ABSTRACT
BACKGROUND AND PURPOSE: The urinary bladder urothelium expresses various receptors and in response to chemical and mechanical stimuli releases mediators, thereby modulating bladder sensory pathways. Transient receptor potential vanilloid 1 (TRPV1) ion channels and nerve growth factor (NGF) in those cells are implicated in this modulatory effect and play a role in sensitizing pain-related afferent pathways during inflammation. In this study, we investigated the interaction between NGF and TRPV1 channels in urothelial cells. EXPERIMENTAL APPROACH: Urothelial cells from female Sprague-Dawley rat bladders were cultured to quantify membrane expression of TRPV1 channels and capsaicin-induced ATP release in the presence of NGF alone or with TrKA or PI3K inhibitors. Pain scores from rats with cyclophosphamide (CYP)-induced bladder inflammation were assessed after treatment with a TrkA antagonist. Bladders (from control and CYP rats) were collected and analysed for NGF content and TRPV1 channel expression. KEY RESULTS: Cultured cells responded to NGF with increased TRPV1 channel expression in the cell membrane and increased release of ATP. Both responses were blocked by either a TrkA antagonist or a PI3K inhibitor. Treatment in vivo with the TrkA antagonist alleviated pain symptoms and reduced CYP-induced NGF overexpression in the mucosa. Furthermore, in urothelial cells from animals with bladder inflammation, expression of TRPV1 channels in the membrane was significantly increased. CONCLUSIONS AND IMPLICATIONS: During bladder inflammation, increased production of NGF in urothelial cells induced increased expression and activity of TRPV1 channels in the cell membrane. This effect was primarily mediated by the PI3K pathway.
Subject(s)
Cystitis/metabolism , Nerve Growth Factor/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , TRPV Cation Channels/metabolism , Adenosine Triphosphate/metabolism , Animals , Capsaicin , Cell Membrane/drug effects , Cell Membrane/metabolism , Cells, Cultured , Cyclophosphamide , Cystitis/chemically induced , Female , Pain/chemically induced , Pain/metabolism , Phosphoinositide-3 Kinase Inhibitors , Rats, Sprague-Dawley , Receptor, trkA/antagonists & inhibitors , Urothelium/drug effects , Urothelium/metabolismABSTRACT
PURPOSE: To study the protection offered by empty liposomes (LPs) alone against acrolein-induced changes in urothelial cell viability and explored uptake of LPs by primary (rat) urothelial cells. METHODS: Acrolein was used as a means to induce cellular damage and reduce urothelial cellular viability. The effect of acrolein or liposomal treatment on cellular proliferation was studied using 5-bromo-2'-deoxy-uridine assay. Cytokine release was measured after urothelial cells were exposed to acrolein. Temperature-dependent uptake study was carried out for fluorescent-labeled LPs using confocal microscopy. RESULTS: Liposome pretreatment protected against acrolein-induced decrease in urothelial cell proliferation. LPs also significantly affected the acrolein-induced cytokine (interferon-gamma) release offering protection to the urothelial cells against acrolein damage. We also observed a temperature-dependent urothelial uptake of fluorescent-labeled LPs occurred at 37 °C (but not at 4 °C). CONCLUSIONS: Empty LPs alone provide a therapeutic efficacy against acrolein-induced changes in urothelial cell viability and may be a promising local therapy for bladder diseases. Hence, our preliminary evidence provides support for liposome-therapy for urothelial protection and possible repair.
Subject(s)
Acrolein/toxicity , Liposomes/pharmacology , Urothelium/drug effects , Urothelium/metabolism , Adenosine Triphosphate/metabolism , Animals , Cell Line , Cell Proliferation/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Female , Interferon-gamma/metabolism , Microscopy, Confocal , Rats , Rats, Sprague-Dawley , Urothelium/cytologyABSTRACT
AIM: To examine function of both cholinergic (muscarinic) and TRPV1 receptors in human bladder urothelial (HBUC) from non-neurogenic overactive bladder (OAB) patients as compared to control subjects. METHODS: Primary HBUC cultures were derived from cystoscopic biopsies from OAB and control subjects. Muscarinic and TRPV1 function was assessed by acetylcholine (5 µm) or capsaicin (0.5 µm) evoked ATP release, measured by luciferase assay. Overall, expression of TRPV1 and muscarinic M3 receptors in bladder urothelial cells was accomplished using western immunoblotting. RESULTS: Our findings revealed that the response to acetylcholine in OAB HBUC cultures (which was blocked by the nonselective muscarinic antagonist, atropine methyl nitrate or AMN) was not significantly different than from controls. The acetylcholine M3 receptor was slightly decreased as compared to control. In contrast, OAB HBUC cultures exhibited a capsaicin hypersensitivity and augmented release of ATP (3.2 fold higher), which was blocked by the antagonist capsazepine. The increase in capsaicin sensitivity correlated with increased urothelial TRPV1 expression. CONCLUSION: Though characterized in a small number of subjects, augmented release of urothelial-derived transmitters such as ATP could 'amplify' signalling between and within urothelial cells and nearby afferent nerves.
