Departments of 1Medicine-Laboratory of Epithelial Cell Biology and 2Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213; and 3Clinical Veterinary Sciences, Ohio State University, Columbus, Ohio 43210
Submitted 10 February 2003 ; accepted in final form 18 May 2003
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ABSTRACT |
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mechanosensitivity; urinary bladder; inositol 1,4,5-triphosphate-sensitive pathways; exocytosis
A number of studies have lent support for the idea that mechanical stimuli can evoke the release of ATP from epithelial cells lining "tubes" or "sacs" such as the urinary bladder (10). In addition, extracellular ATP, most likely of urothelial origin, has been implicated in the distension-evoked activation of bladder afferents (14). Once released from epithelial cells after bladder stretch, ATP is thought to activate purinergic receptors on submucosal afferent fibers and thus may play a role in sensory functions such as nociception (10). In fact, mice lacking the P2X3 purinergic receptor subunit (normally expressed by a subset of bladder afferents) exhibit normal distension-evoked urothelial ATP release but diminished reflex bladder contractions and voiding behavior and a reduction in the behavioral (pain) response to injection of ATP (30). Thus mechanically evoked ATP release from epithelial cells may play a major role in both volume- and pain-mediated reflexes.
Despite the growing interest in the sensory role of ATP, the mechanisms responsible for mediating mechanically evoked ATP release are not well understood. Several lines of evidence suggest that ATP release may involve mechanosensitive ATP channels, ATP transporters, or fusion of ATP-containing vesicles with the plasma membrane (i.e., exocytosis) (6). It has been suggested that the latter may be in part a Ca2+-dependent process, whereby increased intracellular Ca2+ can trigger secretion of ATP and other bioactive mediators.
The release of algogenic agents such as ATP has been demonstrated after tissue injury or inflammation (13). Here, altered release of ATP from either injured or sensitized cells may play a direct role in sensitizing nociceptors and thereby contribute to the initiation of pain and inflammatory responses. In models of rheumatoid arthritis, increased ATP levels are thought to contribute to activation of nociceptors because antagonists of ATP reduce pain behavior (12).
Although altered release of bioactive mediators such as ATP has been demonstrated in pathological bladder conditions such as interstitial cystitis (IC) (26), the mechanism for this altered release is unknown. IC is a chronic pelvic pain syndrome of unknown cause and no generally accepted treatment (24). Symptoms include pain referable to the urinary bladder and increased frequency and urgency of urination. IC may affect more than 700,000 American women and a significant proportion of men with prostatitis or prostatodynia. A comparable disorder has been found in domestic cats, and this syndrome, which exhibits many of the hallmarks of IC in patients, is termed feline interstitial cystitis (FIC) (8). The aims of the present study were to evaluate whether FIC alters swelling-induced ATP release from bladder urothelium. In addition, a number of agents were also evaluated to examine the mechanisms for ATP release and for FIC-induced urothelial hypersensitivity to mechanical stimulation.
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MATERIALS AND METHODS |
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Animals
Healthy and FIC adult cats were used for this study. All cats with FIC were obtained as donations from clients due to a history of chronic recurrent stranguria, hematuria, pollakiuria, and/or urination in inappropriate locations and were evaluated at The Ohio State University (OSU) Veterinary Teaching Hospital. Evaluation consisted of a complete physical examination (including body weight), complete blood count, serum biochemical analysis, urinalysis, urine bacteriological culture, and cystoscopy. Cystoscopy was performed using a 9-F rigid pediatric cystoscope (Karl Storz, Endoscopy America, Culver City, CA) in female cats and a 3-F flexible fiber optic cystoscope (Five Star Medical, San Jose, CA) in male cats. The diagnosis of FIC was based on compatible history and consideration of standard National Institutes of Health inclusion and exclusion criteria after the results of the above laboratory tests were obtained, including the presence of submucosal petechial hemorrhages (glomerulations) at cystoscopy (8). Healthy, age-matched cats obtained from commercial vendors and determined to be free of disease and signs referable to the lower urinary tract according to the same diagnostic criteria as cats with FIC were used as controls. All cats were housed in stainless steel cages in the OSU animal facilities and allowed to acclimate to their environment for at least 3 mo before the study.
Urothelial Cell Culture
Preparation and characterization of urothelial cultures have been described
in previous reports (2).
Briefly, bladders were excised from deeply anesthetized (-chloralose,
60-70 mg/kg; 2% halothane) cats (of either sex), cut open, and gently
stretched (urothelial side up). Anesthesia was determined to be adequate for
surgery by periodic testing for the absence of a withdrawal reflex to a strong
pinch of a hind paw and absence of an eyeblink reflex to tactile stimulation
of the cornea. After tissue removal, all animals were killed via an overdose
of anesthetic. The tissue was incubated overnight in minimal essential medium
(Cellgro, Mediatech, Herndon, VA), penicillin/streptomycin/fungizone, and 2.5
mg/ml dispase (Invitrogen, Rockville, MD). The urothelium was gently scraped
from underlying tissue, treated with 0.25% trypsin, and resuspended in
keratinocyte medium (Invitrogen). The dissociated cell suspension (0.1 ml,
50,000-150,000 cells/ml) was plated on the surface of collagen-coated dishes
and maintained in culture for 1-3 days. Because long-term maintenance of cells
in culture could significantly change the properties of some types of cells
(1), the cells in this study
were examined after a short time in culture. In general, all cells were used
within the first 3 days after plating. All cells in culture were cytokeratin
positive (DAKO, Carpinteria, CA) and, therefore, were presumably of epithelial
origin.
Measurement of ATP Release
In this study, we used exposure to a hypotonic solution as an in vitro
method for evoking mechanical stress. It has been established that hypotonic
stress or swelling shares a number of common characteristics with mechanical
stretch or distension (15). In
fact, our previous studies have demonstrated a similar release of ATP from
urothelium using both hypotonicity- and stretch-evoked paradigms
(4). The extracellular ATP
concentration was measured by using luciferin-luciferase bioluminescence
(5). Cells were seeded on
collagen-coated culture plates, and each plate contained an average of
105 cells before the experiment. The data are presented as
femtomoles ATP released in all cases, and values for each experiment were
normalized per milligram protein. Because a mechanical disturbance has been
demonstrated to alter release of ATP, all cells were carefully washed with
oxygenated physiological solution containing (in mM) 4.8 KCl, 120 NaCl, 1.1
MgCl2, 2.0 CaCl2, 11 glucose, and 10 HEPES (pH 7.4;
25°C; 1 ml/min flow rate) until a stable baseline was achieved. The media
was switched from isotonic to a hypotonic (244-260 mosM) Ringer solution, and
100-µl samples were collected using a Retriever II automated fraction
collector (Isco, Lincoln, NE). The luciferin-luciferase reagent (100 µl;
Adenosine Triphosphate Assay Kit, Sigma) was added to each sample, and
bioluminescence was read using a luminometer (TD-20/20, Sunnyvale, CA). The
detection limit was 10 fmol ATP/sample. The concentration of all agents
tested was chosen based on effectiveness in other cell types (epithelial,
endothelial, or smooth muscle). All antagonists were incubated for a minimum
of 10 min before sample collection. Because phosphates and bicarbonates found
in most physiological media aggressively chelate Gd3+, disabling
its stretch-activated channel (SAC)-blocking ability and often leading to
false-negative results (11),
Gd3+ studies were performed with bicarbonate- and phosphate-free
HEPES-buffered solutions in all experimental and control groups. Unless
otherwise noted, all chemicals were obtained from Sigma and were of reagent
grade or better.
Data Analysis
Pooled data results are given as means ± SE, and statistical
significance was determined using Student's unpaired t-test. Each
figure represents data collected from a minimum of six independent experiments
from a minimum of six different cats. P 0.05 was regarded as
significant.
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RESULTS |
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It has been well established that fluid movement during perfusion can elicit release of ATP due to effects of shear force. In either preparation, slow perfusion elicited a small but not significant (P > 0.05) increase in the amount of ATP over nonperfused baseline. Basal release of ATP was similar for both normal (20 ± 3 fmol ATP) and FIC (35 ± 5 fmol ATP) urothelial cells. In both normal and FIC urothelial cells, constant perfusion of a hypotonic solution (20% decrease in osmolarity; 244-260 mosM) elicited a substantial release of ATP compared with isotonic perfusion at the same flow rate. In contrast to normal urothelial cells, FIC urothelium exposed to the same treatment released a significantly larger amount of ATP [285 ± 45 (FIC) vs. 140 ± 22 fmol ATP (normal)] (Fig. 1, Table 1). No significant differences in hypotonicity-evoked ATP release were detected between cells cultured from 1-3 days in either preparation (data not shown). Multiple applications of hypotonic stimuli (applied to the same cells) elicited similar increases in ATP (range 125-165 fmol ATP in normal; 260-340 fmol ATP in FIC). To test for cell viability and the possible contribution of cell lysis to ATP release after hypotonic tests, two dyes, one of which (0.23 µM ethidium homodimer-1) enters through the damaged membrane of dead cells and binds to nucleic acids (produces a bright red fluorescence in dead cells), and 0.12 µM calcein-AM, which is retained in live cells (producing a green fluorescence in live cells), were added to cultures before hypotonic swelling (Live/Dead viability/cytotoxicity assay kit, Molecular Probes, Eugene, OR). Both calcein and ethidium homodimer-1 can be viewed simultaneously using a conventional fluorescein long-pass filter. Afterward, live and dead cells were counted in three random locations in each well (6 control and 6 after hypotonic stimuli) in every experimental condition. Each experiment typically yielded one to two damaged cells/culture plate, demonstrating that cell lysis after hypotonic swelling is not a contributing factor in ATP release (Fig. 1).
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FIC-Mediated Alterations in Swelling-Evoked ATP Release Are Not Altered by Cell Passage
To evaluate whether the alterations in ATP release in FIC urothelium may be an inherent defect within the urothelium or possibly be a transient in vivo insult to the urothelium that disappears in culture, ATP release with the application of hypotonic swelling was compared after passage and replating of cells (up to 3 passages were tested). In each passage, hypotonic swelling evoked an increase in ATP concentration similar to that in the first culture (Fig. 1). Passaged FIC cells compared with normal cells also displayed a significantly greater release of ATP after swelling. All experiments were performed in similar numbers of cells plated and used within 1-3 days after initial plating.
Modulation of ATP Release
Inhibition of SACs. Amiloride or Gd3+, both of which block certain types of SACs, was tested on hypotonicity-evoked release of ATP. Application of either Gd3+ (10 µM; 20-min incubation) or amiloride (10 µM; 20-min incubation) significantly decreased ATP released by a hypotonic stimulus (60-75% maximal decrease; Fig. 2, Table 1) in both normal and FIC urothelium. Although there was no significant difference in the effect of Gd3+ on FIC compared with normal urothelium, Gd3+ was more effective than amiloride in both preparations in decreasing hypotonicity-evoked ATP release.
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Inhibition of vesicular exocytosis or trafficking. Stretch-activated release of ATP may also be a result of vesicular exocytosis. We evaluated both an inhibitor of vesicle formation, monensin, as well as brefeldin A (BFA), which disrupts the Golgi complex and vesicle trafficking to the cell surface, thereby blocking protein secretion (19). Both monensin (10 µM) as well as BFA (10 µM) blocked hypotonicity-evoked release of ATP from both normal and FIC urothelial cells (Fig. 2, Table 1).
Role of Ca2+ in ATP Release
The inhibitory effect of monensin and BFA suggests that swelling-mediated release of ATP is mediated in part by vesicular exocytosis and raises the possibility that it is a Ca2+-dependent process. This was evaluated by reducing external Ca2+ (0 Ca2+/EGTA buffer; 1-h perfusion). Hypotonicity-evoked ATP release in both normal and FIC urothelium was significantly reduced (mean reduction, 65%) in 0 Ca2+. In contrast, reducing external Ca2+ did not alter basal release in either normal or FIC urothelium. Longer perfusion times in Ca2+-free extracellular media did not further decrease ATP release in either preparation. However, incubation with the Ca2+ chelator BAPTA-AM (2 µM; 30 min; 0 Ca2+) resulted in a complete block of swelling-evoked ATP release (Table 1, Fig. 3A). The effect of elevating intracellular Ca2+ by incubation with the ionophore A-23187 (10 µM; 20 min) was also tested (3). In the presence of Ca2+ but in the absence of stretch or other stimuli, the ionophore alone released a significant amount of ATP (230 ± 49 fmol ATP) from both normal and FIC urothelial cells, with no significant difference between the two preparations.
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The reduction of hypotonicity-evoked ATP release by elimination of external Ca2+ and complete blockade with BAPTA suggests an involvement of internal Ca2+ stores. The role of intracellular Ca2+ concentration was further evaluated by treating urothelial cells with caffeine (10 mM), which depletes intracellular Ca2+ stores (22). Prolonged exposure to caffeine (10 mM; in the absence of external Ca2+) diminished the stretch-evoked ATP concentration in both normal (79% decrease) and FIC (88% decrease) urothelial cells. Similar results were obtained after incubation with ryanodine (10 µM; 30 min) (Table 1, Fig. 3B), a selective blocker of the ryanodine receptor Ca2+ release channel (16). Both agents released a low concentration of ATP (20-40 fmol ATP) in the absence of hypotonic stimulation.
The effects of two agents, heparin and 2-aminoethoxydiphenyl borate (2-APB), known to interfere with inositol 1,4,5-triphosphate (IP3) receptor (IP3R) channels, were also tested. While either agent alone released a low amount of ATP (10-35 ± 7 fmol ATP), incubation with either heparin (10 µM; 30 min) or 2-APB (20 µM, 20 min) significantly decreased hypotonicity-evoked ATP release (Table 1, Fig. 3C). The residual release [likely generated through release via ryanodine receptors (RyRs)] was blocked after incubation with ryanodine (in the presence of 2-APB). These experiments suggest hypotonicity-evoked ATP release involves two intracellular Ca2+ components, release through both IP3 and RyRs.
Enhanced ATP Release in FIC Urothelium May Be Due to Altered IP3 Sensitivity
A brief incubation with the Ca2+ ionophore A-23187 (10-s exposure; in the absence of Ca2+), which did not release ATP alone, potentiated hypotonicity-evoked ATP release in normal urothelium (mean 300%) but did not alter release in FIC urothelium (Fig. 4). To further test whether intracellular Ca2+ or Ca2+ sensitivity is altered in FIC, agents that stimulate IP3 receptors were tested. Administration of the Ca2+ pump inhibitor thapsigargin (10 µM) in the absence of external Ca2+ or mechanical stimulation elicited a significantly larger ATP release in FIC (240 ± 25 fmol ATP) compared with normal (110 ± 18 fmol ATP) urothelium (Table 1, Fig. 4). Another agent, acetylcholine (10 µM), known to stimulate the IP3 pathway, also elicited ATP release (Table 1, Fig. 4). This release was significantly elevated in FIC (190 ± 22 fmol ATP) compared with normal urothelium (85 ± 16 fmol ATP) (Fig. 4), and this release was significantly decreased [78% decrease; 42 ± 6 (FIC) vs. 18 ± 6 fmol ATP (normal)] by 2-APB, suggesting an involvement of IP3R channels in this response.
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DISCUSSION |
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Our results are in agreement with previous reports that hypotonic swelling, a widely used model of mechanical stimulation, releases ATP from various cell types, including epithelial cells (7). Although mechanical stimulation produced by hypotonic swelling and direct stretch may not be entirely equivalent, our characterization of ATP release in urothelial cells has suggested an involvement of SACs. The effectiveness of the SAC antagonist Gd3+ or amiloride to diminish swelling-evoked ATP release in both normal and FIC urothelium supports this view. Lanthanides such as Gd3+ have been demonstrated to inhibit stretch-evoked changes in cell volume and can also inhibit SACs (31), which have been implicated in ATP release from other types of epithelial cells. Although amiloride has been used to block epithelial sodium channels (ENaCs) and Gd3+ has been used to block SACs, both agents in high concentrations have also been demonstrated to exert a nonselective block of voltage-gated and mechanogated ENaCs, Ca2+ channels, and numerous nonselective cation channels (15). Therefore, the interpretation of the blocking effects of these agents in the present experiments might be complex. The effectiveness of both blockers to decrease ATP release provides support for a multistep process linking mechanotransduction and exocytosis in ATP release from bladder urothelium.
A possible role of vesicular exocytosis in ATP release was examined by evaluating the effects of agents that disrupt the Golgi complex and thereby block membrane/protein trafficking. BFA blocks protein secretion from cells by suppressing vesicular transport between the Golgi complex and the endoplasmic reticulum (17). BFA treatment blocked hypotonicity-evoked ATP release from urothelial cells. Monensin, which is a sodium ionophore that inhibits vesicle formation and can also prevent receptor recycling (6), had a similar effect.
Our studies also revealed that removal of extracellular Ca2+ suppressed ATP release, whereas chelation of free intracellular Ca2+ with BAPTA-AM abolished it. In addition, elevation of intracellular Ca2+ with the ionophore A-23187 (10 µM) evoked ATP release as noted in other cell types (25), including endothelium and epithelium. These results suggest that swelling-mediated ATP release is due to both Ca2+ influx and release from intracellular stores.
At least two types of Ca2+ release channels, including those
that are ryanodine and IP3 sensitive, seem to be involved in ATP
release. Hypotonicity-evoked ATP release was attenuated but not completely
blocked by incubation with ryanodine (a selective blocker of RyRs) or
caffeine, an agent that activates ryanodine-sensitive Ca2+ release
channels and depletes Ca2+ stores. Hypotonicity-evoked ATP release
was also reduced but not completely blocked by heparin, a potent blocker of
IP3Rs, as well as by the IP3R antagonist 2-APB. The
latter was more effective in FIC urothelial cells compared with normal
urothelial cells, although a residual response (20% in FIC) still
remained. The residual (IP3 insensitive) component was completely
blocked by ryanodine and thus corresponds to ATP release via RyRs. In some
cell types, heparin activates RyRs, raising the possibility that other
pathways in addition to those sensitive to IP3 are involved in ATP
release.
These findings suggest that the increased ATP release in FIC may involve activation of the IP3 signaling pathway. This was tested by applying thapsigargin, a sarco-endoplasmic reticulum Ca2+-ATPase inhibitor, which causes depletion of intracellular Ca2+ stores. In the absence of external Ca2+ and mechanical stimuli, thapsigargin elicited a robust release of ATP, which was significantly greater in FIC urothelial cells compared with normal cells. Furthermore, the IP3-linked muscarinic agonist acetylcholine produced a significantly greater ATP release in FIC compared with normal urothelium, and this release was inhibited by the IP3R antagonist 2-APB.
A change in the sensitivity of ATP release to intracellular Ca2+ was also evident in FIC cells when the effects of the Ca2+ ionophore A-23187 were examined. Administration of a high concentration of the ionophore alone evoked ATP release to a similar extent in both FIC and normal urothelial cells, suggesting that the sensitivity to Ca2+ is not changed. In contrast, a brief application of the ionophore in the presence of hypotonic swelling potentiated an evoked release of ATP in normal but not in FIC urothelium. These data, taken together with the enhancement of ATP release by thapsigargin or acetylcholine, suggest that FIC may involve alterations in IP3-sensitive pathways. It is possible that this change may occur as a result of environmental conditions or an inherent defect within the urothelial cells. For example, inflammation has been shown to alter the expression of a number of Ca2+-sensor proteins, involved in regulating transmitter exocytosis (18, 29). In the present experiments, the urothelial hypersensitivity does not diminish after cell passage, suggesting that this abnormality may be inherent within the urothelium and not due to an effect of inflammation or injury. While uncovering the mechanism for the alterations in swelling-mediated ATP release requires further experimentation, these results together suggest that FIC results in abnormalities in a Ca2+-dependent exocytotic pathway.
What is the significance of urothelial hypersensitivity leading to altered ATP release? It has been shown that distension or changes in pressure can release ATP from epithelial cells lining various organs such as the urinary bladder and that this has the potential to activate purinergic (P2X3 and/or P2X2/3) receptors on submucosal afferents in close proximity to the epithelium. Distension-evoked activation of bladder afferents can be reduced by purinergic antagonists including suramin or pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (20, 30). In addition, recent studies have demonstrated that ATP administered intravesically induces urinary bladder hyperactivity, most likely by affecting submucosal C fibers (21). These and other data lend support for the idea that bladder afferents, which lie in close proximity to epithelial cells, may play a role in "neural-epithelial" function. Taken together, the results support the idea that increased levels of ATP released from sensitized urothelial cells after distension may activate submucosal purinergic afferents, leading to bladder pain in IC.
Augmented ATP release from urothelial cells could also trigger changes in cell-cell signaling within the urothelium. In a number of cell types, mechanical stimulation induces an immediate and transient elevation of Ca2+ (mediated by P2 purinergic receptors) that quickly spreads to neighboring cells (17). Support for the idea that ATP release may play an autocrine role in urothelial function comes from evidence that urothelial cells also exhibit purinergic (P2) receptors (10). Thus propagation of intercellular Ca2+ "waves" could occur due to release of a diffusible mediator, such as ATP. In fact, triggering of Ca2+ waves within epithelial cells is thought to be a key element in cell signaling, excitability, proliferation, and even cell death (23).
While it has been established that release of Ca2+ from intracellular stores in small discrete regions may have little effect on global cytoplasmic Ca2+ concentrations, it may lead to abnormal local changes in Ca2+ concentration. In some types of cells, this local release of Ca2+, which has been termed Ca2+ "sparks" or "puffs," is known to activate channels/processes in close proximity to the release site. Thus augmented distention-evoked secretion of ATP from urothelial cells in IC may lead to an autocrine activation of urothelial purinergic receptors and the initiation of Ca2+ waves. Further studies are needed to evaluate the role of urothelial ATP release in both neural-epithelial as well as interepithelial signaling within the urinary bladder.
In conclusion, our findings show that IC in cats results in an altered swelling-evoked release of ATP from urothelium. These changes in urothelial hyper-sensitivity may lead to sensory and urothelial deficits and may play a role in bladder pathologies such as IC.
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DISCLOSURES |
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The results of this study have been presented elsewhere in abstract form (9).
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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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REFERENCES |
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