Functional Role for Piezo1 in Stretch-evoked Ca2+ Influx and ATP Release in Urothelial Cell Cultures

The urothelium is a sensory structure that contributes to mechanosensation in the urinary bladder. Here, we provide evidence for a critical role for the Piezo1 channel, a newly identified mechanosensory molecule, in the mouse bladder urothelium. We performed a systematic analysis of the molecular and functional expression of Piezo1 channels in the urothelium. Immunofluorescence examination demonstrated abundant expression of Piezo1 in the mouse and human urothelium. Urothelial cells isolated from mice exhibited a Piezo1-dependent increase in cytosolic Ca²⁺ concentrations in response to mechanical stretch stimuli, leading to potent ATP release; this response was suppressed in Piezo1-knockdown cells. In addition, Piezo1 and TRPV4 distinguished different intensities of mechanical stimulus. Moreover, GsMTx4, an inhibitor of stretch-activated channels, attenuated the Ca²⁺ influx into urothelial cells and decreased ATP release from them upon stretch stimulation. These results suggest that Piezo1 senses extension of the bladder urothelium, leading to production of an ATP signal. Thus, inhibition of Piezo1 might provide a promising means of treating bladder dysfunction.     

TRPA1 mediates the antinociceptive properties of the constituent of Crocus sativus L., safranal

Safranal, contained in Crocus sativus L., exerts anti‐inflammatory and analgesic effects. However, the underlying mechanisms for such effects are poorly understood. We explored whether safranal targets the transient receptor potential ankyrin 1 (TRPA1) channel, which in nociceptors mediates pain signals. Safranal by binding to specific cysteine/lysine residues, stimulates TRPA1, but not the TRP vanilloid 1 and 4 channels (TRPV1 and TRPV4), evoking calcium responses and currents in human cells and rat and mouse dorsal root ganglion (DRG) neurons. Genetic deletion or pharmacological blockade of TRPA1 attenuated safranal‐evoked release of calcitonin gene‐related peptide (CGRP) from rat and mouse dorsal spinal cord, and acute nociception in mice. Safranal contracted rat urinary bladder isolated strips in a TRPA1‐dependent manner, behaving as a partial agonist. After exposure to safranal the ability of allyl isothiocyanate (TRPA1 agonist), but not that of capsaicin (TRPV1 agonist) or GSK1016790A (TRPV4 agonist), to evoke currents in DRG neurons, contraction of urinary bladder strips and CGRP release from spinal cord slices in rats, and acute nociception in mice underwent desensitization. As previously shown for other herbal extracts, including petasites or parthenolide, safranal might exert analgesic properties by partial agonism and selective desensitization of the TRPA1 channel.     

Heat-evoked activation of TRPV4 channels in a HEK293 cell expression system and in native mouse aorta endothelial cells

We have compared activation by heat of TRPV4 channels, heterogeneously expressed in HEK293 cells, and endogenous channels in mouse aorta endothelium (MAEC). Increasing the temperature above 25 degrees C activated currents and increased [Ca(2+)](i) in HEK293 cells transfected with TRPV4 and in MAEC. When compared with activation of TRPV4 currents by the selective ligand 4alphaPDD (alpha-phorbol 12,13-didecanoate), heat-activated currents in both systems showed the typical biophysical properties of currents through TRPV4, including their single channel conductance. Deletion of the three N-terminal ankyrin binding domains of TRPV4 abolished current activation cells by heat in HEK293. In inside-out patches, TRPV4 could not be activated by heat but still responded to the ligand 4alphaPDD. In MAEC, the same channel is activated under identical conditions as in the HEK expression system. Our data indicate that TRPV4 is a functional temperature-sensing channel in native endothelium, that is likely involved in temperature-dependent Ca(2+) signaling. The failure to activate TRPV4 channels by heat in inside-out patches, which responded to 4alphaPDD, may indicate that heat activation depends on the presence of an endogenous ligand, which is missing in inside-out patches.     

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.     

A novel function of capsaicin-sensitive TRPV1 channels: involvement in cell migration

Cell migration relies on a tight temporal and spatial regulation of the intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i in turn depends on Ca2+ influx via channels in the plasma membrane whose molecular nature is still largely unknown for migrating cells. A mechanosensitive component of the Ca2+ influx pathway was suggested. We show here that the capsaicin-sensitive transient receptor potential channel TRPV1, that plays an important role in pain transduction, is one of the Ca2+ influx channels involved in cell migration. Activating TRPV1 channels with capsaicin leads to an acceleration of human hepatoblastoma (HepG2) cells pretreated with hepatocyte growth factor (HGF). The speed rises by up to 50% and the displacement is doubled. Patch clamp experiments revealed the presence of capsaicin and resiniferatoxin (RTX)-sensitive currents. In contrast, HepG2 cells kept in the absence of HGF are not accelerated by capsaicin and express no capsaicin- or RTX-sensitive current. The TRPV1 antagonist capsazepine prevents the stimulation of migration and inhibits capsaicin-sensitive currents. Finally, we compared the contribution of capsaicin-sensitive TRPV1 channels to cell migration with that of mechanosensitive TRPV4 channels that are also expressed in HepG2 cells. A specific TRPV4 agonist, 4alpha-phorbol 12,13-didecanoate, does not increase the displacement. In summary, we assigned a novel role to capsaicin-sensitive TRPV1 channels. They are important Ca2+ influx channels required for cell migration.     

The TRPV4 Cation Channel Mediates Stretch-evoked Ca2+ Influx and ATP Release in Primary Urothelial Cell Cultures

Transient receptor potential channels have recently been implicated in physiological functions in a urogenital system. In this study, we investigated the role of transient receptor potential vanilloid 4 (TRPV4) channels in a stretch sensing mechanism in mouse primary urothelial cell cultures. The selective TRPV4 agonist, 4α-phorbol 12,13-didecanoate (4α-PDD) evoked Ca²⁺ influx in wild-type (WT) urothelial cells, but not in TRPV4-deficient (TRPV4KO) cells. We established a cell-stretch system to investigate stretch-evoked changes in intracellular Ca²⁺ concentration and ATP release. Stretch stimulation evoked intracellular Ca²⁺ increases in a stretch speed- and distance-dependent manner in WT and TRPV4KO cells. In TRPV4KO urothelial cells, however, the intracellular Ca²⁺ increase in response to stretch stimulation was significantly attenuated compared with that in WT cells. Stretch-evoked Ca²⁺ increases in WT urothelium were partially reduced in the presence of ruthenium red, a broad TRP channel blocker, whereas that in TRPV4KO cells did not show such reduction. Potent ATP release occurred following stretch stimulation or 4α-PDD administration in WT urothelial cells, which was dramatically suppressed in TRPV4KO cells. Stretch-dependent ATP release was almost completely eliminated in the presence of ruthenium red or in the absence of extracellular Ca²⁺. These results suggest that TRPV4 senses distension of the bladder urothelium, which is converted to an ATP signal in the micturition reflex pathway during urine storage.Transient receptor potential vanilloid 4 (TRPV4),³ a member of the TRP superfamily of cation channels, is a Ca²⁺-permeable channel activated by a wide variety of physical and chemical stimuli (1, 2). TRPV4 was originally viewed as an osmo- or mechano-sensor, because the channel opens in response to hypotonicity-induced cell swelling (3–5) and shear stress (6). Alternatively, TRPV4 can be activated by diverse chemical stimuli such as synthetic phorbol ester 4α-phorbol 12,13-didecanoate (4α-PDD) (7), a botanical agent (bisandrographolide A), anandamide metabolites such as arachidonic acid and epoxyeicosatrienoic acids, as well as moderate warmth (>27 °C) (8–10). TRPV4 is widely expressed throughout the body, including renal epithelium, auditory hair cells, skin keratinocytes, hippocampus neurons, endothelial cells, and urinary bladder epithelium, thereby contributing to numerous physiological processes such as osmoregulation (11, 12), hearing (13), thermal and mechanical hyperalgesia (14, 15), neural activity in the brain (16), skin barrier recovery (17), and cell volume regulation (18). Therefore, the TRPV4 channel is now considered a multimodal transducer in various tissues and cells.Non-neuronal cells within the urinary bladder wall (notably the transitional epithelial cells (urothelial cells)) function as a barrier against ions, solutes, and infection and also participate in the detection of physical and chemical stimuli (19–21). The urothelium expresses various sensory receptors and channels (bradykinin receptors, adrenergic/cholinergic receptors, nerve growth factor receptors, purinergic receptors, amiloride-sensitive Na⁺ channels, and TRP channels), all of which are substantially implicated in modulating bladder functions (22).Recently, the potential roles of TRP channels have been explored in the bladder. Thus far, expression of TRPV1, TRPV2, TRPV4, TRPA1, and TRPM8 has been reported in different regions of urogenital tracts (21). TRPV1 is reportedly expressed in the epithelial cells lining the urothelium, in interstitial cells, and in sensory nerve terminals. TRPV1-deficient mice displayed a higher frequency of low amplitude non-voiding bladder contractions in comparison with wild-type (WT) mice (22), suggesting that TRPV1 is required for detection of bladder stretch, which involves stretch-evoked release of ATP and nitric oxide. The release of both mediators was reduced in the bladders of TRPV1-deficient mice. In a clinical setting, capsaicin or resiniferatoxin reduces bladder overactivity through desensitization of bladder afferents by acting on TRPV1 (23). Expression of other TRP channels, e.g. TRPM8 and TRPA1, was found in sensory C fibers in the bladder (24–27). The diagnostic ice water test is utilized to determine whether disturbance of bladder function involves neurogenic components, one of which could be related to TRPM8 function, in patients with spinal cord lesion (28). TRPA1 in sensory afferents is activated by several known ligands (allyl isothiocyanate and cinnamaldehyde), thereby inducing bladder overactivity (26). TRPV2 is expressed by several cell types in the rat bladder (29); however, its physiological function has not yet been investigated. TRPV4 is expressed in the urothelium and in smooth muscle cells of the urinary bladder (30, 31). Activation of the channel by specific ligands leads to augmentation of bladder contraction amplitude in cystometry and induction of bladder overactivity in vivo. In a separate cystometry analysis in conjunction with behavioral experiments, the intermicturitional interval was elongated and storage urine volume was increased in TRPV4-deficient mice compared with WT mice (32). Thus, TRPV4 may contribute to bladder function, especially to mediating bladder distention signals to primary afferent nerves during urine storage. However, whether urothelial TRPV4 is required for sensing mechanical stretch, or to what extent urothelial TRPV4 contributes to stretch-evoked ATP release, has not been precisely determined.In the present study, we examined the functional contribution of TRPV4 to stretch-dependent urothelial cell responses and stretch-evoked ATP release in vitro. We first established a primary cell culture for mouse urothelium and retention of TRPV4 expression was confirmed. Because urothelial cells are physically extended during urine storage in vivo, we reproduced this phenomenon in an in vitro experiment using the uni-axial cell stretch system. All the experiments were performed by comparing urothelial cells obtained from WT mice and TRPV4-deficient mice to evaluate the correlation between TRPV4 expression and stretch responses. We demonstrated that urothelial cells sense mechanical stretch stimuli via TRPV4 channels, which induces robust Ca²⁺ influx and contributes to ATP release upon extension.     

Electrophysiological properties of heteromeric TRPV4-C1 channels

We previously reported that TRPV4 and TRPC1 can co-assemble to form heteromeric TRPV4-C1 channels [12]. In the present study, we characterized some basic electrophysiological properties of heteromeric TRPV4-C1 channels. 4α-Phorbol 12,13-didecanoate (4α-PDD, a TRPV4 agonist) activated a single channel current in HEK293 cells co-expressing TRPV4 and TRPC1. The activity of the channels was abrogated by a TRPC1-targeting blocking antibody T1E3. Conductance of the channels was ~95pS for outward currents and ~83pS for inward currents. The channels with similar conductance were also recorded in cells expressing TRPV4-C1 concatamers, in which assembled channels were expected to be mostly 2V4:2C1. Fluorescence Resonance Energy Transfer (FRET) experiments confirmed the formation of a protein complex with 2V4:2C1 stoichiometry while suggesting an unlikeliness of 3V4:1C1 or 1V4:3C1 stoichiometry. Monovalent cation permeability profiles were compared between heteromeric TRPV4-C1 and homomeric TRPV4 channels. For heteromeric TRPV4-C1 channels, their permeation profile was found to fit to Eisenman sequence VI, indicative of a strong field strength cation binding site, whereas for homomeric TRPV4 channels, their permeation profile corresponded to Eisenman sequence IV for a weak field strength binding site. Compared to homomeric TRPV4 channels, heteromeric TRPV4-C1 channels were slightly more permeable to Ca2+ and had a reduced sensitivity to extracellular Ca2+ inhibition. In summary, we found that, when TRPV4 and TRPC1 were co-expressed in HEK293 cells, the predominate assembly type was 2V4:2C1. The heteromeric TRPV4-C1 channels display distinct electrophysiological properties different from those of homomeric TRPV4 channels.     

Carbenoxolone inhibits TRPV4 channel‐initiated oxidative urothelial injury and ameliorates cyclophosphamide‐induced bladder dysfunction

Carbenoxolone (CBX) is a clinically prescribed drug for the treatment of digestive ulcer and inflammation. It is also a widely used pharmacological inhibitor of several channels in basic research. Given that the overactivity of several channels, including those inhibitable by CBX, underlies bladder dysfunction, we tested the potential therapeutic application and mechanism of CBX in the treatment of voiding dysfunction. In a mouse model of cystitis induced by cyclophosphamide (CYP), CBX administration prevented the CYP‐elicited increase in bladder weight, oedema, haemorrhage, and urothelial injury. CBX also greatly improved micturition pattern, as manifested by the apparently decreased micturition frequency and increased micturition volume. Western blot results showed that CBX suppressed CYP‐induced increase in protein carbonyls, COX‐2, and iNOS. Further analysis using cultured urothelial cells revealed that acrolein, the major metabolite of CYP, caused protein oxidation, p38 activation, and urothelial injury. These effects of acrolein were reproduced by TRPV4 agonists and significantly prevented by antioxidant NAC, p38 inhibitor SB203580, TRPV4 antagonist RN‐1734, and CBX. Further studies showed that CBX potently suppressed TRPV4 agonist‐initiated calcium influx and subsequent cell injury. CBX attenuated CYP‐induced cystitis in vivo and reduced acrolein‐induced cell injury in vitro, through mechanisms involving inhibition of TRPV4 channels and attenuation of the channel‐mediated oxidative stress. CBX might be a promising agent for the treatment of bladder dysfunction.     

The Xenopus tropicalis orthologue of TRPV3 is heat sensitive

Thermosensitive members of the transient receptor potential (TRP) family of ion channels (thermal TRP channels) play a crucial role in mammalian temperature sensing. Orthologues of these channels are present in lower vertebrates and, remarkably, some thermal TRP orthologues from different species appear to mediate opposing responses to temperature. For example, whereas the mammalian TRPV3 channel is activated by heat, frog TRPV3 is reportedly activated by cold. Intrigued by the potential implications of these opposing responses to temperature for the mechanism of temperature-dependent gating, we cloned Xenopus laevis TRPV3 and functionally expressed it in both mammalian cell lines and Xenopus oocytes. We found that, when expressed in mammalian cells, the recombinant channel lacks the reported cold sensitivity; rather, it is activated by temperatures >50°C. Furthermore, when expressed in mammalian cells, the frog orthologue shows other features characteristic of mammalian TRPV3, including activation by the agonist 2-aminoethoxydiphenyl borate and an increased response with repeated stimulation. We detected both heat- and cold-activated currents in Xenopus oocytes expressing the recombinant frog TRPV3 channel. However, cold-activated currents were also apparent in control oocytes lacking recombinant TRPV3. Our data indicate that frog TRPV3 resembles its mammalian orthologues in terms of its thermosensitivity and is intrinsically activated by heat. Thus, all known vanilloid receptors are activated by heat. Our data also show that Xenopus oocytes contain endogenous receptors that are activated by cold, and suggest that cold sensitivity of TRP channels established using Xenopus oocytes as a functional expression system may need to be revisited.     

Bisandrographolide from Andrographis paniculata activates TRPV4 channels

Many transient receptor potential (TRP) channels are activated or blocked by various compounds found in plants; two prominent examples include the activation of TRPV1 channels by capsaicin and the activation of TRPM8 channels by menthol. We sought to identify additional plant compounds that are active on other types of TRP channels. We screened a library of extracts from 50 Chinese herbal plants using a calcium-imaging assay to find compounds active on TRPV3 and TRPV4 channels. An extract from the plant Andrographis paniculata potently activated TRPV4 channels. The extract was fractionated further, and the active compound was identified as bisandrographolide A (BAA). We used purified compound to characterize the activity of BAA on certain TRPV channel subtypes. Although BAA activated TRPV4 channels with an EC(50) of 790-950 nm, it did not activate or block activation of TRPV1, TRPV2, or TRPV3 channels. BAA activated a large TRPV4-like current in immortalized mouse keratinocytes (308 cells) that have been shown to express TRPV4 protein endogenously. This compound also activated TRPV4 currents in cell-free outside-out patches from HEK293T cells overexpressing TRPV4 cDNA, suggesting that BAA can activate the channel in a membrane-delimited manner. Another related compound, andrographolide, found in abundance in the plant Andrographis was unable to activate or block activation of TRPV4 channels. These experiments show that BAA activates TRPV4 channels, and we discuss the possibility that activation of TRPV4 by BAA could play a role in some of the effects of Andrographis extract described in traditional medicine.     

Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling

Urothelial cells respond to bladder distension with ATP release, and ATP signaling within the bladder and from the bladder to the CNS is essential for proper bladder function. In other cell types, pannexin 1 (Panx1) channels provide a pathway for mechanically-induced ATP efflux and for ATP-induced ATP release through interaction with P2X₇ receptors (P2X₇Rs). We report that Panx1 and P2X₇R are functionally expressed in the bladder mucosa and in immortalized human urothelial cells (TRT-HU1), and participate in urothelial ATP release and signaling. ATP release from isolated rat bladders induced by distention was reduced by the Panx1 channel blocker mefloquine (MFQ) and was blunted in mice lacking Panx1 or P2X₇R expression. Hypoosmotic shock induced YoPro dye uptake was inhibited by MFQ and the P2X₇R blocker A438079 in TRT-HU1 cells, and was also blunted in primary urothelial cells derived from mice lacking Panx1 or P2X₇R expression. Rinsing-induced mechanical stimulation of TRT-HU1 cells triggered ATP release, which was reduced by MFQ and potentiated in low divalent cation solution (LDPBS), a condition known to enhance P2X₇R activation. ATP signaling evaluated as intercellular Ca²⁺ wave radius was significantly larger in LDPBS, reduced by MFQ and by apyrase (ATP scavenger). These findings indicate that Panx1 participates in urothelial mechanotransduction and signaling by providing a direct pathway for mechanically-induced ATP release and by functionally interacting with P2X₇Rs.     

Calcium regulation by thermo- and osmosensing transient receptor potential vanilloid channels (TRPVs) in human conjunctival epithelial cells

Transient receptor potential vanilloid (TRPV) channels respond to polymodal stresses to induce pain, inflammation and tissue fibrosis. In this study, we probed for their functional expression in human conjunctival epithelial (HCjE) cells and ex vivo human conjunctivas. Notably, patients suffering from dry eye syndrome experience the same type of symptomology induced by TRPV channel activation in other ocular tissues. TRPV gene and protein expression were determined by RT-PCR and immunohistochemistry in HCjE cells and human conjunctivas (body donors). The planar patch-clamp technique was used to record nonselective cation channel currents. Ca(2+) transients were monitored in fura-2 loaded cells. Cultivated HCjE cells and human conjunctiva express TRPV1, TRPV2, and TRPV4 mRNA. TRPV1 and TRPV4 localization was identified in human conjunctiva. Whereas the TRPV1 agonist capsaicin (CAP) (5-20 μM) -induced Ca(2+) transients were blocked by capsazepine (CPZ) (10 μM), the TRPV4 activator 4α-PDD (10 μM) -induced Ca(2+) increases were reduced by ruthenium-red (RuR) (20 μM). Different heating (<40°C or >43°C) led to Ca(2+) increases, which were also reduced by RuR. Hypotonic challenges of either 25 or 50% induced Ca(2+) transients and nonselective cation channel currents. In conclusion, conjunctiva express TRPV1, TRPV2, and TRPV4 channels which may provide novel drug targets for dry eye therapeutics. Their usage may have fewer side effects than those currently encountered with less selective drugs.     

Protease-Activated Receptor 4 Induces Bladder Pain through High Mobility Group Box-1

Pain is the significant presenting symptom in Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS). Activation of urothelial protease activated receptor 4 (PAR4) causes pain through release of urothelial macrophage migration inhibitory factor (MIF). High Mobility Group Box-1 (HMGB1), a chromatin-binding protein, mediates bladder pain (but not inflammation) in an experimental model (cyclophosphamide) of cystitis. To determine if PAR4-induced bladder hypersensitivity depends on HMGB1 downstream, we tested whether: 1) bladder PAR4 stimulation affected urothelial HMGB1 release; 2) blocking MIF inhibited urothelial HMGB1 release; and 3) blocking HMGB1 prevented PAR4-induced bladder hypersensitivity. HMGB1 release was examined in immortalized human urothelial cultures (UROtsa) exposed to PAR4-activating peptide (PAR4-AP; 100 μM; 2 hours) or scrambled control peptide. Female C57BL/6 mice, pretreated with a HMGB1 inhibitor (glycyrrhizin: 50 mg/kg; ip) or vehicle, received intravesical PAR4-AP or a control peptide (100 μM; 1 hour) to determine 1) HMGB1 levels at 1 hour in the intravesical fluid (released HMGB1) and urothelium, and 2) abdominal hypersensitivity to von Frey filament stimulation 24 hours later. We also tested mice pretreated with a MIF blocker (ISO-1: 20 mg/kg; ip) to determine whether MIF mediated PAR4-induced urothelial HMGB1 release. PAR4-AP triggered HMGB1 release from human (in vitro) and mice (in vivo) urothelial cells. Intravesical PAR4 activation elicited abdominal hypersensitivity in mice that was prevented by blocking HMGB1. MIF inhibition prevented PAR4-mediated HMGB1 release from mouse urothelium. Urothelial MIF and HGMB1 represent novel targets for therapeutic intervention in bladder pain conditions.     

Comparative Effects of Heterologous TRPV1 and TRPM8 Expression in Rat Hippocampal Neurons

Heterologous channel expression can be used to control activity in select neuronal populations, thus expanding the tools available to modern neuroscience. However, the secondary effects of exogenous channel expression are often left unexplored. We expressed two transient receptor potential (TRP) channel family members, TRPV1 and TRPM8, in cultured hippocampal neurons. We compared functional expression levels and secondary effects of channel expression and activation on neuronal survival and signaling. We found that activation of both channels with appropriate agonist caused large depolarizing currents in voltage-clamped hippocampal neurons, exceeding the amplitude responses to a calibrating 30 mM KCl stimulation. Both TRPV1 and TRPM8 currents were reduced but not eliminated by 4 hr incubation in saturating agonist concentration. In the case of TRPV1, but not TRPM8, prolonged agonist exposure caused strong calcium-dependent toxicity. In addition, TRPV1 expression depressed synaptic transmission dramatically without overt signs of toxicity, possibly due to low-level TRPV1 activation in the absence of exogenous agonist application. Despite evidence of expression at presynaptic sites, in addition to somatodendritic sites, TRPM8 expression alone exhibited no effects on synaptic transmission. Therefore, by a number of criteria, TRPM8 proved the superior choice for control over neuronal membrane potential. This study also highlights the need to explore potential secondary effects of long-term expression and activation of heterologously introduced channels.     

Macrophage Migration Inhibitory Factor Mediates PAR-Induced Bladder Pain

IntroductionMacrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is constitutively expressed in urothelial cells that also express protease-activated receptors (PAR). Urothelial PAR1 receptors were shown to mediate bladder inflammation. We showed that PAR1 and PAR4 activator, thrombin, also mediates urothelial MIF release. We hypothesized that stimulation of urothelial PAR1 or PAR4 receptors elicits release of urothelial MIF that acts on MIF receptors in the urothelium to mediate bladder inflammation and pain. Thus, we examined the effect of activation of specific bladder PAR receptors on MIF release, bladder pain, micturition and histological changes.MethodsMIF release was measured in vitro after exposing immortalized human urothelial cells (UROtsa) to PAR1 or PAR4 activating peptides (AP). Female C57BL/6 mice received intravesical PAR1- or PAR4-AP for one hour to determine: 1) bladder MIF release in vivo within one hour; 2) abdominal hypersensitivity (allodynia) to von Frey filament stimulation 24 hours after treatment; 3) micturition parameters 24 hours after treatment; 4) histological changes in the bladder as a result of treatment; 5) changes in expression of bladder MIF and MIF receptors using real-time RT-PCR; 6) changes in urothelial MIF and MIF receptor, CXCR4, protein levels using quantitative immunofluorescence; 7) effect of MIF or CXCR4 antagonism.ResultsPAR1- or PAR4-AP triggered MIF release from both human urothelial cells in vitro and mouse urothelium in vivo. Twenty-four hours after intravesical PAR1- or PAR4-AP, we observed abdominal hypersensitivity in mice without changes in micturition or bladder histology. PAR4-AP was more effective and also increased expression of bladder MIF and urothelium MIF receptor, CXCR4. Bladder CXCR4 localized to the urothelium. Antagonizing MIF with ISO-1 eliminated PAR4- and reduced PAR1-induced hypersensitivity, while antagonizing CXCR4 with AMD3100 only partially prevented PAR4-induced hypersensitivity.ConclusionsBladder PAR activation elicits urothelial MIF release and urothelial MIF receptor signaling at least partly through CXCR4 to result in abdominal hypersensitivity without overt bladder inflammation. PAR-induced bladder pain may represent an interesting pre-clinical model of Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS) where pain occurs without apparent bladder injury or pathology. MIF is potentially a novel therapeutic target for bladder pain in IC/PBS patients.     

Hyperglycemia and Diabetes Downregulate the Functional Expression of TRPV4 Channels in Retinal Microvascular Endothelium

Retinal endothelial cell dysfunction is believed to play a key role in the etiology and pathogenesis of diabetic retinopathy. Numerous studies have shown that TRPV4 channels are critically involved in maintaining normal endothelial cell function. In the current paper, we demonstrate that TRPV4 is functionally expressed in the endothelium of the retinal microcirculation and that both channel expression and activity is downregulated by hyperglycaemia. Quantitative PCR and immunostaining demonstrated molecular expression of TRPV4 in cultured bovine retinal microvascular endothelial cells (RMECs). Functional TRPV4 activity was assessed in cultured RMECs from endothelial Ca2+-responses recorded using fura-2 microfluorimetry and electrophysiological recordings of membrane currents. The TRPV4 agonist 4α-phorbol 12,13-didecanoate (4-αPDD) increased [Ca²⁺]_i in RMECs and this response was largely abolished using siRNA targeted against TRPV4. These Ca²⁺-signals were completely inhibited by removal of extracellular Ca²⁺, confirming their dependence on influx of extracellular Ca²⁺. The 4-αPDD Ca²⁺-response recorded in the presence of cyclopiazonic acid (CPA), which depletes the intracellular stores preventing any signal amplification through store release, was used as a measure of Ca²⁺-influx across the cell membrane. This response was blocked by HC067047, a TRPV4 antagonist. Under voltage clamp conditions, the TRPV4 agonist GSK1016790A stimulated a membrane current, which was again inhibited by HC067047. Following incubation with 25mM D-glucose TRPV4 expression was reduced in comparison with RMECs cultured under control conditions, as were 4αPDD-induced Ca²⁺-responses in the presence of CPA and ion currents evoked by GSK1016790A. Molecular expression of TRPV4 in the retinal vascular endothelium of 3 months’ streptozotocin-induced diabetic rats was also reduced in comparison with that in age-matched controls. We conclude that hyperglycaemia and diabetes reduce the molecular and functional expression of TRPV4 channels in retinal microvascular endothelial cells. These changes may contribute to diabetes induced endothelial dysfunction and retinopathy.     

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including by mechanical forces. Certain TRPV4 mutations cause complex hereditary bone or neuronal pathologies in human. Wild-type or mutant TRPV4 transgenes are commonly expressed in cultured mammalian cells and examined by Fura-2 fluorometry and by electrodes. In terms of the mechanism of mechanosensitivity and the molecular bases of the diseases, the current literature is confusing and controversial. To complement existing methods, we describe two additional methods to examine the molecular properties of TRPV4. (1) Rat TRPV4 and an aequorin transgene are transformed into budding yeast. A hypo-osmtic shock of the transformant population yields a luminometric signal due to the combination of aequorin with Ca²⁺, released through the TRPV4 channel. Here TRPV4 is isolated from its usual mammalian partner proteins and reveals its own mechanosensitivity. (2) cRNA of TRPV4 is injected into Xenopus oocytes. After a suitable period of incubation, the macroscopic TRPV4 current is examined with a two-electrode voltage clamp. The current rise upon removal of inert osmoticum from the oocyte bath is indicative of mechanosensitivity. The microAmpere (10^-6 to 10^-4 A) currents from oocytes are much larger than the subnano- to nanoAmpere (10^-10 to 10^-9 A) currents from cultured cells, yielding clearer quantifications and more confident assessments. Microscopic currents reflecting the activities of individual channel proteins can also be directly registered under a patch clamp, in on-cell or excised mode. The same oocyte provides multiple patch samples, allowing better data replication. Suctions applied to the patches can activate TRPV4 to directly assess mechanosensitivity. These methods should also be useful in the study of other types of TRP channels.     

TRPV3 endogenously expressed in murine colonic epithelial cells is inhibited by the novel TRPV3 blocker 26E01

TRPV3 is a Ca²⁺-permeable cation channel, prominently expressed by keratinocytes where it contributes to maintaining the skin barrier, skin regeneration, and keratinocyte differentiation. However, much less is known about its physiological function in other tissues and there is still a need for identifying novel and efficient TRPV3 channel blockers. By screening a compound library, we identified 26E01 as a novel TRPV3 blocker. 26E01 blocks heterologously expressed TRPV3 channels overexpressed in HEK293 cells as assessed by fluorometric intracellular free Ca²⁺ assays (IC₅₀ = 8.6 μM) but does not affect TRPV1, TRPV2 or TRPV4 channels. Electrophysiological whole-cell recordings confirmed the reversible block of TRPV3 currents by 26E01, which was also effective in excised inside-out patches, hinting to a rather direct mode of action. 26E01 suppresses endogenous TRPV3 currents in the mouse 308 keratinocyte cell line and in the human DLD-1 colon carcinoma cell line (IC₅₀ = 12 μM). In sections of the gastrointestinal epithelium of mice, the expression of TRPV3 mRNA follows a gradient along the gastrointestinal tract, with the highest expression in the distal colon. 26E01 efficiently attenuates 2-aminoethoxydiphenyl borate-induced calcium influx in primary colonic epithelial cells isolated from the distal colon. As 26E01 neither shows toxic effects on DLD-1 cells at concentrations of up to 100 μM in MTT assays nor on mouse primary colonic crypts as assessed by calcein-AM/propidium iodide co-staining, it may serve as a useful tool to further study the physiological function of TRPV3 in various tissues.     

Function of transient receptor potential cation channel subfamily V member 4 (TRPV4) as a mechanical transducer in flow-sensitive segments of renal collecting duct system

The TRPV4 Ca(2+)-permeable channel is sensitive to mechanical stimuli. In the current study we have employed immunocytochemical staining in kidney slices and functional assessments (Ca(2+) imaging) in isolated, split-opened, tubule segments to define TRPV4 sites of expression and flow-dependent function in the collecting duct system. Staining patterns revealed strong expression of TRPV4 along the entire collecting duct system with highest levels at the apical (luminal)/subapical region of the principal cells (PCs), the dominant cell type, with more diffuse staining in intercalated cells (ICs). Using fluorescence Ca(2+) imaging and the selective TRPV4 agonist, GSK1016790A, we demonstrated functional TRPV4 channels in PCs and ICs of split-opened cortical collecting ducts and connecting tubules. The agonist was ineffective in inducing a rise in [Ca(2+)](i) in the absence of extracellular Ca(2+) or in tubules from TRPV4-deficient animals. Most importantly, a 10-fold elevation in luminal (apical) fluid flow induced a rapid and sustained influx of Ca(2+) that was abolished by the TRPV channel inhibitor, ruthenium red, or in tubules isolated from TRPV4 deficient animals. We concluded that TRPV4 is highly expressed along the entire collecting duct system where it appears to function as a sensor/transducer of flow-induce mechanical stresses.