EDITORIAL FOCUS
Focus on "Contrasting effects of
cPLA2 on epithelial
Na+ transport"
Peter R.
Smith,
Catherine M.
Fuller,
James S.
Bubien, and
Dale J.
Benos
Department of Physiology and Biophysics, University of Alabama at
Birmingham, Birmingham, Alabama 35294
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ARTICLE |
THE AMILORIDE-SENSITIVE epithelial
Na+ channel (ENaC) plays a central role in the reabsorption
of salt and fluids across Na+-reabsorbing epithelia such as
the distal nephron, distal colon, lungs, and ducts of the exocrine
glands. ENaC is rate limiting for net Na+ reabsorption
because it mediates entry of Na+ from the luminal fluid
during the first stage of electrogenic Na+ transport
(1, 9). Thus ENaC plays a key role in the regulation of
fluid and electrolyte homeostasis and blood pressure. Malfunction or
mutations of ENaC are associated with a number of human diseases such
as Liddle's syndrome, an inherited form of arterial hypertension, pseudohypoaldosteronism type 1, pulmonary edema, cystic fibrosis, and
influenza (1, 11). Because of its central role in
transepithelial fluid transport, the activity of ENaC is highly
regulated by hormones such as aldosterone, vasopressin, and insulin,
extracellular proteases, accessory proteins, and intracellular
mediators such as Na+, Ca2+, pH, and protein
kinases A and C (1, 9). The regulation of ENaC activity
occurs at two major levels: 1) at the level of the membrane
[i.e., modulation of the number of active channels (channel
density)]; and 2) at the level of the channel itself [i.e., modulation of single channel open probability
(Po)].
Cytosolic phospholipase A2 (cPLA2) and
arachidonic acid metabolites have also been implicated in regulating
transepithelial Na+ transport across epithelial tissues.
cPLA2 selectively hydrolyzes phospholipids at the
sn-2 position to release free arachidonic acid
(4). After its release, arachidonic acid is converted by
cyclooxygenases, lipoxygenases, and cytochrome P-450
monoxygenase to prostaglandins, leukotrines, and eicosanoic acids,
products that function as second messengers in signal transduction
pathways (4). In the 1970s, Goodman et al.
(10) demonstrated that the lag period between aldosterone
exposure and stimulation of Na+ transport across the toad
bladder was shortened by the presence of serosal phospholipase A, and
Yorio and Bentley (16) revealed that transepithelial
Na+ transport across the toad urinary bladder stimulated by
the mineralocorticoid hormone aldosterone was markedly attenuated by
mepacrine, a phospholipase A inhibitor. Subsequently, Cantiello and
coworkers (6) showed that activation of cPLA2
and lipoxygenase pathways by the G protein, Gi-3, induced
both an increase in the number (N) and
Po of a poorly selective, 9-pS
amiloride-sensitive Na+ channel expressed in
coverslip-grown Xenopus A6 kidney cells. Aldosterone has
itself been shown to cause increases in channel NPo in A6 cells, although the effect on
Po predominates (12). Thus
considerable circumstantial evidence exists to support a link between
the major salt conservation hormone aldosterone, phospholipase
A2 (PLA2), and Na+ channel
activity. These data establish that lipid metabolism is an important
component in the regulation of transepithelial Na+ transport.
In the current article in focus (Ref. 15, see
p. C147 in this issue), Worrell and associates have extended the
observations of Yorio and Bentley (16) and Cantiello et
al. (6) to ENaC. Using aristolochic acid, a relatively
selective inhibitor cPLA2, and a nonmetabolized analog of
arachidonic acid, 5,8,11,14-eicosatetraynoic acid (ETYA), Worrell et
al. (15) present data indicating that cPLA2
activity and free arachidonic acid are necessary to support transepithelial Na+ transport across confluent monolayers
of Xenopus A6 cells, at least under conditions of chronic
aldosterone exposure, which mimics the situation seen under conditions
of salt deprivation.
Interestingly, activation of short-circuit current as a result of
aristolochic acid inhibition of cPLA2 was only seen after addition of the inhibitor to the apical membrane compartment; addition
of aristolochic acid to the basolateral membrane produced the opposite
effect, i.e., inhibition of transepithelial current. Addition of ETYA
reversed the stimulation due to aristolochic acid at the apical
membrane, but was without effect on the inhibition of current seen on
the basolateral addition of aristolochic acid. Single channel data
revealed that ETYA decreased Po of the highly selective, 4-pS ENaC channel, strongly suggesting that arachidonic acid
and/or its metabolites regulate transepithelial Na+
transport by altering ENaC Po.
These data imply a role for arachidonic acid itself in the
"tune-down" response at the apical membrane, while suggesting that a downstream product of arachidonic acid metabolism could be
responsible for the "tune-up" effect exerted at the basolateral
membrane. The authors (15) postulate that the reduction in
ENaC Po by ETYA reflects the tonic production of
arachidonic acid via a tonically active cPLA2 associated
with the apical membrane.
The most likely downstream effector molecule is the arachidonic acid
metabolite, prostaglandin E2 (PGE2). Because
synthesis of PGE2 can be regulated at the level of
cyclooxygenase, the role of this metabolite in ENaC regulation was
confirmed when the addition of ibuprofen (a cyclooxygenase inhibitor)
had identical effects at the basolateral membrane as did aristolochic
acid, i.e., inhibition of transepithelial current. Although inhibition
of transepithelial Na+ transport could not be reversed by
basolateral application of ETYA, it was reversed by basolateral
addition of the arachidonic acid metabolite, PGE2.
On the basis of these data, the authors postulate that arachidonic acid
from a perinuclear pool of cPLA2 is metabolized to PGE2, and this PGE2, in turn, is necessary to
support transepithelial transport, presumably through autocrine and
paracrine actions. It should be noted, however, that PGE2
has been shown to stimulate transepithelial Na+ transport
across A6 cell monolayers by promoting a cAMP/protein kinase A
(PKA)-mediated increase in ENaC density in the apical membrane
(13). Thus the PGE2-mediated reversal of
aristolochic acid and ibuprofen effects on transepithelial transport
observed by Worrell et al. (15) may not be entirely
related to the cPLA2/arachidonic acid signaling pathway,
but rather they may also reflect activation of the cAMP/PKA pathway via
PGE2.
Worrell and coworkers (15) propose that the apical
downregulation of ENaC via arachidonic acid and the necessity of
PGE2 to support transepithelial Na+ transport
across A6 cell monolayers provide a signaling mechanism to fine-tune
Na+ transport based on the relative activity of the apical
and perinuclear pools of cPLA2. Coupling of a luminal
receptor with apically restricted cPLA2 would provide a
novel transduction mechanism for the regulation of ENaC by luminal
signaling molecules. Although these signaling molecules and their
receptors remain to be identified, candidates include bradykinin,
adenosine, ATP, and endothelin, all of which are present in the luminal
fluid (5). Alternatively, the apical cPLA2
pool may be coupled to a basolateral receptor through a second
messenger pathway. Potential candidates for basolateral signaling
molecules, coupled to either the apical or perinuclear pool of
cPLA2, include epidermal growth factor and endothelin, both
of which have been shown to activate cPLA2 and arachidonic acid release in the distal nephron (5).
Where, then, do the present data fit into the big picture of normal and
dysfunctional ENaC regulation? These new findings seem to contradict
earlier observations that provided evidence for inhibition of
Na+ current via inhibition of PLA2
(16). However, the present studies are the first to
dissect PLA2 signaling pathways in well-polarized monolayers of an established Na+-transporting epithelial
cell line, and, therefore, may more closely reflect the situation in
the kidney under conditions of salt deprivation. The observation that
ETYA inhibited ENaC Po suggests that at least one level of regulation exists either directly at the channel or with a
closely associated regulatory protein. Downstream targets of
arachidonic acid include regulatory enzymes such as calmodulin kinase
II and protein kinase C (PKC); PKC has also been implicated in the
inhibition of amiloride-sensitive Na+ currents in
Xenopus oocytes heterologously expressing ENaC
(3) and so may account for the mechanism underlying the
inhibitory effects of PLA2. What of the stimulatory effects
associated with PGE2 at the basolateral membrane?
The situation here is much less clear, although experiments performed
in other systems have suggested that PGE2 can activate
adenylate cyclase and thus formation of cAMP with subsequent activation
of PKA. Kokko et al. (13) have shown that chronic exposure
to PGE2 at the basolateral membrane of A6 cells resulted in
both an increase in the total number of 4-pS Na+ channels
(presumably synonymous with ENaC) at the apical surface and
accumulation of cellular cAMP, although direct PKA-mediated phosphorylation of ENaC has been harder to demonstrate
(14).
Certainly, increased Na+ absorption in response to
aldosterone is more in keeping with the role of this mineralocorticoid
in salt conservation than is an aldosterone-mediated inhibition of ENaC
current. It seems likely that the net effects of aldosterone to
increase transport are the result of competing inhibitory and stimulatory influences exerted at opposite membrane domains and exerted
both on intrinsic channel activity, as determined by channel Po, and on the total number of active channels
present at the cell surface (N). Other regulatory molecules
may well exert similar inhibitory influences on PLA2 at the
apical membrane, as does aristolochic acid in the experiments described
by Worrell et al. (15) in the present report. As described
above, the renal tubule expresses luminal receptors for a variety of
ligands that are found in tubular fluid after filtration at the
glomerulus (2, 7, 8). Thus Na+ reabsorption,
as handled by the distal convoluted tubule and collecting duct, is by
necessity highly responsive to even slight changes in the internal
environment as transmitted through the glomerulus. The exquisite
sensitivity exhibited by the kidney in terms of Na+
handling is simply reflective of the importance of this ion in whole
body volume homeostasis.
Thus the work of Worrell et al. (15) is important for
reasons that are broader than the specific focus of the paper. These findings show that ion channels in general, and ENaC in particular, interact with their environment in a complex way, a way that is often
times overlooked when considering only the biophysical characteristics of the channel in a single model system. The findings also show directly that the same treatment can have different effects, depending on which side of the cell is exposed to the treatment. Whether or not
arachidonic acid metabolites actually play any significant cellular
physiological role in the regulation of ENaC is perhaps not the main
point of these experiments. Rather, the direct demonstration that these
compounds (and probably a variety of other cellular constituents and
metabolites) can alter channel function means that interpretation of
ion channel function and regulation in intact cells must take into
account the environment in which the experiments are performed.
Although this may be stating the obvious, the obvious is sometimes
overlooked, misunderstood, or worse, even ignored. Does ENaC expressed
by mammalian cells respond similarly? Are the same enzymes present in
the plasma membrane of mammalian cells? Are these enzymes present in
the plasma membrane of oocytes, and do cloned ENaC subunits, when
heterologously expressed, respond similarly to regulation by
cPLA2? It is reasonable to assume that mammalian renal
principal cell membranes are different from Xenopus cell
membranes. One broad question that these experiments trigger is: If
arachidonic acid metabolites and cPLA2 provide some
"background" regulation of ENaC, how is the intrinsic function and
regulation of ENaC altered when these elements are not present? What
portion of ENaC, or the surrounding membrane, is affected by
arachidonic acid metabolites? How do these metabolites prevent the
channels from opening and reduce the time the channel spends in the
open state? Is the thermal responsiveness of the channel altered? Do the metabolites directly interact with the channels, or do they alter
the membrane surrounding the channels? Thus experiments such as those
reported by Worrell et al. (15) need not be directly related to actual cellular events. Their intrinsic value may be to
remind us that ion channel function and regulation are very complex and
depend on many factors. The environment in which biophysical observations are made may have a profound influence on what is actually
observed. Only by experiments such as described by Worrell et al.
(15) and an open mind about the interpretation of such findings will we come closer to a more realistic understanding of ion
channel function and regulation.
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FOOTNOTES |
Address for reprint requests and other correspondence: D. J. Benos, Dept. of Physiology and Biophysics, Univ. of Alabama at Birmingham, 1918 University Blvd., MCLM 704, Birmingham, AL 35294-0005 (E-mail: benos{at}physiology.uab.edu).
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REFERENCES |
1.
Alvarez de la Rosa,
Canessa CM,
Fyfe GK,
and
Zhang P.
Structure and regulation of amiloride sensitive sodium channels.
Annu Rev Physiol
62:
573-594,
2000[ISI][Medline].
2.
Amorim, JB,
and
Malnic G.
V(1) receptors in luminal action of vasopressin on distal K+ secretion.
Am J Physiol Renal Physiol
278:
F809-F816,
2000[Abstract/Free Full Text].
3.
Awayda, MS,
Ismailov II,
Berdiev BK,
Fuller CM,
and
Benos DJ.
Protein kinase regulation of a cloned epithelial Na+ channel.
J Gen Physiol
108:
49-65,
1996[Abstract].
4.
Bonventre, JV.
Phospholipase A2 and signal transduction.
J Am Soc Nephrol
3:
128-150,
1992[Abstract].
5.
Breyer, MD,
and
Ando Y.
Hormonal signaling and the regulation of salt and water transport in the collecting duct.
Annu Rev Physiol
56:
711-739,
1994[ISI][Medline].
6.
Cantiello, HF,
Patenaude CR,
Codina J,
Birnbaumer L,
and
Ausiello DA.
Gi-3 regulated epithelial Na+ channels by activation of phospholipase A2 and lipoxygenase pathways.
J Biol Chem
265:
21624-21628,
1990[Abstract/Free Full Text].
7.
De Jesus Ferreira, MC,
and
Bailly C.
Luminal and basolateral endothelin inhibit chloride reabsorption in the mouse thick ascending limb via a Ca2+-independent pathway.
J Physiol
505:
749-758,
1997[Abstract].
8.
Deetjen, P,
Thomas J,
Lehrmann H,
Kim SJ,
and
Leipziger J.
The luminal P2Y receptor in the isolated perfused mouse cortical collecting duct.
J Am Soc Nephrol
11:
1798-1806,
2000[Abstract/Free Full Text].
9.
Garty, H,
and
Palmer LG.
Epithelial sodium channels: function, structure, and regulation.
Physiol Rev
77:
359-396,
1997[Abstract/Free Full Text].
10.
Goodman, DBP,
Allen JE,
and
Rasmussen H.
Studies on the mechanism of action of aldosterone: hormone-induced changes in lipid metabolism.
Biochemistry
10:
3825-3831,
1971[ISI][Medline].
11.
Guggino, WB,
and
Guggino S.
Amiloride-sensitive sodium channels contribute to the woes of flu.
Proc Natl Acad Sci USA
97:
9827-9829,
2000[Free Full Text].
12.
Kemendy, AE,
Kleyman TR,
and
Eaton DC.
Aldosterone alters the open probability of amiloride-blockable sodium channels in A6 epithelia.
Am J Physiol Cell Physiol
263:
C825-C837,
1992[Abstract/Free Full Text].
13.
Kokko, KE,
Matsumoto PS,
Ling BN,
and
Eaton DC.
Effects of prostaglandin E2 on amiloride-blockable Na+ channels in a distal nephron cell line (A6).
Am J Physiol Cell Physiol
267:
C1414-C1425,
1994[Abstract/Free Full Text].
14.
Shimkets, RA,
Lifton R,
and
Canessa CM.
In vivo phosphorylation of the epithelial sodium channel.
Proc Natl Acad Sci USA
95:
3301-3305,
1998[Abstract/Free Full Text].
15.
Worrell, RT,
Bao H-F,
Denson DD,
and
Eaton DC.
Contrasting effects of cPLA2 on epithelial Na+ transport.
Am J Physiol Cell Physiol
281:
C147-C156,
2001[Abstract/Free Full Text].
16.
Yorio, T,
and
Bentley PJ.
Phospholipase A and the mechanism of action of aldosterone.
Nature
271:
79-81,
1978[ISI][Medline].
Am J Physiol Cell Physiol 281(1):C12-C14
0363-6143/01 $5.00
Copyright © 2001 the American Physiological Society