Vol. 284, Issue 3, F419-F432, March 2003
INVITED REVIEW
Control of epithelial transport via luminal P2 receptors
Jens
Leipziger
Department of Physiology, The Water and Salt Research
Center, Aarhus University, 8000 Aarhus C, Denmark
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ABSTRACT |
P2 membrane receptors are specifically
activated by extracellular nucleotides like ATP, ADP, UTP, and UDP. P2
receptors are subdivided into metabotropic P2Y and ionotropic P2X
receptors. They are expressed in all tissues and induce a variety of
biological effects. In epithelia, they are found in both the
basolateral and the luminal membranes. Their widespread luminal
expression in nearly all transporting epithelia and their effect on
transport are summarized. The P2Y2 receptor is a prominent
luminal receptor in many epithelia. Other luminal P2 receptors include
the P2X7, P2Y4, and P2Y6 receptors.
Functionally, luminal P2Y2 receptor activation elicits
differential effects on ion transport. In nearly all secretory
epithelia, intracellular Ca2+ concentration-activated ion
conductances are stimulated by luminal nucleotides to induce
Cl
, K+, or HCO
secretion.
This encompasses respiratory and various gastrointestinal epithelia or
tissues like the conjunctiva of the eye and the epithelium of sweat
glands. In the distal nephron, all active transport processes
appear to be inhibited by luminal nucleotides. P2Y2
receptors inhibit Ca2+ and Na+ absorption and
K+ secretion. Commonly, in all steroid-sensitive epithelia
(lung, distal nephron, and distal colon), luminal ATP/UTP inhibits
epithelial Na+ channel-meditated Na+
absorption. ATP is readily released from epithelial cells onto their
luminal aspect, where ecto-nucleotidases promote their metabolism. Adenosine generated by the action of 5'-nucleotidase may elicit further
effects on ion transport, often opposite those of ATP. ATP release from
epithelia continues to be poorly understood. Integrated functional
concepts for luminal P2 receptors are suggested: 1) luminal
P2 receptors are part of an epithelial "secretory" defense
mechanism; 2) they may be involved in the regulation of cell
volume when transcellular solute transport is out of balance; 3) ATP and adenosine may be important autocrine/paracrine
regulators mediating cellular protection and regeneration after
ischemic cell damage; and 4) ATP and adenosine have
been suggested to mediate renal cyst growth and enlargement in
polycystic kidney disease.
P2Y; P2X; chloride secretion; sodium absorption; epithelial sodium
channel; potassium secretion
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INTRODUCTION |
EPITHELIAL TRANSPORT IS A regulated
phenomenon involving a large number of hormones and local agonists that
usually bind to their respective transmembrane receptors on the
basolateral membrane. Consequently, this influences intracellular
signaling processes involved in downstream regulation of transport
proteins in either membrane domain. In addition, epithelial transport
can also be influenced by agonists present in the luminal compartment
(25, 125, 139). This has led to the hypothesis that the
luminal membrane contains functionally important hormone receptors.
Obviously, this concept has met with some resistance, because the
source of luminal agonists in particular is often uncertain. Given the fact that many epithelia must be considered "leaky," it can be a
difficult task to investigate the hypothesis of luminal membrane receptors. It must be considered whether leakage of a "luminally" active substance may have occurred and whether the receptor site is
actually on the basolateral membrane. Nonetheless, the use of
immunocytochemistry and sided perfusion of high-resistance epithelia,
like the collecting duct, has unequivocally demonstrated the existence
of luminal membrane receptors and defined some important aspects of
their biological function (34, 125). Certainly, the most
convincing evidence comes from studies in which the same agonist leads
to differential effects when applied to either the luminal or
basolateral side of the epithelium (54, 58, 82, 125). One
prominent and well-established example of the control of epithelial
transport via a luminal agonist and its corresponding luminal receptor
is guanylin. This intestinal peptide hormone binds to the luminal
guanylate cyclase receptor to stimulate cGMP elevations and
subsequently G kinase II-mediated CFTR activation and Cl
secretion (27). The guanylate cyclase receptor is also the target for Escherichia coli heat-stable enterotoxin that
mediates severe secretory diarrhea (26). For the mammalian
nephron, a number of luminal agonists, such as PGE2
(125), vasopressin (52), or ANG II
(104), have been described. Significant attention has recently focused on luminal ANG II and luminal AT1
receptors. Remarkably high intratubular proximal ANG II concentrations
could be identified and are apparently generated by the proximal tubule itself. Luminal ANG II was shown to regulate Na+ and
HCO absorption in the proximal and distal tubule,
and thus ANG II obviously also travels along with the tubular fluid
(104).
This review focuses attention on one specific family of receptors,
namely, those activated by extracellular nucleotides like, e.g., ATP,
ADP, UTP, or UDP. These P2 or purinergic receptors have come to the
awareness of almost every researcher in biology, because they are
ubiquitously expressed and are involved in a myriad of different
cellular functions (113). Mammalian P2 receptors are
subdivided into metabotropic (G protein-coupled) P2Y (P2Y1, P2Y2, P2Y4, P2Y6,
P2Y11, P2Y12, and P2Y13) and
ionotropic P2X (P2X1-7 and P2XM) receptors (28, 113,
151, 161). One prominent feature of P2 receptors is their
extraordinarily frequent expression, especially in the luminal membrane
of epithelia. Extracellular luminal nucleotides have been shown to be
prominent regulators of ion transport. The widespread epithelial P2
receptor expression is the reason for summarizing the present state of
knowledge. This review tries to comprehensively summarize the evidence
for luminal P2 receptors in intact epithelia and their functional effects on the various transport processes. More elaborate attention will be directed toward the mammalian nephron, and finally some integrative functional implications for luminal P2 receptors will be discussed.
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THE DISCOVERY OF LUMINAL P2 RECEPTORS |
Possibly the first experimental description of a luminal P2
receptor-mediated effect was documented in 1969, when Kohn et al.
(65) investigated the transmural potential in the small intestine and showed that luminal (and basolateral) ATP stimulated a
serosal positive-voltage change. The authors concluded that this
reflected "stimulation of ion transport" but assumed an
"intracellular action of ATP." They speculated that "this might
represent a direct stimulation of the electrogenic sodium pump at the
serosal pole of the epithelial cells" but were puzzled by the fact
that they failed to demonstrate a significant depression of the
response in Mg2+-free saline, although there is an absolute
requirement for Mg2+ by the ATPase system. At that time, P2
receptors were yet to be discovered. Starting in the early 1990s, the
work of a number of groups has led to a nearly comprehensive cloning
and characterization of the large and diverse family of P2 receptors
(113). Interestingly, the characterization of the luminal
enterocyte P2 receptors in the small intestine still remains poorly
defined, except that the P2Y4 receptor seems to be one
possible candidate (15).
After the recognition of the P2 receptor family, it was the work of
Wong (159) that led to the discovery that the isolated perfused rat epididymis responds to luminal ATP with Cl
secretion, e.g., movement of NaCl and H2O into the ductal
lumen. Because no effect was detected with basolateral ATP, the authors concluded that a luminal P2 receptor is responsible for this effect. Obviously, the most important question was to establish the source of
luminal ATP in the epididymis. The authors suggested that the high ATP
concentrations present in the spermatozoa could be released, thereby
stimulating Cl secretion and fluidity of the local
environment and thus facilitating sperm transport. Evidence for this is
still pending, but present theories follow these lines and propose that
extracellular ATP in general is a local paracrine/autocrine regulator
(42). Thus the epididymis was the first intact epithelial
tissue in which luminal P2 receptors were described, leading subsequent
investigators to describe the effect of luminal ATP or other
nucleotides in nearly all epithelial tissues. Noteworthy here is an
early study by Simmons (137), who in 1981 described a
luminal ATP-stimulated effect on Cl secretion and
correctly assumed that P2 receptors were localized at "each of the
cellular membranes of this epithelium." Table 1 summarizes the epithelial organs
expressing luminal P2 receptors, the most likely P2 receptor subtype,
and the regulated ion transport process.
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LUMINAL P2 RECEPTORS IN RESPIRATORY EPITHELIUM |
Not long after the results in epididymis appeared
(159), the respiratory epithelium was discovered to be a
remarkably rich source of the expression of luminal P2 receptors
(64, 100). Activation of luminal P2 receptors in
respiratory epithelium has two distinct effects on ion transport:
1) it activates NaCl secretion (46, 53, 100)
and 2) it inhibits electrogenic Na+ absorption
(18, 53, 93). Figure 1 shows
a simplified schematic cell model of a secretory respiratory epithelial
cell. Nucleotide-mediated activation of secretion in the airways was
later also shown to encompass activation of K+ secretion
(10). Rapidly, a number of essential steps forward were
made, driven by the putative therapeutic role of inhaled luminal
nucleotides in the treatment of cystic fibrosis (CF). Luminal P2
receptor stimulation activates mucociliary clearance in three ways: by
their effects on ion transport, resulting in an increased hydration of
the respiratory surface; by stimulation of mucin secretion from goblet
cells (85); and by an increase in ciliary beat frequency
(67). A knockout study confirmed previous pharmacological
data and identified the P2Y2 receptor subtype as the
crucial luminal P2 receptor in respiratory epithelium
(15). In addition to this, but of minor importance, a
luminal P2Y6 receptor is expressed in the luminal membrane
of respiratory epithelia (75). Activation of luminal
P2Y2 (or P2Y6) receptors increases intracellular Ca2+ concentration
([Ca2+]i), and subsequently
Ca2+-activated Cl channels are stimulated,
resulting in Cl secretion. Because in CF this
"alternative Cl channel" is not defective, it may
serve to bypass the secretory defect when epithelium is stimulated with
luminal nucleotides. The important question of the source of ATP in the
luminal epithelial fluid continues to be poorly understood and will be
discussed below. Constitutive basal release of ATP has been described
as possibly generating sufficient concentration to stimulate luminal P2
receptors (73). Physical stimuli are potent stimulators of luminal ATP release in respiratory epithelia, and this release occurs
without an effect on cell viability. Thus a mechanosensitive mechanism
for ATP release could be important and may be a component of the
"cough reflex" (72). The irritant and the cough could stimulate ATP release into the luminal surface liquid, and subsequently the machinery of mucociliary clearance would be activated.
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Fig. 1.
Schematic model of luminal P2Y2
receptor-mediated ion transport regulation in respiratory epithelial
cells. Cl secretion requires
Na+-K+-Cl cotransporter isoform 1 (NKCC1)-mediated, secondarily active Cl uptake on the
basolateral side and extrusion of Cl via either luminal
CFTR or Ca2+-activated Cl channels. Luminal
ATP/UTP stimulates Ca2+-activated Cl channels
and thus Cl secretion. Electrogenic Na+
absorption occurs via luminal epithelial Na+ channels
(ENaCs). Luminal ATP/UTP inhibits Na+ absorption. This also
involves an increase in intracellular Ca2+ concentration
([Ca2+]i).
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LUMINAL P2 RECEPTORS IN GASTROINTESTINAL TRACT EPITHELIA |
In addition to the lung and the epididymis, the activation of ion
secretion is also a hallmark of luminal P2 receptor stimulation in the
gastrointestinal (GI) tract. In the GI tract, this comprises the
activation of K+ secretion in the distal colon
(58) and gallbladder (13); HCO secretion in the gallbladder (11), intrahepatic bile duct (22), and pancreatic duct
(54); and Cl secretion in biliary duct cells
(118, 129), gallbladder (13, 152), small
intestine (15), and cultured pancreatic duct cells (8, 105).
Luminal Nucleotides in GI Tract Glandular Secretion
For salivary (78), bile (22, 118), and
pancreatic (54) juice formation, the following has been
suggested and extends the two-step model (acinar production and ductal
modification) of glandular secretion by a luminal P2 receptor
component. In an initial step, ATP could be secreted by the acinar
cells [salivary gland acini, hepatocytes, or pancreatic acini
(54, 79, 91)]. Second, ATP, as it travels along the duct,
would find luminal P2 receptors and influence ion transport to modify
the specific composition of the digestive juices. In support of this
concept, recent results provide evidence for carbachol-stimulated ATP
release from rat pancreatic acini (138). Thus a
physiological stimulus for the formation of primary pancreatic juice
triggers ATP release for further intraductal pancreatic juice
modification. A stimulating effect of luminal ATP/UTP on
HCO secretion was recently shown in isolated guinea
pig pancreatic ducts (54). Interestingly, addition of
basolateral ATP/UTP inhibited HCO secretion in this
tissue. The luminal P2 receptor in guinea pig pancreatic duct appears
to be the P2Y2 subtype (54). In rat pancreatic
duct, evidence for a P2X7 and P2Y4 receptor
were recently presented (43, 91). It was also proposed
that duct cells themselves participate in luminal ATP release and thus
would regulate secretion in an autocrine or paracrine fashion
(54, 118).
In addition, the stimulation of Na+/H+
exchanger type 3-mediated Na+ absorption via a
P2X7 receptor was proposed in rat submandibular gland ducts
(78). This presently appears to be the only result indicating that luminal P2 receptors activate absorption.
Luminal P2 Receptors in Large and Small Intestine
It is noteworthy that similarly to airway epithelium, luminal
P2Y2/P2Y4 receptor stimulation also
inhibits electrogenic Na+ absorption in the mouse distal
colon (81). The distal mammalian colon is an
aldosterone-sensitive epithelium that will absorb Na+ via
epithelial Na+ channels (ENaCs) in salt-restricted states
(70). In distal colon from normal and CF animals, luminal
ATP/UTP does not activate the alternative Cl secretory
pathway discussed above, because CFTR appears to be the only apical
Cl channel in this tissue (35). Nonetheless,
the stimulation of luminal P2Y receptors in the colon (activation of
K+ secretion and inhibition of Na+ absorption)
and small intestine (activation of Cl secretion) will
result in luminal fluid accumulation. As suggested for the airway
epithelium, luminal P2 receptors may thus serve a purpose in host
defense reactions. Intraluminal intestinal bacterial overgrowth could
be one source of luminal ATP/UTP, which would result in an associated
diarrhea response.
A recent immunocytochemical localization of luminal P2X7
receptors in duodenal villus tip cells suggests yet another functional role of luminal P2 receptors. P2X7 receptors have been
associated with apoptosis in a large variety of cells
(113, 131). Epithelia in the urinary bladder or intestinal
mucosa are rapidly regenerating mammalian tissues. In the intestinal
mucosa, stem cells are located in the crypt base and, after having
moved toward the surface, undergo programmed cell death and get
exfoliated into the intestinal lumen. "Dying" cells eventually will
release ATP and may therefore provide an extracellular death signal.
P2X7 receptors thus appear strategically located to ensure
small intestinal epithelial regeneration (37).
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OTHER EPITHELIA EXPRESSING LUMINAL P2 RECEPTORS |
The ubiquitous nature of luminal P2 receptor expression in nearly
all epithelia is apparent from the long list shown in Table. 1. Another
organ with prominent luminal P2 receptor expression is the scala media
in the inner ear. The entire epithelial lining of the scala media in
the inner ear [vestibular dark cells and strial marginal cells of the
stria vascularis organ (97, 98, 123), the Reissner
membrane epithelium (60), the Henson cells (71), and the sensory outer hair cells (48)]
shows expression of different luminal P2 receptors. For a comprehensive
description, the interested reader is referred to a recent review by
Housley (48). Other epithelia with luminal P2 receptors
not mentioned before include sweat gland acinar cells
(156) and the conjunctival epithelium of the eye
(87). In both tissues, stimulation of these receptors
induce Cl secretion. An understanding of the role of P2
receptors in these and other tissues awaits further studies.
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LUMINAL P2 RECEPTORS ALONG THE NEPHRON |
An increasing number of studies have focused on P2 receptors in
renal epithelia, an issue recently reviewed comprehensively (134). The fact that renal epithelial cells may also
express luminal P2 receptors has been suggested by a number of studies using cultured cells of distal tubular origin (16, 34, 102, 135). In cultured renal epithelial cells, the functional
responses resemble those of the respiratory or other secretory
epithelia. Luminal nucleotides increase
[Ca2+]i, activate Cl secretion,
and inhibit Na+ and Ca2+ absorption (16,
34, 68, 102, 135). Most recently, the luminal expression of a
P2Y2 receptor in the intact isolated perfused cortical
collecting duct of the mouse was demonstrated (17, 80).
Pharmacological screening suggested that this receptor is the only
luminal P2 receptor subtype expressed in the distal nephron.
High-resolution confocal imaging unequivocally demonstrated that the
P2Y2 receptor was located in principal cells. It is
presently uncertain whether rat intercalated cells also express luminal P2 receptors. For rabbit cortical collecting duct, the functional expression of luminal P2Y2/P2Y4 on intercalated
cells has recently been reported (158). The expression of
luminal P2Y2 receptors appears to occur along the entire
distal nephron, down to the inner medullary collecting duct (17,
62, 68, 83). Apparently, mice, rats, and rabbits show this
luminal receptor expression. In the intact collecting duct, luminal
ATP/UTP inhibited electrogenic Na+ transport. A
transepithelial electrical signal indicative of Ca2+-activated Cl secretion, however, was not
observed in the native epithelium (80, 128). It is
therefore assumed that Ca2+-actived Cl
channels are not expressed in native cortical collecting duct principal
cells. This situation is reminiscent of a number of studies in colonic
epithelia, where CFTR is the only luminal Cl channel
mediating Cl secretion (35). However, this
is not the case in cultured colonic epithelia like T84 or HT29 cells,
where in addition to CFTR a Ca2+-activated Cl
conductance also mediates Cl secretion (2, 19,
36). It is therefore speculated that cultured distal tubular
epithelia exhibit a less differentiated state and display
Cl secretory properties normally not seen in the
well-differentiated tissue. In addition, P2Y2 receptor
stimulation in mouse cortical collecting duct principal cells also
inhibited apical ROMK channels and thus K+ secretion
(89). These experiments were performed in split-open tubules and are thus likely to involve luminal P2 receptor stimulation. Presently, it is not known whether transport of urea and
H2O is also influenced by luminal ATP/UTP. Noteworthy here
are two studies that demonstrated that basolateral P2 receptor
stimulation inhibited aquaporin-2 (AQP2)-mediated H2O
transport (61, 121). Thus it may be concluded that luminal
P2Y2 receptor stimulation inhibits at least three, if not
all, relevant transcellular transport processes for salt and water
absorption in collecting duct principal cells. Figure
2 schematically summarizes the effect of
luminal ATP/UTP as an inhibitory transport regulator in distal tubular
principal cells. Given that nearly all epithelia express luminal P2
receptors, it appears likely that other more proximal parts of the
nephron also express luminal P2 receptors. In our own preliminary
experiments using perfused mouse cortical thick ascending limb of
Henle's loop, however, we could not find a luminal ATP-induced
[Ca2+]i increase. However, this may not fully
exclude the presence of luminal P2 receptors in cortical thick
ascending limb of Henle's loop, and further studies are required.
Noteworthy is a recent study in which the so-called
P2Xcilia (possibly P2X4) receptor was shown to
be only capable of activation if extracellular Na+ was low
(92).
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Fig. 2.
Schematic model of luminal P2Y2
receptor-mediated inhibition of transport in distal tubular principal
cell. Luminal ATP/UTP inhibits Na+ and Ca2+
absorption and K+ secretion. Transduction events involved
are not clarified but do not appear to involve
[Ca2+]i. ECaC, epithelial Ca2+
channel; AQP2, aquaporin-2.
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Luminal ATP and Flow-Dependent Regulation of Tubular Transport?
An increase in tubular flow in the nephron is known to increase
K+ secretion (33, 59, 94) and Na+
absorption (127). This effect is thought to reflect in
part the increased delivery of Na+ to more distal nephron
segments and subsequent apical membrane depolarization with the
resulting increase in driving force for K+ exit via luminal
ROMK channels (31). In addition, activation of
maxi-K+ channels mediated by increased tubular flow was
recently shown (144, 157). Regarding Na+
absorption, evidence suggests that an increase in flow could lead to
direct "mechanical" activation of ENaC channels (127). In addition, one may suggest that luminal ATP contributes to
flow-dependent K+ and Na+ transport. Even
though the mechanism of ATP release continues to be obscure, we have
learned that different nonexcitable cells display constitutive ATP
release (73, 145). Assuming that this is also true
for the nephron, one could envisage that an increase in flow could wash
out luminal ATP and thus relieve a proposed tonic inhibition of luminal
ROMK and ENaC channels. In conflict with this is the flow-dependent
increase in K+ secretion via maxi-K+ channels
(157). The authors also showed that elevation of flow increased [Ca2+]i (157). It has
been shown for endothelial cells that an increase in flow can result in
an increase in ATP release (74). It may thus be
speculated that tubular flow increase triggers luminal ATP release and
subsequent activation of maxi-K+ channels. This issue has
recently been addressed from yet another angle. The functional
significance of the rather obscure central luminal cilium was
investigated in Madin-Darby canine kidney cells, and a mechanosensory
role was proposed (112). Increasing superfusion flow or
bending the central cilium directly triggered
[Ca2+]i elevations and hyperpolarized the
cell. Indirect arguments are presented that ATP release is not involved
in this effect. Thus the activation of maxi-K+ channels may
involve a mechanosensory event independently of ATP release. In
summary, K+ secretion induced by increased flow could be
composed of independent but concerted events: 1) increased
delivery of Na+ to more distal nephron segments; and
2) mechanosensitive activation of maxi-K+
channels. In addition, washout of luminal ATP could contribute to this effect.
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PREVALENCE OF LUMINAL P2Y2 RECEPTORS |
It is a prominent finding that nearly all native tissues described
above respond equally well to ATP and UTP. Inspection of the extensive
list of epithelial tissues shown in Table 1 indicates that the
P2Y2 receptor is a very prevalent luminal P2 receptor in
transporting epithelia. Importantly, however, one needs to consider
that the similar "agonist profile" of the P2Y2 and
P2Y4 receptors makes a functional discrimination between
them difficult (113). A recent P2Y2 receptor
knockout study has clarified some issues in this context. Whereas in
respiratory epithelium the P2Y2 receptor indeed appears to
be the critical player in luminal nucleotide-stimulated effects, it is
apparently of no importance for the small intestinal effects
(15). Also, in the stria vascularis of the inner ear it
was previously thought that a luminal P2U receptor mediated inhibition
of K+ secretion (98) with novel evidence
showing a luminal P2Y4 receptor in this tissue (97,
123). An inspection of Table 1 also clearly shows that the
existence of numerous other luminal P2 receptors (P2Y1,
P2Y4, P2Y6, P2X2, P2X4,
P2X5, P2X7) has been suggested. It is therefore
apparent that epithelial cells commonly (if not always) express
multiple P2 receptors (P2X and P2Y) and these may be present in the
same membrane domain (9, 22, 43, 51, 58, 82, 91, 101, 134,
146). In renal epithelium, so far the only luminal P2 receptor
was the P2Y2 subtype (see Table 1). However, not all
epithelia express luminal P2Y2 receptors. Two examples here
are Calu-3 cells (secretory cell line derived from submucosal bronchial
glands) (49) and rat submandibular gland duct cells
(79).
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SOURCE AND FATE OF LUMINAL NUCLEOTIDES |
Source
The luminal expression of P2 receptors has triggered the important
question of the source of luminal nucleotides. In the absence of any
other specific source (e.g., nerve endings), a larger number of
studies have led to the accepted proposal that the epithelial cell
itself is a source of the released nucleotide. ATP release has been
shown for a variety of epithelia and other cells, and in epithelia
release appears to occur preferentially onto the luminal side. A number
of reviews have recently summarized the present state of knowledge
(41, 118, 145). Any mechanical perturbation [touching the
cell with a glass pipette (12, 24), increasing superfusion
flow (74, 99, 124), cell swelling with hyposmolar
solutions (145, 153), or just mechanical shaking of a
culture dish (38, 42, 76)] has been shown to induce ATP
or UTP release without apparent cellular damage. The release pathway
from epithelial cells has been assumed to be either by a conductive
pore (23), a specialized membrane transporter, or
vesicular release and exocytosis (118). CFTR anion
channels and ABC transporters like MDR1 were suggested to
conduct/transport ATP (118, 133), but subsequent studies
could not confirm that CFTR functions directly as an ATP-conductive
pore (38, 86, 115, 154). Importantly, in CF epithelia ATP
release is absent (116), and CFTR has been suggested to
regulate an associated ATP channel in epithelia (57, 142).
A recent study using mutational alterations to change the substrate
specificity of the MDR1 transporter reported no effect on ATP release
(119). Thus the authors argue that MDR1 is not likely to
function as an ATP transporter. However, upregulation of MDR1 augmented
ATP release in hepatoma cells, implying a possible indirect mechanism
of MDR1 on ATP release (119). Whether ATP release can
occur via vesicular fusion and exocytosis will need further studies,
but evidence supporting this concept has been presented (99, 103,
116, 138).
Even though ATP release has been demonstrated to occur preferentially
onto the luminal side of epithelial cells, basolateral ATP release has
also been shown. This basolateral ATP release has been suggested to
occur after "luminal damage" (e.g., a kidney stone in the ureter)
and may convey information to the central nervous system via
P2X2/P2X3 receptors located on sensory nerves. Thus a basolateral release has been suggested to play a role in mechanosensory transduction (7).
The propagation of [Ca2+]i increases from
cell to cell is a widely distributed phenomenon attributed to the
spread of inositol 1,4,5-trisphosphate through gap junctions (30,
39). In addition, in epithelial and nonepithelial cells released
ATP has been discovered to act as an extracellular signal responsible
for "traveling [Ca2+]i waves" (12,
24, 130). ATP release is triggered from a site of initiation
(e.g., by touching a cell with a pipette) and diffuses to neighboring
cells. This, in turn, stimulates P2 receptors, which induce
intracellular [Ca2+]i signals and again ATP
release, resulting in a traveling [Ca2+]i or
extracellular ATP wave. In poorly gap junctional-coupled cell lines,
the expression of different connexons (Cx43, Cx32, Cx26) was recently
shown to greatly augment ATP release (12). At the same
time, it was also noticed that connexons (hemichannels) can be
functional channels and allow the permeation of Lucifer yellow when
lowering extracellular Ca2+ (44). A recent
study in astrocytes presented evidence that ATP is directly conducted
through connexon hemichannels. Dye flux was molecular weight specific,
induced by mechanical stimulation, and blocked by Gd3+ and
flufenamic acid (140). Certainly, these results demand
further rigorous experimental proof, but on the basis of these novel
results, one might speculate that connexon hemichannels could be
localized in the apical membrane of epithelia and thus provide an exit
pathway for secreted ATP. This hypothesis, however, includes a
contradiction because [Ca 2+]i waves mediated
by ATP release were described to be uninfluenced by gap junctional
blockers (12).
Fate
After their release, nucleotides will be metabolized. This
is executed by membrane surface-located ecto-nucleotidases. The field
of extracellular surface enzymes involved in metabolizing extracellular
nucleotides is presently expanding rapidly (163). Ecto-nucleotidases encompass several families with partially
overlapping substrate specificities. The most prominent family
encompasses the ecto-nucleotidase triphosphate diphosphohydrolases
(NTPDases, etc. syn: CD39 or apyrase), which hydrolyze ATP and
ADP to generate AMP (162). Subsequently,
ecto-5'-nucleotidase will generate the nucleoside and phosphate
(77, 163). Other ectonucleotidases encompass the alkaline
phosphatase and the ecto-phosphodiesterase/pyrophosphatase family. A
comprehensive review of this issue is not intended here, and the reader
is directed to pertinent reviews in the field (42, 73, 77,
162-164). However, it should be mentioned that the
metabolism of extracellular nucleotides is more complicated than
initially assumed. Nucleotides cannot only be degraded but also
upgraded (by "ecto-kinases"). The identification of extracellular
nucleoside diphosphokinase has revealed this phenomenon. In the
presence of ATP and UDP, for example, this enzyme can mediate the
formation of ADP and UTP (73). In addition, matters are
further complicated by the recognition that not only nucleotides but
also their metabolizing enzymes (NTPDase, nucleoside
diphosphokinase) can be secreted into the extracellular space as
soluble proteins (21, 163). In the context of this review,
it is important to note that luminal ecto-nucleotidases have been
identified and localized, for example, in respiratory epithelium
(21) and rat pancreatic duct (106). In the
kidney, early data from 1972 indicated that isolated tubule segments
were able to hydrolyze added ATP (120). Later proximal tubule brush-border membrane and basolateral membrane vesicles were
described to exibit ecto-ATPase activity (122). CD39 was recently shown in the renal vasculature but not in medullary nephron segments (84). More comprehensive data are
available for the localization of ecto-5'-nucleotidase in the nephron.
It is noteworthy that ecto-5'-nucleotidase belongs to the
phosphatidylinositol-anchored proteins, which are localized
specifically in the luminal membrane of epithelia (77). It
is thus expressed in the luminal membrane of proximal tubules and
distal tubular intercalated cells but apparently not in the thick
ascending limb of Henle (77). Thus the luminal side of
epithelia including the nephron contains important established
components of the "ATP-signaling machinery," i.e., P2 receptors,
released agonists, and metabolizing enzymes.
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RELEASED LUMINAL ATP AS PRECURSOR FOR ADENOSINE-REGULATED ION
TRANSPORT |
The preceding comments imply that epithelial transport is not only
regulated by the different nucleotides but also by adenosine as a
degradation product of ATP (29). Evidence for this has been presented, for example, in T84 enterocytes, where luminal ATP
exerts a Cl secretory effect via A2 adenosine
receptors and is associated with an increase in cAMP
(141). Evidence for ecto-ATPase activity on the luminal
side of the small intestine has been presented (132), and
most likely a luminal ecto-5'-nucleotidase is also present on the
luminal side of intestinal epithelia (141). A similar
phenomenon was observed in Calu-3 cells, a cell line derived from
submucosal respiratory glands. Calu-3 cells do not express luminal
P2Y2 receptors but release ATP after mechanical
stimulation. Adenosine is subsequently generated, stimulates
A2b adenosine receptors, and then a cAMP-mediated
Cl secetion occurs via CFTR. The authors also present the
interesting finding that cAMP-mediated signaling in response to
A2b receptor activation is localized to the subapical
membrane of these airway epithelial cells (49). In the
kidney, the urinary excretion of adenosine is well documented and
increases strongly during renal ischemia (3, 108).
In the mammalian nephron, no evidence to date suggests that luminal
adenosine modulates epithelial transport (77). In the
Xenopus laevis renal epithelial A6 cell line, however, luminal A1 adenosine receptors have been identified
(1, 20), and it may well be that luminal adenosine
receptors will be discovered in the intact mammalian nephron in the
near future.
| |
FUNCTIONAL IMPLICATIONS FOR LUMINAL P2 RECEPTORS IN EPITHELIA |
Given their ubiquitous expression, one is tempted to search for a
common functional purpose of luminal P2 receptors in epithelia. Present
knowledge makes this task difficult, and different organ systems are
likely to have developed luminal P2 receptors for specific purposes.
The formulation of one common function is certainly hampered by the
lack of essential pieces in this puzzle. Receptor identification is
likely to be incomplete, the molecular release mechanism and its
precise regulation are obscure, and the fate of released ATP processed
by the increasingly growing family of nucleotide-metabolizing enzymes
awaits further specification. Nonetheless, the above-mentioned findings
make it possible to extract common denominators for functional
integrative schemes. Some suggestions for an integrated
functional role of luminal P2 receptors are elaborated below. Emphasis
will be given to renal tissue.
Luminal P2 Receptors Involved in a Nonspecific Epithelial Defense
Mechanism
This concept was originally postulated for the respiratory
epithelium (72) but may extend to other epithelial
tissues. In the respiratory tract, the outer eye, and the intestinal
tract, luminal nucleotides stimulate ion secretion. These epithelia are vulnerable body surfaces, where exogenous harmful particles or bacteria
can produce extensive local damage. Each of these epithelial organs has
specific armaments to counteract potential threats on their luminal
surface. It is proposed that the epithelial defense mechanisms also
involve luminal P2 receptors. A noxious particle would mechanically
trigger nucleotide release. Luminal ATP could also originate from a
bacterial source or from dying defense cells, which have migrated into
the epithelial lumen. Subsequently, P2Y2 receptor-mediated
secretion/inhibition of absorption would be stimulated. Thus larger
amounts of luminal fluid would be generated and therefore would help to
flush away the luminal irritant. For example, large amounts of purulent
sputum in bacterial bronchitis may well be explained by the stimulation
and activation of mucociliary clearance via luminal P2Y2 receptors.
P2 Receptors and the Regulation of Cell Volume
Cell volume regulation is an important property of all cells. It
is likely to be of extraordinary importance in epithelial cells, where
rapid changes in transcellular flux of fluid and solutes occur. It is
easy to conceive that any imbalance of luminal uptake and basolateral
extrusion will compromise cell volume. This is beautifully exemplified
in macula densa cells. Reduction of apical ion influx via the
Na+-K+-Cl cotransporter isoform 2 (NKCC2) reduces cell volume and vice versa (32). A role
for extracellular ATP and P2 receptors has recently been demonstrated
in rat hepatoma (153) and bile duct epithelia
(118). In both cell types, cell swelling was shown to
release ATP, activate P2 receptors and, subsequently,
Ca2+-activated K+ and Cl
conductances. The resulting cellular KCl loss mediates regulatory volume decrease (45). In an extrapolation from these
results, luminal P2 receptors may serve to regulate cellular volume in transporting epithelia. P2 receptor-mediated activation of
K+ and Cl channels is also seen in cultured
renal epithelia (1, 5, 16). However, to date there is no
evidence for Ca2+-activated Cl channels in
intact nephron segments, and it is uncertain whether the mechanism
described applies to renal tubules. However, another mechanism to
regulate cellular volume via luminal P2 receptors can be postulated. A
common functional consequence of extracellular ATP action in the distal
tubule is the inhibition of the major transport processes. Thus
increases in transcellular transport would increase cell volume, elicit
regulated ATP release, and so induce autocrine activation of luminal
(and basolateral) P2 receptors. This would downregulate apical
substrate influx and thus provide a negative-feedback regulation of
cellular volume. This hypothesis requires rigorous testing. One
beautiful example that illustrates the issue of volume regulation, ATP
release, and regulated cell function deserves mentioning here. Using
A1 receptor knockout mice, it has recently been shown that
adenosine is the extracellular mediator of tubuloglomerular feedback
(TGF) (6, 143). In TGF, the macula densa senses the distal
tubular sodium load via the NKCC2 cotransporter. This probably occurs via a change in cellular volume because macula densa cells strongly change their volume in response to luminal electrolyte uptake (32). Cell volume increases induced by increasing luminal
NaCl concentration have recently been shown to stimulate ATP release into the basolateral interstitial space (4). Released ATP
is suggested to be broken down, and the adenosine formed by the
5'-nucleotidase will subsequently constrict preglomerular arterioles
(148). This feedback is disrupted by knockout of
A1 receptors. These results highlight the importance of
cell swelling-induced release of ATP in TGF regulation.
ATP and Ischemic Protection in the Kidney
In the intact distal nephron, a consistent finding has been that
all major transporting activity is downregulated when luminal and/or
basolateral P2Y receptors are activated (61, 68, 80, 89).
For more proximal tubular segments, this has not been studied in any
detail, and it might be a peculiar property of the distal nephron. In
the distal tubule, at least, it is proposed that extracellular (luminal) ATP acts as a luminal signaling molecule and serves to
protect the tubular epithelium under ischemic conditions (Fig. 3). The sequence of events could be as
follows. In ischemia, epithelial cells will suffer energy
depletion and therefore swell. ATP is subsequently released from
epithelial cells by cell swelling (69, 73, 145). This, in
turn, will trigger regulated ATP release and autocrine or paracrine P2
receptor stimulation. The released ATP could enter the tubular fluid
and move along the nephron to autoinhibit energy-consuming transport
processes in more distal tubular segments and so protect them.
Basolateral P2 receptors could mediate a similar process. Obviously,
nucleotides could also originate from the vascular space and enter the
nephron via glomerular filtration.
View larger version (28K):
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|
Fig. 3.
Schematic model of "ischemic protection
hypothesis." Proximal endotubular ATP is proposed to lead to distal
tubular transport inhibition and thus epithelial protection.
|
|
In renal ischemia, increased amounts of adenosine are released
into the urine (108), but it is likely that ATP is the
primary metabolite released from ischemic cells and that it is
then converted to adenosine (see above). In addition, extracellular ATP
acting via P2 receptors has been shown to stimulate cell growth and
division in a number of renal (40, 50, 55, 109) and
nonrenal tissues (47). An intriguing preliminary study
suggests that shortly after renal ischemia, ion transport
processes are downregulated, whereas expression of the P2Y2
receptor itself is upregulated (63). It is therefore
suggested that ATP acts as an "intelligent" extracellular signaling
molecule that can prevent ischemic damage almost before it
occurs but which can also assist in cell recovery and regeneration
after damage has happened.
It has been indicated above that the focus on P2 receptors and ATP
implies a rather crude simplification of matters, because released ATP
will provide the source for the formation of adenosine with numerous
effects mediated via adenosine receptors. Recently, A2a
adenosine receptors were shown to play an important role as a
physiological feedback mechanism for the limitation and termination of
both tissue-specific and systemic inflammatory responses (29, 107). This is worth mentioning in this context because P1 and P2
receptors may serve a common purpose in a concerted effort to limit
tissue damage by noxious events of different origin.
Luminal P2 Receptors in Polycystic Kidney Disease
Luminal P2 receptors have been suggested to play an important role
in the course of polycystic kidney disease (PKD) (135, 155). PKD is associated with the formation of cysts derived from renal tubular epithelia. It is the expansion of cysts that determines the progression of disease and development of renal failure. Expansion of cysts is driven by two processes, cellular proliferation and fluid
secretion, leading to progressive cyst enlargement. Although cysts can
arise from any portion of the renal tubule, evidence from models of
autosomal dominant PKD in both mice (90) and rats
(14) suggests that cysts originate mainly from proximal tubular cells; the same also seems to be true in the recessive form of this disease (96). It has been suggested that a
variety of different locally released factors acting as autocrine or
paracrine regulators stimulate renal cyst growth and expansion
(117). Intriguingly, primary cultured PKD cyst epithelial
cells were recently shown to express luminal P2Y and P2X receptors and
secrete ATP onto the luminal side (i.e., into the cyst lumen)
(135). In PKD, epithelial luminal ATP was also shown to
stimulate Cl secretion (135). Furthermore,
it was shown that cyst fluid contains significant concentrations of ATP
(155). Thus ATP could be one of the local factors involved
in progression of PKD cysts.
| |
ACKNOWLEDGEMENTS |
I appreciate comments on improving this manuscript from Dr. Ivana
Novak, August-Krogh-Institute, Copenhagen, Denmark.
| |
FOOTNOTES |
Address for reprint requests and other correspondence: J. Leipziger, Dept. of Physiology, The Water and Salt Research Center, Aarhus Univ., 8000 Aarhus C, Denmark (E-mail:
leip{at}fi.au.dk).
10.1152/ajprenal.00075.2002
| |
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ABSTRACT
INTRODUCTION
