Shuqi Du

Involvement of Selective Reactive Oxygen Species Upstream of Proapoptotic Branches of Unfolded Protein Response

Cadmium triggers apoptosis of LLC-PK1 cells through induction of endoplasmic reticulum (ER) stress. We found that cadmium caused generation of reactive oxygen species (ROS) and that cadmium-induced ER stress was inhibited by antioxidants. In contrast, suppression of ER stress did not attenuate cadmium-triggered oxidative stress, suggesting that ER stress occurs downstream of oxidative stress. Exposure of the cells to either \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document}, H2O2, or ONOO- caused apoptosis, whereas ER stress was induced only by \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document} or ONOO-. Transfection with manganese superoxide dismutase significantly attenuated cadmium-induced ER stress and apoptosis, whereas pharmacological inhibition of ONOO- was ineffective. Interestingly, transfection with catalase attenuated cadmium-induced apoptosis without affecting the level of ER stress. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document} caused activation of the activating transcription factor 6-CCAAT/enhancer-binding protein-homologous protein (CHOP) and the inositol-requiring ER-to-nucleus signal kinase 1-X-box-binding protein 1 (XBP1) proapoptotic cascades, and overexpression of manganese superoxide dismutase attenuated cadmium-triggered induction of both pathways. Furthermore, phosphorylation of proapoptotic c-Jun N-terminal kinase by \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document} or cadmium was suppressed by dominant-negative inhibition of XBP1. These data elucidated 1) cadmium caused ER stress via generation of ROS, 2) \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document} was selectively involved in cadmium-triggered, ER stress-mediated apoptosis through activation of the activating transcription factor 6-CHOP and inositol-requiring ER-to-nucleus signal kinase 1-XBP1 pathways, and 3) phosphorylation of JNK was caused by \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document}-triggered activation of XBP1.

Differential Expression Profile of Cold (TRPA1) and Cool (TRPM8) Receptors in Human Urogenital Organs

OBJECTIVES To examine the expressions of transient receptor potential (TRP) channel A1 and TRPM8 in the human urogenital organs (urinary bladder and prostate) and how these expressions change in association with bladder outlet obstruction (BOO) or benign prostatic hyperplasia (BPH). In addition to TRPM8, a cool receptor, TRPA1 is recently recognized as a cold receptor. TRPA1 is also a candidate for mechanosensor and/or nociceptor. METHODS Urinary bladder mucosa and muscular layer were taken from 9 controls and 9 patients with BOO. Prostatic specimens were obtained from 5 controls and 6 patients with BPH. Expressions of TRPA1 and TRPM8 messenger RNAs (mRNAs) were quantified by real-time revere transcriptase-polymerase chain reaction. Localization of TRPA1 protein was explored with immunohistochemistry. RESULTS The expression levels of TRPA1 mRNA in the bladder mucosa, bladder muscular layer, and prostate were in the ratio of 639:1:16. TRPA1 mRNA in the bladder mucosa with BOO was significantly upregulated to 2.32 times of control. TRPA1 protein was localized in the epithelial cells of both urinary bladder and prostate gland. The expression of TRPM8 mRNA in the prostate was much higher than that in the bladder mucosa (3024:1), but was not found in the bladder muscle layer. BPH or BOO did not significantly affect the expression of TRPM8. CONCLUSIONS TRPA1 and TRPM8 were differentially expressed in the human urinary bladder and prostate. TRPA1 in the bladder epithelium might be involved in the bladder sensory transduction and the induction process of overactive bladder by BOO.

Suppression of NF-κB by Cyclosporin A and Tacrolimus (FK506) via Induction of the C/EBP Family: Implication for Unfolded Protein Response

Immunosuppressive agents cyclosporin A (CsA) and tacrolimus (FK506) inhibit cytokine production by activated lymphocytes through interfering with calcineurin. However, little is known about their effects on the function of nonlymphoid cells. We found that, in renal tubular cells, induction of MCP-1 by inflammatory cytokines was blunted by CsA and FK506. This suppression was correlated with induction of unfolded protein response (UPR) evidenced by endogenous and exogenous indicators. The induction of UPR by these agents was reversible and observed generally in other nonimmune cells. Furthermore, administration with CsA in reporter mice caused rapid, systemic induction of UPR in vivo. In TNF-α-treated cells, suppression of MCP-1 by CsA or FK506 was associated with blunted responses of NF-κB, the crucial regulator of MCP-1. The suppression of NF-κB was reproduced by other inducers of UPR including AB5 subtilase cytotoxin, tunicamycin, thapsigargin, and A23187. CsA and FK506, as well as other UPR inducers, caused up-regulation of C/EBP family members, especially C/EBPβ and CHOP (C/EBP homologous protein), and overexpression of either C/EBPβ or CHOP significantly attenuated TNF-α-triggered NF-κB activation. Furthermore, down-regulation of C/EBPβ by small interfering RNA substantially reversed the suppressive effect of CsA on TNF-α-induced MCP-1 expression. These results suggested that CsA and FK506 confer insensitiveness to TNF-α on resident cells through UPR-dependent induction of the C/EBP family members.

Rapid, transient induction of ER stress in the liver and kidney after acute exposure to heavy metal: evidence from transgenic sensor mice.

Endoplasmic reticulum (ER) stress‐responsive alkaline phosphatase (ES‐TRAP) serves as a sensitive indicator for ER stress. In response to heavy metals including cadmium, nickel and cobalt, hepatocytes and renal tubular cells expressing ES‐TRAP exhibited ER stress and decreased ES‐TRAP activity. In ES‐TRAP transgenic mice, acute exposure to cadmium showed rapid, transient decreases in the activity of serum ES‐TRAP. It was inversely correlated with the induction of endogenous ER stress markers in the liver and kidney. Our result provides first evidence for the acute, reversible induction of ER stress in vivo after exposure to heavy metal.

Roles of mechanosensitive ion channels in bladder sensory transduction and overactive bladder

Abstract:  In the storage phase, mechanical stretch stimulates bladder afferents. These signals generate sensations and trigger voiding responses, however the precise mechanisms by which mechanical stimuli excite bladder afferents are yet to be explored. For mechanosensory transduction, the presence of mechanosensors is essential in the peripheral sensory systems including sensory nerve endings, urothelium and others. There is increasing evidence that mechanosensitive ion channels, such as degenerin/epithelial Na+ channel (ENaC) and transient receptor potential (TRP) channel families, play key roles in the mechanosensory transduction of the urinary bladder. Pharmacological interventions targeting mechanosensitive ion channels may provide a new strategy for the treatment of bladder dysfunction.

Sex-related differences in activity of lower urinary tract in response to intravesical acid irritation in decerebrate unanesthetized mice

Sex-related differences in lower urinary tract (LUT) activity responding to intravesical infusion of diluted acetic acid (A/A, pH 3.0) were investigated during cystometrograms in decerebrate unanesthetized mice. A/A produced a decrease of intercontraction intervals in both female and male animals, and the extent of the decrease in male mice was much less than in female mice [19 +/- 5% (P = 0.03) vs. 65 +/- 5% (P = 0.03); n = 6 for each], exhibiting a marked difference between the two groups in response to acid irritation of the LUT (P = 0.002). A/A reduced maximal voiding pressure (MVP) (19 +/- 4%, P = 0.03) but had no effect on pressure threshold for inducing voiding contraction (PT) (P = 0.56) in females, whereas A/A did not change MVP (P = 1.00) but increased PT (16 +/- 4%, P = 0.03) in males. A/A decreased bladder compliances of female and male mice in a similar fashion (44 +/- 10% vs. 24 +/- 7%, P = 0.03 for each). In male mice, A/A produced persistent dribbling of fluid after voiding contraction phase, which was virtually not seen in females. The present study demonstrates the differences between female and male mice in response to noxious stimulation in the LUT: the female bladder is more sensitive to the acid irritation, while the male urethra is more irritable to the noxious stimulus. Identification of mechanisms underlying sex-specific characteristics might be helpful for elucidating pathogenesis of painful bladder syndrome.

Bladder outlet obstruction induced expression of prostaglandin E2 receptor subtype EP4 in the rat bladder: a possible counteractive mechanism against detrusor overactivity.

PURPOSE Prostaglandins have been implicated as endogenous modulators of bladder function under physiological and pathological conditions. We examined how the expression of each EP receptor subtype changed in association with bladder outlet obstruction and focused on the functional role of EP4 receptor subtype in the bladder with outlet obstruction. MATERIALS AND METHODS We assessed the gene expression of EP receptor subtypes by reverse transcriptase-polymerase chain reaction. EP4 protein localization was determined by immunohistochemistry. The effect of the selective EP4 agonist ONO-AE1-329 on 50 mM KCl induced contraction of rat bladder strips was examined in vitro. Continuous infusion cystometrograms were done to examine the effect of intravesical perfusion of ONO-AE1-329 on the micturition reflex in urethane anesthetized rats. RESULTS EP4 receptor genes were largely expressed in bladders with outlet obstruction but absent in controls. EP4 receptor proteins were clearly detected in obstructed bladder detrusor smooth muscle and epithelium. ONO-AE1-329 (100 μM) significantly relaxed KCl induced contraction of bladder strips from rats with bladder outlet obstruction. A significant correlation was found between the relaxant effect of ONO-AE1-329 and whole bladder weight. In rats with bladder outlet obstruction intravesical infusion of 10 μM ONO-AE1-329 significantly increased bladder capacity without changing micturition pressure while it had no effect in controls. CONCLUSIONS Activation of the EP4 receptors expressed in bladders with outlet obstruction may suppress detrusor muscle contraction and afferent activity. This might be a compensatory mechanism to counteract the deterioration of storage function in bladders with outlet obstruction.

Emerging Families of Ion Channels Involved in Urinary Bladder Nociception

The expression of multiple ion channels and receptors is essential for nociceptors to detect noxious stimuli of a thermal, mechanical or chemical nature. The peripheral sensory transduction systems of the urinary bladder include sensory nerve endings, urothelial cells and others whose location is suitable for transducing mechanical and chemical stimuli. There is an increasing body of evidence implicating the Deg/ENaC and TRP channel families in the control of bladder afferent excitability under physiological and pathological conditions. Pharmacological interventions targeting these ion channels may provide a new strategy for the treatment of pathological bladder sensation and pain.