serotonergic regulation of distention-induced atp release ......serotonergic regulation of...

Serotonergic regulation of distention-induced ATP release from theurothelium

Kazumasa Matsumoto-Miyai,1,2,3 Erika Yamada,1 Eriko Shinzawa,1 Yoshihisa Koyama,3 Shoichi Shimada,3

Masaru Yoshizumi,1 and Masahito Kawatani11Department of Neurophysiology, Akita University Graduate School of Medicine, Akita, Japan; 2Kansai University of Nursingand Health Sciences, Hyogo, Japan; and 3Department of Neuroscience and Cell Biology, Osaka University Graduate Schoolof Medicine, Osaka, Japan

Submitted 23 January 2015; accepted in final form 9 November 2015

Matsumoto-Miyai K, Yamada E, Shinzawa E, Koyama Y,Shimada S, Yoshizumi M, Kawatani M. Serotonergic regulation ofdistention-induced ATP release from the urothelium. Am J PhysiolRenal Physiol 310: F646–F655, 2016. First published November 18,2015; doi:10.1152/ajprenal.00024.2015.—Serotonin [5-hydroxytryp-tamine (5-HT)] is involved in both motor and sensory functions inhollow organs, especially in the gastrointestinal tract. However, theinvolvement of 5-HT in visceral sensation of the urinary bladderremains unknown. Because distention-induced ATP release from theurothelium plays an essential role in visceral sensation of the urinarybladder, we investigated the regulation of urothelial ATP release bythe 5-HT signaling system. RT-PCR and immunohistochemical anal-yses of the urothelium revealed specific expression of 5-HT1D and5-HT4 receptors. The addition of 5-HT did not affect urothelial ATPrelease without bladder distention, but it significantly reduced disten-tion-induced ATP release by physiological pressure during urinestorage (5 cmH2O). The inhibitory effect of 5-HT on distention-elicited ATP release was blocked by preincubation with the5-HT1B/1D antagonist GR-127935 but not by the 5-HT4 antagonistSB-204070. mRNA encoding tryptophan hydroxylase 1 was detectedin the urinary bladder by nested RT-PCR amplification, and L-tryp-tophan or the selective serotonin reuptake inhibitor citalopram alsoinhibited ATP release, indicating that 5-HT is endogenously synthe-sized and released in the urinary bladder. The addition of GR-127935significantly enhanced the distention-elicited ATP release 40 min afterdistention, whereas SB-204070 reduced the amount of ATP release 20min after distention. These data suggest that 5-HT4 facilitates thedistention-induced ATP release at an earlier stage, whereas 5-HT1D

inhibits ATP release at a later stage. The net inhibitory effect of 5-HTindicates that the action of 5-HT on the urothelium is mediatedpredominantly by 5-HT1D.

5-hydroxytryptamine; 5-hydroxytryptamine1D; 5-hydroxytryptamine4; uri-nary bladder; urine storage

SEROTONIN [5-hydroxytryptamine (5-HT)] is a multifunctionalsignaling molecule in visceromotor and viscerosensory sys-tems. The multiple functions of 5-HT are mediated by variousreceptor subtypes (5-HT1 to 5-HT7) (24, 41). In the gastroin-testinal system, 5-HT can induce bowel contraction or relax-ation by activating intrinsic excitatory or inhibitory motorneurons. Stimulation of 5-HT3 or 5-HT4 receptors on entericcholinergic neurons results in the contraction of smooth mus-cles (6, 17, 51, 55). 5-HT also stimulates 5-HT1A, 5-HT1D, and5-HT4 receptors on inhibitory neurons to release nitric oxide,thereby relaxing smooth muscle (6, 17, 51, 55). Smooth muscle

also expresses 5-HT receptors, including 5-HT2A (mediatingcontraction), 5-HT4, and 5-HT7 subtypes (mediating relax-ation) (6, 17, 51, 55). Furthermore, 5-HT participates in mu-cosal sensory transduction in the gastrointestinal system. En-terochromaffin cells in the mucosa release 5-HT in response tointraluminal pressure (5). Released 5-HT activates extrinsicvagal afferent nerves to transmit sensations of nausea ordiscomfort (51). The mucosal projection of intrinsic primaryafferent neurons is also stimulated by 5-HT, which initiatesperistaltic and secretory reflexes (51).

In addition to the gastrointestinal system, 5-HT regulates thecontraction of smooth muscle in the urinary bladder. 5-HT1A,5-HT2, 5-HT3, and 5-HT7 receptors on the detrusor control itscontractions (11, 13, 28, 29, 39, 45, 47, 48). 5-HT1A, 5-HT2A,5-HT2C, 5-HT3, 5-HT4, and 5-HT7 receptors in the terminals ofautonomic excitatory neurons in the urinary bladder modulateneurogenic contractions of the detrusor (7, 11, 14, 38, 46, 57).The micturition reflex is also regulated by spinal/supraspinal5-HT1A, 5-HT2A, and 5-HT7 receptors (8–10, 15, 25–27, 36,37, 44, 56). However, the function of peripheral 5-HT recep-tors on visceral sensation of the urinary bladder remainsunclear.

Recent studies have revealed that the release of ATP fromthe epithelium of the urinary bladder (urothelium) contributesto visceral sensation (2–4). Distention of the bladder wallduring urine storage evokes the release of ATP from theurothelium (16, 32–35). Studies using purinergic receptorP2X3-deficient mice (12) have suggested that the released ATPtransmits viscerosensory signals to the central nervous systemvia P2X3 at afferent nerve terminals in close proximity to theurothelium. Therefore, in the present study, we investigated theregulation of urothelial ATP release by bladder 5-HT receptorsto reveal the contribution of peripheral 5-HT receptors tobladder visceral sensation.

MATERIALS AND METHODS

Animals. Six- to ten-week-old C57BL/6 male mice were used inthis study. The protocols for the experiments in the present study wereapproved by The Animal Research Committee of Akita Universityand followed the guidelines of the American Physiological Society foranimal research.

RT-PCR. Urinary bladders and brains were dissected from fivemice. The urothelium and urothelium-denuded bladders were dis-sected and separated as follows: opened bladders were incubated indispase solution (2,000 U/ml dispase II, Sanko Junyaku, Tokyo,Japan) prepared in minimum essential medium (Gibco, Invitrogen,Carlsbad, CA) for 1 h at 37°C. The urothelium was gently scrapedfrom the underlying tissues. Total RNA was isolated from the brain,urothelium, and urothelium-denuded bladder using ISOGEN (Nippon

Address for reprint requests and other correspondence: K. Matsumoto-Miyai, Kansai Univ. of Nursing and Health Sciences, 1456-4 Shizuki, Awaji,Hyogo 656-2131, Japan (e-mail: [email protected]).

Am J Physiol Renal Physiol 310: F646–F655, 2016.First published November 18, 2015; doi:10.1152/ajprenal.00024.2015.

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Gene, Tokyo, Japan) according to the manufacturer’s protocol. RNAsamples were treated with deoxyribonuclease I (Sigma, St. Louis,MO) at room temperature for 10 min to exclude artifacts due togenomic DNA contamination. Synthesis of first-strand cDNA andPCR were performed using the Transcriptor One-Step RT-PCR Kit(Roche, Mannheim, Germany). First-strand cDNA was synthesizedusing Oligo(dT) primers at 50°C for 30 min. PCR was performedusing Program Temp Control System PC-320 (ASTEC, Fukuoka,Japan). The PCR involved 35 cycles that consisted of denaturation at94°C for 10 s, annealing at 58°C for 30 s, and extension at 68°C for30 s. GAPDH was used as an internal control. For a nested RT-PCRamplification of tryptophan hydroxylase (TPH)1, a 5 �l of the RT-PCR product using Tph1 outer primers was used as the new templatefor the second round of amplification using the inner primers (Tph1inner) and the same amplification condition. The sequence of theoligonucleotide primers used in this study, their positions on thecorresponding mRNA sequences, and the expected sizes of the PCRproducts are shown in Table 1.

Immunofluorescent staining. After deep anesthesia, C57BL/6 mice(10 wk old) were perfused transcardically with 4% paraformaldehydeand 2.5% glutaraldehyde in 0.1 M PB (pH7.4), and the urinarybladders, brains, and skeletal muscles (quadriceps femoris) were thenisolated. The removed organs were postfixed in the same fixative at4°C overnight, transferred to 30% sucrose in 0.1 M PB for cryopro-tection at 4°C, and then frozen with dry ice. The organs weresectioned at 30 �m, floated in 0.01 M PBS, and then stored 4°C untiluse.

Free-floating sections were rinsed in 0.01 M PBS and treated withblocking solution (0.3% Triton-X and 3% BSA in 0.01 M PBS) toincrease the permeability of the antibody and inhibit nonspecificstaining at room temperature for 1 h. Next, sections were incubatedwith goat anti-5-HT1D polyclonal antibody SR-1D (L-18) or SR-1D(S-18) (1:50, catalog nos. sc-5394 or sc-5393, respectively, Santa

Cruz Biotechnology) or goat anti-5-HT4 polyclonal antibody SR-4(C-18) or SR-4 (N-18) (1:50, catalog nos. sc-32566 or sc-32564,respectively, Santa Cruz Biotechnology) in blocking buffer at 4°Covernight. Sections were then washed three times in 0.01 M PBS andsubjected to a 1 h-incubation with Alexa 488 donkey anti-goatpolyclonal antibody (1:500, Molecular Probe) in 0.01 M PBS at roomtemperature for 1 h. After being washed in 0.01 M PBS, sections weremounted on slides using PermaFluor (Thermo Scientific, Waltham,MA) and visualized by confocal microscopy (Olympus BX61, typeFV1000D, Olympus). An absorption test was performed using 2 �gantibodies incubated with 80 �g of each immunogen blocking peptide(sc-5394 P, sc-5393 P, sc-32566 P, or sc-32564 P, Santa CruzBiotechnology) instead of the primary antibodies.

ATP release assay using the ussing chamber. A urothelial ATPrelease assay was performed as previously described (32, 33). In brief,isolated urinary bladders were opened vertically from the urethra tothe apex. The opened bladder was mounted to act as a diaphragm of7 mm2 between the two halves of a customized small Ussing chamber.The mucosal (urinary) side of the chamber had a volume of 700 �l.Chambers were filled with Krebs solution (117 mM NaCl, 5.9 mMKCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 24.8 mM NaHCO3, 1.2 mMNaH2PO4, and 11.1 mM glucose) with bubbling 95% O2-5% CO2. Inthe chemical stimulation assay, serotonin hydrochloride (Sigma) orvehicle (H2O) was added to the mucosal side for 30 min withouthydrostatic pressure. Fifty microliters of Krebs solution were sampledfrom the mucosal side before and after the addition. ATP release wasassessed by subtracting the ATP content before the addition from thatafter the addition.

In the distention stimulation assay, we applied hydrostatic pressureof 5 cmH2O to the serosal (smooth muscle) side for 60 min, whichreflects the physiological range of pressure during urine storage (49,52). Fifty microliters of Krebs solution were sampled from themucosal side before and 5, 20, 40, and 60 min after application of

Table 1. Oligonucleotide primers for PCR amplification of 5-hydroxytryptamine receptors and tryptophan hydroxylases

Gene Oligonucleotide Sequences Position Expected Product Size, bp

Htr1a Sense: 5=-CAAGATGGCCTTGGCCCGTGAG-3= 1560–1581 441Antisense: 5=-GCCATGGGCTAAGAAGCTGCGT-3= 2000–1979

Htr1b Sense: 5=-CCTGTTGCACTGCTTCCATCATGCA-3= 380–404 324Antisense: 5=-TCCGAGAGCGGGCTTCCACAT-3= 703–683

Htr1d Sense: 5=-GGGCCATCACCGATGCCCTG-3= 1371–1390 266Antisense: 5=-ACTCCGGGCGGCCACGTATA-3= 1636–1617

Htr2a Sense: 5=-GAAGAGGAGAAAGCAGCCAGAGGAG-3= 952–976 627Antisense: 5=-GACGCCGTGGAGAAGAGCACA-3= 1578–1558

Htr2b Sense: 5=-CTCTTTTCAACTGCCTCCATCATGCA-3= 733–758 386Antisense: 5=-GGCACAGTCCACCGTGTTAGGC-3= 1118–1097

Htr2c Sense: 5=-TGCGCCATATCGCTGGACCG-3= 1127–1146 652Antisense: 5=-TGCCTGAACACACATAGCCAATCCA-3= 1778–1754

Htr3a Sense: 5=-CAACAAGACTGATGACTGCTCAGCC-3= 1265–1289 618Antisense: 5=-TGAGCAGTTCCAGGGGCCGTAA-3= 1882–1861

Htr4 Sense: 5=-CCGAGCAGGTGTGGACTGCTT-3= 1081–1101 482Antisense: 5=-CCCCTGACTTCCTCAAATACCGCCTG-3= 1562–1537

Htr5a Sense: 5=-TACAGGGCGGCGAAATTCCGC-3= 1172–1192 453Antisense: 5=-ACCCACTGAGCTACAAGCTATGGGAAG-3= 1624–1598

Htr6 Sense: 5=-TCCTGGGTGCCTGGAGCCTC-3= 468–487 444Antisense: 5=-CGGCCTGAGCTATGCTGGCC-3= 911–892

Htr7 Sense: 5=-CATGTGCTGCACGGCCTCGAT-3= 605–625 446Antisense: 5=-CGTGTTTGAGCAGTCTCGAAAGGTTCG-3= 1050–1024

Tph1 outer Sense: 5=-GGCTTCATCTCAAGCCCACT3= 3246–3265 509Antisense: 5=-TCTTGGAACCCTGACTTGCC-3= 3754–3735

Tph1 inner Sesne: 5=-GCTTCACCCCATGCTCTACA-3= 3334–3353 378Antisense: 5=-GACAAAGTCAAGCCCTTCGC-3= 3711–3692

Tph2 Sense: 5=-CGGGCGTATGGAGCAGGGTT-3= 1247–1266 241Antisense: 5=-TGCTCTGCGTGTAGGGGTTGA-3= 1487–1467

GAPDH Sense: 5=-AGAACATCATCCCTGCATCC-3= 655–674 367Antisense: 5=-TCCACCACCCTGTTGCTGTA-3= 1021–1002

Htr, 5-hydroxytryptamine receptor; Tph, tryptophan hydroxylase.

F6475-HT CONTROLS UROTHELIAL ATP RELEASE

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pressure. Drugs modulating serotonergic signaling were added to themucosal side 30 min before the application of pressure. The concen-trations of drugs used in this study were determined according toprevious studies (1, 19, 21, 31).

The ATP content in 50 �l Krebs solution on the mucosal side of thechamber was assayed with the luciferin-luciferase method (Kik-koman, Tokyo, Japan) according to the manufacturer’s protocol.Standard curves were constructed in each experiment using 3 � 10�7,3 � 10�8, 3 � 10�9, and 3 � 10�10 M ATP. Distention-elicitedchanges in ATP content were calculated by subtracting the valuebefore pressure was initiated. Since the changes in ATP content atlater time points tend to be overestimated because of the gradualreduction in the total volume of Krebs solution, we compared thechanges in ATP content under different conditions only at the sametime point. Statistically significant differences were detected using anunpaired t-test. All data are expressed as means � SE.

RESULTS

Expression of 5-HT receptor subtypes in the urothelium. Weperformed RT-PCR with total RNA from the urothelium,urothelium-denuded bladder, and brain and investigated themRNA expression profile of 5-HT receptor subtypes. Asshown in Fig. 1, the urothelium exhibited a distinct mRNAexpression pattern compared with the urothelium-denudedbladder. Intense bands corresponding to 5-HT1D and 5-HT4

receptors were detected in the urothelium (Fig. 1, top). Faintbands corresponding to 5-HT2A and 5-HT6 receptors was alsodetected. On the other hand, the urothelium-denuded bladdershowed the expression of broader set of 5-HT receptor sub-types: 5-HT1A, 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2B, 5-HT2C,5-HT3A, 5-HT4, and 5-HT7 (Fig. 1, middle). mRNA expressionof all subtypes except for the peripheral subtype 5-HT2B wasdetected in the brain, which served as a positive control tissue(Fig. 1, bottom). A positive 5-HT2B PCR product was ampli-fied from colorectal tissue (data not shown). These data show

that all primer pairs worked well to amplify the 5-HT receptorsubtypes in the present study.

To confirm the protein-level expression of 5-HT1D and5-HT4 in the urothelium, immunofluorescent staining using aspecific antibody for 5-HT1D [SR-1D (L-18)] or 5-HT4 [SR-4(C-18)] was performed. The intense immunofluorescent-posi-tive signals for 5-HT1D in the urinary bladder wall (Fig. 2A)were significantly reduced in the absorption test using blockingimmunogen peptide (Fig. 2B), confirming the specificity of theantibody. Plasma membrane-associated immunoreactivity for5-HT1D was observed in the urothelium (Fig. 2C) and detrusor(Fig. 2D). The arcuate nucleus of the hypothalamus in the brain(positive control tissue) also exhibited neuronal cell mem-brane-associated immunofluorescent signals (Fig. 2E), whereasfaint fluorescence was found in skeletal muscle (quadricepsfemoris, negative control tissue; Fig. 2F). Immunofluorescentstaining using another anti-5-HT1D antibody, SR-1D (S-18),showed similar results (data not shown).

The bladder wall was also intensely immunostained againstthe anti-5-HT4 antibody [SR-4 (C-18); Fig. 3A]. Immunofluo-rescent signals were observed on urothelial cells (Fig. 3C,urothelium) in a similar membrane-associated manner. Thesignificant immunoreactivity for 5-HT4 was also observed inthe detrusor (Fig. 3D). The neuronal cell membrane-associatedimmunoreactivity in the arcuate nucleus of the hypothalamus(Fig. 3E) and the subtle fluorescence in the absorption test (Fig.3B) and in the musclus quadriceps femoris (Fig. 3F) confirmedthe specificity of the antibody. Similar results were obtained byimmunofluorescent staining using another anti-5-HT4 antibody[SR-4 (N-18); data not shown].

Exogenous 5-HT inhibits distention-induced urothelial ATPrelease via 5-HT1D. As expression of 5-HT1D and 5-HT4

receptors was confirmed in the urothelium, we investigated theeffect of exogenously applied 5-HT on urothelial ATP release.

Fig. 1. mRNA expression of serotonin [5-hydroxytryptamine (5-HT)] receptors in the urinary bladder. RT-PCR analysis of the indicated 5-HT receptor subtypesusing total RNA isolated from the mouse urothelium (top), urothelium-denuded bladder (middle), and brain (bottom) is shown. 5-HT1D and 5-HT4 mRNAs weresignificantly expressed in the urothelium. As a positive control, RT-PCR of GAPDH was performed. The positions of marker bands (100-bp ladder) are indicatedto the right of the gels. The expected sizes of PCR products are shown in Table 1.

F648 5-HT CONTROLS UROTHELIAL ATP RELEASE




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The application of 5-HT (10 �M) without distention tended toreduce constitutive ATP release (�0.04 � 0.03 nM), but thedifference from vehicle (H2O; 0.01 � 0.01 nM) was notstatistically significant (P � 0.23; Fig. 4A). On the other hand,exogenous 5-HT produced a significant inhibitory effect ondistention-induced ATP release by physiological pressure dur-ing urine storage (5 cmH2O). The addition of 5-HT (10 �M)significantly reduced distention-induced ATP release to�33.3% (0.08 � 0.03 nM with 5-HT vs. 0.23 � 0.03 nM withvehicle, P � 0.05 by an unpaired t-test; Fig. 4B). To elucidatewhich subtype of 5-HT receptor contributes to the inhibition ofATP release, we investigated the effects of the 5-HT1B/1D

antagonist GR-127935 and the 5-HT4 antagonist SB-204070on the inhibitory effect of 5-HT. As shown in Fig. 4B, inhibi-

tion of distention-induced ATP release by 5-HT was dimin-ished by preincubation with GR-127935 (0.20 � 0.04 nM, P �0.05 vs. 5-HT by an unpaired t-test). In contrast, SB-204070slightly enhanced the inhibitory effect of 5-HT (0.05 � 0.03nM; Fig. 4B). These results indicate that the inhibitory effect of5-HT was mediated by 5-HT1D.

To examine whether the urothelium is the target for 5-HTeffects or not, we investigated the effect of exogenous 5-HT ondistention-induced ATP release from urothelium-denudedbladder walls. The 5 cmH2O-induced ATP release from theurothelium-denuded bladder (0.043 � 0.012 nM at 20 min,n � 8) was less than that from intact bladder walls, indicatingthat the most of ATP was released from the urothelium.Exogenous 5-HT (10 �M) did not affect distention-induced

Fig. 3. Localization of 5-HT4 in the urinary bladder. Immunofluorescentstaining for 5-HT4 using the anti-5-HT4 antibody SR-4 (C-18) is shown. A:urinary bladder wall [mucosal side (top) and serosal side (bottom)]. Significantimmunoreactivity was observed. B: absorption test in the urinary bladder wall.The intensity of immunofluorescent signals was greatly reduced. C: high-power field in the urothelium. Plasma membrane-associated immunoreactivitywas detected on urothelial cells. D: high-power field in the detrusor. Smoothmuscle also exhibited positive immunoreactivity. E: arcuate nucleus of thehypothalamus in the brain (positive control tissue). The neuronal cell bodyshowed immunoreactivity in a membrane-associated manner. F: musclusquadriceps femoris (negative control tissue). Little immunofluorescence wasfound. Scale bars � 100 �m in A, B, E, and F and 50 �m in C and D.

Fig. 2. Localization of 5-HT1D in the urinary bladder. Immunofluorescentstaining for 5-HT1D using the anti-5-HT1D antibody SR-1D (L-18) is shown. A:urinary bladder wall [mucosal side (top) and serosal side (bottom)]. Significantimmunoreactivity was observed. B: absorption test in the urinary bladder wall.The intensity of immunofluorescent signals was greatly reduced. C: high-power field in the urothelium (UT). Plasma membrane-associated immunore-activity was detected on urothelial cells. D: high-power field in the detrusor.Smooth muscle also exhibited positive immunoreactivity. E: arcuate nucleus ofthe hypothalamus in the brain (positive control tissue). The neuronal cell bodyshowed immunoreactivity in a membrane-associated manner. F: musclusquadriceps femoris (negative control tissue). Little immunofluorescence wasfound. Scale bars � 100 �m in A, B, E, and F and 50 �m in C and D.





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ATP release from urothelium-denuded bladder walls (0.063 �0.028 nM at 20 min, n � 8, P � 0.82 vs. 5-HT by an unpairedt-test).

TPH1 mRNA expression in the urinary bladder and theeffect of L-tryptophan or selective serotonin reuptake inhibitoron distention-induced ATP release. The endogenous synthesis,release, and reuptake of 5-HT in the urinary bladder wereexamined next. First, the expression of mRNAs for TPH1 andTPH2 was investigated by a single or nested RT-PCR methods.A single RT-PCR using Tph1 inner primers or Tph2 primersrevealed TPH1 mRNA expression in the small intestine andTPH2 mRNA expression in the brain (Fig. 5A). On the otherhand, neither TPH1 nor TPH2 transcripts were detected in theurinary bladder (Fig. 5A). Similarly, single RT-PCR usingTph1 outer primers generated no visible band for TPH1 tran-scripts in the urinary bladder (data not shown). However, asignificant level of TPH1 mRNA was detected by nestedRT-PCR amplification using Tph1 inner and outer primers(Fig. 5A).

We then investigated the effect of L-tryptophan, a 5-HTprecursor, on distention-induced ATP release (Fig. 5B). Simi-lar to 5-HT, 1 mM L-tryptophan significantly reduced disten-tion-induced ATP release (0.16 � 0.06 nM) compared with thevehicle, hydrochloric acid (final concentration: 1 mM, 0.42 �0.10 nM, P � 0.05 by an unpaired t-test). Because hydrochlo-ric acid may activate transient receptor potential vanilloid(TRPV)1 channels and then induce ATP release, we assessedthe effects of hydrochloric acid and L-tryptophan on ATPrelease in the absence of pressure. Hydrochloric acid (1 mM)induced ATP release in the absence of pressure (0.14 � 0.08nM), but L-tryptophan did not alter hydrochloric acid-evokedATP release (0.12 � 0.05 nM). These results suggest thatfacilitation of 5-HT synthesis inhibited ATP release due tophysiological pressure but not hydrochloric acid.

Next, we evaluated the effect of citalopram, a selectiveserotonin reuptake inhibitor (SSRI), on distention-inducedATP release. As shown in Fig. 5C, the addition of citalopram(10 �M) significantly decreased ATP release (0.09 � 0.04 nM)to �39% of the vehicle control (0.23 � 0.03 nM, P � 0.05 byan unpaired t-test). The extent of inhibition was quite similar tothat by exogenous 5-HT (Fig. 4B), suggesting that 5-HT wasendogenously released in the urinary bladder at sufficientlevels to fully activate 5-HT receptors in the urothelium.

Distinct functions of 5-HT1D and 5-HT4 in distention-in-duced urothelial ATP release. Because Gi/o-coupled 5-HT1D

and Gs-coupled 5-HT4 receptors have opposite effects on theactivities of adenylyl cyclases, the functions of these receptorsin distention-induced urothelial ATP release may be different.To elucidate the possible distinct functions of these receptors,we assessed the effect of addition of GR-127935 or SB-204070without 5-HT. These antagonists exhibited opposite effects ondistention-induced ATP release at different times. Twentyminutes after distention (Fig. 6A), blockade of 5-HT4 withSB-204070 significantly reduced urothelial ATP release to51.5% compared with the vehicle (0.12 � 0.04 nM withSB-204070 vs. 0.23 � 0.03 nM with the vehicle, P � 0.05 byan unpaired t-test). However, the reducing effect of GR-127935 was small and not statistically significant (0.19 � 0.03nM, P � 0.41). On the other hand, as shown in Fig. 6B, theeffects of antagonists 40 min after distention were quite dif-ferent from those at 20 min. Inhibition of 5-HT1D by GR-127935 significantly enhanced distention-induced urothelialATP release by �156% at 40 min (0.39 � 0.03 nM withGR-127935 vs. 0.25 � 0.03 nM with vehicle, P � 0.05 by anunpaired t-test), whereas SB-204070 did not induce any sig-nificant change (0.22 � 0.06 nM). These results suggest thatendogenous 5-HT facilitated distention-induced ATP release

Fig. 4. Serotonergic inhibition of distention-induced urothelial ATP release via 5-HT1B/D. A: effects of 5-HT (10 �M) or vehicle (H2O) on constitutive ATPrelease on the mucosal side of the chamber. The y-axis indicates changes in the ATP content 20 min after the addition. 5-HT did not induce any significant changein ATP content in the absence of distention of the bladder wall. Values are means � SE; n � 5 for the vehicle and 7 for 5-HT. B: effects of 5-HT on physiologicaldistention-induced ATP release from the urothelium and the effect of 5-HT1B/1D or 5-HT4 antagonist (GR-127935 or SB-204070, respectively) on 5-HT-inducedmodulation of ATP release. Physiological distention of the bladder wall was induced by 5 cmH2O of hydrostatic pressure. The y-axis on the graph shows thechange in ATP content on the mucosal side of the chamber 20 min after distention. The addition of 5-HT (10 �M) significantly reduced ATP release comparedwith vehicle (H2O). Preincubation with GR127935 (1 �M) restored the distention-induced increase in ATP content to normal levels (P � 0.05 vs. 5-HT by anunpaired t-test), whereas pretreatment with SB-204070 (1 �M) did not affect the inhibitory effect of 5-HT on ATP release. Values are means � SE; n � 24 forvehicle, 7 for 5-HT, 6 for 5-HT GR-127935, and 6 for 5-HT SB-204070.





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via 5-HT4 at an early phase (�20 min) and inhibited it via5-HT1D at a late phase (�40 min).

The facilitatory effect of 5-HT4 on distention-induced ATPrelease indicated that blockade of 5-HT4 may enhance theextrinsic 5-HT-induced inhibition of ATP release. To investi-gate this possibility, we compared the effect of 5-HT with thatof a combination of 5-HT and the 5-HT4 antagonist SB-204070. As shown in Fig. 6C, the inhibitory effect of 5-HT ondistention-induced ATP release was restricted to the relativelyearly phase. A significant decrease in ATP release by 5-HTwas detected 20 min after distention, but no significant differ-ence was observed 40 min and later. However, a combinationof 5-HT and SB204070 prolonged the inhibitory effect until 60min after distention. A significant reduction in urothelial ATPrelease by 5-HT and SB-204070 was detected 40 and 60 min inaddition to 20 min compared with vehicle. This result con-firmed the inhibitory effect of 5-HT1D and the facilitatory

effect of 5-HT4 and suggest that the action of 5-HT waspredominantly mediated by 5-HT1D.

DISCUSSION

In the present study, we demonstrated the specific expres-sion of 5-HT receptor subtypes in the urothelium and theirfunctional role in distention-induced urothelial ATP release.Expression of mRNAs encoding 5-HT1D, 5-HT2A, 5-HT4, and5-HT6 receptors was detected in the mouse urothelium (Fig. 1).In addition, plasma membrane-associated localization of5-HT1D and 5-HT4 was shown by the immunofluorescentstaining using specific antibodies (Figs. 2 and 3).

The peripheral 5-HT1D expression may be characteristic ofthe urothelium. To our knowledge, the expression and functionof 5-HT1D have not been reported in the urinary bladder,gastrointestinal tract, or other hollow organs, except for vaso-

Fig. 5. mRNA expression of tryptophan hydroxylase (TPH) in the urinary bladder and the effect of L-tryptophan or selective serotonin reuptake inhibitor ondistention-induced ATP release. A: single or nested RT-PCR analysis of TPH1 and TPH2. TPH1 and TPH2, single RT-PCR amplification using Tph1 innerprimers and Tph2 primers, respectively; TPH (Inner), nested PCR amplification using Tph1 outer and inner primers. cDNA fragments representing TPH1 in thesmall intestine and TPH2 in the brain were detected by single RT-PCR. mRNA for the TPH1 transcript was detected in the urinary bladder, brain, and smallintestine by nested RT-PCR method. As a positive control, single RT-PCR for GAPDH was performed. The positions of marker bands (100-bp ladder) areindicated to the right of the gel. The expected sizes of PCR products are shown in Table 1. B: effect of L-tryptophan (1 mM) or vehicle (HCl) on ATP releaseon the mucosal side of the chamber. The y-axis on the graph shows the change in ATP content on the mucosal side of the chamber 20 min after distention. Thetwo left bars show the effects of L-tryptophan on physiological pressure (5 cmH2O)-induced ATP release from the urothelium; the effects of L-tryptophan onconstitutive ATP release without distention are indicated in the two right bars (no pressure). The addition of L-tryptophan (1 mM) significantly reduced 5cmH2O-induced ATP release compared with vehicle (HCl, P � 0.05 vs. L-tryptophan by an unpaired t-test) but did not alter constitutive ATP release withoutdistention. Values are means � SE; n � 13 for vehicle under 5 cmH2O 12 for L-tryptophan under 5 cmH2O, 10 for vehicle without pressure, and 10 forL-tryptophan without pressure. C: modulation of hydrostatic pressure (5 cmH2O)-induced urothelial ATP release by the selective serotonin reuptake inhibitorcitalopram (10 �M) or vehicle (H2O), which were added 30 min before pressure application. The y-axes on the graphs show the changes in ATP content on themucosal side of the chamber 20 min after pressure application. Blockade of 5-HT reuptake significantly inhibited ATP release (P � 0.05 vs. vehicle by anunpaired t-test). Values are means � SE; n � 24 for vehicle and 8 for citalopram.





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constriction of mucosal vessels (18). In the urothelium, 5-HT1D

was the dominant 5-HT receptor mediating the inhibitory effecton distention-induced ATP release. ATP release from theurothelium transmits visceral sensations such as bladder walldistention or bladder pain (12, 58). Therefore, activation of5-HT1D may inhibit visceral sensation and extend the urinestorage period. Intriguingly, the involvement of 5-HT1D in thefunctions of the micturition reflex in the central nervous systemhas not been reported, whereas the essential contribution ofspinal or supraspinal 5-HT1A to the micturition reflex has beenwell investigated (8–10, 26, 56). These data suggest that5-HT1D is quite specific to the peripheral visceral sensation ofthe urinary bladder, indicating that 5-HT1D may be a noveltarget for drug therapy for disorders involving hypersensitivityof the lower urinary tract such as overactive bladder andinterstitial cystitis.

5-HT1D is a Gi/o-coupled receptor, and its activation resultsin inhibition of adenylyl cyclase and a reduction in cAMP. Wehave previously demonstrated that activation of adenylyl cy-clase and an increase in cAMP facilitate urothelial ATP release(34). Therefore, the inhibitory effect of 5-HT1D on urothelialATP release may be mediated by a decrease in cAMP.

Expression of 5-HT4 receptors has been detected in theepithelium of other hollow organs. 5-HT4 is present in themucosa of the esophagus, stomach, and intestine (23, 43, 60),epithelium of the airway (40), and ovarian epithelium (22).Activation of 5-HT4 in the peripheral colorectum results in theinhibition of visceral hypersensitivity (20, 23). Gastric disten-tion-evoked visceral pain is also inhibited by a 5-HT4 agonist(50). These data indicate that 5-HT4 may be an epithelial-type5-HT receptor that is involved in visceral sensation. Of note, anincrease in distention-induced ATP release from the urothe-

Fig. 6. Regulatory function of 5-HT1D or 5-HT4 in urothelial ATP release. A and B: modulation of hydrostatic pressure (5 cmH2O)-induced urothelial ATP releaseby 5-HT receptor antagonists. The 5-HT1B/1D antagonist GR-127935 (1 �M), 5-HT4 antagonist SB-204070 (1 �M), or vehicle (H2O) was added 30 min beforepressure application. The y-axes on the graphs show the changes in ATP content on the mucosal side of the chamber 20 min (A) or 40 min (B) after pressureapplication. A: 20 min after pressure application, blockade of 5-HT4 significantly inhibited ATP release (P � 0.05 vs. vehicle by an unpaired t-test), whereasinhibition of 5-HT1B/1D had no significant effect. B: in contrast, blockade of 5-HT1B/1D significantly enhanced urothelial ATP release 40 min after distention (P �0.05 vs. vehicle by an unpaired t-test). At this time point, the 5-HT4 antagonist did not show any difference from the vehicle. Values are means � SE; n � 24for vehicle, 9 for GR-127935, and 13 for SB-204070. C: temporal change in ATP release during hydrostatic pressure (5 cm H2O)-induced distention. The x-axisshows the distention time, and the y-axis indicates changes in ATP contents on the mucosal side of the chamber. 5-HT significantly reduced ATP release at 20min, but the reducing effect was eliminated at 40 min and later. On the other hand, the combination of 5-HT and the 5-HT4 antagonist SB-204070 prolongedthe reducing effect from 20 to 60 min. Values are means � SE; n � 24 for vehicle (H2O), 7 for 5-HT, and 6 for 5-HT SB-204070. *P � 0.05 vs. vehicleby an unpaired t-test.





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lium by 5-HT4 activation (Fig. 6) may lead to enhancedvisceral sensation, which is opposite of what occurs in thegastrointestinal tract. In addition to the urothelium, 5-HT4 isexpressed in autonomic nerve terminals in the urinary bladderand facilitates neurogenic contraction of the detrusor (7, 38,57). These data suggest that peripheral 5-HT4 facilitates void-ing by enhancing both visceromotor and viscerosensory func-tions of the urinary bladder. Therefore, the combination of5-HT1D activation and 5-HT4 inhibition may exert a furthersignificant therapeutic effect on lower urinary tract symptoms,as shown in Fig. 6C. Interestingly, the time windows for theeffects of endogenous 5-HT1D and 5-HT4 were different (5-HT1D functioned at a later stage and 5-HT4 functioned at anearlier stage; Fig. 6, A and B), which implies that 5-HT is aswitching molecule from the initial perception of the need tourinate to prolongation of urine storage.

5-HT2A mRNA expression is also found in native human urothe-lium but not in human urothelium-derived cell lines (42). However,the function of 5-HT2A seems to not be essential in the urothelium, assuggested by its weak expression and the present result showing thatthe functionally dominant 5-HT receptor subtype was 5-HT1D.

Our data on 5-HT receptor expression are partially incon-sistent with another previous study. Chetty and colleagues (11)reported 5-HT3A and 5-HT3B mRNA expression in the mousebladder mucosa, whereas we detected 5-HT3A expression onlyin the urothelium-denuded urinary bladder. Although such adiscrepancy may be due to the different positions of the primerpairs, the primer pairs for 5-HT3A in the present study ampli-fied mouse 5-HT3A mRNA in the brain (Fig. 1) and themucosa-denuded colorectum (data not shown). An alternativeexplanation is that the different methods by which the urothe-lium was removed may have caused the discrepancy. If thebladder mucosa in the study by Chetty and colleagues includedthe lamina propria, the discrepancy could be accounted for bythe 5-HT3A expression in the lamina propria, because thelamina propria was left in the urothelium-denuded bladder inthe present study.

We have shown a small but significant amount of TPH1mRNA expression in the urinary bladder by nested RT-PCR(Fig. 5A), suggesting that 5-HT could be endogenously syn-thesized. The present result using L-tryptophan or an SSRI(Fig. 5, B and C) also imply the endogenous synthesis andrelease in the isolated urinary bladder. Although the cell typethat synthesizes and releases 5-HT remains unknown, severalstudies have implied that mast cells may be a source of 5-HTin the urinary bladder. TPH is expressed in mast cells in humanand normal rat gastrointestinal tracts (61), and mast cellssynthesize 5-HT from 5-hydroxytryptophan (59). Mast cellsare predominantly located around submucosal and adventitialblood vessels with infiltration of the lamina propria and mus-cularis propria in the urinary bladder of normal female rats(30). Although further studies are necessary to identify thesource of 5-HT in the urinary bladder, the significant effect of5-HT1D or 5-HT4 antagonist alone on distention-induced ATPrelease (Fig. 6, A and B) indicates that the amount of endog-enously-released 5-HT is sufficient to exert its functionsthrough these receptors.

In summary, we identified specific expression of 5-HT1D and5-HT4 receptors in the urothelium and their functions in dis-tention-evoked ATP release from the urothelium. 5-HT1D wasthe functionally dominant receptor mediating serotonergic in-

hibition of urothelial ATP release, which leads to inhibition ofvisceral sensation, whereas 5-HT4 facilitated urothelial ATPrelease. 5-HT1D is likely to be a urothelium-specific receptorsubtype in the system that regulates micturition. Since urothe-lial ATP release was significantly elevated in interstitial cystitis(54) and was correlated with urinary frequency in overactivebladder patients (53), activation of 5-HT1D may be a novelspecific therapy for the treatment of disorders involving fre-quent urination or bladder pain.

ACKNOWLEDGMENTS

The authors are grateful to the Center of Medical Research and Education,Graduate School of Medicine, Osaka University, for technical support.

GRANTS

This work was supported by Japan Society for the Promotion of ScienceGrants-In-Aid for Scientific Research (C) KAKENHI 21600001, 23590707,24590722, and 26460694.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).The authors acknowledge all funding sources supporting this work and all

institutional or corporate affiliations. The authors certify that they had nocommercial associations that might pose a conflict of interest in connectionwith the submitted article, and the authors accept full responsibility for theconduct of this study, had full access to all of the data, and controlled thedecision to publish.

AUTHOR CONTRIBUTIONS

Author contributions: K.M.-M. and M.K. conception and design of re-search; K.M.-M., E.Y., E.S., and Y.K. performed experiments; K.M.-M., E.Y.,E.S., Y.K., and S.S. analyzed data; K.M.-M., Y.K., S.S., M.Y., and M.K.interpreted results of experiments; K.M.-M. and Y.K. prepared figures;K.M.-M. and M.K. drafted manuscript; K.M.-M. and M.K. edited and revisedmanuscript; K.M.-M., E.Y., E.S., Y.K., S.S., M.Y., and M.K. approved finalversion of manuscript.

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