Departments of 1 Internal Medicine and 2 Molecular Genetics, University of Cincinnati School of Medicine, Cincinnati, Ohio 45267; 3 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892; and 4 Department of Cell Biology, Institute of Anatomy, University of Aarhus, Aarhus, DK-8000 Denmark
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ABSTRACT |
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Physiological and pharmacological studies have demonstrated that extracellular ATP, acting through P2Y2 purinoceptor, modulates water permeability of renal medullary collecting duct cells and the secretion of ions, mucin, and surfactant phospholipids by respiratory epithelia. Here we provide direct molecular evidence for the expression of P2Y2 purinoceptor in these cells. RT-PCR confirmed P2Y2 purinoceptor mRNA expression in rat lung and kidney and demonstrated expression in renal collecting ducts. Northern analysis showed that both lung and kidney express one 3.6-kb P2Y2 purinoceptor mRNA transcript. Immunoblots using peptide-derived polyclonal antibody to P2Y2 purinoceptor showed that inner medullary collecting ducts (IMCD) express two distinct and specific products (47 and 105 kDa) and account for the majority of the receptor expression in inner medulla, whereas the 105-kDa form is predominant in lung. Immunoperoxidase labeling on cryosections showed localization of receptor protein in the apical and basolateral domains of IMCD principal cells and in the secretory cells (Clara cells and goblet cells) of the terminal respiratory bronchioles.
collecting duct; water transport; Clara cells; goblet cells; type II pneumocytes
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INTRODUCTION |
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P2 PURINERGIC RECEPTORS are distributed in a
variety of cells and tissues, where they are involved in vital
functions, such as platelet aggregation, maintenance of vascular tone,
neurotransmission, cardiac and smooth muscle contraction and epithelial
chloride secretion (7). The P2Y2 purinoceptor (previously
known as P2u or P2n) is a G protein-coupled nucleotide receptor, the
agonist (ATP or UTP) occupancy of which activates a membrane-bound
phosphoinositide-specific phospholipase C, resulting in enhanced
production of inositol triphosphate and the subsequent release of
intracellular calcium. The potency order of the agonists for
P2Y2 purinoceptor typically is UTP = ATP > adenosine-5'-O-(3-thiotriphosphate) (ATPS) > 2-(methylthio)-ATP (2-MeS-ATP) >
,
-methylene-ATP
(
,
-MeATP) (23). Receptors that conform to this order of potency
have been cloned from several tissues or cells from mouse, rat, and
human (3, 19, 25, 26, 29).
Our knowledge of the role of P2 purinoceptors in general and
P2Y2 subtype in particular, in renal function is very
limited compared with that of other systems such as cardiovascular,
nervous, muscular, etc. Studies have implicated extracellular ATP in
the regulation of renal microvascular function, tubuloglomerular
feedback response, and tubular transport (9, 11, 21). Previously, we
demonstrated by physiological techniques the existence of a nucleotide
receptor in the terminal inner medullary collecting duct (IMCD) of rat,
which triggers a rapid rise in intracellular calcium when occupied by
ATP or UTP but not by ADP or 2-MeS-ATP or ,
-MeATP, suggesting a
classic P2Y2 purinoceptor (8). More recently, using a
similar approach, Endou and co-workers (2) presented evidence for the
existence of both P2Y1 and P2Y2 purinoceptors in rat nephron and collecting duct segments (2). Since an
agonist-stimulated rise in intracellular calcium in the terminal IMCD
is known to be associated with a decrease in the osmotic water
permeability (Pf), we examined the effect of ATP
and related nucleotides on the arginine vasopressin (AVP)-stimulated
Pf of in vitro microperfused rat terminal IMCD. We
also determined whether the effects of ATP are mediated through changes
in intracellular cAMP levels. Our studies demonstrated that 1)
agonist occupancy of the P2Y2 purinoceptor in the terminal
IMCD causes an inhibition of AVP-stimulated Pf; and
2) this effect is due to a decrease in cellular cAMP levels, most likely resulting from the activation of protein kinase C (13).
Similar effects of extracellular ATP on AVP-induced
Pf were observed in rabbit cortical collecting
tubules by other investigators (28). Thus extracellular ATP, acting
through the P2Y2 purinoceptor, may play an important role
in the regulation of water permeability in collecting ducts, and
consequently in the urinary concentration-dilution processes.
Precise regulation of fluid transport also occurs in the lung.
Homeostasis of the fluids that line the surface of lung is vitally
important for lung defense (1). P2Y2 purinoceptor has been
shown to regulate important secretory functions in respiratory epithelia. These include regulation of Na+,
Cl, and HCO
3
transport by Clara cells (33), secretion of mucin by goblet cells (12),
and secretion of surfactant phospholipids by type II alveolar cells or
pneumocytes (27).
Although a great deal has been learned regarding the role of P2Y2 purinoceptor in the regulation of transport processes in the kidney and lung using pharmacological and functional approaches, a more comprehensive understanding of the P2Y2 purinoceptor-mediated regulation in these tissues will depend on the development of molecular tools for the investigation of P2Y2 purinoceptor gene expression at mRNA and protein levels. In this report, we introduce such tools and exploit them to study P2Y2 purinoceptor expression and localization in rat renal medulla and lung. We demonstrate the expression of P2Y2 purinoceptor mRNA in rat lung, kidney, and collecting ducts by RT-PCR. We characterize the P2Y2 purinoceptor mRNA transcript in rat lung and kidney by Northern analysis. We demonstrate the expression of P2Y2 purinoceptor protein in medullary collecting ducts and type II alveolar cells of lung by immunoblotting. Using immunoperoxidase labeling, we localize P2Y2 purinoceptor at cellular level in rat inner medulla and lung.
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METHODS |
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Experimental animals. Unless otherwise mentioned, male Sprague-Dawley rats (150-250 g body wt) obtained from either Taconic Farms (Germantown, NY) or Harlan Sprague-Dawley (Indianapolis, IN) were used in these studies. The rats were maintained in pathogen-free state and were fed ad libitum with commercial rodent diet and had free access to drinking water.
Microdissection of collecting duct segments. Rats were killed and kidneys were prepared for microdissection as described previously (15). Briefly, after decapitation, the left kidney was perfused quickly with ice-cold HEPES-buffered isotonic solution. This was followed by perfusion with collagenase-hyaluronidase solution that was prewarmed to 37°C. The kidney was removed and sliced, and the pieces were incubated in the collagenase-hyaluronidase solution at 37°C with continuous oxygenation for 10-60 min. Cortical and medullary collecting duct segments were identified and dissected by free hand using Dumont no. 5 forceps. The HEPES-buffered dissection solution contained vanadyl ribonuclease complex (VRC), as a ribonuclease inhibitor. Lengths of 1-2 mm of these microdissected segments were washed with BSA (0.1%) in VRC-free dissection solution and then transferred into PCR tubes containing Triton X-100 (1%), placental RNase inhibitor, and dithiothreitol in diethyl pyrocarbonate (DEPC)-treated water and frozen immediately. Thus the tubules were permeabilized by exposing to hypotonic shock in the presence of detergent. To monitor RNA or cDNA contamination of reagents, blanks were prepared with 2 µl of the wash solution in lieu of tubules.
Primers. Gene-specific primers for rat P2Y2 purinoceptor were designed with the aid of computer program OLIGO 5.0 (National Bioscience, Plymouth, MN). The chief criteria used were specificity, Tm close to 60°C, lack of predicted internal structure. The sense primer sequence (5'CGA ATC ACC TGC CAC GAC 3') corresponds to nt 896-913 in the open reading frame and the anti-sense primer (5'TAT CAG CCC CTT TGA ACA AGC 3') corresponds to nt 1536-1556 in the 3'-untranslated region (3'-UTR) of the rat P2Y2 purinoceptor cDNA sequence as reported by Rice et al. (26). The predicted size of the PCR product is 661 bp and includes > 50% of the coding region of the P2Y2 purinoceptor mRNA. Primers specific for aquaporin-2 (AQP2) water channel were described previously (14).
RT-PCR amplifications. Total RNA was extracted from different
tissues or organs and from microdissected IMCD segments using RNAzol B
(Tel-Test, Friendswood, TX) per the manufacturer's protocol. RT-PCR
was performed as described previously (14, 15). Reverse transcription
was initiated by poly(dT)15 priming using avian myeloblastosis virus (AMV) reverse transcriptase (Boehringer-Mannheim). For reverse transcriptase-negative (RT) control reactions, the enzyme was substituted with an equal volume of DEPC-treated water. The
reaction was carried out for 60 min at 55°C followed by
inactivation of the enzyme for 1 min at 95°C. PCR was carried out
using gene-specific primers for P2Y2 purinoceptor and
Ampli-Taq DNA polymerase (Perkin-Elmer PCR kit). Parallel
amplifications were carried out for AQP2 mRNA to confirm the adequacy
of permeabilization of tubules and the efficacy of the reverse
transcription reaction. Blank tubes without tubules were analyzed in
parallel amplifications to demonstrate the absence of cDNA
contamination of reagents or pipettes. The samples were overlaid with
mineral oil and processed for 29-32 cycles [94°C, 1 min
(3 min for the initial cycle); 58°C, 1 min; 72°C, 1 min].
The elongation period in the last cycle was extended to 7 min. Aliquots
of PCR products were electrophoresed on 2% agarose gels and visualized
under ultraviolet light after staining with ethidium bromide. Remaining
PCR products were purified using QIAquick PCR purification kit (Qiagen,
Santa Clara, CA). The purified PCR products were sequenced at the
University of Cincinnati DNA Core facility by an automated sequencing
method (Applied Biosystems, model 373) with fluorescent dye terminator
chemistry. Sequences were analyzed using BLASTN (National Center for
Biotechnology Information). The purified PCR product was used as cDNA
probe for Northern hybridization.
Northern analysis. Northern hybridization was performed to
determine the relative size of the P2Y2 purinoceptor mRNA
transcript in rat lung, whole kidney, and inner medulla. Total RNA was
extracted from rat lung, whole kidney, and inner medulla using
Tri-Reagent (Molecular Research Center, Cincinnati, OH) per the
manufacturer's protocol. Thirty micrograms of total RNA was size
fractionated on a 1% formaldehyde agarose gel in 1× MOPS buffer
for 4.5 h at 96 V. An RNA molecular weight standard was used to
determine RNA size (GIBCO-BRL). RNA was transferred to Hybond
N+ nylon membrane overnight using capillary transfer in
10× SSC and ultraviolet cross linked. Equality of sample loading
was determined by matching the density of the 28S rRNA band visualized
by ethidium bromide. The total RNA blot was prehybridized in 15 ml of
ExpressHyb (Clontech) for 30 min at 65°C, hybridized with 1.5 × 106 cpm/ml of 32P random-labeled (High
Prime DNA Label, Boehringer-Mannheim) rat P2Y2 purinoceptor
partial cDNA probe for 2 h at 65°C. Membranes were washed twice at
RT for 15 min each in 2× SSC and 0.05% SDS, then 2× at
50°C for 15 min each in 0.1× SSC and 0.1% SDS, and exposed
to X-ray film (MS film, Kodak) overnight at 70°C with a
Kodak MS intensifying screen.
Polyclonal antibodies. A peptide-derived rabbit polyclonal antibody against the P2Y2 purinoceptor was raised by standard methods reported from our laboratories (15). The peptide sequence CSISSDDSRRTESTPAGSETKDIRL (with added amino-terminal cysteine) corresponds to the carboxy terminus residues 351-374 of rat P2Y2 purinoceptor published by Rice et al. (26). The peptide was conjugated to maleimide-activated keyhole limpet hemocyanin (KLH) by thio-ether linkage through the amino-terminal cysteine. Two rabbits (L246, L247) were immunized with this peptide-KLH conjugate using a combination of Freund's complete and incomplete adjuvants. Both rabbits developed ELISA titers of greater than 1:32,000. The antisera were affinity purified using a column on which 2 mg of the same immunizing peptide was immobilized via covalent linkage to agarose beads (Sulfo-Link Immobilization kit no. 2; Pierce, Rockford, IL). An IgG fraction of the preimmune serum was purified on a protein A column (Pierce) for use in control experiments. Peptide-derived polyclonal antibodies to AQP1 and AQP2 were produced and characterized previously (6, 31).
Preparation of membranes from IMCD and non-IMCD enriched fractions. Fractions enriched in IMCD and non-IMCD elements were prepared from rat kidney inner medullas as described previously (15). The procedure involves collagenase and hyaluronidase digestion of the inner medulla to separate structures into isolated elements. Subsequent low-speed centrifugations and washes separate the IMCD from the rest of the elements (thin limbs and vasculature) in the inner medulla. The fractions obtained as above were processed for preparation of crude membrane fractions as described previously (15). Briefly, IMCD suspensions were suspended in ice-cold isolation solution containing protease inhibitors and homogenized using a tissue homogenizer. The homogenates were initially spun at low speed (1,000 g) for 10 min at 4°C to pellet incompletely homogenized fragments and nuclei. The pellets were suspended in isolation solution and then rehomogenized and spun again at 1,000 g for 10 min at 4°C. The supernatants were combined and spun at 17,000 g for 20 min at 4°C to obtain fractions enriched in plasma membranes. The pellets were suspended in isolation solution with protease inhibitors, and the total protein concentration was measured. The membrane fractions thus isolated were solubilized at 60°C for 20 min in Laemmli sample buffer.
Preparation of type II alveolar cells for immunoblotting. Freshly isolated type II cells from rat lungs were provided by Dr. Francis X. McCormack of the Pulmonary and Critical Care Division, University of Cincinnati Medical Center. The isolation and characterization of these cells have been described previously (20). The cells were lysed and solubilized by hypotonic shock and detergent (0.5% Triton X-100) treatment in the presence of protease inhibitors. Total protein content was determined, and the preparation was further solubilized at 60°C for 20 min in Laemmli sample buffer.
Electrophoresis and immunoblotting of membrane proteins. These were carried out as described previously (15). Membrane proteins were electrophoresed on 12% polyacrylamide minigels (Novex, San Diego, CA) under denaturing conditions. The separated proteins were electrophoretically transferred to nitrocellulose membranes using Bio-Rad transfer apparatus (Bio-Rad Laboratories, Hercules, CA). The membranes were blocked with milk proteins and probed with the affinity-purified polyclonal antibody to P2Y2 purinoceptor (L246) at an IgG concentration of 0.2 µg/ml. For preadsorption controls, the antibody solution was mixed with 2 mg of immunizing peptide and incubated overnight at 4°C before use. Affinity-purified anti-AQP1 antibody (L266) and anti-AQP2 antibody (L126) were used at a concentration of 0.5 µg/ml each. The secondary antibody was donkey anti-rabbit IgG conjugated to horseradish peroxidase (Pierce No. 31458) used at a concentration of 0.16 µg/ml. Sites of antigen-antibody reaction were visualized using SuperSignal Substrate (Pierce) before exposing the blot to light-sensitive imaging film (Kodak no. 165-1579 Scientific Imaging Film).
Immunocytochemistry. Male Wistar rats (200-300 g body wt) were purchased from the Møllegaard Breeding Center, Denmark. The rats had free access to water and standard rat chow. Rats were anesthetized by injecting pentobarbital sodium intraperitoneally, and the kidneys and lungs were perfusion fixed with ice-cold fixative (4% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4). The perfusion-fixed organs were then removed and prepared for immunocytochemistry. Briefly, tissue blocks prepared from inner medulla and lungs were postfixed for 2 h in 4% paraformaldehyde and infiltrated with 2.3 M sucrose/2% paraformaldehyde for 30 min. The blocks were then mounted on holders and rapidly frozen in liquid nitrogen. Thin (0.85 µm) cryosections, cut on a Reichert Ultracut FCS cryoultramicrotome, were incubated with the affinity-purified antibody against P2Y2 purinoceptor (L246; 0.7-2.8 µg/ml). The labeling was visualized by using horseradish peroxidase-conjugated secondary antibody (DAKO P448, 1:100 dilution; DAKO, Glostrup, Denmark). Controls were performed using preimmune IgG fraction, nonimmune IgG, or omission of primary or secondary antibody.
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RESULTS |
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Expression of P2Y2 purinoceptor mRNA in kidney and
lung. An RT-PCR approach was used to examine the expression of
P2Y2 purinoceptor mRNA. Figure
1A shows that the P2Y2
purinoceptor mRNA is expressed in all three regions of rat kidney
(cortex and outer and inner medulla). In addition, the receptor mRNA is
expressed in liver, lung, heart, and cerebral cortex. In all cases, the
amplification products were single bands corresponding to the predicted
size (661 bp) of the target sequence. Because the mRNA for
P2Y2 purinoceptor is expressed in all three regions of the
kidney, further RT-PCR experiments were carried out to examine whether
the mRNA is expressed specifically in collecting ducts. Figure
1B shows the results of RT-PCR amplifications performed
directly on microdissected collecting duct segments. With this
approach, microdissected tubules were transferred directly into the PCR
tubes and permeabilized prior to reverse transcription reaction.
Consequently, unlike the conventional approaches, the starting material
contains both DNA and RNA. Since the P2Y2 purinoceptor gene
is intron-less, the PCR primers could not be engineered to generate
products of different sizes in cDNA vs. genomic DNA. Because of these
limitations, the RT samples showed very faint amplification
bands of the same size as the RT+ samples (Fig. 1B). However,
the robust signals in RT+ samples in contrast to the very faint
amplifications in RT
samples demonstrate the presence of
P2Y2 purinoceptor mRNA in the collecting duct segments from
all the three regions of kidney (cortex, outer medulla, and inner
medulla). To further confirm the expression of P2Y2
purinoceptor mRNA in medullary collecting ducts, we also carried out
RT-PCR amplifications on total RNA extracted from microdissected IMCD
segments (Fig. 1C). The sample size used in the microextraction
procedure was 20 mm of IMCD segments (~12,600 cells). RT-PCR
amplifications were carried out on RNA samples that correspond to 2 mm
of original tubule length. As shown in Fig. 1C, these
amplifications also produced single bands of expected molecular mass.
However, due to the inherent technical limitations involved in
extracting RNA from tiny amounts of starting material, the
RT
samples showed very feeble signals, indicating possible
genomic DNA contamination. Nevertheless, on the basis of the relative
intensities of signals from RT
and RT+ samples in Fig. 1,
B and C, it is reasonable to conclude that the
collecting ducts express P2Y2 purinoceptor mRNA.
Furthermore, in all these amplifications, blanks showed no cDNA
contamination. In addition, our RT-PCR amplifications on microdissected
nephron segments showed P2Y2 receptor mRNA expression in
glomeruli, proximal convoluted and straight tubules, descending and
ascending thin limbs of loop of Henle, and medullary and cortical thick
ascending limbs (not illustrated).
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The specificity of amplification of the target mRNA was assessed by direct sequencing of PCR products obtained from RT-PCR amplifications of total RNA isolated from rat lung, kidney, inner medulla, and IMCD. Sequence comparison of the amplified PCR products with sequences available in GenBank using the BLASTN alignment algorithm showed that these sequences are 98% identical to the cDNA sequences of G protein-coupled P2Y2 purinoceptor cloned from rat type II alveolar cells (26), aortic smooth muscle cells (29), and pituitary cells (3). In addition, significant nucleotide homology was found among these PCR products and mouse (94%) and human (82%) P2Y2 purinoceptor cDNAs.
P2Y2 purinoceptor mRNA transcript size in rat kidney
and lung. The sizes of the P2Y2 purinoceptor mRNA
transcript in rat whole kidney, inner medulla, and lung were determined
by Northern analysis. The partial cDNA probe used for Northern analysis
was the purified PCR product, the specificity of which had been
confirmed by sequencing as above. Our Northern analysis shows that both
rat lung and kidney express one transcript of ~3.6 kb (Fig.
2).
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Specificity of P2Y2 purinoceptor antibody. Figure
3 shows the specificity of our
P2Y2 purinoceptor antibody. Figure 3A shows immunoblots of crude membrane preparations (17,000 g pellets) from IMCD fractions from rat inner medulla and probed with either affinity-purified antibody alone (left lane) or the same
antibody preincubated with an excess of the immunizing peptide
(right lane). The antibody labeled a distinct 47-kDa band,
consistent with the predicted molecular mass of glycosylated rat
P2Y2 purinoceptor protein. In addition, the antibody
labeled a distinct band at 105 kDa and another at a higher molecular
mass at the limit of gel resolution. Preincubation with the immunizing
peptide completely ablated the 47-kDa and 105-kDa bands, but did not
completely ablate the higher molecular mass band. Thus the 47-kDa form
is likely to be P2Y2 purinoceptor monomer, and the 105-kDa
form is a possible complex containing the P2Y2 protein.
Since more than two apparently specific bands were detected in
immunoblots using IMCD preparations, we tested our antibody on
preparations of alveolar type II cells, from which the rat
P2Y2 purinoceptor was originally cloned (26). Figure
3B shows an immunoblot performed with type II cells and crude
membrane preparations from rat kidney medulla. As evident from the
immunoblot, the pattern of bands is similar in both type II cells and
renal medulla. The major difference, however, is the predominance of
the 105-kDa form of protein in type II cells.
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Expression of P2Y2 purinoceptor protein in IMCD and
non-IMCD elements of inner medulla. To examine the expression and
relative abundance of P2Y2 purinoceptor protein in
collecting ducts and noncollecting duct elements of the inner medulla,
we fractionated the collagenase-digested whole inner medulla into IMCD
and non-IMCD enriched fractions. Figure 4
shows three immunoblots of crude membrane fractions (17,000 g
pellets) from whole inner medulla, IMCD-rich, and non-IMCD-rich
fractions analyzed by SDS-PAGE. As shown in Fig. 4, B and
C, AQP1 protein was enriched in non-IMCD fractions, whereas
AQP2 protein was enriched in IMCD fractions relative to the whole inner
medulla, indicating that our enrichment procedure was effective. The
blot probed with anti-P2Y2 purinoceptor antibody (Fig.
4A) shows that the 47-kDa and 105-kDa proteins were clearly enriched in the IMCD-rich fractions compared with the
non-IMCD-rich fractions. The 47-kDa protein in IMCD-rich fraction was
also more prominent compared with the corresponding protein in the
whole inner medullary preparation.
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Immunocytochemical localization of P2Y2 purinoceptor in
inner medulla and lung. To localize the P2Y2
purinoceptor in the kidney inner medulla and lung at the cellular
level, we performed immunoperoxidase labeling of the receptor protein
on cryosections. Figure 5 shows the
immunocytochemical localization of P2Y2 purinoceptor
protein in inner medulla. Compared with the preadsorption controls
shown in Fig. 5B, Fig. 5A shows that the antibody
labeled collecting duct principal cells, as well as thin limbs and
vascular structures in the inner medulla. In the principal cells, the
labeling is seen uniformly both on apical and basolateral domains,
except at the areas marked by arrows. The arrows in Fig. 5A,
show patches of intense labeling in the apical membrane, compared with
the rest of the cell membrane. Based on these observations, we conclude that in the principal cells, the labeling is seen predominantly over
the apical domains and to a lesser extent on the basolateral aspects.
Figure 6 shows the labeling of
P2Y2 purinoceptor in terminal respiratory bronchioles of
rat lung. The labeling is restricted to nonciliated secretory cells,
namely, the Clara cells (Fig. 6A) and the goblet cells (Fig.
6B). In the Clara cells, the labeling is intense
on the dome-shaped apical domain and weaker on the basolateral aspects
(Fig. 6A). Diffuse labeling of the P2Y2 protein is
seen in the goblet cells (Fig. 6B).
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DISCUSSION |
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Renal collecting duct water transport is regulated by a complex
interplay among different intracellular signaling pathways that are
under the control of membrane receptors for hormones such as AVP, as
well as the autacoids like prostaglandin E2, endothelin, and extracellular nucleotides. These membrane receptors
act as modulators of collecting duct water transport and to date have not been fully studied. One such potential receptor in the kidney is
the P2Y2 purinoceptor. Pharmacological and functional
studies have demonstrated that extracellular nucleotides, acting
through P2Y2 purinoceptor, downregulate the AVP-stimulated
osmotic water permeability in collecting duct and thus may play an
important role in urinary concentration-dilution processes (8, 13, 28).
Functional studies have also shown that P2Y2 purinoceptor regulates important secretory functions in respiratory epithelia (1).
The evidence accumulated so far indicates that this receptor is
expressed in nonciliated secretory cells of the terminal respiratory bronchioles (Clara cells and goblet cells) and the type II alveolar cells (pneumocytes). In Clara cells, this receptor has been shown to
regulate ion (Na+, Cl, and
HCO
3) transport across the epithelium (33). In the goblet cells, the P2Y2 purinoceptor regulates
the secretion of mucin (12), and in type II alveolar cells this receptor is involved in the secretion of surfactant phospholipids (27).
However, our knowledge of P2Y2 purinoceptor expression in
the kidney, lung, and other organs, tissues, or cells thus far is based
on pharmacological and functional studies. In this report, we provide
molecular (mRNA and protein) evidence for the expression of
P2Y2 purinoceptor in rat kidney and lung. We also characterize the cellular distribution of P2Y2 purinoceptor
protein in renal medulla and terminal respiratory bronchioles. To
achieve these goals, we used RT-PCR, Northern analysis, and
immunochemical methods.
The rat P2Y2 purinoceptor cDNA cloned from type II alveolar cells has an open reading frame of 1,125 base pairs (359-1483 bp), and the gene has no introns (26). The open reading frame encodes a putative protein of 374 amino acid residues with a predicted molecular weight of 42,275 in the unglycosylated state (26). This protein contains seven transmembrane-spanning domains, characteristic of G protein-coupled receptors. The rat P2Y2 purinoceptor protein shares considerable degree of sequence homology with mouse (>95%) and human (87%) P2Y2 purinoceptor proteins.
Using an RT-PCR approach, Rice et al. (26) reported the expression of P2Y2 purinoceptor mRNA in heart, kidney, lung, spleen, and testis. However, they did not find evidence for the expression of this receptor in rat brain and liver. In contrast, our RT-PCR amplifications show abundant expression of P2Y2 purinoceptor mRNA in rat liver and cerebral cortex. Furthermore, we have demonstrated that the P2Y2 purinoceptor mRNA is expressed throughout the rat kidney collecting duct system. Sequence analysis of PCR fragments obtained by amplifications on total RNA from lung, kidney inner medulla, and microdissected IMCD segments showed a high degree of identity (98%) to the P2Y2 purinoceptor cDNA cloned from different rat tissues.
A partial P2Y2 purinoceptor cDNA PCR product obtained from rat IMCD was used as a probe in Northern analysis to determine the size of the receptor transcript from lung and kidney. This cDNA probe (661 bp) includes 52% of the coding region and extends 75 bp into 3'-UTR. Our Northern analysis shows that both lung and kidney express a single transcript of ~3.6 kb. Using a similar approach, Seye et al. (29) reported a transcript size of ~3 kb for P2Y2 purinoceptor mRNA in adult rat aorta, which agrees with our findings.
In immunoblots, our peptide-derived antibody recognizes two distinct protein bands (47 and 105 kDa), which appear to be specific as revealed in preadsorption controls. It is reasonable to assume that the 47-kDa protein is the glycosylated monomer of the P2Y2 purinoceptor. The 105-kDa form may represent a duplex of the native monomer, or a complex of P2Y2 purinoceptor and some other proteins, which might perhaps play a regulatory role. One such protein could be one of the arrestins, a family of 48-kDa proteins that promote the processes of desensitization, internalization, downregulation, and resensitization of G protein-coupled receptors (18). The nature of the third band (>105 kDa) that is partially ablated by preadsorption with the peptide requires further investigation. Our fractionation experiments with kidney inner medulla revealed that collecting ducts account for the major fraction of the 47-kDa protein and almost all of the 105-kDa protein in the inner medulla. It is interesting to note that freshly isolated rat type II pneumocytes predominantly express the high-molecular-weight form (105 kDa) of the protein.
Thus our RT-PCR and immunoblotting studies demonstrate that the P2Y2 purinoceptor mRNA and protein are expressed in rat kidney and collecting duct system. The latter is consistent with earlier pharmacological and functional studies (8, 13). To localize P2Y2 purinoceptor at the cellular level, we performed immunoperoxidase labeling for the receptor protein on ultrathin cryosections. Our immunocytochemical observations on inner medulla confirm the findings of our immunoblotting studies. In addition, the immunocytochemical data further demonstrate that the P2Y2 purinoceptor protein is expressed predominantly on the apical aspect and to a lesser degree on the basolateral domains of the collecting duct principal cells. We consider this a very important finding, as it suggests that the physiological significance of this receptor is more complex than what we were able to unravel in our earlier microperfusion experiments, which were aimed at the examination of the basolateral domain, but not the luminal aspect (13). This observation will open further vistas for the study of the role of P2Y2 purinoceptor in water transport in medullary collecting ducts.
The immunocytochemical labeling of nonciliated secretory cells of
terminal respiratory bronchioles (Clara cells and goblet cells) is
striking and is consistent with the known functions of these cells in
response to extracellular nucleotides. It is well established that in
response to extracellular nucleotides, the Clara cells secrete
Cl, HCO
3, and
Na+, and the goblet cells release mucin (12, 33).
Furthermore, although it is not well established, there is a
possibility that this receptor may exist as a component of the ATP
binding cassette (ABC) family of proteins along with cystic fibrosis
transmembrane conductance regulator (CFTR) and outwardly rectifying
chloride channel (ORCC) in the lung. In this model, ATP permeates
through CFTR and activates P2Y2 purinoceptor, which in turn
opens ORCC (5). This model also explains the observations that
extracellular nucleotides are effective chloride secretagogues in
airway epithelial cells in patients with cystic fibrosis that do not
express functional CFTR (17, 30). In addition, CFTR mRNA and protein
are also expressed in the kidney in both proximal and distal nephrons
(4, 22). Studies have also identified CFTR expression and/or
Cl channels with properties similar to CFTR in
proximal tubules and IMCD (10, 32). Although direct evidence is
lacking, it is likely that CFTR may also play an important role in
fluid and electrolyte transport in the kidney in conjunction with
P2Y2 purinoceptor and ORCC.
Finally, the future studies should address the involvement and significance of P2Y2 purinoceptor in pathophysiological conditions. The preliminary data obtained by us in a rat model of renal ischemic reperfusion injury (IRI) showed that P2Y2 purinoceptor mRNA is markedly increased with a concomitant decrease in AQP2 water channel expression in medulla, thus indicating the probable involvement of this receptor in the diuretic condition seen in IRI (16). These findings, coupled with the recent report by Paller et al. (24) that purinergic receptors mediate cell proliferation and enhanced recovery from renal ischemia by exogenous ATP, open further avenues for the investigation of the role of P2Y2 purinoceptor in ischemic acute renal failure.
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ACKNOWLEDGEMENTS |
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We thank Dr. Anil Menon for helpful suggestions and Drs. Maurice Burg and Kenneth Spring for critical reading of the manuscript. We also thank Dr. Francis X. McCormack for kindly providing the rat type II alveolar cells and Darren DiIulio for the technical assistance.
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FOOTNOTES |
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This work was supported by the University of Cincinnati Academic Development Fund (B. K. Kishore) and the intramural budget of the National Heart, Lung, and Blood Institute (NHLBI) (S. M. Ginns and M. A. Knepper), as well as the Danish Research Academy, the Novo Nordic Foundation, the Danish Medical Council, and the University of Aarhus Research Foundation (S. Nielsen). C. M. Krane is recipient of a new investigator award in the NHLBI, National Institutes of Health Program of Excellence in Molecular Biology of Heart and Lung.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: B. K. Kishore, Division of Nephrology and Hypertension, Univ. of Cincinnati Medical Center, 231 Bethesda Ave., ML 0585, Cincinnati, OH 45267-0585 (E-mail: BK.Kishore{at}uc.edu).
Received 2 April 1999; accepted in final form 27 July 1999.
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