Feline interstitial cystitis results in mechanical hypersensitivity and altered - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:44 |只看该作者 |倒序浏览 |打印

作者：L. A. Birder,, S. R. Barrick, J. R. Roppolo, A. J. Kanai,, W. C. de Groat, S. Kiss, and C. A. Buffington作者单位：Departments of 1 Medicine-Laboratory of EpithelialCell Biology and 2 Pharmacology, University ofPittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213; and 3 Clinical Veterinary Sciences, Ohio State University,Columbus, Ohio 43210


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      【摘要】: q' c& ~6 O7 X+ k8 ^% D
   ATP can be released from a variety of cell types by mechanical stimulation;however, the mechanism for this release and the influence of pathology are notwell understood. The present study examined intracellular signaling mechanismsinvolved in swelling-evoked (exposure to a hypotonic solution) release of ATPin urothelial cells from normal cats and cats diagnosed with interstitialcystitis (feline interstitial cystitis; FIC). Using the luciferin-luciferasebioluminescent assay, we demonstrate that swelling-evoked ATP release issignificantly elevated in FIC cells. In both normal and FIC cells, ATP releasewas significantly decreased (mean 70% decrease) by application of blockers ofstretch-activated channels (amiloride or gadolinium), as well as brefeldin Aand monensin (mean 90% decrease), suggesting that ATP release occurs whenATP-containing vesicles fuse with the plasma membrane. Swelling-evoked releasewas reduced after removal of external calcium (65%), and release was blocked by incubation with BAPTA-AM or agents that interfere with internal calciumstores (caffeine, ryanodine, heparin, or 2-aminoethoxydiphenyl borate). Inaddition, agents known to act through inositol 1,4,5-triphosphate(IP 3 ) receptors (thapsigargin, acetylcholine) release significantlymore ATP in FIC compared with normal urothelium. Taken together, these resultssuggest that FIC results in a novel hypersensitivity to mechanical stimulithat may involve alterations in IP 3 -sensitive pathways.+ |2 h, y9 q9 t/ R! h* i: A

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mechanosensitivity; urinary bladder; inositol 1,4,5-triphosphate-sensitive pathways; exocytosis * g( J' w0 G, P
      【关键词】 interstitial cystitis mechanical hypersensitivity urothelium9 a+ E4 ~  m# a3 E3 d5 g6 m
   EPITHELIAL CELLS ARE OFTEN subjected to mechanical disturbances, such as shear force, changes in cell volume due to alterations in osmolarity,and mechanical stretch or distension( 14, 27 ). For example, epithelialcells that form the lining of the urinary bladder (uroepithelial cells) areconsistently subjected to mechanical stretch as well as changes in osmolarity during the course of a normal micturition cycle( 28 ). These changes may leadto a coordinated endocytosis and exocytosis at the cell membrane, changes inepithelial morphology, and release of bioactive substances such as nitricoxide or ATP ( 4, 28 ).

A number of studies have lent support for the idea that mechanical stimulican 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 inthe distension-evoked activation of bladder afferents( 14 ). Once released fromepithelial cells after bladder stretch, ATP is thought to activate purinergic receptors on submucosal afferent fibers and thus may play a role in sensoryfunctions such as nociception( 10 ). In fact, mice lackingthe P2X 3 purinergic receptor subunit (normally expressed by asubset of bladder afferents) exhibit normal distension-evoked urothelial ATPrelease but diminished reflex bladder contractions and voiding behavior and areduction in the behavioral (pain) response to injection of ATP( 30 ). Thus mechanically evokedATP release from epithelial cells may play a major role in both volume- andpain-mediated reflexes.
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Despite the growing interest in the sensory role of ATP, the mechanismsresponsible for mediating mechanically evoked ATP release are not wellunderstood. Several lines of evidence suggest that ATP release may involvemechanosensitive ATP channels, ATP transporters, or fusion of ATP-containingvesicles with the plasma membrane (i.e., exocytosis)( 6 ). It has been suggested thatthe latter may be in part a Ca 2  -dependent process, whereby increased intracellular Ca 2  can trigger secretion of ATP and otherbioactive mediators.% w6 f+ p! z+ d( X
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The release of algogenic agents such as ATP has been demonstrated aftertissue injury or inflammation( 13 ). Here, altered release ofATP from either injured or sensitized cells may play a direct role insensitizing nociceptors and thereby contribute to the initiation of pain andinflammatory responses. In models of rheumatoid arthritis, increased ATPlevels are thought to contribute to activation of nociceptors becauseantagonists of ATP reduce pain behavior( 12 ).  i- P. [- p9 O) i" Q. F

Although altered release of bioactive mediators such as ATP has beendemonstrated in pathological bladder conditions such as interstitial cystitis(IC) ( 26 ), the mechanism forthis altered release is unknown. IC is a chronic pelvic pain syndrome ofunknown cause and no generally accepted treatment( 24 ). Symptoms include painreferable to the urinary bladder and increased frequency and urgency ofurination. IC may affect more than 700,000 American women and a significantproportion of men with prostatitis or prostatodynia. A comparable disorder hasbeen found in domestic cats, and this syndrome, which exhibits many of thehallmarks of IC in patients, is termed feline interstitial cystitis (FIC)( 8 ). The aims of the presentstudy were to evaluate whether FIC alters swelling-induced ATP release from bladder urothelium. In addition, a number of agents were also evaluated toexamine the mechanisms for ATP release and for FIC-induced urothelialhypersensitivity to mechanical stimulation.8 V9 |; z5 P6 X% O& X
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MATERIALS AND METHODS

All procedures were conducted in accordance with Institutional Animal Careand Use Committee policies.
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Healthy and FIC adult cats were used for this study. All cats with FIC wereobtained as donations from clients due to a history of chronic recurrentstranguria, hematuria, pollakiuria, and/or urination in inappropriatelocations and were evaluated at The Ohio State University (OSU) VeterinaryTeaching Hospital. Evaluation consisted of a complete physical examination(including body weight), complete blood count, serum biochemical analysis, urinalysis, urine bacteriological culture, and cystoscopy. Cystoscopy wasperformed using a 9-F rigid pediatric cystoscope (Karl Storz, EndoscopyAmerica, Culver City, CA) in female cats and a 3-F flexible fiber opticcystoscope (Five Star Medical, San Jose, CA) in male cats. The diagnosis ofFIC was based on compatible history and consideration of standard NationalInstitutes of Health inclusion and exclusion criteria after the results of theabove laboratory tests were obtained, including the presence of submucosalpetechial hemorrhages (glomerulations) at cystoscopy ( 8 ). Healthy, age-matched catsobtained from commercial vendors and determined to be free of disease andsigns referable to the lower urinary tract according to the same diagnosticcriteria as cats with FIC were used as controls. All cats were housed instainless steel cages in the OSU animal facilities and allowed to acclimate totheir environment for at least 3 mo before the study.

Urothelial Cell Culture

Preparation and characterization of urothelial cultures have been describedin previous reports ( 2 ).Briefly, bladders were excised from deeply anesthetized ( -chloralose,60-70 mg/kg; 2% halothane) cats (of either sex), cut open, and gentlystretched (urothelial side up). Anesthesia was determined to be adequate forsurgery by periodic testing for the absence of a withdrawal reflex to a strongpinch of a hind paw and absence of an eyeblink reflex to tactile stimulationof the cornea. After tissue removal, all animals were killed via an overdoseof anesthetic. The tissue was incubated overnight in minimal essential medium (Cellgro, Mediatech, Herndon, VA), penicillin/streptomycin/fungizone, and 2.5mg/ml dispase (Invitrogen, Rockville, MD). The urothelium was gently scrapedfrom underlying tissue, treated with 0.25% trypsin, and resuspended inkeratinocyte medium (Invitrogen). The dissociated cell suspension (0.1 ml,50,000-150,000 cells/ml) was plated on the surface of collagen-coated dishesand 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 studywere 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 cytokeratinpositive (DAKO, Carpinteria, CA) and, therefore, were presumably of epithelialorigin.' Y3 c; y" M" Y* Y( x! j5 O4 H

Measurement of ATP Release& S1 @2 K4 w( M3 Q5 l2 \& M

In this study, we used exposure to a hypotonic solution as an in vitromethod for evoking mechanical stress. It has been established that hypotonicstress or swelling shares a number of common characteristics with mechanicalstretch or distension ( 15 ). Infact, our previous studies have demonstrated a similar release of ATP fromurothelium using both hypotonicity- and stretch-evoked paradigms( 4 ). The extracellular ATPconcentration was measured by using luciferin-luciferase bioluminescence ( 5 ). Cells were seeded oncollagen-coated culture plates, and each plate contained an average of10 5 cells before the experiment. The data are presented asfemtomoles ATP released in all cases, and values for each experiment werenormalized per milligram protein. Because a mechanical disturbance has beendemonstrated to alter release of ATP, all cells were carefully washed with oxygenated physiological solution containing (in mM) 4.8 KCl, 120 NaCl, 1.1MgCl 2, 2.0 CaCl 2, 11 glucose, and 10 HEPES (pH 7.4;25°C; 1 ml/min flow rate) until a stable baseline was achieved. The mediawas switched from isotonic to a hypotonic (244-260 mosM) Ringer solution, and100-µl samples were collected using a Retriever II automated fractioncollector (Isco, Lincoln, NE). The luciferin-luciferase reagent (100 µl; Adenosine Triphosphate Assay Kit, Sigma) was added to each sample, andbioluminescence was read using a luminometer (TD-20/20, Sunnyvale, CA). Thedetection limit was 10 fmol ATP/sample. The concentration of all agentstested 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 foundin most physiological media aggressively chelate Gd 3 , disablingits stretch-activated channel (SAC)-blocking ability and often leading tofalse-negative results ( 11 ),Gd 3  studies were performed with bicarbonate- and phosphate-freeHEPES-buffered solutions in all experimental and control groups. Unlessotherwise noted, all chemicals were obtained from Sigma and were of reagent grade or better.
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Data Analysis

Pooled data results are given as means ± SE, and statistical significance was determined using Student's unpaired t -test. Eachfigure represents data collected from a minimum of six independent experimentsfrom a minimum of six different cats. P & s& f) k# h* ^' O6 c4 r3 ^
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RESULTS4 A9 \4 C2 I& h0 A
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FIC Alters Swelling-Evoked, But Not Basal, ATP Release FromUrothelial Cells
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It has been well established that fluid movement during perfusion canelicit 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 ATPwas similar for both normal (20 ± 3 fmol ATP) and FIC (35 ± 5 fmol ATP) urothelial cells. In both normal and FIC urothelial cells, constantperfusion of a hypotonic solution (20% decrease in osmolarity; 244-260 mosM)elicited a substantial release of ATP compared with isotonic perfusion at thesame 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 significantdifferences in hypotonicity-evoked ATP release were detected between cells cultured from 1-3 days in either preparation (data not shown). Multipleapplications of hypotonic stimuli (applied to the same cells) elicited similarincreases 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 ethidiumhomodimer-1) enters through the damaged membrane of dead cells and binds tonucleic acids (produces a bright red fluorescence in dead cells), and 0.12µM calcein-AM, which is retained in live cells (producing a greenfluorescence 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 aconventional fluorescein long-pass filter. Afterward, live and dead cells werecounted in three random locations in each well (6 control and 6 afterhypotonic stimuli) in every experimental condition. Each experiment typically yielded one to two damaged cells/culture plate, demonstrating that cell lysisafter hypotonic swelling is not a contributing factor in ATP release( Fig. 1 ).
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Fig. 1. Swelling-evoked ATP release in normal and feline interstitital cystitis(FIC) urothelial cells. Graph depicts amount of swelling-evoked ATP release(fmol ATP release/mg protein) from isolated normal or FIC urinary bladderurothelial cells ( A ) as well as after passage and replating of cells[2 passages are depicted ( B and C )]. Values are means± SE from recordings in a minimum of 6 independent experiments.Photomicrograph ( B ) depicts a group of urothelial cells afterexposure to hypotonic stimuli. Arrow indicates a cell with damaged membrane,which exhibited a bright red ethidium homodimer-1-induced fluorescence. Inremaining cells, calcein dye is retained and produces a green fluorescence inlive cells. Ethidium homodimer-1 is excluded by the intact membrane of livecells. * P 0 w) `1 W. i) w' `7 E8 k
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Table 1. ATP release in normal or FIC urothelium
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FIC-Mediated Alterations in Swelling-Evoked ATP Release Are NotAltered by Cell Passage5 d4 u6 f2 e! Y

To evaluate whether the alterations in ATP release in FIC urothelium may bean inherent defect within the urothelium or possibly be a transient in vivoinsult to the urothelium that disappears in culture, ATP release with theapplication of hypotonic swelling was compared after passage and replating ofcells (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 cellscompared with normal cells also displayed a significantly greater release ofATP after swelling. All experiments were performed in similar numbers of cellsplated and used within 1-3 days after initial plating.
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Modulation of ATP Release2 H8 E# w% Y, {/ Y# s

Inhibition of SACs. Amiloride or Gd 3 , both of whichblock certain types of SACs, was tested on hypotonicity-evoked release of ATP.Application of either Gd 3  (10 µM; 20-min incubation) oramiloride (10 µM; 20-min incubation) significantly decreased ATP releasedby a hypotonic stimulus (60-75% maximal decrease; Fig. 2, Table 1 ) in both normal and FICurothelium. Although there was no significant difference in the effect of Gd 3  on FIC compared with normal urothelium, Gd 3  wasmore effective than amiloride in both preparations in decreasing hypotonicity-evoked ATP release.
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Fig. 2. Swelling-evoked ATP release from normal ( A ) or FIC urothelial( B ) cells is decreased by stretch-activated channel blockers. Graphdepicts stretch-evoked ATP release (Hypo; fmol ATP released/mg protein) afterincubation with amiloride (Amil; 10 µM; n = 6), Gd 3  (10 µM; n = 6), and inhibitors of transport brefeldin A (BFA; 10µM; n = 6) or monensin (MN; 10 µM; n = 6). Values aremeans ± SE. * P
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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 asbrefeldin A (BFA), which disrupts the Golgi complex and vesicle trafficking tothe cell surface, thereby blocking protein secretion( 19 ). Both monensin (10 µM)as well as BFA (10 µM) blocked hypotonicity-evoked release of ATP from bothnormal and FIC urothelial cells ( Fig.2, Table 1 ).

Role of Ca 2  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 thepossibility that it is a Ca 2  -dependent process. This was evaluatedby reducing external Ca 2  (0 Ca 2  /EGTA buffer; 1-hperfusion). Hypotonicity-evoked ATP release in both normal and FIC urotheliumwas significantly reduced (mean reduction, 65%) in 0 Ca 2 . Incontrast, reducing external Ca 2  did not alter basal release ineither normal or FIC urothelium. Longer perfusion times inCa 2  -free extracellular media did not further decrease ATP releasein either preparation. However, incubation with the Ca 2  chelatorBAPTA-AM (2 µM; 30 min; 0 Ca 2  ) resulted in a complete block ofswelling-evoked ATP release ( Table1, Fig.3 A ). The effect of elevating intracellular Ca 2  by incubation with the ionophore A-23187 (10 µM; 20 min)was also tested ( 3 ). In thepresence of Ca 2  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 differencebetween the two preparations.

Fig. 3. Role of Ca 2  in ATP release. A : swelling-evoked ATPrelease in normal or FIC urothelium is diminished in the absence ofextracellular Ca 2  {extracellular Ca 2  concentration([Ca 2  ] o ); 100 µM EGTA} and ablated by 2 µMBAPTA-AM. B : swelling-evoked ATP release in normal or FIC urotheliumis significantly reduced after incubation with ryanodine (Ryan; 10 µM) orcaffeine (Caf; 10 mM) in the absence of extracellular Ca 2 . C : swelling-evoked ATP release in normal or FIC urothelium issignificantly decreased after incubation with 2-aminoethoxydiphenyl borate(2-APB; 20 µM) or heparin (Hep; 10 µM) in the absence of extracellularCa 2 . Values are means ± SE from recordings in a minimum of6 independent experiments. * P : c. d- B0 P; `6 c- `" e1 p

The reduction of hypotonicity-evoked ATP release by elimination of externalCa 2  and complete blockade with BAPTA suggests an involvement ofinternal Ca 2  stores. The role of intracellular Ca 2  concentration was further evaluated by treating urothelial cells with caffeine(10 mM), which depletes intracellular Ca 2  stores( 22 ). Prolonged exposure tocaffeine (10 mM; in the absence of external Ca 2  ) diminished thestretch-evoked ATP concentration in both normal (79% decrease) and FIC (88辌rease) urothelial cells. Similar results were obtained after incubation with ryanodine (10 µM; 30 min) ( Table1, Fig.3 B ), a selective blocker of the ryanodine receptorCa 2  release channel ( 16 ). Both agents released alow concentration of ATP (20-40 fmol ATP) in the absence of hypotonicstimulation.

The effects of two agents, heparin and 2-aminoethoxydiphenyl borate(2-APB), known to interfere with inositol 1,4,5-triphosphate (IP 3 )receptor (IP 3 R) channels, were also tested. While either agentalone released a low amount of ATP (10-35 ± 7 fmol ATP), incubationwith either heparin (10 µM; 30 min) or 2-APB (20 µM, 20 min)significantly decreased hypotonicity-evoked ATP release( Table 1, Fig. 3 C ). The residual release [likely generated through release via ryanodine receptors (RyRs)] wasblocked after incubation with ryanodine (in the presence of 2-APB). Theseexperiments suggest hypotonicity-evoked ATP release involves two intracellularCa 2  components, release through both IP 3 and RyRs.5 G' L/ ]% }& ]; I4 U8 v* j3 P
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Enhanced ATP Release in FIC Urothelium May Be Due to AlteredIP 3 Sensitivity
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A brief incubation with the Ca 2  ionophore A-23187 (10-sexposure; in the absence of Ca 2  ), 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 testwhether intracellular Ca 2  or Ca 2  sensitivity isaltered in FIC, agents that stimulate IP 3 receptors were tested.Administration of the Ca 2  pump inhibitor thapsigargin (10 µM)in the absence of external Ca 2  or mechanical stimulation elicited a significantly larger ATP release in FIC (240 ± 25 fmol ATP) comparedwith normal (110 ± 18 fmol ATP) urothelium ( Table 1, Fig. 4 ). Another agent,acetylcholine (10 µM), known to stimulate the IP 3 pathway, alsoelicited ATP release ( Table 1, Fig. 4 ). This release wassignificantly elevated in FIC (190 ± 22 fmol ATP) compared with normalurothelium (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 aninvolvement of IP 3 R channels in this response.
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Fig. 4. Enhanced ATP release may be due to altered inositol 1,4,5-triphosphatesensitivity. A : brief addition of a Ca 2  ionophore(A-23187; 2 µM; 2 min) potentiates swelling-evoked ATP release in normalbut not in FIC urothelium. B : effect of either thapsigargin (Thap; 10µM) or acetylcholine (Ach; 10 µM) alone, in the absence of swelling orextracellular Ca 2 , released significantly greater ATP in FICcompared with normal urothelium. Values are means ± SE from recordingsin a minimum of 6 independent experiments. * P , A8 }5 P  J, o1 i; x  [
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DISCUSSION

This study established that urothelial cells from cat urinary bladderrelease ATP in response to hypotonic swelling and that this release isupregulated in cells from cats with FIC. An augmentation in ATP release evokedby mechanical stretching has been reported( 26 ). The similarity in theseobservations lends further support for the proposal that FIC is a naturally occurring model of human IC. Although the mechanism(s) by which mechanicalstimuli can evoke the release of ATP from urothelial or other epithelial celltypes is unclear, our findings demonstrate that ATP release occurs in part bya Ca 2  -dependent vesicular exocytotic process that involves anIP 3 signaling pathway which may be altered in FIC.$ d% R+ T* j; L$ s% N
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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 celltypes, including epithelial cells( 7 ). Although mechanicalstimulation produced by hypotonic swelling and direct stretch may not beentirely equivalent, our characterization of ATP release in urothelial cellshas suggested an involvement of SACs. The effectiveness of the SAC antagonistGd 3  or amiloride to diminish swelling-evoked ATP release in bothnormal and FIC urothelium supports this view. Lanthanides such asGd 3  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. Althoughamiloride has been used to block epithelial sodium channels (ENaCs) andGd 3  has been used to block SACs, both agents in highconcentrations have also been demonstrated to exert a nonselective block ofvoltage-gated and mechanogated ENaCs, Ca 2  channels, and numerousnonselective cation channels ( 15 ). Therefore, theinterpretation of the blocking effects of these agents in the presentexperiments might be complex. The effectiveness of both blockers to decreaseATP release provides support for a multistep process linkingmechanotransduction and exocytosis in ATP release from bladder urothelium.
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A possible role of vesicular exocytosis in ATP release was examined byevaluating the effects of agents that disrupt the Golgi complex and therebyblock membrane/protein trafficking. BFA blocks protein secretion from cells bysuppressing vesicular transport between the Golgi complex and the endoplasmicreticulum ( 17 ). BFA treatmentblocked hypotonicity-evoked ATP release from urothelial cells. Monensin, whichis a sodium ionophore that inhibits vesicle formation and can also preventreceptor recycling ( 6 ), had asimilar effect.9 }% _% |: L/ Q' q, i

Our studies also revealed that removal of extracellular Ca 2  suppressed ATP release, whereas chelation of free intracellular Ca 2  with BAPTA-AM abolished it. In addition, elevation of intracellular Ca 2  with the ionophore A-23187 (10 µM) evoked ATPrelease as noted in other cell types( 25 ), including endotheliumand epithelium. These results suggest that swelling-mediated ATP release isdue to both Ca 2  influx and release from intracellular stores.

At least two types of Ca 2  release channels, including those that are ryanodine and IP 3 sensitive, seem to be involved in ATPrelease. Hypotonicity-evoked ATP release was attenuated but not completelyblocked by incubation with ryanodine (a selective blocker of RyRs) orcaffeine, an agent that activates ryanodine-sensitive Ca 2  releasechannels and depletes Ca 2  stores. Hypotonicity-evoked ATP releasewas also reduced but not completely blocked by heparin, a potent blocker ofIP 3 Rs, as well as by the IP 3 R antagonist 2-APB. Thelatter was more effective in FIC urothelial cells compared with normalurothelial cells, although a residual response ( 20% in FIC) stillremained. The residual (IP 3 insensitive) component was completelyblocked by ryanodine and thus corresponds to ATP release via RyRs. In somecell types, heparin activates RyRs, raising the possibility that otherpathways in addition to those sensitive to IP 3 are involved in ATPrelease.

These findings suggest that the increased ATP release in FIC may involveactivation of the IP 3 signaling pathway. This was tested byapplying thapsigargin, a sarco-endoplasmic reticulum Ca 2  -ATPaseinhibitor, which causes depletion of intracellular Ca 2  stores. Inthe absence of external Ca 2  and mechanical stimuli, thapsigarginelicited a robust release of ATP, which was significantly greater in FICurothelial cells compared with normal cells. Furthermore, theIP 3 -linked muscarinic agonist acetylcholine produced asignificantly greater ATP release in FIC compared with normal urothelium, andthis release was inhibited by the IP 3 R antagonist 2-APB.2 {1 d8 x; U3 Z9 ^  l
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A change in the sensitivity of ATP release to intracellular Ca 2  was also evident in FIC cells when the effects of the Ca 2  ionophore A-23187 were examined. Administration of a high concentration of theionophore alone evoked ATP release to a similar extent in both FIC and normalurothelial cells, suggesting that the sensitivity to Ca 2  is notchanged. In contrast, a brief application of the ionophore in the presence ofhypotonic swelling potentiated an evoked release of ATP in normal but not inFIC urothelium. These data, taken together with the enhancement of ATP releaseby thapsigargin or acetylcholine, suggest that FIC may involve alterations inIP 3 -sensitive pathways. It is possible that this change may occuras a result of environmental conditions or an inherent defect within theurothelial cells. For example, inflammation has been shown to alter theexpression of a number of Ca 2  -sensor proteins, involved inregulating transmitter exocytosis( 18, 29 ). In the presentexperiments, the urothelial hypersensitivity does not diminish after cell passage, suggesting that this abnormality may be inherent within theurothelium and not due to an effect of inflammation or injury. Whileuncovering the mechanism for the alterations in swelling-mediated ATP releaserequires further experimentation, these results together suggest that FICresults in abnormalities in a Ca 2  -dependent exocytoticpathway.5 [  I" T( d5 e5 m7 h* t' ?

What is the significance of urothelial hypersensitivity leading to alteredATP release? It has been shown that distension or changes in pressure canrelease ATP from epithelial cells lining various organs such as the urinarybladder and that this has the potential to activate purinergic(P2X 3 and/or P2X 2/3 ) receptors on submucosal afferentsin close proximity to the epithelium. Distension-evoked activation of bladderafferents can be reduced by purinergic antagonists including suramin orpyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid( 20, 30 ). In addition, recentstudies have demonstrated that ATP administered intravesically induces urinarybladder hyperactivity, most likely by affecting submucosal C fibers ( 21 ). These and other datalend support for the idea that bladder afferents, which lie in close proximityto epithelial cells, may play a role in "neural-epithelial"function. Taken together, the results support the idea that increased levelsof ATP released from sensitized urothelial cells after distension may activate submucosal purinergic afferents, leading to bladder pain in IC.
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Augmented ATP release from urothelial cells could also trigger changes incell-cell signaling within the urothelium. In a number of cell types,mechanical stimulation induces an immediate and transient elevation ofCa 2  (mediated by P2 purinergic receptors) that quickly spreads toneighboring cells ( 17 ). Support for the idea that ATP release may play an autocrine role in urothelialfunction comes from evidence that urothelial cells also exhibit purinergic(P2) receptors ( 10 ). Thuspropagation of intercellular Ca 2  "waves" could occurdue to release of a diffusible mediator, such as ATP. In fact, triggering of Ca 2  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 Ca 2  fromintracellular stores in small discrete regions may have little effect on global cytoplasmic Ca 2  concentrations, it may lead to abnormal local changes in Ca 2  concentration. In some types of cells, thislocal release of Ca 2 , which has been termed Ca 2  "sparks" or "puffs," is known to activatechannels/processes in close proximity to the release site. Thus augmenteddistention-evoked secretion of ATP from urothelial cells in IC may lead to an autocrine activation of urothelial purinergic receptors and the initiation ofCa 2  waves. Further studies are needed to evaluate the role ofurothelial ATP release in both neural-epithelial as well as interepithelialsignaling within the urinary bladder.
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In conclusion, our findings show that IC in cats results in an alteredswelling-evoked release of ATP from urothelium. These changes in urothelialhyper-sensitivity may lead to sensory and urothelial deficits and may play arole in bladder pathologies such as IC.) o2 B' ?1 Y0 G% }- E! \
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DISCLOSURES
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This work was supported by National Institutes of Health Grants R01-DK-54824 and R01-DK-57284 (L. A. Birder), R01-NS-045078 (J. R. Roppolo),and R01-DK-47938 (C. A. Buffington).. y; U. x$ H3 a( c. F9 t
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The results of this study have been presented elsewhere in abstract form( 9 ).
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ACKNOWLEDGMENTS

The authors thank C. Tai for assistance.5 \$ E, V1 i- V' {$ d3 H
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