Weiqun Yu

Expression and distribution of transient receptor potential (TRP) channels in bladder epithelium

The urothelium is proposed to be a sensory tissue that responds to mechanical stress by undergoing dynamic membrane trafficking and neurotransmitter release; however, the molecular basis of this function is poorly understood. Transient receptor potential (TRP) channels are ideal candidates to fulfill such a role as they can sense changes in temperature, osmolarity, and mechanical stimuli, and several are reported to be expressed in the bladder epithelium. However, their complete expression profile is unknown and their cellular localization is largely undefined. We analyzed expression of all 33 TRP family members in mouse bladder and urothelium by RT-PCR and found 22 specifically expressed in the urothelium. Of the latter, 10 were chosen for closer investigation based on their known mechanosensory or membrane trafficking functions in other cell types. Western blots confirmed urothelial expression of TRPC1, TRPC4, TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, TRPML1, and polycystins 1 and 2 (PKD1 and PKD2) proteins. We further defined the cellular and subcellular localization of all 10 TRP channels. TRPV2 and TRPM4 were prominently localized to the umbrella cell apical membrane, while TRPC4 and TRPV4 were identified on their abluminal surfaces. TRPC1, TRPM7, and TRPML1 were localized to the cytoplasm, while PKD1 and PKD2 were expressed on the apical and basolateral membranes of umbrella cells as well as in the cytoplasm. The cellular location of TRPV1 in the bladder has been debated, but colocalization with neuronal marker calcitonin gene-related peptide indicated clearly that it is present on afferent neurons that extend into the urothelium, but may not be expressed by the urothelium itself. These findings are consistent with the hypothesis that the urothelium acts as a sentinel and by expressing multiple TRP channels it is likely it can detect and presumably respond to a diversity of external stimuli and suggest that it plays an important role in urothelial signal transduction.

Expression and distribution of ectonucleotidases in mouse urinary bladder.

Background Normal urinary bladder function requires bidirectional molecular communication between urothelium, detrusor smooth muscle and sensory neurons and one of the key mediators involved in this intercellular signaling is ATP. Ectonucleotidases dephosphorylate nucleotides and thus regulate ligand exposure to P2X and P2Y purinergic receptors. Little is known about the role of these enzymes in mammalian bladder despite substantial literature linking bladder diseases to aberrant purinergic signaling. We therefore examined the expression and distribution of ectonucleotidases in the mouse bladder since mice offer the advantage of straightforward genetic modification for future studies. Principal Findings RT-PCR demonstrated that eight members of the ectonucleoside triphosphate diphosphohydrolase (NTPD) family, as well as 5′-nucleotidase (NT5E) are expressed in mouse bladder. NTPD1, NTPD2, NTPD3, NTPD8 and NT5E all catalyze extracellular nucleotide dephosphorylation and in concert achieve stepwise conversion of extracellular ATP to adenosine. Immunofluorescent localization with confocal microscopy revealed NTPD1 in endothelium of blood vessels in the lamina propria and in detrusor smooth muscle cells, while NTPD2 was expressed in cells localized to a region of the lamina propria adjacent to detrusor and surrounding muscle bundles in the detrusor. NTPD3 was urothelial-specific, occurring on membranes of intermediate and basal epithelial cells but did not appear to be present in umbrella cells. Immunoblotting confirmed NTPD8 protein in bladder and immunofluorescence suggested a primary localization to the urothelium. NT5E was present exclusively in detrusor smooth muscle in a pattern complementary with that of NTPD1 suggesting a mechanism for providing adenosine to P1 receptors on the surface of myocytes. Conclusions Ectonucleotidases exhibit highly cell-specific expression patterns in bladder and therefore likely act in a coordinated manner to regulate ligand availability to purinergic receptors. This is the first study to determine the expression and location of ectonucleotidases within the mammalian urinary bladder.

Spontaneous voiding by mice reveals strain-specific lower urinary tract function to be a quantitative genetic trait

Lower urinary tract (LUT) symptoms become prevalent with aging and affect millions; however, therapy is often ineffective because the etiology is unknown. Existing assays of LUT function in animal models are often invasive; however, a noninvasive assay is required to study symptom progression and determine genetic correlates. Here, we present a spontaneous voiding assay that is simple, reproducible, quantitative, and noninvasive. Young female mice from eight inbred mouse strains (129S1/SvImJ, A/J, C57BL/6J, NOD/ShiLtJ, NZO/H1LtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ) were tested for urination patterns on filter paper. Repeat testing at different times of the day showed minimal within-individual and within-strain variations, but all parameters (spot number, total volume, percent area in primary void, corner voiding, and center voiding) exhibited significant variations between strains. Calculation of the intraclass correlation coefficient, an estimate of broad-sense heritability, for each time of day and for each voiding parameter revealed highly significant heritability [spot number: 61%, percent urine in primary void: 90%, and total volume: 94% (afternoon data)]. Cystometrograms confirmed strong strain-specific urodynamic characteristics. Behavior-voiding correlation analysis showed no correlation with anxiety phenotypes. Diagnostically, the assay revealed LUT symptoms in several systems, including a demonstration of voiding abnormalities in older C57BL/6J mice (18-24 mo), in a model of protamine sulfate-induced urothelial damage and in a model of sucrose-induced diuresis. This assay may be used to derive pathophysiological LUT readouts from mouse models. Voiding characteristics are heritable traits, opening the way for genetic studies of LUT symptoms using outbred mouse populations.

Loss of β1-integrin from urothelium results in overactive bladder and incontinence in mice: a mechanosensory rather than structural phenotype

Bladder urothelium senses and communicates information about bladder fullness. However, the mechanoreceptors that respond to tissue stretch are poorly defined. Integrins are mechanotransducers in other tissues. Therefore, we eliminated β1‐integrin selectively in urothelium of mice using Cre‐LoxP targeted gene deletion. β1‐Integrin localized to basal/ intermediate urothelial cells by confocal microscopy. β1‐Integrin conditional‐knockout (β1‐cKO) mice lacking urothelial β1‐integrin exhibited down‐regulation and mislocalization of α3‐ and α5‐integrins by immunohistochemistry but, surprisingly, had normal morphology, permeability, and transepithelial resistance when compared with Cre‐negative littermate controls. β1‐cKO mice were incontinent, as judged by random urine leakage on filter paper (4‐fold higher spotting, P<0.01; 2.5‐fold higher urine area percentage, P<0.05). Urodynamic function assessed by cystometry revealed bladder overfilling with 80% longer intercontractile intervals (P<0.05) and detrusor hyperactivity (3‐fold more prevoid contractions, P<0.05), but smooth muscle contractility remained intact. ATP secretion into the lumen was elevated (49 vs. 22 nM, P<0.05), indicating abnormal filling‐induced purinergic signaling, and short‐circuit currents (measured in Ussing chambers) revealed 2‐fold higher stretch‐activated ion channel conductances in response to hydrostatic pressure of 1 cmH2O (P<0.05). We conclude that loss of integrin signaling from urothelium results in incontinence and overactive bladder due to abnormal mechanotransduction; more broadly, our findings indicate that urothelium itself directly modulates voiding.—Kanasaki, K., Yu, W., von Bodungen, M., Larigakis, J. D., Kanasaki, M., Ayala de la Pena, F., Kalluri, R., Hill, W.G. Loss of β1‐integrin from urothelium results in overactive bladder and incontinence in mice: a mechanosensory rather than structural phenotype. FASEB J. 27, 1950–1961 (2013). www.fasebj.org

Cellular Expression Profile for Interstitial Cells of Cajal in Bladder - A Cell Often Misidentified as Myocyte or Myofibroblast

Background Interstitial cells of Cajal (ICC) have been identified in urinary bladder of several species, but their presence in mice remains uncertain. Meanwhile, dozens of reports indicate that dysregulation of connexin 43 plays an important role in bladder overactivity, but its localization has not been clearly defined, with reports of expression in either the smooth muscle or in myofibroblasts. We recently identified a population of ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) positive cells that resemble ICC and are distinct from smooth muscle, fibroblasts, myofibroblasts and neurons. Thus we sought to define more clearly the molecular signature of ICC and in doing so resolve some of these uncertainties. Principle findings Immunofluorescent localization revealed that NTPDase2-positive cells lie closely adjacent to smooth muscle but are separate from them. NTPDase2 positive cells exhibited co-localization with the widely accepted ICC marker - c-kit. They were further shown to co-localize with other ICC markers CD34 and Ano1, but not with mast cell marker tryptase. Significantly, they show convincing co-localization with connexin 43, which was not present in smooth muscle. The identity of these cells as ICC was further confirmed by the presence of three mesenchymal markers – vimentin, desmin, and PDGFβ receptor, which indicates their mesenchymal origin. Finally, we observed for the first time, the presence of merlin/neurofibromin 2 in ICC. Normally considered a neuronal protein, the presence of merlin suggests ICC in bladder may have a role in neurotransmission. Conclusions NTPDase2 positive cells in mice bladder are ICC, which can be defined by the presence of c-Kit, CD34, Ano1, NTPDase2, connexin 43, vimentin, desmin, PDGFβ receptor and merlin/NF2. These data establish a definitive molecular expression profile, which can be used to assist in explorations of their functional roles, and the presence of NTPDase2 suggests that purinergic signaling plays a role in regulation of ICC function.

Extracellular UDP enhances P2X-mediated bladder smooth muscle contractility via P2Y6 activation of the phospholipase C/inositol trisphosphate pathway

Bladder dysfunction characterized by abnormal bladder smooth muscle (BSM) contractions is pivotal to the disease process in overactive bladder, urge incontinence, and spinal cord injury. Purinergic signaling comprises one key pathway in modulating BSM contractility, but molecular mechanisms remain unclear. Here we demonstrate, using myography, that activation of P2Y6 by either UDP or a specific agonist (MRS 2693) induced a sustained increase in BSM tone (up to 2 mN) in a concentration‐dependent manner. Notably, activation of P2Y6 enhanced ATP‐mediated BSM contractile force by up to 45%, indicating synergistic interactions between P2X and P2Y signaling. P2Y6‐activated responses were abolished by phospholipase C (PLC) and inositol trisphosphate (IP3) receptor antagonists U73122 and xestospongin C, demonstrating involvement of the PLC/IP3 signal pathway. Mice null for Entpd1, an ectonucleotidase on BSM, demonstrated increased force generation on P2Y6 activation (150%). Thus, in vivo perturbations to purinergic signaling resulted in altered P2Y6 activity and bladder contractility. We conclude that UDP, acting on P2Y6, regulates BSM tone and in doing so selectively maximizes P2X1‐mediated contraction forces. This novel neurotransmitter pathway may play an important role in urinary voiding disorders characterized by abnormal bladder motility.—Yu, W., Sun, X., Robson, S. C., Hill, W. G. Extracellular UDP enhances P2X‐mediated bladder smooth muscle contractility via P2Y6 activation of the phospholipase C/inositol trisphosphate pathway. FASEB J. 27, 1895–1903 (2013). www.fasebj.org

Evaluation of voiding assays in mice: impact of genetic strains and sex.

Void spot assays (VSA) and cystometry are two of the most common tests performed in mice to assess lower urinary tract function. Assay protocols and methodology vary greatly among laboratories, and little is known about reproducibility of results generated by different laboratories. We performed VSA in four mouse strains, comparing males with females and comparing results between two independent laboratories. Unique aspects of the current study include direct comparison of results of VSA performed in a similar manner in two locations and comparison of cystometry performed using two different rates of infusion in these two laboratories. Both assays were performed in male and female 129S1/SvImJ, C57BL/6J, NOD/ShiLtJ, and CAST/EiJ mice, and cystometry was performed under urethane anesthesia (10/group). Assays were performed and results analyzed as previously described. Results obtained in female mice were compared with previously reported values. Results of lower urinary tract function testing in mice vary in a consistent manner with strain and sex. Variables in husbandry, testing techniques, and analysis of results can significantly affect conclusions, particularly those obtained by cystometry. Although VSA results were remarkably similar between the two laboratories, consistent methods for performing lower urinary tract function testing in mice are required to compare results among studies with confidence.

Intact urothelial barrier function in a mouse model of ketamine-induced voiding dysfunction

Ketamine is a popular choice for young drug abusers. Ketamine abuse causes lower urinary tract symptoms, with the underlying pathophysiology poorly understood. Disruption of urothelial barrier function has been hypothesized to be a major mechanism for ketamine cystitis, yet the direct evidence of impaired urothelial barrier function is still lacking. To address this question, 8-wk-old female C57BL/6J mice were injected intraperitoneally with 30 mg·kg(-1)·day(-1) ketamine for 12 wk to induce ketamine cystitis. A spontaneous voiding spot assay showed that ketamine-treated mice had increased primary voiding spot numbers and smaller primary voiding spot sizes than control mice (P < 0.05), indicating a contracted bladder and bladder overactivity. Consistently, significantly increased voiding frequency was observed in ketamine-treated mice on cystometrograms. These functional experiments indicate that ketamine induces voiding dysfunction in mice. Surprisingly, urothelial permeability in ketamine-treated mice was not changed when measured using an Ussing chamber system with isotopic urea and water. Mouse urothelial structure was also not altered, and intact umbrella cell structure was observed by both transmission and scanning electron microscopy. Furthermore, immunostaining and confocal microscopy confirmed the presence of a well-defined distribution of zonula occuldens-1 in tight junctions and uroplakin in umbrella cells. In conclusion, these data indicate that ketamine injection induces voiding dysfunction in mice but does not necessarily disrupt mouse bladder barrier function. Disruption of urothelial barrier function may not be the major mechanism in ketamine cystitis.

Polarized ATP distribution in urothelial mucosal and serosal space is differentially regulated by stretch and ectonucleotidases

Purinergic signaling is a major pathway in regulating bladder function, and mechanical force stimulates urothelial ATP release, which plays an important role in bladder mechanotransduction. Although urothelial ATP release was first reported almost 20 years ago, the way in which release is regulated by mechanical force, and the presence of ATP-converting enzymes in regulating the availability of released ATP is still not well understood. Using a set of custom-designed Ussing chambers with the ability to manipulate mechanical forces applied on the urothelial tissue, we have demonstrated that it is stretch and not hydrostatic pressure that induces urothelial ATP release. The experiments reveal that urothelial ATP release is tightly controlled by stretch speed, magnitude, and direction. We have further shown that stretch-induced urothelial ATP release is insensitive to temperature (4°C). Interestingly, stretch-induced ATP release shows polarized distribution, with the ATP concentration in mucosal chamber (nanomolar level) about 10 times higher than the ATP concentration in serosal chamber (subnanomolar level). Furthermore, we have consistently observed differential ATP lifetime kinetics in the mucosal and serosal chambers, which is consistent with our immunofluorescent localization data, showing that ATP-converting enzymes ENTPD3 and alkaline phosphatase are expressed on urothelial basal surface, but not on the apical membrane. In summary, our data indicate that urothelial ATP release is finely regulated by stretch speed, magnitude, and direction, and extracellular ATP signaling is likely to be differentially regulated by ectonucleotidase, which results in temporally and spatially distinct ATP kinetics in response to mechanical stretch.

ADP-induced bladder contractility is mediated by P2Y12 receptor and temporally regulated by ectonucleotidases and adenosine signaling

Purinergic signaling comprises one key pathway in modulating bladder smooth muscle (BSM) contractility, disorders of which become highly prevalent with aging. ADP was first observed to modulate BSM contractility >40 yr ago, yet the underlying molecular mechanism still remains unclear. Here, we demonstrate, using myography, that ADP and ADPβS dose‐dependently induce mouse BSM contraction, and ADP‐induced BSM contraction is blocked by a selective P2Y12 receptor (P2Y12R) antagonist, PSB 0739 (25 μM), but is unaffected by P2Y1 and P2Y13 receptor antagonists. P2Y12R in BSM exhibits distinct pharmacological properties that are different from P2Y12R in platelets. After an immediate contraction, prolonged exposure to ADP causes BSM to become refractory to further ADP‐mediated contraction. However, in mice lacking ectonucleotidases Entpd1 (ATP→ADP→AMP) or Nt5e (AMP→adenosine), or by inhibiting adenosine signaling, the refractory response was altered, resulting in repeated BSM contractions in response to repeated ADP (0.1‐1 mM) stimulation. Our data indicate that P2Y12R undergoes slow desensitization; ADP‐P2Y12 signaling is tightly regulated by Entpd1/Nt5e activity and adenosine receptors; and ADP‐adenosine signaling play an important role in modulating P2X‐mediated BSM contraction. The identification of P2Y12R in BSM, and the current clinical availability of P2Y12R inhibitors, such as clopidogrel, offers potentially novel treatment strategies for bladder contractility disorders.—Yu, W., Sun, X., Robson, S. C., Hill, W. G., ADP‐induced bladder contractility is mediated by P2Y12 receptor and temporally regulated by ectonucleotidases and adenosine signaling. FASEB J. 28, 5288–5298 (2014). www.fasebj.org