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RECEPTORS AND SIGNAL TRANSDUCTION
1Renal-Electrolyte Division and Laboratory of Epithelial Cell Biology, 3Department of Cell Biology and Physiology, and 2Department of Pharmacology and Center for Clinical Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania
Submitted 20 January 2006 ; accepted in final form 19 March 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1 cm2 tissue area) of 
30% or 24%, respectively, after 5 h. Although A1, A2a, and A3 selective agonists all stimulated membrane capacitance after being administrated serosally, only the A1 agonist caused large increases in capacitance after being administered mucosally. Adenosine receptor antagonists as well as adenosine deaminase had no effect on stretch-induced capacitance increases, but adenosine potentiated the effects of stretch. Treatment with U-73122, 2-aminoethoxydiphenylborate, or xestospongin C or incubation in calcium-free Krebs solution inhibited adenosine-induced increases in capacitance. These data indicate that the uroepithelium is a site of adenosine biosynthesis, that adenosine receptors are expressed in the uroepithelium, and that one function of these receptors may be to modulate exocytosis in umbrella cells. capacitance; exocytosis
Adenosine may regulate normal bladder function. Message for all four adenosine receptor subtypes is detected in whole rat bladders by RT-PCR analysis (10), and Northern blot analysis confirms that A2b receptors are abundantly expressed in the detrusor muscle (33); however, the tissue distribution of A1, A2a, and A3 receptors within the bladder has not been defined. Studies thus far indicate that adenosine inhibits contraction of isolated rat detrusor muscle exposed to carbachol, acetylcholine, potassium depolarization, or field stimulation, a function that has been variously ascribed to A2a and/or A2b receptor activation (8, 18, 22, 26, 40). The involvement of adenosine in the inhibition of muscle contraction may be the consequence of membrane hyperpolarization as a result of ATP-sensitive K+ channel activation (15), a downstream effect of A2a activation. In contrast, A1 receptor activation may have a role in stimulated detrusor muscle contraction in cat bladder muscle (41). Adenosine also acts to regulate bladder function by inhibiting the contraction of the detrusor muscle via A1 receptors present at the neuromuscular junctions and in the central nervous system via activation of A1 and A2 receptors (13, 27, 32). The latter act to increase the urine volume necessary to induce volume-evoked micturition reflex in rats (32). Whether adenosine affects other bladder functions is unknown.
The interface between the urine and the underlying nervous tissue and musculature of the bladder is the uroepithelium (2). This stratified tissue, comprised of basal, intermediate, and umbrella cell layers, not only forms a dynamic barrier that maintains the composition of the urine but also may communicate the contents of the urine and the degree of bladder filling to sensory afferents that underlie the epithelium (2). The uroepithelium can release ATP (11, 39), but it is unknown whether this contributes to the high concentrations of adenosine found in urine. As the bladder fills, the apical surface of umbrella cells becomes flattened and cytoplasmic discoidal/fusiform vesicles fuse with the apical plasma membrane, which increases the luminal surface area of the bladder and may allow the bladder to accommodate increased urine volumes (35). Beyond bladder filling and its associated mechanotransduction pathways that increase cAMP and Ca2+ within the uroepithelium (35, 38), few physiological regulators of umbrella cell discoidal/fusiform vesicle exocytosis are known. We recently showed (39) that ATP, released from the bladder epithelium, binds to cell surface P2X receptors on uroepithelium and acts as an autocrine regulator of membrane traffic in the umbrella cell.
Our studies indicate that the uroepithelium is a source of adenosine biosynthesis, that all four adenosine receptors are expressed in the uroepithelium, and that adenosine/adenosine agonists stimulate umbrella cell exocytosis, possibly through a Ca2+-dependent signaling pathway. These observations reveal a previously undescribed role for adenosine as an autocrine/paracrine modulator of exocytosis in the umbrella cell layer.
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MATERIALS AND METHODS |
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Animals. Animals used in this study were female New Zealand White rabbits (3–4 kg; Myrtle's Rabbitry, Thompson Station, TN), female Sprague-Dawley rats (weighing 250–300 g), and female C57BL/6J mice (3–4 mo old). Rabbits were euthanized by injection of 300 mg of pentobarbital sodium into the ear vein, whereas rats and mice were euthanized by inhalation of 100% CO2. After euthanasia and thoracotomy the bladders were rapidly excised and processed as described below. All animal studies were carried out with the approval of the University of Pittsburgh Animal Care and Use Committee.
Antibodies and labeled probes. Affinity-purified rabbit polyclonal anti-rat adenosine A1 receptor antibody and corresponding control peptides were purchased from Novus Biologicals (Littleton, CO). Rabbit polyclonal antibodies to the A2a, A2b, and A3 receptors and antigenic peptides were purchased from Chemicon International (Temecula, CA). Secondary goat anti-rabbit antibodies conjugated to FITC or horseradish peroxidase were purchased from Jackson Immunoresearch Laboratories (West Grove, PA). Topro-3 and TRITC-phalloidin were purchased from Invitrogen-Molecular Probes (Carlsbad, CA).
Western blot analysis of adenosine receptors in uroepithelium. Rat bladder uroepithelial tissue was obtained by gently scraping the epithelium with a 17-mm cell scraper (Sarstedt, Newton, NC). The collected cells were lysed in 0.5% SDS lysis buffer [mM: 100 NaCl, 50 triethanolamine, and 5 EDTA with 0.5% (wt/vol) SDS] containing a protease inhibitor cocktail (5 µg/ml leupeptin, 5 µg/ml antipain, 5 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride). Proteins were resolved by SDS-PAGE and transferred to Immobilon-P, and the blots were probed with adenosine receptor-specific antibodies with our previously described techniques (35, 39). In some experiments, primary antibody was combined with antigenic peptide for 2 h at room temperature before incubation with the transferred proteins. Images were scanned with an ArtixScan 1800f flatbed scanner (Microtek International, Carson, CA), the contrast was corrected with Photoshop (Adobe Systems, San Jose, CA), and the images were imported into FreeHand (Macromedia, San Francisco, CA).
Immunofluorescence analysis of adenosine receptors in uroepithelium. Excised bladders were fixed in 4% (wt/vol) paraformaldehyde dissolved in 100 mM sodium cacodylate (pH 7.4) buffer for 2 h at room temperature, and the tissue was cut into small pieces with a razor blade and then cryoprotected, sectioned, and incubated with primary antibodies (1:250–1:500 dilution) preincubated with or without immunogenic peptide for 2 h at room temperature as described previously (35). After being washed, the sections were incubated with a mixture of FITC-conjugated goat-anti rabbit secondary antibody (diluted 1:100), rhodamine-phalloidin (1:50), and Topro-3 (1:1,000). The sections were washed with PBS, postfixed with 4% (wt/vol) paraformaldehyde, and mounted under coverslips with p-diaminobenzidine-containing mounting medium (35).
Scanning laser confocal analysis of fluorescently labeled cells. Imaging was performed on a TCS-SL confocal microscope equipped with argon and green and red helium-neon lasers (Leica, Dearfield, IL). Images were acquired by sequential scanning with a x100 (1.4 numerical aperture) planapochromat oil objective and the appropriate filter combination. Settings were as follows: photomultipliers set to 600–800 V, 1 Airy disk, and Kalman filter (n = 4). Serial (z) sections were captured with a 0.25-µm step size. The images (512 x 512 pixels) were saved as TIFF files. The OpenLab program (Improvision, Lexington, MA) was used to project the serial sections into one image. The contrast level of the final images was adjusted in Photoshop, and the contrast-corrected images were imported into FreeHand.
Mounting of rabbit uroepithelium in Ussing stretch chambers. Excised rabbit bladders were cut open longitudinally and then washed in Krebs solution and mounted on custom-made Teflon racks with the mucosal side down. The smooth muscle layers were removed with sharp scissors and forceps. The remaining mucosal tissue, containing the uroepithelium, was mounted on plastic rings with a 2-cm2 opening and then was sandwiched between two halves of a modified Ussing stretch chamber as described previously (38). Each hemichamber (mucosal and serosal) was filled with 12.5 ml of Krebs solution, the serosal hemichamber was bubbled with gas containing 5% (vol/vol) CO2 and 95% (vol/vol) air, and the tissue was equilibrated for 30–60 min. In some experiments, the hydrostatic pressure in the mucosal hemichamber was increased as described previously (38).
Measurement of extracellular adenosine.
After mounting in Ussing stretch chambers and a 30-min period of equilibration, the tissue was isovolumetrically washed three times with 60 ml of Krebs solution. After an additional 30-min incubation, hydrostatic pressure was increased as described above. Aliquots (1,000 µl) were taken with replacement from the serosal and mucosal hemichambers at the designated time points (0, 5, 30, 60, and 120 min). Adenosine, AMP, and inosine were measured with an LCMS assay on a ThermoFinnigan LCQ mass spectrometer with electrospray ionization. To 60 µl of sample, 10 µl of 140 pg/µl internal standard (9-
-D-arabinofuranoside) was added, and 50 µl was injected for analysis. Purines were resolved on a C-18 reverse-phase column (Eclipse Zorbax XDB, 4.6 mm x 150 mm) with water-methanol-0.1% formic acid as the mobile phase at a flow rate of 0.5 ml/min. The mobile phase was held at 100% water for 1.5 min, changed to 90:10 water-methanol over 0.5 min, and held at this composition for an additional 8 min. The analytes were monitored with single ion monitoring: for AMP, mass-to-charge ratios (m/z) = 348; for adenosine and adenine 9--D-arabinofuranoside (internal standard), m/z = 268; and for inosine, m/z = 291.
Capacitance measurements.
Changes in capacitance primarily reflect changes in the apical cell surface area of the umbrella cells, where 1 µF 1 cm2 of membrane area (38). Capacitance was measured as described previously (38).
Data analysis. Nonlinear regression analysis built into the GraphPad Prism program (San Diego, CA) was used to determine EC50 values for adenosine and adenosine agonists. Statistically significant differences between means were determined by Student's t-test, or when multiple comparisons were made significant differences were assessed by ANOVA with Bonferroni's correction.
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RESULTS |
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38 kDa was detected with an anti-A1 receptor antibody (Fig. 1A), which is close to the calculated molecular mass of the A1 receptor (36.5 kDa). Two protein species were detected with an A2a receptor antibody (Fig. 1B). The higher-molecular-mass species coincided with the reported molecular mass of the protein (44.7 kDa), whereas the other, lower-molecular-mass species (38 kDa) was reported previously and may represent proteolytic processing of the larger precursor (20, 25, 29). The A2b receptor antibody recognized a single molecular mass species of 52 kDa, which is greater than the calculated mass of 36.3 kDa (Fig. 1C) but is identical to the reported mass of A2b receptors detected in bovine corneal endothelium and kidney vasculature (20, 34). The A3-specific antibody detected a protein species of 52 kDa, consistent with previous reports (20), and a species of 36.2 kDa, the nominal molecular mass of the protein. The signals for each antibody reaction were significantly attenuated in the presence of antigenic peptide, confirming the specificity of the reactions. Together, these results indicate that all four adenosine receptor species are expressed in the uroepithelium.
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Consistent with the Western blot analysis, all four adenosine receptors were localized within the uroepithelium (Fig. 2). The A1 receptor antibody strongly labeled the apical plasma membrane of umbrella cells. In contrast, less staining was observed along the basolateral plasma membrane of the umbrella cell layer, and there was little staining of the underlying intermediate and basal cell layers (Fig. 2A). Signal for the A1 receptor was also observed in the underlying submucosal connective tissue (data not shown). The A2a receptor antibody labeled the cytoplasm of all three types of uroepithelial cells and also strongly labeled a layer of tissue elements below the epithelium, possibly connective tissue cells, myofibroblasts, or blood vessels (Fig. 2B). By immunofluorescence it was difficult to tell whether A2a was localized to the plasma membrane. The A2b receptor antibody stained the apical cytoplasm and the basolateral plasma membrane of the umbrella cells and also appeared to label the cytoplasm of the intermediate/basal cells (Fig. 2C). Consistent with previous reports of A2b gene expression in the detrusor muscle (33), intense A2b receptor staining was also observed in bladder smooth muscle tissue (data not shown). The A3 receptor antibody specifically labeled the basolateral plasma membrane of umbrella cells and the plasma membranes of the underlying epithelial cell layers (Fig. 2D). Staining of the submucosa or detrusor muscle was not observed. In all cases, antibody signal for all four receptors was blocked on addition of the immunogenic peptide (data not shown). A similar distribution of adenosine receptors was observed in mouse uroepithelium (data not shown).
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1 cm2 of tissue area). In isolated rabbit uroepithelium, capacitance primarily reflects the apical plasma membrane surface area of the umbrella cell layer (38) and increased capacitance correlates well with other measures of exocytosis (35). In control tissue, not exposed to adenosine, no change in capacitance was observed after 5 h (Fig. 4). However, addition of 1 µM adenosine to either the mucosal or serosal hemichamber resulted in an increase in membrane capacitance (Fig. 4). The increase in capacitance was linear over 5 h, with a change of 30% observed with addition to the serosal hemichamber (Fig. 4A) and a change of 24% with addition to the mucosal hemichamber (Fig. 4B). The EC50 for adenosine-induced changes in capacitance was similar for both serosal and mucosal surfaces of the tissue, 0.17 and 0.14 µM, respectively.
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In summary, the above results indicate that the uroepithelium is responsive to adenosine and that adenosine may increase exocytosis in the uroepithelium.
Multiple adenosine receptors may modulate umbrella cell exocytosis.
To assess which adenosine receptors were responsible for the changes in capacitance described above, we measured the ability of the following adenosine receptor-selective agonists to induce changes in membrane capacitance: the A1-selective agonist CCPA (A1 KD 0.2–1.3 nM), the A2a-selective agonist CGS-21680 (A2a KD 25 nM), and the A3-selective receptor agonist Cl-IB-MECA (A3 KD 1–10 nM) (12). At present, no A2b-selective agonists are available. Consistent with the immunofluorescence analysis presented above, the A1-selective agonist CCPA caused the most marked increase in capacitance on mucosal addition (40%), with an EC50 of 3 nM (Fig. 5A), a value similar to the reported KD (1 nM) this agonist has for A1 receptors (12). The A2a-selective agonist CGS-21680 induced an 10% change in capacitance, with an EC50 of 30 nM. Again, the observed EC50 was close to the KD (25 nM) reported for A2a receptors (Fig. 5B; Ref. 12). The A3-selective agonist Cl-IB-MECA was the least potent agonist, with an EC50 value of 700 nM, which is significantly larger than the reported KD (1–10 nM) (12). Cl-IB-MECA treatment resulted in an 10% capacitance change when added to the mucosal hemichamber (Fig. 5C).
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0.3 nM at the serosal surface vs. 3.0 nM at the mucosal surface) (Fig. 5D). However, the maximal change in capacitance was less than that observed on mucosal addition (20% vs. 40%). The most effective serosal agonist was CGS-21680, which caused an 30% change in capacitance with an EC50 of 35 nM (Fig. 5E). Cl-IB-MECA treatment resulted in a maximal 25% change in capacitance, with an EC50 of 190 nM (Fig. 5F). Again, the measured EC50 for CCPA and CGS-21680 approximated the measured KD of these agonists for A1/A2a receptors, whereas the EC50 for Cl-IB-MECA was significantly greater than the reported KD for this agonist and A3 receptors.
To further define the receptor subtypes responsible for the adenosine-dependent changes in capacitance, specific adenosine receptor antagonists were added in combination with adenosine (1 µM) to either the serosal or mucosal hemichamber. The receptor-selective antagonists used were DPCPX (A1 selective; Ki = 0.3–4.0 nM), ZM-241385 (A2a selective; Ki = 0.5 nM), MRS-1754 (A2b selective; Ki = 2.0 nM), and VUF 5574 (A3 selective; Ki = 4.0 nM) (12, 37). The Gaddum equation was used to calculate the concentration of antagonist that would ideally give a 
90% reduction in adenosine-induced changes in capacitance. The most effective antagonist, when added in combination with adenosine at the serosal surface, was the A2a-selective agonist ZM-241385, which inhibited adenosine-induced changes in capacitance by 50% (Fig. 6A). DPCPX, MRS-1754, and VUF 5574 also significantly decreased adenosine-induced changes in membrane capacitance by 25%. Whereas mucosal addition of ZM-241385, MRS-1754, and VUF 5574 did not significantly inhibit adenosine-induced changes in capacitance at the 5-h time point, the A1 receptor-selective antagonist DPCPX significantly decreased adenosine-induced effects by 80% (Fig. 6B).
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Stretched-induced changes in capacitance are modulated by, but do not require, adenosine.
We previously showed (35, 39) that the stretch associated with bladder filling is a physiologically relevant stimulus for exocytosis in the umbrella cell layer and that ATP release is an important upstream signal in this process. Whether other upstream stimuli modulate stretch-induced membrane turnover in the umbrella cells is unknown. We exposed isolated uroepithelium to increased hydrostatic pressure in the presence or absence of 1 µM CGS-15943, a broad-spectrum adenosine receptor antagonist (12). Increased pressure stimulated changes in capacitance of 50% (Fig. 7A). However, treatment with CGS-15943 had no significant effect on pressure-induced capacitance changes, and no significant effect was noted when the receptor-selective agonists DPCPX, ZM-241385, MRS-1754, and VUF 5574 were tested (Fig. 7A). Furthermore, deaminase had no significant effect on capacitance when added during the pressure stimulus (Fig. 7B). Intriguingly, when adenosine (1 µM) was added to either the serosal or mucosal surface of tissue exposed to increased pressure, it significantly potentiated both the kinetics of the resulting capacitance change and the plateau levels after 5-h incubation (Fig. 7C). These data indicate that although adenosine signaling is not required for pressure-induced changes in capacitance, adenosine may act to modulate the rate and the extent of these surface area changes.
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10% after 5 h (Fig. 8C), indicating that under these conditions agonists shifted the equilibrium between exocytosis and endocytosis so that the latter predominated. We also observed a potential role for extracellular Ca2+ in this process. When tissue was incubated in nominally Ca2+-free Krebs buffer, the adenosine-induced capacitance changes in capacitance were completely inhibited (Fig. 8, D and E). However, if Ca2+ was added back to the Ca2+-free Krebs solution after 4 h, there was a rapid increase in capacitance during the subsequent 1 h, indicating that the inhibition observed in Ca2+-free medium was rapidly reversible. In summary, both intracellular and extracellular Ca2+ may be essential for adenosine receptor-modulated umbrella cell exocytosis.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Adenosine biosynthesis occurs in uroepithelium.
Although adenosine is found in the urine (17, 36), it was unknown whether the bladder mucosa contributes to the pool of urinary adenosine. Our data indicate that adenosine may be produced in significant quantities by the uroepithelium. We observed adenosine biosynthesis under baseline conditions, which significantly increased in response to stretch. Under the latter condition maximum concentrations of adenosine in the mucosal chamber approached 50 nM, whereas in the serosal chamber they approached 200 nM. The actual concentration of adenosine near the uroepithelial cell membranes is difficult to discern because of the relatively large volume of our chamber system, unstirred layer effects, and the action of nucleoside transporters that may have caused us to underestimate adenosine biosynthesis at either or both tissue surfaces. Although our results indicate that the epithelium is the primary source of adenosine, there are scattered endothelial cells, fibroblasts, immune cells, and smooth muscle cells in the connective tissue underlying the epithelium that may contribute to the serosal adenosine release in vivo.
The mechanism of adenosine biosynthesis in the uroepithelium is unknown. Although the role of nucleoside transporters cannot be ruled out, it seems unlikely that hydrolysis of ATP by ecto/exonucleotidases plays a major role. We previously showed (39) that increased pressure stimulates ATP release from both surfaces of the uroepithelium, but mucosal release is 50-fold greater than serosal release. In contrast, we found that the majority of adenosine biosynthesis occurred at the serosal surface of pressure-treated tissue. Increased pressure stimulated inosine biosynthesis at both cell surfaces, but the mechanism of inosine biosynthesis is not clear as the experiments were performed in the presence of the adenosine deaminase inhibitor EHNA. It is possible that the inhibitory activity of EHNA was not complete, or that inosine was produced by some other mechanism, e.g., release from nucleotide transporters at the cell surface. Pressure also stimulated the biosynthesis of AMP at the mucosal surface of the tissue, possibly via the extracellular cAMP-adenosine pathway (19). In this pathway, cAMP that is produced in response to adenylyl cyclase activation is released by the cell and converted to adenosine by the serial action of ectophosphodiesterase (cAMP to AMP) and ecto-5'-nucleotidase (AMP to adenosine). Interestingly, cAMP production is rapidly increased within the uroepithelium in response to increased hydrostatic pressure, and cAMP is an important second messenger that stimulates exocytosis in the umbrella cell (35). Future experiments will define whether pressure stimulates release of cAMP into the extracellular fluid of umbrella cells and whether the extracellular cAMP-adenosine pathway contributes to AMP or adenosine biosynthesis by the uroepithelium.
Uroepithelium expresses multiple adenosine receptors. Gene expression of all four adenosine receptors has been described previously in the bladder (10), but the distribution of these receptors was unexplored until this analysis. A1 receptors were expressed at the apical pole of the umbrella cells and, to a lesser extent, at the basolateral surface of the umbrella cell and in the connective tissue underlying the uroepithelium. As noted below, functional A1 receptors may also be present on the serosal surface of the uroepithelium. Studies in cat indicate that A1 receptors may also be present on the detrusor muscle (41). Although we did not observe significant staining for A1 receptors in the muscle tissue of rat or mouse bladders (unpublished observations), we cannot rule out that other antibodies may give different staining patterns or that the distribution of A1 receptors is different in cat bladders.
In a similar fashion, functional A2a receptors have been detected on guinea pig detrusor (15), but our analysis indicated that A2a receptors were found predominantly on the uroepithelium and the underlying connective tissue elements but not the detrusor muscle. Again, this could reflect the antibodies used in this analysis and/or species differences. We observed significant A2b receptor expression along the basolateral surface of the umbrella cells, and, consistent with previous Northern blot analysis (33), we observed significant A2b protein expression in the detrusor muscle. Surprisingly, there are little data implicating A2b receptors in bladder detrusor function. A3 receptors were predominantly expressed along the umbrella cell basolateral membrane and, to a lesser extent, in the subepithelial connective tissue. The apical distribution of A1 receptors, the cytoplasmic distribution of A2a receptors, and the basolateral distribution of A2b and A3 receptors we observed in the umbrella cell is consistent with previous reports that A1 receptors are found on the apical surface of polarized Madin-Darby canine kidney cells, and the other receptors are found intracellularly or at the basolateral surface of these cells (30).
Regulation of exocytosis in uroepithelium by adenosine. Other than a small number of studies implicating adenosine receptor signaling in detrusor muscle contraction and innervation (15, 18, 22, 26, 41), little else is known about the function of adenosine in the bladder. By monitoring changes in capacitance we observed that adenosine, as well as adenosine receptor agonists, all stimulated exocytosis in the umbrella cell layer.
Consistent with the distribution of A1 receptors on the apical surface of the umbrella cell layer, the A1-selective agonist CCPA was the most potent stimulator of exocytosis when added to the mucosal surface of the tissue (EC50 3 nM). Furthermore, the A1-selective antagonist DPCPX was most effective at inhibiting adenosine-induced capacitance changes when added to the mucosal surface of the tissue. We also observed that the A2a-selective agonist CGS-21680 stimulated modest changes in capacitance (10%) with an EC50 of 30 nM, consistent with the reported KD for this receptor agonist (12). The lack of A3 receptor expression at the apical surface of the umbrella cells, the very high EC50 measured for the A3-selective agonist Cl-IB-MECA (700 nM), several hundredfold higher than the reported KD for this agonist (1–10 nM) (12), and the lack of effect of VUF 5574 make it unlikely that A3 receptors play a significant role in modulating exocytosis at the apical surface of the umbrella cells. Overall, the data indicate that A1 receptors are the predominant regulators of exocytosis at the mucosal surface and that A2a receptors may play a lesser role.
The most effective serosal agonist was the A2a-selective agonist CGS-21680, which caused an 30% change in capacitance. Furthermore, ZM-241385 was the most effective inhibitor of adenosine-induced capacitance at the serosal surface of the tissue. Surprisingly, the A1-selective agonist CCPA was the most potent agonist, with an EC50 of 0.3 nM. However, the maximal change in capacitance was less than that observed on mucosal addition (20% vs. 40%), and CCPA was less effective than CGS-21680 at promoting capacitance changes. One possibility is that there are smaller numbers of A1 receptors on the serosal surface of the tissue, which would explain why they were not readily detectable by immunofluorescence and why they are less effective at stimulating exocytosis at the serosal surface of the tissue. The A2b-selective antagonist MRS-1754 caused a significant inhibition of adenosine-induced capacitance changes at the serosal surface, implicating this receptor in adenosine-mediated regulation of exocytosis. The A3-selective agonist Cl-IB-MECA caused an 25% change in capacitance. However, the EC50 value for Cl-IB-MECA was significantly higher than the reported KD (200 nM vs. 1–10 nM) (12), possibly indicating that A3 receptors are not effective transducers of adenosine-mediated changes in exocytosis. The functional role of A3 receptors in the uroepithelium remains to be defined. In summary, A2a receptors are the major contributors to adenosine-induced changes in exocytosis at the serosal surface of tissue, with a lesser input from A1 and possibly A2b receptors.
An important issue is the apparent discrepancy between the measured KD of A1 receptors for adenosine (10 nM) in rat cortical membranes (21) and the EC50 of 140 nM we measured when adenosine was added to the mucosal surface of the uroepithelial tissue. One possibility is that there are multiple adenosine receptors active at the mucosal surface that give an aggregate response that is greater than the KD of an individual receptor. This is possible because both A1- and A2a-selective agonists gave responses when added to the mucosal surface. An alternative possibility is that the KD for rabbit A1 receptors is different from that for the rat receptor. In fact, there are known species differences in agonist affinity (12). An additional possibility is that adenosine turnover may decrease the effective concentration of the agonist in the bath. Although A1 receptors are resistant to downregulation, the high concentrations of adenosine in extracellular fluids (in the 50–200 nM range) and urine (17, 36) would likely lead to chronic activation and/or desensitization/downregulation of the A1 receptors unless significant adenosine turnover occurred in the tissue. Although the other receptors have lower affinities for adenosine, a similar requirement for adenosine turnover may exist.
Although A1/A3 receptors and A2a/A2b receptors often have opposing effects on cellular function (e.g., generation of cAMP), activation of A1, A2a, or A3 receptors in the uroepithelium resulted in increased capacitance, indicating that a common secondary messenger cascade acted downstream of receptor activation to regulate exocytosis. All four receptors are known to couple to PLC, which generates IP3 and can result in increased cytoplasmic Ca2+ (12, 23). Consistent with this mechanism we observed that inhibitors of PLC or IP3 receptor-dependent Ca2+ release pathways blocked adenosine-induced exocytosis. However, our data indicate that extracellular Ca2+ may also play a role, as incubation in Ca2+-free Krebs solution inhibited adenosine-induced exocytosis. Although adenosine is generally thought to inhibit voltage-sensitive channels in many tissues (12), in the uroepithelium adenosine could act to depolarize the cell, activating voltage-sensitive Ca2+ channels, which, in turn, would result in Ca2+-dependent Ca2+ release. Alternatively, depletion of Ca2+ from intracellular stores could active Ca2+ influx via plasma membrane-associated store-operated channels, a pathway commonly found in nonexcitable cells that couples PLC/IP3 pathways to influx of extracellular Ca2+ (28).
We also assessed whether adenosine played a role in the pressure-induced changes in exocytosis we measured previously (35, 38, 39). Although the uroepithelial tissue is responsive to low concentrations of adenosine and its agonists and the tissue can produce adenosine (especially in the presence of hydrostatic pressure), adenosine did not seem to be important for the basal levels of pressure-induced changes in capacitance. However, addition of exogenous adenosine promoted increased rates of capacitance change above those observed for tissue exposed to hydrostatic pressure alone. This additive effect could be explained if adenosine induced second messengers distinct from those generated by stretch or if the effects of stretch and adenosine may act in a synergistic manner to raise the amount of second messengers such as Ca2+ to levels greater than stretch or adenosine alone.
Finally, we note that the serosal surface of the tissue has numerous cell types (e.g., uroepithelial cells, endothelial cells, fibroblasts, macrophages, smooth muscle cells, and myofibroblasts), and we cannot rule out that adenosine binds to and stimulates release of secretagogues from these cell types that then indirectly stimulate exocytosis in the umbrella cell layer.
Adenosine as autocrine/paracrine regulator of uroepithelial function. Other than bladder filling, and the attendant activation of downstream signaling cascades such as cAMP and Ca2+ (35, 38), the nature of the physiological stimuli that modulate membrane traffic in the umbrella cell is poorly understood. We recently identified (39) extracellular ATP (released from the uroepithelium) as an important autocrine factor that acts as a proximal signal for both pressure-induced exocytosis and endocytosis. The data presented in this paper indicate that adenosine, acting through cell surface receptors, may also act in an autocrine/paracrine manner to regulate exocytosis in the umbrella cell layer both in response to, and independently of, bladder filling.
Exocytic and related endocytic pathways are crucial because they regulate turnover of the apical membrane and proteins that comprise the apical membrane barrier of the umbrella cell (2). Furthermore, exocytosis/endocytosis in umbrella cells may modulate the sensory function of the uroepithelium, a recently described function of the epithelium that allows it to communicate the state of the external environment to the underlying nervous system and possibly the musculature as well (4, 9). The uroepithelium is now known to release various transmitters including nitric oxide, acetylcholine, and ATP, and the uroepithelium expresses several neurotransmitter receptors including adrenergic, nicotinic, muscarinic, neurokinin, transient receptor potential channel 1 (VR1), P2X, and P2Y receptors (3–7, 9, 11) and now adenosine receptors (this paper). An important function of adenosine-induced membrane turnover could be to regulate the release of transmitters and modulate the density of receptors at the surface of the umbrella cell. Finally, although we have identified one function for adenosine in the uroepithelium, modulation of exocytic traffic, it is likely that adenosine will regulate other functions of the uroepithelium and bladder including ion transport, uroepithelial-afferent nerve signaling, and bladder contraction.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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