Cellular localization of P2Y2 purinoceptor in rat renal inner medulla and lung

Bellamkonda K. Kishore1, Seth M. Ginns3, Carissa M. Krane2, Søren Nielsen4, and Mark A. Knepper3

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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) (ATPgamma S) > 2-(methylthio)-ATP (2-MeS-ATP) > alpha ,beta -methylene-ATP (alpha ,beta -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 alpha ,beta -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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 1.   Detection of P2Y2 purinoceptor mRNA expression in different tissues or organs and renal collecting ducts by RT-PCR. Oligo(dT)15 primed reverse transcription with (RT+) or without (RT-) avian myeloblastosis leukemia virus (AMV) reverse transcriptase was performed on samples of total RNA (1-2 µg/tube), followed by PCR amplifications of RT products using primers specific for P2Y2 purinoceptor (661 bp). Blanks contained no RNA or tubule samples. Amplifications of aquaporin-2 (AQP2) water channel (562 bp) were run in parallel as a positive control for reverse transcription and efficiency of permeabilization of tubules. PCR products were electrophoresed on 2% agarose gels and visualized after staining with ethidium bromide. A: expression of P2Y2 purinoceptor mRNA in different tissues or organs. B: expression of P2Y2 purinoceptor mRNA in renal collecting ducts. RT-PCR amplifications were performed directly on microdissected and permeabilized collecting ducts (1.4 to 2.0 mm total length for sample). For AQP2 amplifications, 1.3 mm of inner medullary collecting duct (IMCD) segments were used. C: RT-PCR amplifications of P2Y2 purinoceptor were performed with total RNA extracted from microdissected (2 mm) IMCD segments. Results shown are representative of 2-4 different sets of amplifications. MW, molecular weight; OM, outer medulla; IM, inner medulla; CCD, cortical collecting duct.

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).


View larger version (113K):
[in this window]
[in a new window]
 
Fig. 2.   Determination of molecular masses of P2Y2 purinoceptor mRNA transcripts in rat lung, whole kidney (WK), and inner medulla (IM) by Northern analysis. A: 30 µg of total RNA per sample were electrophoresed and then transferred to nylon membranes and hybridized with 32P-labeled rat P2Y2 purinoceptor partial cDNA (661 bp). B: equality of loading and the quality of fractionation were assessed by the relative abundance of the 28S and 18S rRNA bands visualized by ethidium bromide. Results shown are representative of two independent Northern hybridizations.

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.


View larger version (61K):
[in this window]
[in a new window]
 
Fig. 3.   Determination of the specificity of anti-P2Y2 purinoceptor antibody (L246) by immunoblotting. Proteins were size fractionated by SDS-PAGE on 12% minigels and transferred to nitrocellulose membranes. A: 15 µg of membrane protein (17,000 g pellet) from fractions enriched in IMCD segments were used in Western analysis with either the affinity-purified antibody to P2Y2 purinoceptor (left lane) at a concentration of 0.2 µg/ml or with the same antibody preadsorbed with the immunizing peptide (right lane). B: 15 µg of protein from freshly isolated, lysed and solubilized type II alveolar cells of rat lungs (left lane, T-II) or of plasma membrane fractions (17,000 g pellet) from renal medulla (right lane, KM) were used in Western analysis using affinity-purified P2Y2 purinoceptor antibody (L246; 0.2 µg/ml). Results shows are representative of 2-4 different immunoblotting experiments.

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.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 4.   Determination of relative abundance of P2Y2 purinoceptor protein in fractions enriched in IMCD or non-IMCD elements compared with whole inner medullary preparations. Collagenase-digested inner medullary tissue was separated into whole inner medullary preparation and fractions enriched in IMCD or non-IMCD elements of inner medulla. Fifteen micrograms of membrane protein (17,000 g pellets) from these 3 preparations were size fractionated by SDS-PAGE and transferred to nitrocellulose membranes. The latter were probed with anti-P2Y2 purinoceptor antibody (L246) at an IgG concentration of 0.2 µg/ml (A). Control blots were generated using 2 µg of membrane protein/lane and were probed with either an AQP1 antibody (L266, 0.5 µg/ml; B) as a marker for thin limbs and vascular elements or an AQP2 antibody (L126, 0.5 µg/ml; C) as a marker for collecting ducts. Results shown are representative of 2 different immunoblotting experiments.

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).


View larger version (108K):
[in this window]
[in a new window]
 
Fig. 5.   Immunocytochemical localization of P2Y2 purinoceptor protein in inner medulla. A: labeling in collecting duct principal cells (arrows). Labeling is also seen in thin limbs and vascular structures. Within collecting duct principal cells, labeling is seen predominantly on apical (arrows) and to a lesser degree on basolateral domains. B: preadsorption control where the antibody was preincubated with the immunizing peptide. Pictures shown are representative of at least 4 sections examined.



View larger version (113K):
[in this window]
[in a new window]
 
Fig. 6.   Immunocytochemical localization of P2Y2 purinoceptor protein in rat lung: sections from terminal respiratory bronchioles. A: intense labeling of Clara cells (arrowheads). B: labeling of goblet cells (arrowheads). None of the ciliated epithelial cells in A or B shows any labeling under these conditions (arrows). C: a preadsorption control where the antibody was preincubated with the immunizing peptide. Pictures shown are representative of at least 4 sections examined.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Boucher, R. C. Human airway ion transport. Am. J. Respir. Crit. Med. 150: 271-281, 1994[ISI][Medline].

2.   Cha, S. H., T. Sekine, and H. Endou. P2 purinoceptor localization along rat nephron and evidence suggesting existence of subtypes P2Y1 and P2Y2. Am. J. Physiol. Renal Physiol. 274: F1006-F1014, 1998[Abstract/Free Full Text].

3.   Chen, Z. P., N. Krull, S. Xu, A. Levy, and S. L. Lightman. Molecular cloning and functional characterization of a rat pituitary G protein-coupled adenosine triphosphate (ATP) receptor. Endocrinology 137: 1833-1840, 1996[Abstract].

4.   Crawford, I., P. C. Maloney, P. L. Zeitlin, W. B. Guggino, S. C. Hyde, H. Turley, K. C. Gatter, A. Harris, and C. F. Higgins. Immunocytochemical localization of the cystic fibrosis gene product CFTR. Proc. Natl. Acad. Sci. USA 88: 9262-9266, 1991[Abstract].

5.   Devidas, S., and W. B. Guggino. The cystic fibrosis transmembrane conductance regulator and ATP. Curr. Opin. Cell. Biol. 9: 547-552, 1997[ISI][Medline].

6.   DiGiovanni, S. R., S. Nielsen, E. I. Christensen, and M. A. Knepper. Regulation of collecting duct water channel expression by vasopressin in Brattleboro rat. Proc. Natl. Acad. Sci. USA 91: 8984-8988, 1994[Abstract].

7.   Dubyak, G. R., and C. El-Moatassim. Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 266: F577-F606, 1993.

8.   Ecelbarger, C. A., Y. Maeda, C. C. Gibson, and M. A. Knepper. Extracellular ATP increases intracellular calcium in rat terminal collecting duct via a nucleotide receptor. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 267: F998-F1006, 1994[Abstract/Free Full Text].

9.   Friedlander, G., and C. Amiel. Extracellular nucleotides as modulators of renal tubular transport. Kidney Int. 47: 1500-1506, 1995[ISI][Medline].

10.   Husted, R. F., K. A. Volk, R. D. Sigmund, and J. B. Stokes. Anion secretion by the inner medullary collecting duct. Evidence for involvement of the cystic fibrosis transmembrane conductance regulator. J. Clin. Invest. 95: 644-650, 1995[ISI][Medline].

11.   Inscho, E. W., K. D. Mitchell, and G. Navar. Extracellular ATP in the regulation of renal microvascular function. FASEB J. 8: 319-328, 1994[Abstract/Free Full Text].

12.   Kim, K. C., and B. C. Lee. P2 purinoceptor regulation of mucin release by airway goblet cells in primary culture. Br. J. Pharmacol. 103: 1053-1056, 1991[Abstract].

13.   Kishore, B. K., C.-L. Chou, and M. A. Knepper. Extracellular nucleotide receptor inhibits AVP-stimulated water permeability in inner medullary collecting duct. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 269: F863-F869, 1995[Abstract/Free Full Text].

14.   Kishore, B. K., B. Mandon, N. B. Oza, S. R. DiGiovanni, R. A. Coleman, N. L. Ostrowski, J. B. Wade, and M. A. Knepper. Rat renal arcade segment expresses vasopressin-regulated water channel and vasopressin V2 receptor. J. Clin. Invest. 97: 2763-2771, 1996[Abstract/Free Full Text].

15.   Kishore, B. K., J. B. Wade, K. Schorr, T. Inoue, B. Mandon, and M. A. Knepper. Expression of synaptotagmin VIII in rat kidney. Am. J. Physiol. Renal Physiol. 275: F131-F142, 1998[Abstract/Free Full Text].

16.   Kishore, B. K., Z. Wang, H. Rabb, M. Haq, and M. Soleimani. Upregulation of P2Y2 purinoceptor during ischemic reperfusion injury (IRI): possible relevance to diuresis of IRI (Abstract). J. Am. Soc. Nephrol. 9: 581, 1998.

17.   Knowles, M. R., L. L. Clarke, and R. C. Boucher. Activation by extracellular nucleotides of chloride secretion in the airway epithelia of patients with cystic fibrosis. N. Engl. J. Med. 325: 533-538, 1991[Abstract].

18.   Krupnick, J., and J. L. Benovic. The role of receptor kinases and arrestins in G protein-coupled receptor regulation. Annu. Rev. Pharmacol. Toxicol. 38: 289-319, 1998[ISI][Medline].

19.   Lustig, K. D., A. K. Shiau, A. J. Brake, and D. Julius. Expression cloning of an ATP receptor from mouse neuroblastoma cells. Proc. Natl. Acad. Sci. USA 90: 5113-5117, 1993[Abstract].

20.   McCormack, F. X., M. Damodarasamy, and B. M. Elhalwagi. Deletion mapping of N-terminal domains of surfactant protein A. The N-terminal segment is required for phospholipid aggregation and specific inhibition of surfactant secretion. J. Biol. Chem. 274: 3173-3181, 1999[Abstract/Free Full Text].

21.   Mitchell, K. D., and G. Navar. Modulation of tubuloglomerular feedback responsiveness by extracellular ATP. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 264: F458-F466, 1993[Abstract/Free Full Text].

22.   Morales, M. M., T. P. Carroll, T. Morita, E. M. Schwiebert, O. Devuyst, P. D. Wilson, A. G. Lopes, B. A. Stanton, H. C. Dietz, G. R. Cutting, and W. B. Guggino. Both wild type and functional isoform of CFTR are expressed in kidney. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 270: F1038-F1048, 1996[Abstract/Free Full Text].

23.   O'Connor, S. E. Recent developments in the classification and functional significance of receptors for ATP and UTP, evidence for nucleotide receptors. Life Sci. 50: 1657-1664, 1992[ISI][Medline].

24.   Paller, M. S., E. J. Schnaith, and M. E. Rosenberg. Purinergic receptor mediated cell proliferation and enhanced recovery from renal ischemia by adenosine triphosphate. J. Lab. Clin. Med. 131: 174-183, 1998[ISI][Medline].

25.   Parr, C. E., D. M. Sullivan, A. M. Paradiso, E. R. Lazarowski, L. H. Burch, J. C. Olsen, L. Erb, G. A. Weisman, R. C. Boucher, and J. T. Turner. Cloning and expression of a human P2u nucleotide receptor, a target for cystic fibrosis pharmacotherapy. Proc. Natl. Acad. Sci. USA 91: 3275-3279, 1994[Abstract].

26.   Rice, W. R., F. M. Burton, and D. T. Fiedeldey. Cloning and expression of the alveolar type II cell P2u-purinergic receptor. Am. J. Respir. Cell. Mol. Biol. 12: 27-32, 1995[Abstract].

27.   Rice, W. R., and F. M. Singleton. P2-purinoceptors regulate surfactant secretion from isolated alveolar type II cells. Br. J. Pharmacol. 89: 485-491, 1986[Abstract].

28.   Rouse, D., M. Leite, and W. N. Suki. ATP inhibits the hydrosmotic effect of AVP in rabbit CCT: evidence for a nucleotide P2u receptor. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 267: F289-F295, 1994[Abstract/Free Full Text].

29.   Seye, C. I., A.-P. Gadeau, D. Daret, F. Dupuch, P. Alzieu, L. Capron, and C. Desgranges. Overexpression of the P2Y2-purinoceptor in intimal lesions of the rat aorta. Arterioscler. Thromb. Vasc. Biol. 17: 3602-3610, 1997[Abstract/Free Full Text].

30.   Stutts, M. J., T. C. Chinet, S. J. Mason, J. M. Fullton, L. L. Clarke, and R. C. Boucher. Regulation of Cl- channels in normal and cystic fibrosis airway epithelial cells by extracellular ATP. Proc. Natl. Acad. Sci. USA 89: 1621-1625, 1992[Abstract].

31.   Terris, J., C. A. Ecelbarger, S. Nielsen, and M. A. Knepper. Long-term regulation of four renal aquaporins in rats. Am. J. Physiol. Renal Fluid Electrolyte Physiol. 271: F414-F422, 1996[Abstract/Free Full Text].

32.   Vandorpe, D., N. Kizer, F. Ciampolillo, V. A. Memoli, W. B. Guggino, and B. Stanton. cAMP stimulates CFTR (cystic fibrosis transmembrane conductance regulator) chloride channels in inner medullary collecting duct. Am. J. Physiol. Cell Physiol. 269: C683-C689, 1995[Abstract].

33.   Van Scott, M. R., C. W. Davis, and R. C. Boucher. Na+ and Cl- transport across rabbit nonciliated bronchiolar epithelial (Clara) cells. Am. J. Physiol. Cell Physiol. 256: C893-C901, 1989[Abstract/Free Full Text].


Am J Physiol Renal Physiol 278(1):F43-F51