to a partial reversal with a mean channelP o of 0.30 ± 0.03%.
Inhibition of cPLA2 from the basolateral side.
Unlike the results of cPLA2 inhibition from the apical side of the monolayer, basolateral application of aristolochic acid produced a decrease in transepithelial current.1 Figure7 A shows the results of basolateral application of 200 μM aristolochic acid on open-circuit current. Relative current is plotted. Starting current amplitude was 5.2 ± 0.09 μA/cm2 for both the control and treatment groups. The replacement of basolateral media results in a slow rundown of transepithelial current, with current being reduced to 3.1 ± 0.1 μA/cm2 after 20 min. The inclusion of aristolochic acid in the basolateral media results in a significant decrease (over control) in transepithelial current. The decrease over control is first noted and significant (P < 0.05) at ∼3 min and continues to develop for the 20-min time course presented. The time course is thus slower than that of the apical effect shown in Fig. 1 A that was complete at 1 min. Currents at the 5-min time point were 4.7 ± 0.1 μA/cm2 for control and 3.7 ± 0.1 μA/cm2 for basolateral aristolochic acid. This difference was greater at the 20-min time point where currents were 3.1 ± 0.1 and 1.3 ± 0.08 μA/cm2 for control and basolateral aristolochic acid-treated groups, respectively. Transepithelial resistance rose from 1.29 ± 0.07 KΩ control to 1.56 ± 0.08 KΩ at the 15-min time point.
Inhibition of transepithelial open-circuit current by basolateral application of aristolochic acid was not reversed by the basolateral application of ETYA. This differs from the effect of ETYA on the increase in current observed with apical application of aristolochic acid (see Fig. 5). Figure 7 B shows the results when ETYA is added to the basolateral media subsequent to basolateral aristolochic acid application. Control open-circuit current was 3.8 ± 0.2 μA/cm2. Current was reduced to 1.8 ± 0.1 μA/cm2 with a 10-min basolateral application of aristolochic acid. Subsequently, a 2-min application of basolateral ETYA (40 μM) significantly reduced currents further to 1.0 ± 0.06 μA/cm2 (P < 0.01). This indicates that the inhibition of current seen with basolateral application of aristolochic acid is not due to free fatty acid and implies that a metabolic product of AA may be involved in maintaining transepithelial transport.2
The effect of basolateral application of aristolochic acid was also measured under short-circuit current conditions and is shown in Fig.8. The results did not differ from those observed in the open-circuit conditions (Fig. 7 A). Current began at 27 ± 6 μA/cm2 for both control and experimental groups. Unlike the open-circuit current conditions, current level under short-circuit current was maintained throughout the experiment for the control group (26 ± 6 μA/cm2 at 10 min). Addition of basolateral aristolochic acid (200 μM) resulted in a marked decrease in short-circuit current, with a significant decrease clearly observed at 2 min (P < 0.05) and a large decrease observed at 10 min, 7 ± 2 μA/cm2with aristolochic acid vs. 26 ± 6 μA/cm2 for the control group. Addition of 1 × 10−5 M amiloride reduced currents to similar levels of 4.2 ± 0.9 and 4.1 ± 0.9 μA/cm2 for control and experimental groups, respectively.
Perhaps the most probable AA metabolite to mediate support of transepithelial transport is the cyclooxygenase product of AA, PGE2. Chronic application of basolateral PGE2has been reported to increase Na+ channel number in the apical membrane of frog skin (17) and in A6 cells (27, 30). If PGE2 was necessary for transport, then inhibition of cPLA2 might produce an inhibition of transport as observed in Figs. 7 and 8, and the current inhibition should be prevented by the addition of exogenous PGE2 to the basolateral side of the monolayer. Such experiments are summarized in Fig. 9. Open-circuit current was measured under three conditions: basolateral aristolochic acid alone, basolateral PGE2 alone, and simultaneous application of basolateral aristolochic acid and PGE2. Starting currents were 3.2 ± 1.2 μA/cm2 for each group. As shown previously, basolateral application of aristolochic acid led to a significant reduction in transepithelial current (1.6 ± 0.2 μA/cm2 at 15 min). The basolateral application of 10 μM PGE2 alone resulted in the stimulation of transepithelial current (5.8 ± 0.2 μA/cm2 at 5 min). The addition of basolateral PGE2 with basolateral aristolochic acid prevented the basolateral inhibition of transepithelial current caused by basolateral aristolochic acid application alone. Current levels in the presence of both basolateral PGE2 and aristolochic acid were 4.8 ± 0.3 at 5 min and 6.7 ± 0.5 μA/cm2at 15 min.
Direct inhibition of cyclooxygenase by basolateral ibuprofen leads to an inhibition of transepithelial open-circuit current similar to that seen with cPLA2 inhibition by basolateral aristolochic acid (Fig. 9 B). Starting currents were 5.2 ± 0.2 μA/cm2 for each group. Transepithelial current was reduced to 3.5 ± 0.2 and 2.9 ± 0.2 μA/cm2with basolateral application of aristolochic acid or ibuprofen, respectively. Current reduction in both cases was reversed with basolateral addition of 10 μM PGE2. Current 5 min after basolateral PGE2 was 4.8 ± 0.3 μA/cm2in monolayers treated with basolateral aristolochic acid and 6.6 ± 0.4 μA/cm2 for monolayers treated with basolateral ibuprofen. The difference in current magnitude after PGE2application is interesting and is addressed in discussion.
Combined effect of cPLA2 inhibition from the apical side and basolateral PGE2 addition.
The increase in transepithelial current observed with the apical application of aristolochic acid is additive, with the stimulation of current observed with the basolateral application of PGE2. Figure 10 shows transepithelial open-circuit current measurements from monolayers treated simultaneously with apical aristolochic acid (200 μM) and basolateral PGE2 (10 μM). Starting current was 8.9 ± 0.3 μA/cm2. Combined application of apical aristolochic acid and basolateral PGE2 resulted in a peak increase in current of 21.2 ± 1.3 μA/cm2 at 10 min. Peak current magnitudes observed with apical application of aristolochic acid alone (Fig. 1 A) were 14.2 ± 0.6 μA/cm2, whereas those observed with basolateral PGE2 were 6.7 ± 0.5 μA/cm2 (Fig. 9, A and B). The sum of these current magnitudes, 20.9 μA/cm2, is not significantly different from the 21.2 ± 1.3 μA/cm2observed in Fig. 10.
The data presented demonstrate that cPLA2 is tonically active in transporting A6 cells and that this activity is necessary for the support of transepithelial transport. Interestingly, however, a sidedness to cPLA2 inhibition results in a divergent effect on transepithelial transport, with inhibition from the apical side resulting in an increase in current and inhibition from the basolateral side resulting in a decrease in current. The increase in transepithelial current observed with apical side inhibition of cPLA2 results from the downmodulation of ENaC by free AA. The decrease in transepithelial current observed with basolateral side inhibition of cPLA2 results in part from a decrease in the metabolic product of AA, PGE2. Furthermore, the sidedness of cPLA2 inhibition suggests there is a local apical membrane-associated pool of cPLA2 at or near the apical Na+ channel in addition to commonly found perinuclear and endoplasmic reticular localization of cPLA2. These data provide a novel and intriguing mechanism whereby transepithelial Na+ transport and thus fluid reabsorption can be modulated over a relatively rapid time course, as well as provide for differential modulation by apical or basolateral signaling mechanisms.
The apical effect.
Free AA or free unsaturated fatty acids have been shown to affect a number of ion channels (31), producing either an increase (11, 12, 39) or a decrease (23) in single channel P o. The data presented herein not only demonstrates that free fatty acid reduces the P oof the apical Na+ channel, ENaC, but also shows that under normal conditions of transport (e.g., aldosterone stimulated), free AA is produced at the apical membrane and exerts a downmodulatory effect on ENaC, thus slowing transport. Inhibition of cPLA2, by aristolochic acid from the apical side of transporting A6 cell monolayers, produced an approximate twofold increase in transepithelial Na+ transport and a 58% decrease in the rate of AA liberated at the apical membrane. Apical ETYA reversed the stimulatory effect of apical aristolochic acid on transepithelial current, indicating that free AA itself was acting to downmodulate transport at the apical membrane. Although apical AA application produced similar results (not shown), these results were more variable and could be misleading due to cellular metabolism of AA into a variety of signaling molecules. ETYA provided an advantage in that it is an analog of AA and has similar effects on ion channels as AA (12); however, ETYA is not subject to cellular metabolism. Apical ETYA might also inhibit PGE2 production, resulting in a reduction of current as with the basolateral effect of aristolochic acid or ibuprofen. However, if this were the principal means of the apical ETYA reduction of current, one would not expect current inhibition by AA or an additive effect of basolateral PGE2 and apical aristolochic acid as is observed. In single channel recordings, ETYA reduced the P o of ENaC. The reduction in ENaC activity with ETYA supports the notion that tonic production of free AA at or near the apical membrane downmodulates ENaC activity and thus transepithelial transport of Na+. The fact that an increase in transepithelial transport is observed with the inhibition of cPLA2 from the apical side supports the presence of tonically active cPLA2 at or near the apical Na+ channel in the transporting A6 cells. Alternatively, AA liberated at a locale distant to ENaC within the cell might be responsible for downmodulation of ENaC activity. This alternative hypothesis is less likely because inhibition of cPLA2 by basolateral application of aristolochic acid does not produce the same affect as apical application.
The basolateral effect.
Inhibition of cPLA2 from the basolateral side of the A6 cell monolayer produced an effect on transepithelial current that was opposite from inhibition from the apical side. Basolateral application of aristolochic acid produced an inhibition of transepithelial current with a time course somewhat slower than that which was observed for the increase in current with apical aristolochic acid. Unlike that of the apical side effect, ETYA did not relieve the inhibition of current seen with basolateral side inhibition of cPLA2 but rather led to a greater inhibition of current. This effect of ETYA is consistent with the notion that free AA is tonically produced within the cell and is being metabolized to products that act to support transepithelial transport. Basolateral addition of the cyclooxygenase product of AA metabolism, PGE2, was able to prevent as well as reverse the inhibitory effect of basolateral aristolochic acid application. In support of PGE2 involvement, basolateral application of the cyclooxygenase inhibitor, ibuprofen, produced transepithelial current inhibition similar to that of aristolochic acid. The effect of ibuprofen was also reversed by the addition of PGE2. Interestingly, PGE2 produced a more robust response subsequent to ibuprofen than subsequent to aristolochic acid. Inhibition of cPLA2 would result in a decrease in all metabolic products of AA, whereas cyclooxygenase inhibition would only decrease prostaglandin production. Thus the more robust response with PGE2 after ibuprofen may indicate the involvement of other AA metabolites involved in the support of transport. Indeed, the lipoxygenase product of AA, LTD4, has been shown to increase Na+ channel activity in A6 cells (8).
The inhibitory effect on transport by basolateral side inhibition of cPLA2 supports the notion that cPLA2 within transporting A6 cells is active, and along with cyclooxygenase activity, is necessary to support Na+ transport. Indeed, basolateral PGE2 has previously been shown to increase short-circuit current and Na+ conductance in A6 cells (27, 30) and frog skin (17).
Model for the divergent effect.
The divergent effect on transepithelial current with inhibition of cPLA2 from the apical and basolateral sides suggests discrete intracellular pools of cPLA2. Although cPLA2 has predominately been localized to the perinuclear and endoplasmic reticular membranes within cells (35), several studies have reported evidence that suggests that cPLA2 also can occur at the plasma membrane in confluent bovine endothelial cells (36), in fibroblasts (7), in glomerular epithelial cells (28), and in bradykinin-stimulated Madin-Darby canine kidney cells (26). Perhaps the most convincing evidence for cPLA2 presence in the plasma membrane derives from inside-out patch-clamp experiments in GH3 cells, where functionally, cPLA2 occurs in the same membrane patch as the Ca2+-activated K+ channel (BK channel) (14). Ion channels occur in the plasma membrane at relatively low abundance. At minimum, if one cPLA2 was associated with one BK channel, one might expect the plasma membrane abundance of cPLA2 to also be relatively low, particularly compared with the nuclear envelope.
A receptor-coupled apical membrane-situated cPLA2 could act as the transduction mechanism for intraluminal signaling molecules. The stimulation of Cl− current in colonic epithelia by 5′-(N-ethylcarboxamido)adenosine (an adenosine analog) was found to correlate with the release of [3H]AA and was abolished by the PLA2 inhibitor 4-bromophenacyl bromide, suggesting that adenosine was acting by increasing PLA2activity, and the resulting free AA was affecting transepithelial Cl− current (1). Similarly, Smallridge and Gist (38) have shown that the ATP stimulation of125I− efflux in the thyroid cell line FRTL-5 was correlated with an increased release of [3H]AA, both of which were abolished by the PLA2 inhibitor U-73122. It is interesting to think of these results in light of the connection between ATP, Cl− secretion, and Na+reabsorption, where it is speculated that external ATP, somehow dependent on active cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel, acts to inhibit Na+reabsorption and the Na+ channel (15). Endothelin-1 has also been shown to increase free AA levels (24) in smooth muscle and to affect Na+transport in epithelia (20). The emerging precedence from these data suggests that receptor-mediated signaling through cPLA2 and AA release can have an effect on ion transport processes. Receptors to adenosine, ATP, and endothelin-1 have been identified in renal collecting duct epithelia either directly (10, 16, 34, 37) or by inference from pharmacological effects (see below). Moreover, as might be expected for intratubular signaling, adenosine, ATP, and endothelin-1 are present in normal urine and have been shown to vary during certain pathological conditions (24, 32). Their occurrence, particularly in the cases of adenosine and endothelin-1, are proposed to protect against ischemic damage by inhibiting the transport of Na+(2, 6, 18). As briefly mentioned earlier, the amiloride-sensitive Na+ channel is inhibited by extracellular ATP, the level of which is partially determined by CFTR activity (15), thus allowing for cellular self-regulation of apical Na+ reabsorption. Additionally, apical application of adenosine in A6 cells, via a type A2receptor, produces an inhibition of Na+ channel activity (29). Basolateral application of adenosine, on the other hand, produces an increase in Na+ transport (9). Endothelin-1 applied basolaterally, most likely ETA receptor mediated, also produces a stimulation of apical Na+ channel activity (20), thus suggesting a precedence that compounds, which may act through cPLA2, produce opposite effects when applied to the apical membrane vs. the basolateral membrane. Studies that determine the effects and dependence of apical agonists on cPLA2activity, free AA release at the apical membrane, and Na+channel activity are currently under way.
On the basis of the data presented, we propose that 1) AA from cPLA2 activity at or near the apical membrane is primarily responsible for downmodulation of transepithelial current, and 2) AA from cPLA2 activity combined with cyclooxygenase activity within the cell, presumably perinuclear, produces PGE2, which is necessary to support transport. Together, the apical effect of AA downmodulation of ENaC and the necessity of the AA product PGE2 to support transport provide a cellular signaling mechanism to fine-tune Na+transport, based on the relative activity of cPLA2 at the apical membrane and the perinuclear cPLA2 and cyclooxygenase activity. With fixed cPLA2 activity, manipulation of cyclooxygenase activity would result in relative changes of free AA. With greater cyclooxygenase activity, more PGE2 would be produced, resulting in less free AA, which would tend to maximize transport. In contrast, less cyclooxygenase activity would yield less PGE2 and result in more free AA, which would tend to minimize transport. Increased cPLA2activity at or near the apical membrane would tend to decrease transport, whereas increased cPLA2 near an active cyclooxygenase enzyme would tend to increase transport. Indeed, as early as 1975, an increased phospholipid turnover was suggested to occur with stimulation of Na+ transport in toad bladder (22). This increase was attributed to cellular increase in phospholipase activity (42). This report provides further evidence as well as a model for the increased rate of phospholipid turnover and establishes the increased turnover as important in the regulation of Na+ transport. Specifically, AA and PGE2 act in an antagonistic manner in A6 cells to modulate Na+ transport rate.
We are grateful to B. J. Duke for plating and maintaining the 2F3 cell cultures. All short-circuit current experiments were made in the laboratory of Bonnie Blazer-Yost (Dept. of Biology, Indiana Univ. Purdue Univ. at Indianapolis). We are most appreciative of her support in providing equipment and the expertise of her laboratory personnel, Carla J. Faletti, Amy Hartman, and Diane Hoover. Without this support, the short-circuit current measurements would have been much more difficult.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-09215 (to R. T. Worrell), National Science Foundation Grant IBN-9603837 (to D. D. Denson), NIDDK Grants DK-37963 and DK-50268 (to D. C. Eaton), and by the Center for Cell and Molecular Signaling at Emory.
Address for reprint requests and other correspondence: R. T. Worrell, Dept. of Physiology, Center for Cell and Molecular Signaling, Emory Univ., Atlanta, Georgia 30322 (E-mail:).
↵1 It is interesting to note that long-term (>15–20 min) application of aristolochic acid to the apical side also resulted in an inhibition of transepithelial current subsequent to current stimulation. The most likely explanation is that aristolochic acid is diffusing through or across the cell and reaching the sites that are more readily accessible from the basolateral membrane (the basolateral effect but with a lag time to the onset). All apical application experiments were performed with <10 min of aristolochic application to avoid this secondary effect. Transepithelial current increase was never observed subsequent to the decrease in current with basolateral aristolochic acid, thus suggesting a necessity for cPLA2 activity in the support of transport.
↵2 Experiments with basolateral ETYA alone also produced inhibition of current similar to basolateral aristolochic acid. This arises from ETYA inhibition of the enzymes that metabolize AA (i.e., cyclooxygenase). ETYA was used in this experiment as a means of distinguishing between a free fatty acid effect vs. an AA metabolite effect.
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