Expansion and Long-Term Culture of Differentiated Normal Rat Urothelial Cells in Vitro

Zhang, Xu Dong; Ludwikowski, Barbara; Hurst, Robert E.; Frey, Peter

doi:10.1290/1071-2690(2001)037<0419:ealtco>2.0.co;2

Cited by 36 publications

(21 citation statements)

References 21 publications

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“…Preparation of Primary Urothelial Cell Cultures-Whole bladders were taken from anesthetized mice, and the urothelial cells were prepared by modified procedures (33)(34)(35). Briefly, the bladder was inverted by pushing the dome downward through the bladder neck with a blunt 18-gauge needle.…”

Section: Methodsmentioning

confidence: 99%

“…After replacing with PBS, urothelial cells were harvested and collected with a cell scraper. After pipetting, all urothelial cells were centrifuged (1000 rpm for 5 min), and the cell pellet was resuspended with keratinocyte serum-free medium (KSFM, Invitrogen) (34,36). This medium was supplemented with epidermal growth factor (5 ng/ml), bovine pituitary extract (50 g/ml), cholera toxin (30 ng/ml), penicillin (100 units/ml), and streptomycin (1 g/ml) (all from Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

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

Mochizuki

Sokabe

Araki

et al. 2009

Journal of Biological Chemistry

260

281

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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, 4␣-phorbol 12,13-didecanoate (4␣-PDD) evoked Ca 2؉ influx in wild-type (WT) urothelial cells, but not in TRPV4-deficient (TRPV4KO) cells. We established a cellstretch system to investigate stretch-evoked changes in intracellular Ca 2؉ concentration and ATP release. Stretch stimulation evoked intracellular Ca 2؉ increases in a stretch speed-and distance-dependent manner in WT and TRPV4KO cells. In TRPV4KO urothelial cells, however, the intracellular Ca 2؉ increase in response to stretch stimulation was significantly attenuated compared with that in WT cells. Stretch-evoked Ca 2؉ 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 4␣-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 Ca 2؉ . 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 Ca 2ϩ -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 4␣-phorbol 12,13-didecanoate (4␣-PDD) (7), a botanical agent (bisandrographolide A), anandamide metabolites such as arachidonic acid and epoxyeicosatrienoic acids, as well as moderate warmth (Ͼ27°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 channe...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Mochizuki

Sokabe

Araki

et al. 2009

Journal of Biological Chemistry

260

281

View full text Add to dashboard Cite

show abstract

“…We adopted and modified the protocol to isolate bladder epithelium from male mice (42). Briefly, after the whole bladder was excised, it was everted to expose the mucosal surface.…”

Section: Methodsmentioning

confidence: 99%

767: NRF2 is Essential for the Chemopreventive Efficacy of Oltipraz Against Urinary Bladder Carcinogenesis

Iida¹,

Itoh²,

Kumagai³

et al. 2005

Journal of Urology

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The induction of phase 2 detoxifying enzymes, such as UDP-glucuronosyltransferases (UGTs), in response to an array of naturally occurring and synthetic agents, such as oltipraz (4-methyl-5-[2-pyrazinyl]-1,2-dithiole-3-thione), provides an effective means of protection against a variety of carcinogens. Transcription factor Nrf2 is an essential regulator of the inducible expression of detoxifying enzyme genes by chemopreventive agents. In this study, we investigated in Nrf2-deficient mice the susceptibility to the urinary bladder-specific carcinogen N-nitrosobutyl(4-hydroxybutyl)amine (BBN) and the chemopreventive efficacy of oltipraz. The incidence of urinary bladder carcinoma by BBN was significantly higher in Nrf2 ؊/؊ mice than in wild-type mice; invasive carcinoma was found in 24.0 and 38.5% of wild-type and Nrf2 ؊/؊ mice, respectively. Oltipraz induced the phase 2 enzymes responsible for BBN detoxification in the liver and urinary bladder in an Nrf2-dependent manner. As expected, therefore, oltipraz decreased the incidence of urinary bladder carcinoma by BBN in wild-type mice but had little effect in Nrf2 ؊/؊ mice. In wild-type mouse liver, oltipraz significantly induced BBN glucuronidation and decreased the urinary concentration of N-nitrosobutyl(3-carboxypropyl)amine, a proximate carcinogen of BBN. Importantly, BBN was found to suppress the expression of UGT1A specifically in the urinary bladder. This suppression was counteracted by oltipraz in wild-type mice but not in Nrf2 ؊/؊ mice. These results show that Nrf2 and its downstream target genes are responsible for BBN detoxification. Furthermore, oltipraz prevents carcinogenesis by BBN by enhancing detoxification of this carcinogen in the liver and urinary bladder.

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“…These urine-derived cells also showed the ability to differentiate into various cell lineages as described below, and were capable of growing for at least 14 passages in vitro. Cells from human urine specimens could be consistently cultured long-term using a medium that we originally developed for culture of rat urothelium 61 . However, the phenotype of the cultured human urine-derived cells was not that of primary urothelial cells.…”

Section: Urinementioning

confidence: 99%

Mesenchymal Stem Cells for Cell Therapy and Tissue Regeneration in Urology

Shi¹,

Zhang²

2011

Regenerative Medicine and Tissue Engineering - Cells and Biomaterials

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Expansion and Long-Term Culture of Differentiated Normal Rat Urothelial Cells in Vitro

Cited by 36 publications

References 21 publications

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

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

767: NRF2 is Essential for the Chemopreventive Efficacy of Oltipraz Against Urinary Bladder Carcinogenesis

Mesenchymal Stem Cells for Cell Therapy and Tissue Regeneration in Urology

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