JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 JPETThis Fast article Forward. has not been Published copyedited andon formatted. May 22, The 2008 final asversion DOI:10.1124/jpet.108.139295 may differ from this version.
JPET #139295
Title Page
GSK1016790A, a Novel and Potent TRPV4
Channel Agonist Induces Urinary Bladder
Contraction and Hyperactivity: Part I Downloaded from
Kevin S. Thorneloe, Anthony C. Sulpizio, Zuojun Lin, David J. Figueroa, Angela
K. Clouse, Gerald P. McCafferty, Tim P. Chendrimada, Erin S.R. Lashinger, Earl jpet.aspetjournals.org
A. Gordon, Louise Evans, Blake A. Misajet, Douglas J. DeMarini, Josephine H.
Nation, Linda N. Casillas, Robert W. Marquis, Bartholomew J. Votta, Steven A. at ASPET Journals on September 27, 2021 Sheardown, Xiaoping Xu, David P. Brooks, Nicholas J. Laping, and Timothy D.
Westfall
Cardiovascular and Urogenital Centre of Excellence in Drug Discovery (KST,
ACS, ZL, DJF, AKC, GPM, TM, ESRL, EAG, LE, BAM, XX, CPB, NJL, TDW)
GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA,
USA, 19406;
Oncology Centre of Excellence in Drug Discovery (DJD, LNC, RWM, BJV)
GlaxoSmithKline Pharmaceuticals, 1250 South Collegeville Road, Collegeville,
PA, USA, 19426;
Transgenics, Genotyping and Viral Vectors (JHN, SAS), GlaxoSmithKline
Pharmaceuticals, Harlow, UK CM19 5AD
1
Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
Running Title Page
Running Title: TRPV4 activator GSK1016790A induces bladder overactivity
Correspondence: Kevin S. Thorneloe
GlaxoSmithKline Pharmaceuticals
709 Swedeland Road
King of Prussia, PA 19406 Downloaded from
Tel: (610) 270-6621
Fax (610) 270-5681 jpet.aspetjournals.org email: [email protected]
Pages: 39 at ASPET Journals on September 27, 2021
Tables: 2
Figures: 8
References: 40
Abstract: 247 words
Introduction: 483 words
Discussion: 1494 words
Abbreviations: UBSM – urinary bladder smooth muscle
TRPV4 – transient receptor potential vanilloid 4
4α−PDD - 4α-phorbol 12,13-didecanoate
2 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
Abstract
The transient receptor potential vanilloid 4 (TRPV4) member of the TRP superfamily has recently been implicated in numerous physiological processes.
Here we describe a small molecule TRPV4 channel activator, GSK1016790A, which we have utilized as a valuable tool in investigating the role of TRPV4 in the urinary bladder. GSK1016790A elicited Ca2+ influx in mouse and human TRPV4
expressing HEK cells (EC50 values of 18 and 2.1 nM, respectively), and evoked a Downloaded from dose-dependent activation of TRPV4 whole-cell currents at concentrations above
1 nM. In contrast the TRPV4 activator 4α-phorbol 12,13-didecanoate (4α−PDD) jpet.aspetjournals.org was 300-fold less potent than GSK1016790A in activating TRPV4 currents.
TRPV4 mRNA was detected in urinary bladder smooth muscle (UBSM) and
+/+
urothelium of TRPV4 mouse bladders. Western blotting and at ASPET Journals on September 27, 2021 immunohistochemistry demonstrated protein expression in both the UBSM and urothelium that was absent in TRPV4-/- bladders. TRPV4 activation with
GSK1016790A contracted TRPV4+/+ mouse bladders in vitro, both in the presence and absence of the urothelium, an effect that was undetected in
TRPV4-/- bladders. Consistent with effects on TRPV4 HEK whole-cell currents
4α−PDD demonstrated a weak ability to contract bladder strips when compared to GSK1016790A. In vivo, urodynamics in TRPV4+/+ and TRPV4-/- mice revealed an enhanced bladder capacity in the TRPV4-/- mice. Infusion of GSK1016790A into the bladders of TRPV4+/+ mice induced bladder overactivity with no effect in
TRPV4-/- mice. Overall TRPV4 plays an important role in urinary bladder function
3 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295 that includes an ability to contract the bladder as a result of the expression of
TRPV4 in the UBSM.
Downloaded from
jpet.aspetjournals.org
at ASPET Journals on September 27, 2021
4 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
Introduction
Transient Receptor Potential Vanilloid 4 (TRPV4), a member of the TRP superfamily of cation channels, has been implicated in a number of physiological processes including: osmoregulation (Liedtke and Friedman, 2003; Mizuno et al.,
2003), hearing (Tabuchi et al., 2005), thermal and mechanical hyperalgesia
(Todaka et al., 2004; Grant et al., 2007; Alessandri-Haber et al., 2006), cell
volume regulation in epithelial cells (Arignes et al., 2004), and maintenance of Downloaded from the alveolar septal barrier (Alvarez et al., 2006). These observations have been largely based on observations from TRPV4-/- mice, with only two pharmacological jpet.aspetjournals.org activators of TRPV4 reported to date, a natural product bisandrographolide
(Smith et al., 2006) and the more widely used synthetic ligand 4α-phorbol 12,13-
α
didecanoate (4 -PDD; Watanabe et al., 2002a). In addition, ruthenium red has at ASPET Journals on September 27, 2021 been used as a TRPV4 channel blocker but is relatively non-selective.
TRPV4 conducts Ca2+ and Na+ across the plasma membrane of various cell types with a ~6:1 selectivity under physiological conditions (Strotmann et al.,
2000; Watanabe et al., 2002a; Watanabe et al., 2002b; Plant and Strotmann,
2007) normally enhancing cellular excitability. TRPV4 is activated by increased temperature with a threshold of ~27°C (Guler et al., 2002; Watanabe et al.,
2002b), thereby demonstrating significant constitutive activity at physiological temperatures (Vriens et al., 2004; Shibasaki et al., 2007). TRPV4 is also activated by physical cell stress, such as that induced by hypotonic challenge
(Strotmann et al., 2000; Gao et al., 2003; Liedtke and Friedman, 2003). Overall
TRPV4 activation appears to be a multimodal and complex graded process that
5 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295 likely serves to finely tune TRPV4 activity in different physiological and pathophysiological states.
TRP channels have been implicated in urinary bladder function, with a documented role for the TRPV1 family member in bladder sensation as a function of its expression on the urothelium and bladder afferent nerves (Birder et al., 2001; Birder et al., 2002). TRPV1 has been exploited clinically to desensitize
bladder afferents and reduce bladder overactivity (Avelino and Cruz, 2006). More Downloaded from recently, TRPV4 expression has been shown in the urothelium, and implicated in the regulation of urothelial ATP release modulating the sensitivity of bladder jpet.aspetjournals.org afferent nerves (Birder et al., 2007; Gevaert et al., 2007). Urinary bladder smooth muscle (UBSM) elicits spontaneously occurring phasic contractions of myogenic origin that directly result from UBSM action potentials (Hashitani and Brading, at ASPET Journals on September 27, 2021
2003). A number of ion channels have been shown to modulate UBSM action potentials, including depolarizing L-type Ca2+ channels, and various K+ channels that hyperpolarize the membrane potential (Heppner et al., 1997; Petkov et al.,
2001; Hashitani and Brading, 2003; Thorneloe and Nelson, 2003; Thorneloe et al., 2005). In addition, non-selective cation channels, like those of the TRP family, can contribute to depolarization and bladder contraction (Thorneloe and
Nelson, 2004).
Here we describe a novel TRPV4 channel activator GSK1016790A with significantly greater potency than those previously reported, and demonstrate a functional role for TRPV4 channels expressed in UBSM as regulators of urinary bladder activity.
6 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
Methods
Generation of TRPV4-/- Mice. Genomic fragments homologous to the TRPV4 locus were cloned by PCR from the E14.1 ES cell line using the Expand long template PCR kit (Roche). Primers for the 5' homology arm were VR4_5F (5'
TTC TTG TTG ACC CAC AAG AAG CGC CT) and VR4_5R (5' ATG GTG TCG
TTG CGC CCG TTG CTT AGG TT) spanning coding exons 3-4; for the 3' arm,
VR4_3F (5' TTC TTC CAG CCC AAG GAT GAG GGA GGC T) and VR4_3R (5' Downloaded from
AGA TGC CGG GTG TCC TCA TCT GTC ACC T) spanning coding exons 5-7.
The homology arms were inserted into the vector by homologous recombination jpet.aspetjournals.org in yeast as previously described (De Marini et al., 2000) such that they flanked the positive selection cassette containing an IRES-LacZ reporter, a PGK-neo selectable marker and the yeast URA3 selectable marker (supplemental Fig. 1A), at ASPET Journals on September 27, 2021 positioned to disrupt exons 4 and 5 thereby interrupting the coding sequence at
TM1 and frameshifts the remainder of the transcript. The NotI-linearised targeting vector (25ug) was electroporated into 107 E14.1 ES cells using a Bio-Rad
(Hercules, CA) Gene Pulser system set at 500uF, 230v. Homologous recombination in G418 resistant ES cells was confirmed by Southern blot of
EcoRV digested genomic DNA using a 3' external probe generated by PCR using primers VR4_3’PF (5’ AGC CCT GTT CAC TTA GC) and VR4_3’PR (5’ GCC
CTG CAC AAT CGT) which detects 3.75kb and 3.3kb bands at the wild-type and targeted locus respectively (supplemental Fig. 1B). Correct integration at the 5' end was confirmed by long PCR (data not shown). Four correctly targeted ES cell clones were identified by Southern blot analysis out of 400 clones in total.
7 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
Targeted clones were injected into C57B1/6J-derived blastocysts and gave rise to germline transmitting chimaeric mice. Male chimaeras were crossed with
C57B16/J females to give heterozygous offspring. These heterozygous mice were further backcrossed and interbred to generate homozygous mutants. The absence of wild type transcript from homozygous KO mice was confirmed by RT-
PCR (Fig. 3A). Genotypic analysis was performed by PCR using DNA isolated
from tails, neo-specific primers NeoF1.3 (5' GCT GCT CTG ATG CCG CCG TGT Downloaded from
TC) and NeoR1.3 (5' CTT CGC CCA ATA GCA GCC AGT CC), which produce a
202bp product from the targeted allele, and wild type-specific primers jpet.aspetjournals.org VR4204L19 (5’ CAG ACA GCA ATG GGC GAT G) and VR42U19 (5’ CAC CGG
CAA CAT GCG TGA A), which produce a 221bp product from the wild type allele. Interbred mice produced the expected Mendelian ratio of homozygotes to at ASPET Journals on September 27, 2021 heterozygotes to wild types. Mice utilized in these studies were back crossed for at least six generations, weighed 18-26 g and were housed under a 12-h light/dark cycle with free access to food and water. All experiments were conducted according to the requirements of the United Kingdom Animals
(Scientific Procedures) Act (1986) and strictly conformed to the ethical standards of the Institutional Animal Care and Use Committee (IACUC) of GlaxoSmithKline
Pharmaceuticals.
Reverse transcriptase polymerase chain reaction (RT-PCR) and Taqman.
Total RNA was isolated from TRPV4+/+ and TRPV4-/- bladders using TRI Reagent
(Ambion, Austin, TX). RT-PCR was performed with M-MLV reverse transcriptase and SuperTaq DNA polymerase enzymes (Ambion) in conjunction with TRPV4
8 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295 selective primers (Earley et al., 2005). RT-PCR products were separated on a
1% agarose gel with 1-kb ladder (Invitrogen, Carlsbad, CA) utilized as a standard. Images were obtained using a Kodak Image Station 440. For Taqman studies cDNA was synthesized using MultiScribe Reverse Transcriptase (Applied
Biosystems, Foster City, CA). All probe/primer sets were purchased as ‘assay on demand’ (Applied Biosystems). GAPDH was utilized as the housekeeper. The
urothelium and UBSM layers were separated by sharp dissection. Successful Downloaded from separation of these two layers was verified by Taqman amplifying uroplakin 3A and SM22 as urothelial and SM markers, respectively. Urothelial and UBSM jpet.aspetjournals.org expression was presented relative to the average expression in UBSM.
Membrane Preparation and Western Blotting. A crude membrane preparation was prepared from frozen tissues, which were homogenized in pH 7.0 buffer at ASPET Journals on September 27, 2021 containing 4mM HEPES, 320mM sucrose and protease inhibitors (10 ml per gram tissue). The homogenate was centrifuged at 2000 x g for 10min and the supernatant was saved. Another equal volume of buffer was added to the pellet, which was re-homogenized and centrifuged. The two supernatants were pooled and centrifuged for 1h at 100,000 x g. The resultant pellet was re-suspended in buffer and total protein was quantified according to Bradford (Biorad). 40ug of membrane protein from TRPV4+/+ or TRPV4-/- urothelium and UBSM were subjected to SDS-PAGE gel electrophoresis with protein lysates from TRPV4 expressing HEK293 cells as a positive control. Proteins were then transferred to nitrocellulose membranes, which were probed with a TRPV4 polyclonal antibody
(1:500 dilution) raised against CDGHQQGYPRKWRTDDAPL of the human
9 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
TRPV4 C-terminus (Delany et al., 2001), followed by incubation with a secondary goat anti-rabbit (926-32211, Licor). Images of the Western blots were acquired using the Odyssey® Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE).
Immunohistochemistry. Bladders from TRPV4+/+ and TRPV4-/- mice were dissected, embedded in O.C.T. compound (Fisher Scientific, Pittsburgh, PA) and frozen on dry ice. A polyclonal antibody specific for a synthetic peptide identical
to the first sixteen amino acids of rat TRPV4, MADPGDGPRAAPGDVA, was Downloaded from prepared in rabbits and affinity purified (Invitrogen). Eight micron thick cryostat sections were thaw mounted onto SuperFrost Plus slides and fixed in 3% jpet.aspetjournals.org Formaldehyde in PBS. Sections were rinsed in PBS, blocked in 10% normal donkey serum (Jackson Immunoresearch, West Grove, PA) to eliminate non- specific binding, and incubated with the TRPV4 antibody (2.0 ug/ml) in PBS for at ASPET Journals on September 27, 2021 two hours at room temperature. Bound antibody was detected using a Texas red conjugated donkey anti-rabbit IgG (Jackson Immunoresearch). TRPV4 immunoreactive urothelial cells were detected by simultaneous incubation of the sections with the TRPV4 antibody and an Uroplakin III specific antibody (Santa
Cruz, Santa Cruz, CA). Immunoreactivity was visualized by a combination of the appropriate fluorescein isothiocyanate or Texas red–labeled donkey secondary antisera (Jackson Immunoresearch, West Grove, PA). Sections were counter stained with 4′6-diamidino-2phenylindole (DAPI; Molecular Probes, Eugene, OR) and images were acquired using a Nikon E1000 microscope (Nikon, Tokyo,
Japan), a DXM 1200C camera (Nikon) and Elements BR imaging program
(Nikon).
10 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
TRPV4 Ca2+ Influx. HEK (MSRII) cells were transduced with a murine TRPV4 containing bacmam virus (6 particles/cell), then plated at 50,000 cells per well in a 96-well plate and allowed to adhere O/N. The cells were then dye loaded with
Fura-2 (Molecular Probes). Briefly, cells were incubated for 1 hr at 37ºC in the presence of 3 uM Fura-2 in TG buffer (mM: 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2,
10 glucose, 10 HEPES, pH = 7.4) containing 0.02% Pluronic acid (Sigma). The Downloaded from dye containing solution was then removed and replaced with fresh TG buffer and the cells incubated for an additional 30 min at 37ºC to allow for hydroysis of the jpet.aspetjournals.org dye. The buffer was then replaced with TG buffer containing 1% bovine serum albumen (Sigma, St. Louis, MO) and the assay plate was loaded into a
FlexStation (Molecular Devices, Sunnyvale, CA). The intracellular calcium at ASPET Journals on September 27, 2021 concentration was monitored as the change in Fura-2 fluorescence following addition of a TRPV4 agonist. The response obtained using untransduced or Null- virus transduced HEK cells was minimal.
Electrophysiology. Whole-cell voltage-clamp recordings were performed on
HEK293 cells expressing human TRPV4 channels and on untransfected HEK cells. The pipette solution contained (in mM): 130 CsCl, 5 EGTA, 5.5 MgCl2,
5 Na2ATP, 0.1 Na-GTP, 5 HEPES; pH = 7.2. The external solution contained (in mM): 135 NaCl, 5 CsCl, 1 MgCl2, 1 CaCl2, 10 Glucose, 10 HEPES; pH = 7.4.
Membrane currents were recorded at room temperature using a ramp voltage- clamp protocol repeated every 10-s (holding potential of -60 mV, depolarizing
11 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295 from -60 mV to +60 mV in 760-ms). Peak drug responses were measured at +60 mV and normalized by the cell membrane capacitance yielding current densities.
Urinary Bladder Contractility. TRPV4+/+ and TRPV4-/- mice were euthanized by
CO2 asphyxiation. A midline abdominal incision was made and the bladder was dissected free from the animal. The bladders were cleaned of adherent connective tissue/fat and cut into two equal halves from bladder dome to the
urethral opening. A length of 4-0 suture was tied and double knotted at the top Downloaded from and another at the base of each tissue. The tissue was then suspended vertically in a 15 ml organ bath by securing one suture to a stationary holder and the other jpet.aspetjournals.org to an isometric force transducer. The organ bath contained Krebs solution (38-
39°C) and was constantly aerated by bubbling with 95% oxygen / 5% carbon dioxide. One gram of tension was applied to each tissue and the bladder strips at ASPET Journals on September 27, 2021 were allowed to equilibrate for 60 min. During equilibration the Krebs solution was changed and the tension readjusted to 1 gram every 20 min. For electrical field stimulation experiments the tissues were electrically stimulated at a frequency of 8 Hz delivered every 60 seconds. The stimulation parameters were:
60 V; 0.5 msec pulse duration; 1 sec train duration. The 8 Hz stimulation was repeated until a stable contractile response was obtained (10-20 stimulations).
The stimulation was discontinued and the tissues were allowed to recover for 60 minutes. A frequency–contraction curve to 1, 2, 4, 8, 16, 32, and 64 Hz was then generated in each tissue. Three consecutive responses, separated by 60 seconds each, were obtained for each frequency and the average of the 3 contraction amplitudes was measured. In experiments involving urothelium-free
12 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295 bladder halves, TRPV4+/+ bladders were cut in half, the urothelium was removed by sharp dissection under a dissecting microscope from one half, and compared to the paired urothelium-intact half. Force was measured in mN or as an integrated area under the curve. Phasic contractile activity was assessed by measuring the phasic area (area under the curve minus baseline).
Conscious Cystometry. Mouse cystometry was performed as described
previously (Thorneloe et al., 2005). Mice were anesthetized using iso-flurane Downloaded from
(Abbott Laboratories, N.Chicago, IL) and the urinary bladder exposed by an abdominal midline incision. A purse string suture was made in the dome of the jpet.aspetjournals.org bladder into which a saline filled bladder catheter (PE-10) was inserted and secured. The catheter was routed subcutaneously to the back of the neck and stored in a skin pouch. After a minimum 7 day recovery, and under light at ASPET Journals on September 27, 2021 isoflurane anesthesia, the end of the bladder catheter was retrieved from the skin pouch. Saline filled polyethylene (PE-50) tubing was used to extend the externalised catheter and connect it to the syringe pump on a Small Animal
Cystometry Lab Station (MED Associates Inc., St. Albans, VT). Mice were infused with saline (25 µl/min) via the bladder catheter for a 30 min equilibration period, followed by 30 min of basal, control urodynamics that were utilized for genotype comparison. Mice were then challenged by infusion with saline containing 0.25% acetic acid or 10 µM GSK1016790A for an additional hour.
Infused maximum (volume infused to induce voiding), void volume (urine voided), pressure threshold (pressure prior to micturition), average filling pressure, minimum filling pressure and peak pressure (micturition peak) were recorded.
13 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
Non-voiding contractions were assessed as increases in bladder pressure of greater than 5 mmHg during the filling phase, and were analyzed using Mini
Analysis software (Synaptosoft, Decatur, GA). On completion of cystometry mice were euthanized by carbon dioxide inhalation followed by diaphragm disruption.
Statistical Analysis. Data is expressed as the mean ± S.E.M. GraphPad Prism
Version 4 (GraphPad Software Inc., San Diego, CA) was used for graphing and
statistical analysis. Two-way RM ANOVA was used for multiple group Downloaded from comparisons. Student’s paired or unpaired t-tests were used as appropriate. P <
0.05 was deemed statistically significant. jpet.aspetjournals.org
at ASPET Journals on September 27, 2021
14 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
Results
Identification of the TRPV4 activator GSK1016790A. As part of a small molecule screening effort GSK1016790A (N-((1S)-1-{[4-((2S)-2-{[(2,4- dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3- methylbutyl)-1-benzothiophene-2-carboxamide, was identified as a novel TRPV4 channel activator (Fig. 1A). GSK1016790A potently induced Ca2+ influx in human
embryonic kidney (HEK) cells expressing mouse TRPV4 channels with an EC50 Downloaded from value of 18 nM (supplemental Fig. 2). GSK1016790A demonstrated a similar potency at human TRPV4 channels (EC50 = 2.1 nM), and was ineffective in null- jpet.aspetjournals.org transduced HEK cells. GSK1016790A was approximately 10 fold more potent activating TRPV4 than activating TRPV1 channels (see companion paper,
Willette et al., 2008), which based on sequence homology is the closest TRP at ASPET Journals on September 27, 2021 superfamily member to TRPV4. In addition, we tested GSK1016790A against human TRPM8 and TRPA1 channels at concentrations up to 20 uM demonstrating no modulation of Ca2+ influx. GSK1016790A elicited a steep, dose-dependent activation of whole-cell currents in TRPV4 transfected HEK cells at concentrations of 3 nM and above, reaching a current density of 710 ± 144 pA/pF measured at +60 mV with 10 nM GSK1016790A (n=6, Fig. 2A, C and D).
By comparison, the TRPV4 activator 4α-PDD was ineffective at 0.3 uM evoking current at 1 uM and above, reaching 323 ± 88 pA/pF at 10 uM (n=6, Fig. 2A, C and D). Whereas 10 nM GSK1016790A did not activate currents in untransfected
HEK cells, 10 uM 4α-PDD elicited a small non-specific current (Fig. 2B and D, 55
15 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
± 11 pA/pF). This suggests that 4α-PDD at a concentration of 10 uM may modulate other ion channel activities in addition to TRPV4.
TRPV4 expression in the urinary bladder. RT-PCR performed on total RNA isolated from intact TRPV4+/+ bladders yielded a ~ 500 bp product (Fig. 3A) consistent with the predicted 478 bp TRPV4 product. The TRPV4 product was absent from TRPV4+/+ bladder samples when the reverse transcriptase enzyme
-/- was omitted from the reaction, and when total RNA from TRPV4 mouse Downloaded from bladders was used as substrate (Fig. 3A). Taqman for TRPV4 on isolated urothelium and UBSM tissues demonstrated that TRPV4 is expressed in both of jpet.aspetjournals.org these tissues and that mRNA levels are ~ 20 fold higher in the urothelium than in the UBSM (Fig. 3B). As expected, Taqman on TRPV4-/- urothelium and UBSM yielded no amplification products. Western blotting for TRPV4 on crude at ASPET Journals on September 27, 2021 membranes isolated from TRPV4 expressing HEK cells demonstrated two bands just above 98 kDa (Fig. 3C), which was not present in untransfected cells.
Similarly this doublet was observed in crude membranes prepared from both
TRPV4+/+ UBSM and urothelial tissues, but not in TRPV4-/- UBSM and urothelial membranes (Fig. 3C). Immunohistochemistry was performed on fresh frozen sections of urinary bladders isolated from TRPV4+/+ and TRPV4-/- mice. TRPV4 immunofluorescence was detected in both the urothelium and UBSM layers of the bladder wall in TRPV4+/+ sections, but not in the sub-urothelial space between these layers, and was absent in TRPV4-/- sections (Fig. 4A). Co- labelling demonstrated overlap with the urothelial cell marker uroplakin III and
TRPV4 in the urothelium, indicating that TRPV4 is expressed on urothelial cells
16 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
(Fig. 4B). In addition, TRPV4 immunofluroescence was observed on spindle shaped cells within the UBSM layer typical of smooth muscle cell morphology
(Fig. 4C). Consistent with this co-labelling for the smooth muscle marker smooth muscle alpha-actin and TRPV4 demonstrated significant overlap (Supplemental
Figure 3). Immunofluoresence intensity was higher in the urothelium compared to the UBSM (Fig. 4A), consistent with the significantly higher mRNA expression
levels determined by Taqman in the urothelium (Fig 3B). Supporting a functional Downloaded from presence of TRPV4 in mouse UBSM cells, GSK1016790A activated cation currents from freshly isolated guinea-pig UBSM cells (10 nM, n=16) that were jpet.aspetjournals.org inhibited by the TRPV4 channel blocker ruthenium red (10 uM, n=6,
Supplemental Methods and Supplemental Figure 4). These results indicate that
TRPV4 is expressed in the UBSM and urothelium of TRPV4+/+ bladders and at ASPET Journals on September 27, 2021 confirm an absence of TRPV4 expression in TRPV4-/- bladders.
Role of TRPV4 in in vitro bladder contractility. An in vitro contractile response was observed in TRPV4+/+ bladders with GSK1016790A (10-100 nM, Fig. 5). This effect was absent in TRPV4-/- mouse bladders. GSK1016790A (100 nM; Fig. 6A and B) and carbachol (1 uM, n=4, data not shown) contractions were not effected in paired bladder halves upon removal of the urothelium. These GSK1016790A- induced contractions were also not different when normalized to the carbachol response, 29±4% and 38±6%, respectively, in the urothelium free and intact bladder halves. Therefore, urothelial TRPV4 channels did not appear to contribute to contractions induced by GSK1016790A. GSK1016790A-induced contractions were reversed ~ 80% by 1 uM nifedipine in both urothelium intact
17 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295 and urothelium free bladder halves (Fig. 6C). Consistent with the weak ability of
4α-PDD to evoke TRPV4 currents in TRPV4 HEK cells, 4α-PDD demonstrated a poor ability to contract bladder halves eliciting only small contractile responses at
10 and 100 uM (Fig. 6D). This is in sharp contrast to the larger contractions observed with a 100-fold lower concentration of GSK1016790A (100 nM).
GSK1016790A-induced contractions were blocked by ruthenium red pre-
treatment (10 uM, n=3), but were insensitive to ω-conotoxin GVIA (0.3 uM, n=3), Downloaded from tetrodotoxin (1 uM), atropine (1 uM, n=5), or desensitization of TRPV1 and purinergic P2X channels with capsaicin (10 uM, n=5) and αβ-methylene ATP (10 jpet.aspetjournals.org
µM, n=4) respectively. In addition, the amplitude and frequency dependence of nerve-mediated bladder contractions evoked by electrical field stimulation (1-64
+/+ -/- Hz) were not different in TRPV4 and TRPV4 mice (n=4 mice/genotype). at ASPET Journals on September 27, 2021
Taken together these results suggest the contractile effects of GSK1016790A are due to a direct action on the UBSM and not an indirect effect on neurotransmitter release.
Urodynamics in TRPV4-/- mice. Conscious cystometry was performed to evaluate the bladder function of unrestrained, age, weight and sex-matched
TRPV4+/+ and TRPV4-/- mice during intravesical saline infusion. The urodynamic threshold, peak micturition and average filling pressures were not significantly different between genotypes (Table 1). However, the bladder capacity of TRPV4-
/- mice was greatly enhanced compared to TRPV4+/+ mice. This was indicated by
TRPV4-/- mice requiring significantly larger infused volumes to initiate a micturition event and demonstrating larger micturition void volumes (Fig. 7; Table
18 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
1). Non-voiding contraction frequency and amplitude, measured as bladder pressure oscillations during the filling phase of cystometrograms, were not different between TRPV4+/+ and TRPV4-/- mice (TRPV4+/+ 0.013±0.002 Hz n=15,
TRPV4-/- 0.013±0.003 Hz n=11, p=0.98; TRPV4+/+ 9.1±0.4 mmHg n=15, TRPV4-/-
8.0±0.6 mmHg n=11, p=0.11). Cystometric measurements were made during infusion of GSK1016790A into the bladders of TRPV4+/+ and TRPV4-/- mice. No
significant effect was observed with 1 µM GSK1016790A (n=3), however, 10 uM Downloaded from
GSK1016790A evoked a robust reduction in infused and voided volumes, and enhanced the average and minimum filling pressures in TRPV4+/+ mice (n=8, Fig. jpet.aspetjournals.org 8, Table 2). Effects of GSK1016790A on bladder volumes and bladder pressures were completely absent in TRPV4-/- mice (Fig. 8; Table 2). In additional studies
+/+ -/-
TRPV4 (n=7) and TRPV4 (n=11) mice were infused intravesicularly with at ASPET Journals on September 27, 2021 saline followed by 0.25% acetic acid to induce bladder overactivity (Supplemental
Table 1). Bladder overactivity was apparent as acetic acid greatly reduced bladder capacity, indicated by reductions in infused and voided volumes in both
TRPV4+/+ and TRPV4-/- mice. In addition, average filling and threshold pressures were increased by acetic acid for TRPV4+/+ and TRPV4-/- mice, although the increase in threshold pressure did not reach significance for TRPV4+/+ (p=0.10).
A comparison of the normalized response to acetic acid in TRPV4+/+ and
TRPV4-/- mice revealed no significant differences between genotypes
(Supplemental Table 1).
19 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
Discussion
GSK1016790A is a novel TRPV4 channel activator that is ~300-fold more potent than the commonly used TRPV4 activator 4α-PDD. We have utilized, molecular techniques, TRPV4-/- mice, in vitro contractility and in vivo urodynamics in conjunction with GSK1016790A, to support a role for TRPV4 in
UBSM. TRPV4 mRNA was amplified by PCR, and protein expression was demonstrated by Western blotting and immunohistochemistry, in UBSM and Downloaded from urothelium. GSK1016790A contracted bladder independent of the urothelium and induced bladder overactivity in vivo. Effects of GSK1016790A on contractility and jpet.aspetjournals.org urodynamics were absent in TRPV4-/- mice, indicating the responses to be
TRPV4 mediated. Inhibition of TRPV4 function, via knocking out of the TRPV4
gene, resulted in an enhancement of bladder capacity without alterations in at ASPET Journals on September 27, 2021 bladder pressures. These data demonstrate the functional selectivity of the
TRPV4 channel activator GSK1016790A, and implicate the functional expression of TRPV4 channels in UBSM as regulators of bladder contractility.
GSK1016790A elicits Ca2+ influx through mouse and human TRPV4 channels in HEK cells with EC50 values of 18 and 2.1 nM, respectively. These values are consistent with our electrophysiological recordings demonstrating a 3 nM threshold for TRPV4 activation. In contrast we show that under identical conditions ≥ 1 uM 4α-PDD is required to activate TRPV4, demonstrating that
GSK1016790A has at least 300-fold greater potency for activating TRPV4 than
4α-PDD. This was confirmed in functional bladder contractility studies in which
~1000 fold higher concentrations of 4α-PDD (10 uM) were required to provide a
20 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295 similar potentiation of force as with GSK1016790A (10 nM). A previous report measuring Ca2+ entry and TRPV4 current activation with 4α-PDD suggested an
EC50 of 0.3 uM (Watanabe et al., 2002a). Our electrophysiological data would suggest a ~10-fold weaker potency (Fig. 2C). These differing values for 4α-PDD may be attributable to differences in recording conditions and/or expression systems. GSK1016790A is also more potent than the natural product
bisandrographolide that activates TRPV4 with an EC50 value of 0.95 uM (Smith et Downloaded from al., 2006). To our knowledge GSK1016790A is by far the most potent TRPV4 channel activator reported to date. jpet.aspetjournals.org We have detected TRPV4 mRNA expression in mouse UBSM and urothelium, with ~20 fold higher levels in the urothelium. In addition, we have demonstrated TRPV4 protein expression in both tissues by Western blotting and at ASPET Journals on September 27, 2021 immunohistochemistry. In all cases mRNA and protein expression was absent in
TRPV4-/- samples demonstrating the TRPV4 specificity of the antibody signals and PCR amplifications. TRPV4 immunofluoresence was more robust in the urothelium than in the UBSM, consistent with our quantitative PCR results. This study provides the first evidence supporting TRPV4 protein expression and function in UBSM. A recent report using semi-quantitative RT-PCR demonstrated that TRPV4 mRNA is present in rat urothelium, and at a lower level in UBSM
(Birder et al., 2007). They detected TRPV4 by immunohistochemistry in rat urothelium however TRPV4 protein expression in UBSM was not documented.
Subsequently, TRPV4 immunofluoresence was confirmed in rat and mouse urothelium, however, no specific UBSM TRPV4 protein expression was observed
21 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
(Gevaert et al., 2007). The use of different TRPV4 antibodies and/or stringency conditions may account for discrepancies between the current study demonstrating UBSM TRPV4 protein expression and previous reports not detecting TRPV4 protein in UBSM (Birder et al., 2007; Gevaert et al., 2007).
GSK1016790A contracted bladder with a dependence on TRPV4 expression as the response was absent in TRPV4-/- bladders. GSK1016790A
contractions were unaffected by urothelium removal. Pharmacological Downloaded from modulators inhibiting neurotransmitter release from nerve terminals, or blockade/desensitization of cholinergic/purinergic receptors had no effect on jpet.aspetjournals.org GSK1016790A contractions. This suggests a lack of a role for TRPV4 expressed in the urothelium and/or in nerves within the bladder in mediating GSK1016790A contractions, and implicates the TRPV4 protein expression detected in UBSM as at ASPET Journals on September 27, 2021 the contractile mediator. TRPV4 contraction of airway smooth muscles has also been reported (Jia et al., 2004). The ability of TRPV4 activation to contract
UBSM is consistent with identified constitutive and stretch-activated non- selective cation channels in UBSM cells (Wellner and Isenberg, 1993; Thorneloe and Nelson, 2004), as TRPV4 channels demonstrate constitutive activity
(Watanabe et al., 2002b; Vriens et al., 2004) and sensitivity to stretch (Strotmann et al., 2000; Gao et al., 2003; Liedtke et al., 2000). In addition, we have observed a small contractile response to 4α-PDD at concentrations ≥ 10 uM in TRPV4+/+ mouse bladders, concentrations consistent with those shown to activate TRPV4 in electrophysiological recordings. Despite the conclusions presented by Birder et al. (2007) that concentrations up to 100 uM 4α-PDD did not affect basal tone of
22 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295 rat bladder strips, it appears from the data presented in figure 4 in their publication that 10 uM and above did elicit a small dose-dependent increase in baseline force, which is consistent with our 4α-PDD data on mouse bladder strips.
Nifedipine-sensitive, L-type Ca2+ channel (Cav1.2) mediated Ca2+ entry is the major contributor to mouse UBSM contractions (Wegner et al., 2004). In
-/- -/- contrast to Cav1.2 mice (Wegner et al., 2004), TRPV4 mice do not Downloaded from demonstrate an overt loss in UBSM contractility, as indicated by normal contractile responses to electrical field stimulation and carbachol in vitro, and jpet.aspetjournals.org normal peak voiding pressures in vivo. This is consistent with a modulatory role for TRPV4 in bladder contractility in vitro and bladder function in vivo. Nifedipine reversed GSK1016790A contractions indicating a prominent role for L-type Ca2+ at ASPET Journals on September 27, 2021 channels in mediating TRPV4 contractions, and suggesting that a direct supply of
Ca2+ through TRPV4 channels is likely to play a minor role, if any, in the contraction. It appears that TRPV4 channel activation depolarizes the UBSM membrane potential, thereby activating L-type Ca2+ channel entry of contractile
Ca2+. However, we can not completely eliminate a contributory role for an expression of TRPV4 in other cell types present within the UBSM layer including fibroblasts and interstitial cells (Davidson and McCloskey, 2005). Conceivably
TRPV4 activation in these cells, could also play a role and indirectly modulate
UBSM contractility, via electrical and/or chemical signalling that leads to nifedipine-sensitive contraction. These data suggest that TRPV4 plays a role in regulating the UBSM membrane potential, and perhaps responds to urinary
23 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295 bladder distension during bladder filling, as TRPV4 channels are activated by stretch (Strotmann et al., 2000; Gao et al., 2003; Liedtke and Friedman, 2003).
Our urodynamic characterization of TRPV4-/- mice demonstrated an increased bladder capacity based on larger voided urine volumes and a requirement of a larger infused volume in order to elicit micturition events when compared to TRPV4+/+ mice. This phenotype is similar to that described in a
-/- distinct TRPV4 mouse (Gevaert et al., 2007). However, we do not feel that the Downloaded from urodynamics in TRPV4-/- mice represent a manifestation of an incontinent phenotype as concluded by Gevaert et al., 2007. In fact our interpretation of both jpet.aspetjournals.org the bladder phenotype described here and by Gevaert et al., 2007 is consistent with a beneficial effect on bladder function, with the loss of TRPV4 resulting in an enhancement of bladder capacity equating to a reduced frequency of urination. at ASPET Journals on September 27, 2021
TRPV4 channels are expressed in sensory afferent neurons and have been implicated in thermal and mechanical hyperalgesia, as well as pressure and heat sensation (Todaka et al., 2004; Grant et al., 2007; Alessandri-Haber et al.,
2006; Suzuki et al., 2003; Lee et al., 2005). This suggests a potential for expression of TRPV4 in bladder-specific sensory afferent nerves functioning as regulators of in vivo bladder activity. In addition, TRPV4-mediated ATP release from the urothelium is proposed to sensitize sensory bladder afferents, as a mechanism contributing to TRPV4-induced bladder hyperactivity in vivo (Birder et al., 2007; Gevaert et al., 2007). We have shown that acetic acid sensitization of bladder afferents remains unaltered in TRPV4-/- mice. This finding would not support a prominent role for TRPV4 in in vivo bladder afferent signalling under
24 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295 conditions of acidic/chemical inflammatory bladder irritation. However, our data is consistent with the lack of an altered response to acetic acid reported in urodynamic characterizations of TRPV1-/- mice (Birder et al., 2002). TRPV4 dependent release of ATP from the urothelium functioning to sensitize bladder afferents is not functional in in vitro studies of bladders denuded of the urothelium, in which we have clearly identified a functional role for TRPV4
regulating contraction. Therefore, we would propose that the GSK1016790A- Downloaded from induced bladder overactivity in vivo, absent in TRPV4-/- mice is a result of the functional role of TRPV4 in both the urothelium and the UBSM, causing a release jpet.aspetjournals.org of ATP from the urothelium sensitizing bladder afferents (Birder et al., 2007;
Gevaert et al., 2007) and direct contraction of UBSM, respectively.
In summary, we have described a novel TRPV4 activator GSK1016790A at ASPET Journals on September 27, 2021 that is at least 300-fold more potent than the TRPV4 activator 4α−PDD.
GSK1016790A along with TRPV4 antibodies has enabled a clear identification of the expression and function of TRPV4 channels in UBSM. Modulation of TRPV4 channel activity demonstrates the ability to negatively and positively regulate in vivo bladder function, as TRPV4 channel activation with GSK1016790A reduces bladder capacity, and loss of TRPV4 channels in TRPV4-/- mice enhances bladder capacity. We propose that TRPV4 plays a modulatory role regulating bladder contractility and compliance during filling.
25 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
References
Alessandri-Haber N, Dina OA, Joseph EK, Reichling D and Levine JD (2006) A transient receptor potential vanilloid 4-dependent mechanism of hyperalgesia is engaged by concerted action of inflammatory mediators. J Neurosci 26:3864-
3874.
Downloaded from
Alvarez DF, King JA, Weber D, Addison E, Liedtke W and Townsley MI (2006)
Transient receptor potential vanilloid 4-mediated disruption of the alveolar septal jpet.aspetjournals.org barrier: a novel mechanism of acute lung injury. Circ Res 99:988-995.
Arniges M, Vazquez E, Fernandez-Fernandez JM and Valverde MA (2004) at ASPET Journals on September 27, 2021 Swelling-activated Ca2+ entry via TRPV4 channel is defective in cystic fibrosis airway epithelia. J Biol Chem 279:54062-54068.
Avelino A and Cruz F (2006) TRPV1 (vanilloid receptor) in the urinary tract: expression, function and clinical applications. Naunyn Schmiedebergs Arch
Pharmacol. 373:287-299.
Birder LA, Kanai AJ, de Groat WC, Kiss S, Nealen ML, Burke NE, Dineley KE,
Watkins S, Reynolds IJ and Caterina MJ. (2001) Vanilloid receptor expression suggests a sensory role for urinary bladder epithelial cells. Proc Natl Acad Sci U
S A 98:13396-13401.
26 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
Birder L, Kullmann FA, Lee H, Barrick S, de Groat W, Kanai A and Caterina M
(2007) Activation of urothelial transient receptor potential vanilloid 4 by 4alpha- phorbol 12,13-didecanoate contributes to altered bladder reflexes in the rat. J
Pharmacol Exp Ther 323:227-235.
Birder LA, Nakamura Y, Kiss S, Nealen ML, Barrick S, Kanai AJ, Wang E, Ruiz
G, De Groat WC, Apodaca G, Watkins S and Caterina MJ (2002) Altered urinary Downloaded from bladder function in mice lacking the vanilloid receptor TRPV1. Nat Neurosci
5:856-860. jpet.aspetjournals.org
Davidson RA and McCloskey KD (2005) Morphology and localization of interstitial cells in the guinea pig bladder: structural relationships with smooth at ASPET Journals on September 27, 2021 muscle and neurons. J Urol 173:1385-1390.
Delany NS, Hurle M, Facer P, Alnadaf T, Plumpton C, Kinghorn I, See CG,
Costigan M, Anand P, Woolf CJ, Crowther D, Sanseau P and Tate SN (2001)
Identification and characterization of a novel human vanilloid receptor-like protein, VRL-2. Physiol Genomics 4:165-174.
DeMarini DJ, Creasy CL, Lu Q, Mao J, Sheardown SA, Sathe GM and Livi GP.
(2000) Oligonucleotide-mediated, PCR-independent cloning by homologous recombination. Biotechniques 30:520-523.
27 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
Earley S, Heppner TJ, Nelson MT and Brayden JE (2005) TRPV4 forms a novel
Ca2+ signaling complex with ryanodine receptors and BKCa channels. Circ Res
97:1270-1279.
Gao X, Wu L and O'Neil RG (2003) Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives
through protein kinase C-dependent and -independent pathways. J Biol Chem Downloaded from
278:27129-27137.
jpet.aspetjournals.org Gevaert T, Vriens J, Segal A, Everaerts W, Roskams T, Talavera K, Owsianik G,
Liedtke W, Daelemans D, Dewachter I, Van Leuven F, Voets T, De Ridder D and
Nilius B (2007) Deletion of the transient receptor potential cation channel TRPV4 at ASPET Journals on September 27, 2021 impairs murine bladder voiding. J Clin Invest 117:3453-3462.
Grant AD, Cottrell GS, Amadesi S, Trevisani M, Nicoletti P, Materazzi S, Altier C,
Cenac N, Zamponi GW, Bautista-Cruz F, Lopez CB, Joseph EK, Levine JD,
Liedtke W, Vanner S, Vergnolle N, Geppetti P and Bunnett NW (2007) Protease- activated receptor 2 sensitizes the transient receptor potential vanilloid 4 ion channel to cause mechanical hyperalgesia in mice. J Physiol 578:715-733.
Guler AD, Lee H, Iida T, Shimizu I, Tominaga M and Caterina M (2002) Heat- evoked activation of the ion channel, TRPV4. J Neurosci 22:6408-6414.
28 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
Hashitani H and Brading AF (2003) Ionic basis for the regulation of spontaneous excitation in detrusor smooth muscle cells of the guinea-pig urinary bladder. Br J
Pharmacol 140:159-169.
Heppner TJ, Bonev AD and Nelson MT (1997) Ca2+-activated K+ channels regulate action potential repolarization in urinary bladder smooth muscle. Am J
Physiol 273:C110-117. Downloaded from
Jia Y, Wang X, Varty L, Rizzo CA, Yang R, Correll CC, Phelps PT, Egan RW and jpet.aspetjournals.org Hey JA (2004) Functional TRPV4 channels are expressed in human airway smooth muscle cells. Am J Physiol 287:L272-278.
at ASPET Journals on September 27, 2021
Lee H, Iida T, Mizuno A, Suzuki M and Caterina MJ (2005) Altered thermal selection behavior in mice lacking transient receptor potential vanilloid 4. J
Neurosci 25:1304-1310.
Liedtke W, Choe Y, Martí-Renom MA, Bell AM, Denis CS, Sali A, Hudspeth AJ,
Friedman JM and Heller S (2000) Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor. Cell 103:525-535.
Liedtke W and Friedman JM (2003) Abnormal osmotic regulation in trpv4-/- mice.
Proc Natl Acad Sci U S A 100:13698-13703.
29 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
Mizuno A, Matsumoto N, Imai M and Suzuki M (2003) Impaired osmotic sensation in mice lacking TRPV4. Am J Physiol 285:C96-101.
Petkov GV, Heppner TJ, Bonev AD, Herrera GM and Nelson MT (2001) Low levels of K(ATP) channel activation decrease excitability and contractility of urinary bladder. Am J Physiol 280:R1427-1433.
Downloaded from
Plant TD and Strotmann R (2007) TRPV4. Handb Exp Pharmacol 179:189-205.
jpet.aspetjournals.org Shibasaki K, Suzuki M, Mizuno A and Tominaga M (2007) Effects of body temperature on neural activity in the hippocampus: regulation of resting membrane potentials by transient receptor potential vanilloid 4. J Neurosci at ASPET Journals on September 27, 2021
27:1566-1575.
Smith PL, Maloney KN, Pothen RG, Clardy J and Clapham DE (2006)
Bisandrographolide from Andrographis paniculata activates TRPV4 channels. J
Biol Chem 281:29897-29904.
Strotmann R, Harteneck C, Nunnenmacher K, Schultz G and Plant TD (2000)
OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity. Nat Cell Biol 2:695-702.
30 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
Suzuki M, Mizuno A, Kodaira K and Imai M (2003) Impaired pressure sensation in mice lacking TRPV4. J Biol Chem 278:22664-22668.
Tabuchi K, Suzuki M, Mizuno A and Hara A (2005) Hearing impairment in TRPV4 knockout mice. Neurosci Lett 382:304-308.
Thorneloe KS, Meredith AL, Knorn AM, Aldrich RW and Nelson MT (2005) Downloaded from
Urodynamic properties and neurotransmitter dependence of urinary bladder contractility in the BK channel deletion model of overactive bladder. Am J Physiol jpet.aspetjournals.org 289:F604-610.
Thorneloe KS and Nelson MT (2003) Properties and molecular basis of the at ASPET Journals on September 27, 2021 mouse urinary bladder voltage-gated K+ current. J Physiol 549:65-74.
Thorneloe KS and Nelson MT (2004) Properties of a tonically active, sodium- permeable current in mouse urinary bladder smooth muscle. Am J Physiol
286:C1246-1257.
Todaka H, Taniguchi J, Satoh J, Mizuno A and Suzuki M (2004) Warm temperature-sensitive transient receptor potential vanilloid 4 (TRPV4) plays an essential role in thermal hyperalgesia. J Biol Chem 279:35133-35138.
31 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
Vriens J, Watanabe H, Janssens A, Droogmans G, Voets T and Nilius B (2004)
Cell swelling, heat, and chemical agonists use distinct pathways for the activation of the cation channel TRPV4. Proc Natl Acad Sci U S A 101:396-401.
Watanabe H, Davis JB, Smart D, Jerman JC, Smith GD, Hayes P, Vriens J,
Cairns W, Wissenbach U, Prenen J, Flockerzi V, Droogmans G, Benham CD and
Nilius B. (2002a) Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol Downloaded from derivatives. J Biol Chem 277:13569-13577.
jpet.aspetjournals.org Watanabe H, Vriens J, Suh SH, Benham CD, Droogmans G and Nilius B (2002b)
Heat-evoked activation of TRPV4 channels in a HEK293 cell expression system and in native mouse aorta endothelial cells. J Biol Chem 277:47044-47051. at ASPET Journals on September 27, 2021
Wellner MC and Isenberg G (1993) Properties of stretch-activated channels in myocytes from the guinea-pig urinary bladder. J Physiol 466:213-227.
Wegener JW, Schulla V, Lee TS, Koller A, Feil S, Feil R, Kleppisch T, Klugbauer
N, Moosmang S, Welling A and Hofmann F (2004) An essential role of Cav1.2 L- type calcium channel for urinary bladder function. FASEB J 18:1159-1161.
Willette RN, Bao W, Nerurkar S, Yue T, Doe CP, Stankus G, Ju H, Thomas H,
Fishman CE, Sulpizio AC, Behm DJ, Hoffman S, Lin Z, Lozinskaya I, Casillas LN,
Lin M, Trout REL, Votta BJ, Thorneloe KS, Lashinger ESR, Figueroa DJ, Marquis
32 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
R and Xu X (2008) Systemic Activation of the Transient Receptor Potential V4
Channel Causes Endothelial Failure and Circulatory Collapse: Part II. J
Pharmacol Exp Ther (companion paper).
Downloaded from
jpet.aspetjournals.org
at ASPET Journals on September 27, 2021
33 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
Footnotes
This work was supported by GlaxoSmithKline Pharmaceuticals.
Downloaded from
jpet.aspetjournals.org
at ASPET Journals on September 27, 2021
34 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
Legends for Figures
Figure 1: A. Chemical structure of the TRPV4 channel activator GSK1016790A, (N-((1S)-
1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1- piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide.
Figure 2: Comparison of TRPV4 current activation by GSK1016790A and
4α-PDD. A and B. Example current density/voltage relationship induced by a Downloaded from depolarizing ramp voltage-clamp protocol in TRPV4 expressing HEK cells (A) and in untransfected HEK cells (B), before and after TRPV4 channel activation jpet.aspetjournals.org with 10 nM GSK1016790A (left) and 10 uM 4α-PDD (right). C. Concentration- dependent increases of current density measured at +60 mV by GSK1016790A and 4α-PDD in HEK293 cells expressing TRPV4, n values are indicated for each at ASPET Journals on September 27, 2021 dose. D. Comparison of current density activated by 10 nM GSK1016790A or 10
µM 4α-PDD at +60 mV in HEK293 cells expressing TRPV4 and untransfected
HEK293 cells, n values are indicated for each.
Figure 3: TRPV4 expression in mouse UBSM and urothelium. A. RT-PCR performed on RNA isolated from TRPV4+/+ and TRPV4-/- bladders. TRPV4 mRNA
(478 bp) is amplified from TRPV4+/+, but is not amplified from TRPV4-/- bladders
(+) or in the absence of reverse transcriptase (-). B. Taqman performed on
UBSM and urothelium (URO) tissues of TRPV4+/+ and TRPV4-/- mice. C. Western blotting of crude membrane preparations from TRPV4 transfected HEK cells, and
UBSM and urothelium tissues of TRPV4+/+ and TRPV4-/- mice.
35 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
Figure 4: TRPV4 urinary bladder immunohistochemistry A. TRPV4 immunofluorescence detected in TRPV4+/+ urothelium and UBSM but not in the suburothelial space. Absence of immunofluoresence in TRPV4-/- bladder sections. B. Co-labeling for TRPV4 and the urothelial cell marker uroplakin III. C.
TRPV4 immunofluorescence in UBSM cells of TRPV4+/+ bladder sections, but not
-/- in TRPV4 bladder sections. Downloaded from
Figure 5: TRPV4 activation with GSK1016790A contracts mouse urinary jpet.aspetjournals.org bladder. A. Effect of GSK1016790A (10, 30 and 100 nM) on bladder halves from
TRPV4+/+ and TRPV4-/- mice. B. Average change in force as a percentage of baseline, baseline force ~10 mN (n=6 per group; 2 way RM ANOVA, p=0.009, at ASPET Journals on September 27, 2021
**P<0.01 vs. TRPV4-/-).
Figure 6: TRPV4 activation contracts the bladder independent of the urothelium. A. Representative contractile effect of GSK1016790A and relaxant effect of nifedipine on urothelium removed (-Uro) and intact (+Uro) bladder halves isolated from the same TRPV4+/+ mouse bladder. B. Average % increase in force from baseline (baseline ~10 mN) produced by 100 nM GSK1016790A in
–Uro and +Uro bladder halves. C. Average % relaxation of GSK1016790A contractions by 1 uM nifedipine. D. Percent increase in contractile force of
TRPV4+/+ bladders elicited by various concentrations of GSK1016790A and
4α−PDD.
36 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
Figure 7: TRPV4-/- mice have an enhanced bladder capacity. A.
Representative cystometric profile from TRPV4+/+ (top) and TRPV4-/- (bottom) mice. B. Average volume infused to elicit micturition (infused) in TRPV4+/+ and
TRPV4-/- mice. C. Average volume voided (void volume) from TRPV4+/+ and
TRPV4-/- mice. (*significantly different, unpaired students t-test, TRPV4+/+ vs.
-/- TRPV4 ). Downloaded from
Figure 8: Intravesicularly infused GSK1016790A reduces the bladder jpet.aspetjournals.org capacity of TRPV4+/+, but not TRPV4-/- mice. A and B. Cystometric profile from a TRPV4+/+ and a TRPV4-/- mouse, respectively, under control conditions (Top), and in the presence of intravesicularly infused TRPV4 agonist GSK1016790A (10 at ASPET Journals on September 27, 2021
µM) (Bottom). Arrows indicate voiding events. C and D. Average infused bladder volumes in the absence and presence of GSK1016790A in TRPV4+/+ (n=8) and
TRPV4-/- (n=3) mice, respectively.
37 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
Tables
Table 1: Basal urodynamics and body weights of TRPV4+/+ and TRPV4-/-
mice
Threshold Peak Average Infused Void Body
Pressure Pressure Pressure Volume Volume Weight Downloaded from
(mmHg) (mmHg) (mmHg) (ul) (ul) (grams)
+/+
TRPV4 (n=15) 17 ± 1 35 ± 2 11.6 ± 0.8 152 ± 18 134 ± 25 29.8 ± 0.6 jpet.aspetjournals.org
TRPV4-/- (n=11) 19 ± 2 34 ± 2 12.1 ± 2.2 259 ± 41* 254 ± 41* 28.2 ± 0.5
at ASPET Journals on September 27, 2021 (*p<0.05 vs TRPV4+/+, unpaired t-test)
38 JPET Fast Forward. Published on May 22, 2008 as DOI: 10.1124/jpet.108.139295 This article has not been copyedited and formatted. The final version may differ from this version.
JPET #139295
Table 2: Effect of GSK1016790A (10 uM) on TRPV4+/+ and TRPV4-/- bladder
pressures (mmHg) and volumes (ul)
Threshold Threshold Peak Peak
Pressure Pressure Pressure Pressure
(Control) (‘790) (Control) (‘790)
Downloaded from TRPV4+/+(n=8) 13.0 ± 0.5 15.3 ± 1.8 32 ± 3 33 ± 2
TRPV4-/- (n=3) 9.9 ± 1.9 9.8 ± 1.7 28 ± 3 28 ± 3 jpet.aspetjournals.org
Average Average Minimum Minimum
Pressure Pressure Pressure Pressure
(Control) (‘790) (Control) (‘790) at ASPET Journals on September 27, 2021
TRPV4+/+(n=8) 10.3 ± 0.6 13.6 ± 1.8 6.6 ± 0.6 10.1 ± 1.5*
TRPV4-/- (n=3) 8.4 ± 1.4 7.5 ± 1.1 3.8 ± 0.8 3.5 ± 0.3
Infused Infused Void Void
Volume Volume Volume Volume
(Control) (‘790) (Control) (‘790)
TRPV4+/+(n=8) 148 ± 21 60 ± 18* 102 ± 18 25 ± 11*
TRPV4-/- (n=3) 255 ± 16 280 ± 29 250 ± 18 287 ± 36
(*p<0.05 vs same genotype control, paired t-test)
39 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. JPET FastForward.PublishedonMay22,2008asDOI:10.1124/jpet.108.139295 A
O OH O O H N S N N S N O H O Cl Cl
GSK1016790A
Figure 1
Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 27, 2021 27, September on Journals ASPET at A + TRPV4 600 600 Control 10 µM 4alpha-PDD 400 400 (pA/pF)
200 (pA/pF) 200 Control Current density 10 nM GSK1016790 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. Current density 0 0 -60 -40 -20 0 20 40 60 -60 -40 -20 0 20 40 60 -200 Voltage (mV) -200 Voltage (mV) JPET FastForward.PublishedonMay22,2008asDOI:10.1124/jpet.108.139295
-400 -400
B -TRPV4 600 600
400 400 Control
(pA/pF) Control 10 nM GSK1016790 (pA/pF) 200 200 10 µM 4alpha-PDD Current density Current density 0 0 -60 -40 -20 0 20 40 60 -60 -40 -20 0 20 40 60 -200 Voltage (mV) -200 Voltage (mV)
-400 -400 C D 900 1000 6
6 800 - TRPV4 600 + TRPV4 600 6 4 GSK1016790A 4 alphaPDD 400 300 6
4 200 6 5 5 3 Activated current (pA/pF) current Activated 0 3 3 0 Figure 2 (pA/pF) density current TRPV4 1E-3 0.01 0.1 1 10 10 nM GSK1016790 10 µM 4 alphaPDD
[Drug] (µM)
Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 27, 2021 27, September on Journals ASPET at TRPV4+/+ TRPV4-/- 25 A B + - + - 20
15 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. JPET FastForward.PublishedonMay22,2008asDOI:10.1124/jpet.108.139295 1018 bp 1 517 bp Relative Expression Relative 0
-/- -/- +/+ +/+
TRPV4 - TRPV4 - TRPV4 - M S URO- TRPV4 URO B UBSM U C UBSM UBSM UROURO UBSMUBSM UROURO +/++/+ -/--/- -/--/- +/+ +/+ BlankBlank TRPV4 HEKTRPV4 HEK TRPV4 TRPV4 TRPV4 TRPV4 TRPV4 TRPV4 TRPV4 TRPV4 TRPV4 kDa TRPV4 TRPV4 188 -
98 - Figure 3
62 -
Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 27, 2021 27, September on Journals ASPET at A This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. JPET FastForward.PublishedonMay22,2008asDOI:10.1124/jpet.108.139295 C
TRPV4+/+ TRPV4 -/-
TRPV4+/+ TRPV4-/- B
TRPV4 Uroplakin Merge
Figure 4
Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 27, 2021 27, September on Journals ASPET at A TRPV4+/+
30 100 nM
10 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. JPET FastForward.PublishedonMay22,2008asDOI:10.1124/jpet.108.139295
2 mN TRPV4-/- 10 min
B 40
30 ** TRPV4+/+ (n = 6) 20 TRPV4-/- (n = 6)
10
0
-10 % Change in% ForceChange 10 nM 30 nM 100 nM -20
Figure 5 GSK1016790A
Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 27, 2021 27, September on Journals ASPET at A B - URO (n=4) Nifedipine (1µM) GSK1016790A (100 nM) 140 + URO (n=4)
120
100 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. -Uro 2 mN JPET FastForward.PublishedonMay22,2008asDOI:10.1124/jpet.108.139295 80
15 min. 60
40
+ Uro Force Force Force in in in Increase Increase Increase % % % 20
0 D C 0 120 GSK1016790A 100 4alphaPDD -25 80
60 -50 40
20 -75 %%% Increase Increase Increase in in in Force Force Force Nifedipine response response response Nifedipine Nifedipine Nifedipine 0
(%(%(% GSK1016790A GSK1016790A GSK1016790A Contraction) Contraction) Contraction) 0.01 0.1 1 10 100 -100 Concentration (uM)
Figure 6
Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 27, 2021 27, September on Journals ASPET at A B Infused 300 * TRPV4+/+ p=0.015 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.
200 JPET FastForward.PublishedonMay22,2008asDOI:10.1124/jpet.108.139295
100 Volume (ul) Volume
0 TRPV4+/+ (n=15) TRPV4-/- (n=11)
TRPV4-/- 10 mm Hg C Void Volume 300 5 min * p=0.015
200
100 Volume (ul) Volume
0 TRPV4+/+ (n=15) TRPV4-/- (n=11)
Figure 7
Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 27, 2021 27, September on Journals ASPET at ABTRPV4+/+ TRPV4-/- This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. JPET FastForward.PublishedonMay22,2008asDOI:10.1124/jpet.108.139295
CDTRPV4+/+ TRPV4-/- 175 350 150 300 125 250 100 p=0.02 200 p=0.02 75 * 150 *
Volume (ul) Volume 50 (ul) Volume 100 25 50 0 0
Figure 8 Control GSK1016790A Control GSK1016790A
Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 27, 2021 27, September on Journals ASPET at