Contrasting effects of cPLA2 on
epithelial Na+ transport
Roger T.
Worrell1,
Hui-Fang
Bao1,
Don D.
Denson2, and
Douglas C.
Eaton1
Departments of 1 Physiology and 2 Anesthesiology,
Center for Cell and Molecular Signaling, Emory University, Atlanta,
Georgia 30322
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ABSTRACT |
Activity of the epithelial Na+ channel
(ENaC) is the limiting step for discretionary Na+
reabsorption in the cortical collecting duct. Xenopus laevis kidney 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; cyclooxygenase; A6 cells; cytosolic phospholipase A2
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INTRODUCTION |
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 cytochrome
P-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.
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MATERIALS AND METHODS |
Cell culture.
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
K
) 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. 1A and 7A).
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).

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Fig. 1.
Inhibition of cytosolic phospholipase A2
(cPLA2) from the apical side increases transepithelial
Na+ transport. A: open-circuit transepithelial
current measurement with apical application of the cPLA2
inhibitor, aristolochic acid (200 µM; ), and
transepithelial currents from control monolayers ( ) are
shown. Inhibition of cPLA2 from the apical side increased
transepithelial current ~2-fold within 1 min (P < 0.001). B: effect of apical addition of aristolochic acid is
fully reversible. Relative transepithelial current with addition of
aristolochic acid to the apical side (time 0) followed by
washout (Wash) of aristolochic acid (arrow, at 3 min) is shown.
Relative current after washout was not significantly different from
control current. For each group of 2F3 cells, n = 12.
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AA efflux.
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 M
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).
Reagents.
[3H]AA was obtained from Amersham. Aristolochic acid,
ibuprofen, 5,8,11,14-eicosatetraynoic acid (ETYA),
7,7-dimethyleicosadienoic acid (DEDA), and PGE2
were obtained from Biomol. All other reagents were obtained from either
GIBCO BRL or Sigma.
Significance tests.
Data are presented as means ± SE. Significance was estimated
using the Student's t-test with P < 0.05 indicating a significant difference.
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RESULTS |
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. 1A). 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 K
in control to
0.95 ± 0.06 K
. The increase in current with apical
aristolochic acid was fully reversible, as shown in Fig. 1B.
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.

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Fig. 2.
Short-circuit measurement of transepithelial current with
apical application of 200 µM aristolochic acid ( ) and
control monolayers ( ). Apical aristolochic acid
application produced a significant change in transepithelial current
within 20 s of addition (P < 0.01). Current
stimulation saturated at ~2 min. Addition of 1 × 10 5 amiloride (5-min time point) reduced transepithelial
current to near 0 both for control and aristolochic acid-treated
groups. Currents in control and treated groups after amiloride were not
significantly different. For each group of A6 cells, n = 4.
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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.

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Fig. 3.
Apical application of the secretory PLA2
inhibitor 7,7-dimethyleicosadienoic acid (DEDA) does not change
transepithelial current. Apical application of 10 µM DEDA (time
0) produced no significant change in transepithelial open-circuit
current, whereas in the same set of monolayers, apical application of
the cPLA2 inhibitor aristolochic acid (100 µM, arrow, at
5 min) produced a significant change (P < 0.001). For
2F3 monolayers, n = 12.
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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. Figure
4A 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. 4B). 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).

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Fig. 4.
Apical application of aristolochic acid reduces apical release of
free arachidonic acid. A: apical efflux of free
[3H]arachidonic acid ([3H]AA) with apical
application of 200 µM aristolochic acid ( , added at
arrow) and under control conditions ( ). Data are
presented as the % counts remaining in the cell for each time point.
B: mean apical side [3H]AA efflux rates from
individual monolayers. Efflux rates are from the 10- to 20-min time
interval. Although the efflux rates are small, apical application of
aristolochic acid (Arist) produced a significant reduction in the rate
of [3H]AA release (*P < 0.05, n = 12 for 2F3 monolayers).
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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 K
to 1.16 ± 0.06 K
. 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.

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Fig. 5.
The apical aristolochic acid effect is reversed by apical
addition of the unsaturated free fatty acid, 5,8,11,14-eicosatetraynoic
acid (ETYA). After apical addition of 200 µM aristolochic acid
(time 0), apical addition of 40 µM ETYA (arrow, at 5 min)
reduces transepithelial open-circuit current to control levels.
Relative current with ETYA was not significantly different from control
(n = 6 for 2F3 monolayers).
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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. Figure
6 shows the results of cell-attached
patch-clamp recordings on A6 cells, specifically ENaC activity. Typical
single channel records are shown in Fig. 6A. 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. 6B, 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. 6A. 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 6C shows the mean results obtained from eight
patches. ENaC open probability (Po) 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 channel
Po of 0.30 ± 0.03%.

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Fig. 6.
The unsaturated free fatty acid ETYA reduces apical epithelial
Na+ channel (ENaC) open probability
(Po). A: representative cell-attached
single channel recordings in A6 cells of ENaC under control, 40 µM
ETYA, and washout conditions. Bath application of ETYA reduces the
amount of time the channel spends in the open state. This effect is
partially reversible on washout of the ETYA from the bath.
B: current-voltage relations of ENaC in the presence and
absence of ETYA. The apparent reduction in single channel amplitude
with ETYA observed in A does not represent a change in
single channel conductance (slope of relation, see text for details).
C: ENaC Po from 8 patches similar to
those in A. Bath application of 40 µM ETYA results in a
52% reduction in Po (*P < 0.01). This reduction in ENaC Po is partially
reversible on ETYA washout (#P < 0.05 vs.
ETYA treated).
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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 Figure
7A 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. 1A 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 K
control to
1.56 ± 0.08 K
at the 15-min time point.

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Fig. 7.
Inhibition of cPLA2 from basolateral side
inhibits transepithelial Na+ transport. A:
open-circuit transepithelial current measurement with basolateral
application of the cPLA2 inhibitor aristolochic acid (200 µM; ). Relative transepithelial currents from control
monolayers are shown ( ). Inhibition of
cPLA2 from the basolateral side inhibited transepithelial
current beginning at 2 min and reaching maximal inhibition at ~15 min
(P < 0.001 vs. control, 15-min time point for each,
n = 12 for 2F3 cell monolayers). Under open-circuit
current measurement conditions used, control currents showed some
degree of decline within the observation time period (see
MATERIALS AND METHODS). Current at time 0 was
~7 µA/cm2. B: effect of basolateral addition
of aristolochic acid is not reversed by basolateral application of
ETYA. Relative transepithelial current with the addition of
aristolochic acid to the basolateral side (time 0) followed
by basolateral application of ETYA (arrow, at 10 min). Relative current
was reduced further by basolateral application of ETYA
(P < 0.01, n = 6 for 2F3 cell
monolayers).
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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 7B 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. 7A). 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/cm2
with 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.

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Fig. 8.
Short-circuit measurement of transepithelial current with
basolateral application of 200 µM aristolochic acid
( ). Current from untreated monolayers is shown
( ). Basolateral aristolochic acid application produced
a significant inhibition of transepithelial current within 2 min of
addition (P < 0.05) and more substantial inhibition at
10 min (P < 0.001). Addition of 1 × 10 5 amiloride (last data point, past 10 min) reduced
transepithelial current to near 0. For each group, n = 4 A6 cell monolayers.
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Perhaps the most probable AA metabolite to mediate support of
transepithelial transport is the cyclooxygenase product of AA, PGE2. Chronic application of basolateral PGE2
has 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/cm2
at 15 min.

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Fig. 9.
Inhibition of open-circuit transepithelial current by
basolateral application of aristolochic acid is prevented by
basolateral application of prostaglandin E2
(PGE2). A: transepithelial current measured with
basolateral application of aristolochic acid (at time 0,
darker circles), basolateral PGE2 (at time 0,
triangles), or both aristolochic acid and PGE2 (at
time 0, lighter circles). Basolateral PGE2 alone
increased transepithelial current (P < 0.01) and was
able to prevent the inhibition of current with basolateral application
of aristolochic acid (P < 0.001, 15-min time point).
For each group, n = 6 2F3 monolayers. B:
basolateral application of the cyclooxygenase inhibitor ibuprofen (at
time 0, triangles) inhibits current to a similar degree as
does basolateral application of aristolochic acid (at time
0, circles). Both effects are reversed by basolateral application
of PGE2 (arrow, at 5 min). For each group,
n = 6 2F3 monolayers.
|
|
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. 9B). 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/cm2
with 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/cm2
in 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 PGE2
application 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. 1A) 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/cm2
observed in Fig. 10.

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Fig. 10.
The combination of apical aristolochic acid and
basolateral PGE2 produces an additive effect on
transepithelial open-circuit current. Apical application of
aristolochic acid produced a current level of 14.2 ± 0.6 µA/cm2 (see Fig. 1A). Basolateral application
of PGE2 produced a current level of 6.7 ± 0.5 µA/cm2 (see Fig. 9A). In combination, current
levels, as shown in this figure, peaked at 21.2 ± 1.3 µA/cm2 (n = 6 2F3 monolayers). The
combined effect is not significantly different from the sum of the
individual effects.
|
|
 |
DISCUSSION |
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 Po. The data presented herein not only
demonstrates that free fatty acid reduces the Po
of 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 Po 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 PLA2
activity, and the resulting free AA was affecting transepithelial
Cl
current (1). Similarly, Smallridge and
Gist (38) have shown that the ATP stimulation of
125I
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 A2
receptor, 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 cPLA2 activity, 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 cPLA2 activity 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.
 |
ACKNOWLEDGEMENTS |
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.
 |
FOOTNOTES |
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:
rworrel{at}emory.edu).
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.
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.
Received 29 June 2000; accepted in final form 14 February 2001.
 |
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