The role of ATP signalling in response to mechanical stimulation studied in T24 cells using new microphysiological tools.

The capacity to store urine and initiate voiding is a valued characteristic of the human urinary bladder. To maintain this feature, it is necessary that the bladder can sense when it is full and when it is time to void. The bladder has a specialized epithelium called urothelium that is believed to be important for its sensory function. It has been suggested that autocrine ATP signalling contributes to this sensory function of the urothelium. There is well-established evidence that ATP is released via vesicular exocytosis as well as by pannexin hemichannels upon mechanical stimulation. However, there are still many details that need elucidation and therefore there is a need for the development of new tools to further explore this fascinating field. In this work, we use new microphysiological systems to study mechanostimulation at a cellular level: a mechanostimulation microchip and a silicone-based cell stretcher. Using these tools, we show that ATP is released upon cell stretching and that extracellular ATP contributes to a major part of Ca2+ signalling induced by stretching in T24 cells. These results contribute to the increasing body of evidence for ATP signalling as an important component for the sensory function of urothelial cells. This encourages the development of drugs targeting P2 receptors to relieve suffering from overactive bladder disorder and incontinence.

Localization of P2X receptor subtypes 2, 3 and 7 in human urinary bladder.

BACKGROUND: Voiding dysfunctions are a common problem that has a severe negative impact on the quality of life. Today there is a need for new drug targets for these conditions. The role of ATP receptors in bladder physiology has been studied for some time, primarily in animal models. The aim of this work is to investigate the localization of the ATP receptors P2X2, P2X3 and P2X7 and their colocalization with vimentin and actin in the human urinary bladder. METHODS: Immunohistochemical analysis was conducted on full-thickness bladder tissues from fundus and trigonum collected from 15 patients undergoing open radical cystectomy due to chronic cystitis, bladder cancer or locally advanced prostate cancer. Colocalization analyses were performed between the three different P2X subtypes and the structural proteins vimentin and actin. Specimens were examined using epifluorescence microscopy and correlation coefficients were calculated for each costaining as well as the mean distance from the laminin positive basal side of the urothelium to the vimentin positive cells located in the suburothelium. RESULTS: P2X2 was expressed in vimentin positive cells located in the suburothelium. Less distinct labelling of P2X2 was also observed in actin positive smooth muscle cells and in the urothelium. P2X3 was expressed in vimentin positive cells surrounding the smooth muscle, and in vimentin positive cells located in the suburothelium. Weaker P2X3 labelling was seen in the urothelium. P2X7 was expressed in the smooth muscle cells and the urothelium. In the suburothelium, cells double positive for P2X2 and vimentin where located closer to the urothelium while cells double positive for P2X3 and vimentin where located further from the urothelium. CONCLUSION: The results from this study demonstrate that there is a significant difference in the expression of the purinergic P2X2, P2X3 and P2X7 receptors in the different histological layers of the human urinary bladder.

Secondary stimulation from Bacillus Calmette-Guérin induced macrophages induce nitric oxide independent cell-death in bladder cancer cells.

The anti-tumour mechanisms following Bacillus Calmette-Guérin (BCG) treatment of bladder-cancer remain largely unknown. Previous studies have shown involvement of nitric-oxide (NO) formation in the BCG-mediated effect. We analyzed the effects of macrophage secreted factors (MSFs) from BCG-stimulated RAW264.7 cells on the bladder-cancer cell line MBT2. Direct treatment with BCG did not induce NO in MBT2-cells whereas supernatant from BCG-stimulated macrophages increased NOS2 mRNA and protein expression, NO concentrations and cell-death. Blocking NO-synthesis with the NOS-inhibitor L-NAME did not affect levels of cell-death suggesting cytotoxic pathways involving other signalling molecules than NO. Several such candidate genes were identified in a microarray.

Nitric oxide as a marker for evaluation of treatment effect of cyclosporine A in patients with bladder pain syndrome/interstitial cystitis type 3C.

OBJECTIVE: Bladder pain syndrome/interstitial cystitis (BPS/IC) is a chronic inflammatory disease and to date few treatments or tools for investigating the activity of the disease are available. This study evaluated whether luminal nitric oxide (NO) could be used as a marker for evaluation of therapeutic outcome in BPS/IC type 3C treated with the immunosuppressive agent cyclosporine A (CsA). MATERIAL AND METHODS: Ten patients with BPS/IC type 3C were given CsA for 16 weeks, initially at 3 mg/kg/day, and after 12 weeks the dose was scaled down. Formation of NO was measured in the urinary bladder with a silicone catheter, and symptom and bother score related to the disease were evaluated with the Interstitial Cystitis Symptom and Problem Index, every second week. RESULTS: All patients had elevated NO levels in the bladder initially and NO levels decreased during treatment with CsA. When the dose of CsA was lowered NO formation increased and after 2 weeks without medication, the NO formation was the same as before the study began. CONCLUSIONS: The results indicate that measurement of NO is a tool for evaluating the response to anti-inflammatory treatment in patients with BPS/IC type 3C. NO could serve as a marker for assessing the activity of the inflammation.