Departments of 1 Pediatrics and 3 Medicine, Mount Sinai School of Medicine, New York, New York 10029-6574; and 2 Department of Medicine, Universidade Federal do Rio de Janeiro, Hospital Universitario Clementino Fraga Filho, 21949-900 Rio de Janeiro, Brazil
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ABSTRACT |
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Nucleotide binding to purinergic P2
receptors contributes to the regulation of a variety of physiological
functions in renal epithelial cells. Whereas P2 receptors have been
functionally identified at the basolateral membrane of the cortical
collecting duct (CCD), a final regulatory site of urinary
Na+, K+, and acid-base excretion, controversy
exists as to whether apical purinoceptors exist in this segment. Nor
has the distribution of receptor subtypes present on the unique cell
populations that constitute Ca2+ the CCD been established.
To examine this, we measured nucleotide-induced changes in
intracellular Ca2+ concentration
([Ca2+]i) in fura 2-loaded rabbit CCDs
microperfused in vitro. Resting [Ca2+]i did
not differ between principal and intercalated cells, averaging ~120
nM. An acute increase in tubular fluid flow rate, associated with a
20% increase in tubular diameter, led to increases in
[Ca2+]i in both cell types. Luminal perfusion
of 100 µM UTP or ATP--S, in the absence of change in flow rate,
caused a rapid and transient approximately fourfold increase in
[Ca2+]i in both cell types (P < 0.05). Luminal suramin, a nonspecific P2 receptor antagonist,
blocked the nucleotide- but not flow-induced [Ca2+]i transients. Luminal perfusion with a
P2X (
,
-methylene-ATP), P2X7 (benzoyl-benzoyl-ATP),
P2Y1 (2-methylthio-ATP), or
P2Y4/P2Y6 (UDP) receptor agonist had no effect
on [Ca2+]i. The nucleotide-induced
[Ca2+]i transients were inhibited by the
inositol-1,4,5-triphosphate receptor blocker 2-aminoethoxydiphenyl
borate, thapsigargin, which depletes internal Ca2+ stores,
luminal perfusion with a Ca2+-free perfusate, or the L-type
Ca2+ channel blocker nifedipine. These results suggest that
luminal nucleotides activate apical P2Y2 receptors in the
CCD via pathways that require both internal Ca2+
mobilization and extracellular Ca2+ entry. The flow-induced
rise in [Ca2+]i is apparently not mediated by
apical P2 purinergic receptor signaling.
microperfusion; fura 2; purinergic receptor; principal cell; intercalated cell; intracellular calcium concentration
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INTRODUCTION |
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CUMULATIVE EVIDENCE INDICATES that ion transport across epithelia is regulated by binding of extracellular nucleotides to purinergic receptors present on cell surfaces. Two main groups of purinergic receptors have been identified based on their pharmacological properties. P2 purinergic receptors are activated by ATP, ADP, UTP, and UDP, whereas P1 purinoceptors respond preferentially to AMP and adenosine, the breakdown products of ATP hydrolysis (14). Within the P2 purinoceptor family, P2X receptors are intrinsic ion channels that mediate depolarization and influx of Ca2+, whereas P2Y receptors are coupled to heterotrimeric G proteins, phospholipases, and phosphoinositol signaling pathways (14, 46, 57). Specific subtypes of P2X or P2Y receptors can be identified based on their response to specific nucleotide agonists (14, 46, 57).
The P2Y receptor family is comprised of at least five distinct molecular subtypes (P2Y1,2,4,6,11; reviewed in Ref. 57). Binding of ATP to the G protein-coupled P2Y receptor activates phospholipase C (PLC), leading to inositol-1,4,5-triphosphate (IP3) production and mobilization of internal Ca2+ stores (14, 46, 57). P2Y receptor activation has also been shown to stimulate production of diacylglycerol and protein kinase C, modulate adenylate cyclase activity and cAMP production, and stimulate the formation and release of prostaglandins (1, 14, 30, 46, 57, 68). P2X receptors, comprising at least seven subtypes (P2X1-7) (46, 57), form Ca2+-permeable nonselective cation channels that, on activation, allow for Ca2+ entry from the extracellular milieu into the cell (6, 60).
The cortical collecting duct (CCD) of the mammalian nephron contributes to the final renal regulation of Na+, K+, acid-base, and water homeostasis. The CCD is a heterogeneous epithelium comprising two morphologically and functionally distinct cell types. Whereas principal cells reabsorb Na+ and water (in the presence of vasopressin) and secrete K+, intercalated cells transport acid-base and can, under certain conditions, absorb K+ (10, 29, 52, 56, 59). Although these cells reside directly adjacent to each other within the CCD, they are considered not to be coupled, maintaining different resting intracellular pH (54).
Functional studies of CCD cells grown in culture (30, 62), isolated tubules (9, 34), and established cell lines expressing properties typical of CCD principal cells (36, 41) provide evidence for the presence of P2Y receptors in this segment. However, few studies have been directed at delineating the polarity of these receptors to the apical or basolateral membranes. A basolateral localization of P2Y2 (previously known as P2U) receptors has been supported by the observations that peritubular ATP and UTP cause a rapid increase in intracellular Ca2+ concentration ([Ca2+]i) in nonperfused rat CCDs (9) and inhibit the hydrosmotic action of vasopressin in microperfused rabbit CCDs (49). Although apical and basolateral P2Y2 receptors have been identified in primary cultures of rabbit CCD cells (30), Deetjen et al. (12) found no functional apical P2 receptors in microperfused rabbit CCDs. Recently, Kishore et al. (28) detected P2Y2 receptor mRNA in microdissected rat CCDs. Although immunoreactive P2Y2 receptor was identified along the apical, and to a lesser extent basolateral, membranes of collecting duct principal cells in the inner medulla, the localization of the protein in the CCD was not explored (28).
The purpose of the present study was to 1) determine whether functional P2 receptors are present on the apical surfaces of rabbit CCD cells and, if so, 2) identify the distribution of receptor subtypes present on the unique cell populations that constitute the CCD. On the basis of our results, we also sought to identify the source of Ca2+ giving rise to the nucleotide-induced [Ca2+]i transients. To accomplish these aims, we used the Ca2+-sensitive fluorescent dye fura 2 to measure changes in principal and intercalated cell [Ca2+]i in isolated CCDs microperfused with nucleotide analogs. As an incidental finding, we noted that epithelial stretch induced by rapid increases in tubular fluid flow rate led to [Ca2+]i transients in the CCD. The role of apical P2 purinergic signaling in this response was investigated.
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MATERIALS AND METHODS |
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Animals. Adult female New Zealand White rabbits were obtained from Covance (Denver, PA) and housed in the Mount Sinai School of Medicine animal care facility. The animals were fed standard rabbit chow and given free access to food and water. Animals were killed by intraperitoneal injection of a lethal dose of pentobarbital sodium (100 mg/kg). All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (Washington, DC: National Academy Press, 1996).
In vitro microperfusion. The kidneys were removed via a midline incision, sliced into 2-mm coronal sections, and single tubules were dissected freehand in cold (4°C) dissection solution containing (in mM) 145 NaCl, 2.5 K2HPO4, 2.0 CaCl2, 1.2 MgSO4, 4.0 Na lactate, 1.0 Na3 citrate, 6.0 L-alanine, and 5.5 D-glucose, pH 7.4, 290 ± 2 mosmol/kgH2O (51). A single tubule was studied from each animal.
Each isolated tubule was immediately transferred to a temperature and O2/CO2-controlled specimen chamber, assembled with a No. 1 coverslip (Corning) painted with a 1-µl drop of Cell-Tak (Collaborative Biomedical Products, Bedford, MA). The CCD was mounted on concentric glass pipettes, cannulated, and then positioned directly on the Cell-Tak to immobilize the segment for the duration of the experiment. We previously showed that Cell-Tak does not alter the accessibility of the CCD basolateral membrane to the extracellular medium (10, 58). Tubules were perfused and bathed at 37°C with Burg's solution, which resembled the dissection solution except that 25 mM NaCl was replaced by NaHCO3 and the solution was gassed with 95% O2-5% CO2 at room temperature to reach a pH of 7.4 (51). In some experiments, CCDs were perfused with Burg's solution prepared without Ca2+ (Ca2+-free perfusate). During the 60-min equilibration period and thereafter, the perfusion chamber was continuously suffused with a gas mixture of 95% O2-5% CO2 to maintain pH at 7.4 at 37°C. The bathing solution was continuously exchanged at a rate of 10 ml/h using a syringe pump (Razel, Stamford, CT).Measurement of
[Ca2+]i.
After equilibration, tubules were loaded with 20 µM of the
acetoxymethyl ester of fura 2 (Molecular Probes, Eugene, OR) added to
the bath for 20 min. In several experiments, rhodamine-labeled peanut
lectin (PNA; Vector Labs, Burlingame, CA) was added to the luminal
perfusate for 5 min to identify intercalated cells; rabbit principal
cells do not bind PNA (53). With the use of a Nikon
Eclipse TE300 inverted epifluorescence microscope linked to a cooled
Pentamax charge-coupled device camera (Princeton Instruments) interfaced with a digital imaging system (MetaFluor, Universal Imaging,
Westchester, PA), [Ca2+]i was measured in
individually identified fura 2-loaded cells visualized using a Nikon S
Fluor ×40 objective (numeric aperture 0.9, working
distance 0.3). Autofluorescence was not detectable at the
camera gains used. Cells were alternately excited at 340 and 380 nm,
and the images were digitized for subsequent analysis. Images were
acquired every 2 to 10 s. An intracellular calibration was
performed at the conclusion of each experiment according to the
technique of Grynkiewicz (24). The 340/380-nm fluorescence ratio was determined initially in the presence of a
Ca2+-free bath plus 10 µM EGTA-AM (Rmin) and
then in a 2 mM Ca2+ bath containing ionomycin (10 µM;
Rmax). The equation used to calculate experimental values
of [Ca2+]i was
[Kd(R Rmin)/(Rmax
R)](Sf2/Sb2), where R is the observed ratio of
emitted light, Kd is the dissociation constant
for fura 2 and Ca2+, assumed to be 224 nM, and
Sf2 and Sb2 are the fluorescence signals of
free and bound dye at 380 nm, respectively (24). Two to
six cells were analyzed in each CCD.
Pharmacological classification of P2 receptor subclass.
To identify the specific classes of purinergic receptors expressed on
the apical membrane of the CCD, fura 2-loaded cells in CCDs perfused at
flow rates of ~1-2
nl · min1 · mm
1 were
monitored for changes in [Ca2+]i induced by
luminal perfusion of the following selective agonists (100 µM): UTP,
UDP, ATP and its nonhydrolyzable analog adenosine 5'-O-(3-thiotriphosphate) (ATP-
-S), 2-methylthio-ATP,
,
-methylene ATP, and benzoyl-benzoyl-ATP (BzBz-ATP). These
ligands are considered to be the highest affinity agonists for their
respective receptor subtypes (14, 46, 57). A rank order
potency of ATP = UTP > ATP-
-S > ADP is consistent
with the pharmacology of the P2Y2 receptor subtype
(14, 46).
,
-Methylene-ATP and BzBz-ATP bind to P2X
and P2X7 receptors, whereas 2-methylthio-ATP and UDP are
selective for P2Y1 and P2Y4/P2Y6
receptors, respectively (14, 46, 57). Care was taken not
to acutely increase tubular fluid flow rate and luminal diameter during
exchanges of the luminal perfusate in these experiments (e.g., on
addition of luminal nucleotides). In some studies, CCDs were pretreated
with luminal suramin (100 µM), a nonspecific P2 receptor antagonist
(except for P2Y4) (18, 57), to confirm that
the nucleotide-induced [Ca2+]i transients
were mediated by this class of purinergic receptors.
Effect of ATP/UTP on gap junctional intercellular communication. Isolated CCDs were split open to expose the apical surfaces of all cells and placed on a coverslip to which Cell-Tak had been previously applied. Microinjection pipettes (Sterile Femtotips II, Eppendorf, Hamburg, Germany) with a tip diameter of 2 µm were filled with Lucifer yellow (20% prepared in normal saline). Randomly identified individual cells were injected with this fluorescent compound using a microinjection system (Micromanipulator 5171, Eppendorf). After injection, the tubule was washed with phosphate-buffered saline. Baseline fluorescence images of the microinjected CCDs were obtained at 5-min intervals for 15 min at room temperature. Thereafter, 100 µM UTP or ATP was added to bathing solution and fluorescence was again monitored for 30 min.
Statistical analysis. Results are expressed as means ± SE; n equals the number of animals, unless otherwise indicated. Significant differences were determined by paired or unpaired t-tests, as appropriate, using the software program SigmaStat (SPSS). Significance was asserted if P < 0.05.
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RESULTS |
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Steady-state [Ca2+]i
and response to an increase in flow rate.
Intercalated cells, which were differentiated from principal cells in
the rabbit CCD by their selective apical binding of rhodamine PNA (Fig.
1), appear more brightly fluorescent
under epifluorescence illumination compared with principal cells.
Steady-state [Ca2+]i did not differ between
principal and intercalated cells [110 ± 14 vs. 130 ± 17 nM, respectively; P = not significant (NS); n = 28].
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Response of [Ca2+]i to
luminal UTP, ATP, or ATP--S.
Luminal perfusion of 5 of 10 CCDs with 100 µM ATP induced a rapid and
transient increase followed by a slow return of
[Ca2+]i toward basal values over the next 5 min in intercalated (Fig. 4A)
and principal (data not shown) cells. Similar increases in [Ca2+]i were observed in principal (Fig.
4B) and intercalated cells (data not shown) in all eight
CCDs perfused with 100 µM UTP. In the CCDs that responded to luminal
ATP and all segments perfused with UTP, the peak
[Ca2+]i averaged approximately fourfold above
baseline in both cell types (Fig. 5). We
consider that the inconsistent response of CCDs to luminal ATP reflects
the presence of ecto-5'-nucleotidase, an enzyme that catalyzes the
breakdown of ATP into adenosine, along the apical cell membrane of
intercalated cells in the CCD (32). Luminal perfusion with
the poorly hydrolyzable ATP analog ATP-
-S (n = 3)
elicited a biphasic increase in [Ca2+]i
similar to that observed in response to UTP. The ATP-
-S-induced increases in principal and intercalated cell
[Ca2+]i over resting levels were not
statistically different from those observed in CCDs that responded to
ATP and all segments perfused with UTP (Fig. 5; P = NS). A scatterplot showing the increases in individual principal and
intercalated cell [Ca2+]i elicited by luminal
perfusion with UTP or ATP/ATP-
-S is shown in Fig.
6. Pretreatment of CCDs with luminal
suramin blocked the [Ca2+]i transient induced
by luminal perfusion with ATP (n = 3) or UTP
(n = 3) (Fig. 5).
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Effect of ,
-methylene-ATP, benzoyl-benzoyl-ATP,
2-methylthio-ATP, or UDP on
[Ca2+]i.
To determine whether P2X, P2X7, P2Y1, or
P2Y6 receptors exist on the apical membrane of principal or
intercalated cells, the response of individually identified cells to
luminal exposure to
,
-methylene-ATP, BzBz-ATP, 2-methylthio-ATP,
or UDP was examined. No significant changes in
[Ca2+]i were seen after luminal perfusion
with 100 µM
,
-methylene-ATP (n = 3), BzBz-ATP
(n = 4), 2-methylthio-ATP (n = 3), or
UDP (n = 4) in either cell type. Representative
tracings of these experiments are shown in Fig. 4,
C-F.
Contribution of extracellular and intracellular Ca2+ to nucleotide-induced [Ca2+]i transients. An increase in [Ca2+]i could be due to Ca2+ release from intracellular stores and/or external Ca2+ entry. To evaluate whether the nucleotide-induced increase in [Ca2+]i was due to Ca2+ release from internal stores, the effect of luminal UTP on [Ca2+]i was examined in CCDs pretreated with either thapsigargin, which depletes internal stores, or 2-APB, an IP3 receptor antagonist. These studies were performed with UTP because luminal perfusion of this nucleotide consistently led to [Ca2+]i transients in the CCD.
Pretreatment of CCDs (n = 5) with thapsigargin (100 nM) added to the bathing medium for 30 min led to significant (P < 0.05) increases in resting [Ca2+]i from a baseline of 89 ± 7 to 230 ± 43 nM in principal cells and 124 ± 33 to 284 ± 31 nM in intercalated cells within 3-5 min, consistent with release of internal Ca2+ stores. Luminal perfusion of these thapsigargin-treated CCDs with UTP failed to induce an increase in [Ca2+]i in either principal (to peak of 260 ± 51 nM; P = NS compared with pre-UTP values) or intercalated (to peak of 253 ± 32 nM; P = NS compared with pre-UTP values) cells. Figure 7A shows a representative tracing of the effect of UTP on [Ca2+]i in a thapsigargin-treated CCD. These data suggest that release of intracellular Ca2+ stores contributes to the purinergic response.
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Effect of ATP/UTP on gap junctional intercellular communication.
The similar [Ca2+]i transients observed in
principal and intercalated cells in response to luminal nucleotides
suggests that either purinergic receptors are present on both cell
types or receptor signaling leads to intercellular spread of
Ca2+ (or other signaling molecules) between individual
collecting duct cells. To determine whether principal and intercalated
cells are directly coupled and/or gap junctions are opened in response to nucleotide binding, individual CCD cells in split-open CCDs were
microinjected with the cell-impermeant fluorescent dye Lucifer yellow.
Under control conditions (Figs. 8, A and
B), Lucifer yellow fluorescence was restricted to the cells in which the tracer was microinjected. Addition of UTP had no apparent effect on intercellular spread in cells monitored for up to 30 min (Fig. 8C;
n = 2 CCDs).
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DISCUSSION |
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Our observations that luminal perfusion of CCDs with ATP, UTP, or
ATP--S leads to a suramin-sensitive transient increase in
[Ca2+]i in both principal and intercalated
cells in the mammalian CCD (Figs. 4-6) suggests that functional
P2Y2 receptors are localized to the apical membranes of
both cell types. This is in accordance with the detection of functional
P2Y2 receptors on the apical membrane of MDCK cells
(17, 69), A6 distal cells (2, 36, 37),
primary cultures of rabbit CCD cells (30), and
immortalized rabbit distal convoluted tubular cells (4,
50). Although P2Y4 receptors also have a high
affinity for ATP and UTP, the lack of response to UDP and sensitivity
of the [Ca2+]i response to suramin suggests
that functional P2Y4 receptors are not present along the
apical membrane of the CCD (5, 64). The absence of an
effect of luminal
,
-methylene-ATP (Fig. 4C), BzBz-ATP
(Fig. 4D), 2-methylthio-ATP (Fig. 4E), and UDP
(Fig. 4F) on [Ca2+]i further
suggests that functional P2X, P2X7, P2Y1, and
P2Y6 receptors, respectively, are absent on the apical
membrane of both cell types.
In contrast to our findings, Deetjen et al. (12) failed to demonstrate apical ATP/UTP-induced effects on [Ca2+]i in isolated perfused rabbit CCDs. They did, however, detect functional P2Y2 receptors in the mouse CCD in the same study. We consider the discrepant results to reflect species and/or methodological differences between the microperfusion assays used by us and those reported by Deetjen. Specifically, the present studies were performed in CCDs isolated from New Zealand White rabbits and equilibrated in 95% O2-5% CO2 at 37°C for 60 min, a treatment period that our laboratory (55, 66) and others (7) have shown necessary for isolated microperfused segments to attain a stable transepithelial voltage and rate of transport. In addition, we found that the response to luminal nucleotides was dependent on flow rate. Specifically, neither principal or intercalated cells responded to luminal nucleotides with an increase in [Ca2+]i when the CCDs were initially perfused at fast flow rates. Whether the rabbit tubules studied by Deetjen et al. (12) were perfused at comparable flow rates to those used in the present study is uncertain.
The concomitant nucleotide-induced rise in [Ca2+]i in both principal and intercalated cells can be interpreted to reflect either the presence of apical receptors on both cell types or a nucleotide-induced increase in cell-cell coupling. Whereas principal and intercalated cells have been considered not to be functionally coupled (31, 54), recent studies report the presence of transcripts encoding the gap junctional protein connexin-42 and immunodetectable protein in the CCD (3, 25). These results suggest that, under certain conditions, cell-cell coupling may be activated. The absence of apparent dye coupling between cells in split-open CCDs (Fig. 9) suggests that coupling is absent under baseline conditions. Nor do gap junctions appear to be opened in response to nucleotide exposure.
Of particular note and interest was our incidental finding that an acute increase in tubular fluid flow rate (Figs. 2 and 3) led to a transient increase in [Ca2+]i, a response apparently not mediated by apical P2 purinergic receptor signaling. Praetorius and Spring (42) recently reported that the primary apical cilium in Madin-Darby canine kidney (MDCK) cells is mechanically sensitive, responding to flow with an increase in [Ca2+]i. The Ca2+ signal then spreads to adjacent MDCK cells by diffusion of a second messenger through gap junctions (42). Whereas mechanical perturbation of the apical cilium (15) in the microperfused CCD could account for our detection of a flow-stimulated increase in principal cell [Ca2+]i, the mechanism underlying the flow-induced [Ca2+]i transient in intercalated cells, which are devoid of an apical cilium and appear not to be coupled to adjacent principal cells, remains to be explained.
Mechanical stress results in release of ATP and UTP in polarized airway epithelia across both apical and basolateral membranes (26). Nucleotide release across the apical membrane is proposed to coordinate airway mucociliary clearance responses, including water secretion and ciliary beat frequency, whereas basolateral release represents a paracrine mechanism by which mechanical stresses signal adjacent epithelial cells (26). To the extent that mechanical stress induced by tubular fluid flow results in bidirectional nucleotide release, the absence of effect of luminal suramin on the flow-induced [Ca2+]i transient is compatible with the possibility that nucleotides released at the basolateral membrane bind to and activate basolateral P2 purinergic receptors. Although luminal activation of apical P2X receptors could also account for the flow-induced response, our data suggest that this class of receptors is nonfunctional, if present, on the apical membrane. Also possible, but not explored in the present study, is that P1 purinergic receptor signaling contributes to the flow-induced responses.
We believe that the response to luminal perfusion with nucleotides reflects activation of luminal P2Y2 receptors and is distinct from the response to increasing flow/tubule stretch because of the following observations. First, perfusion with suramin inhibited the [Ca2+]i response to luminal nucleotides but not tubule stretch. Second, repetitive increases in tubular flow rate led to multiple transient increases in [Ca2+]i, whereas tubules were insensitive to more than one sequential challenge with luminal nucleotide (data not shown).
Our data suggests that the apical purinergic receptor-induced [Ca2+]i transient comprises at least two interdependent components: a rapid mobilization of Ca2+ from IP3-sensitive stores and luminal Ca2+ entry through nifedipine-sensitive Ca2+ channels. The inhibition of the nucleotide-induced response by thapsigargin (Fig. 7A) and 2-APB (Fig. 7B) is consistent with stimulation of the IP3 receptor and internal Ca2+ mobilization. The absence of a response in cells subjected to prolonged exposure to Ca2+-free luminal perfusate (Fig. 7C), which presumably depletes internal stores, provides additional evidence for the participation of this pathway in apical purinergic signaling. Whether the nifedipine-sensitive extracellular Ca2+ influx step is mediated by a Ca2+-release-activated Ca2+ channel, an L-type Ca2+ channel or Ca2+-permeable cation channels (8, 16) remains to be established. The inhibition of this Ca2+ entry step by 2-APB alone (Fig. 7B) is compatible with nucleotide-induced Ca2+ entry via store-operated channels (SOCs), activated by a reduction in Ca2+ in the endoplasmic reticulum lumen (35, 38, 45) and suggests that the IP3 receptor is essential for maintaining coupling between store depletion and activation of SOCs. Ma et al. (35) recently suggested that store emptying promotes a reversible docking of the endoplasmic reticulum with the plasma membrane to activate SOCs (40, 44).
The physiological relevance of apical P2 receptors in the CCD depends on delivery of the appropriate ligand to the extracellular space and the local activity of ecto-nucleotidases, which catalyze the catabolism of ATP. Collecting duct cells may constitutively release ATP into the extracellular space in concentrations sufficient to activate P2 receptors (13, 23, 27, 33, 39, 43, 47, 48, 57, 61, 63, 65). The exclusive localization of ecto-5'-nucleotidase at the luminal membrane of intercalated but not principal cells (21) suggests that ATP, if released locally into the lumen of the CCD, may participate in P1 receptor signal transduction.
An increasing number of functional studies demonstrate that
activation of apical purinergic receptors in the kidney leads to
significant alterations in ion and solute transport. Relevant to the
CCD are the observations that apical (or basolateral) ATP inhibits
electrogenic amiloride-sensitive Na+ absorption in rabbit
(30) and M-1 mouse (11) CCD cell lines. Apical (or basolateral) ATP stimulates Cl secretion in
M-1 mouse CCD cells (11) and MDCK cells (67). Furthermore, it has been shown that nucleotides activate the inwardly rectifying ~70-pS K channel in MDCK cells (19, 20, 41)
and ~32-pS K channel in A6 cells (37), but inhibit the
apical small conductance K channels in mouse CCD (34).
Finally, basolateral nucleotides inhibit vasopressin-induced water
transport in perfused rabbit CCDs (49). Whereas our
identification of functional P2Y receptors on principal cells is
compatible with their proposed role in regulating Na+,
K+, and Cl
transport, the detection of these
purinergic receptors on the apical surfaces of intercalated cells
raises the possibility that H+/HCO
In conclusion, functional P2Y2 receptors are present on the apical surfaces of both principal and intercalated cells in the microperfused rabbit CCD. The [Ca2+]i transients observed in these cells in response to luminal nucleotides is apparently not due to intercellular coupling. In addition, the [Ca2+]i transients induced by acute increases in luminal flow rate/epithelial stretch is not mediated by apical suramin-sensitive purinergic activation. The localization of functional P2 receptors to the apical membrane of both principal and intercalated cells suggests that luminal ATP and/or UTP may play a significant regulatory role in electrolyte, acid-base, and water transport in the CCD.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK; DK-38470 to L. M. Satlin) and an American Heart Association Grant-in-Aid (to L. M. Satlin). C. B. Woda was supported by NIDDK Grant T32-HD-07537 (L. M. Satlin, PI).
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FOOTNOTES |
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Abstracts of this work were presented at the Annual Meeting of the American Society of Nephrology in 2000, Toronto, Ontario, Canada.
Address for reprint requests and other correspondence: L. M. Satlin, Box 1664, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029-6574 (E-mail: lisa.satlin{at}mssm.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 14, 2002;10.1152/ajprenal.00316.2001
Received 12 October 2001; accepted in final form 12 March 2002.
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