The urothelium has two distinct ATP release mechanisms that contribute differently to bladder function

Beckel J1, Silberfeld A1, Chavez B1, Gianakas C1, Tandoc B1, De Groat W C1

Research Type

Basic Science / Translational

Abstract Category

Female Lower Urinary Tract Symptoms (LUTS) / Voiding Dysfunction

Abstract 456
Basic Science: Overactive Bladder and Pain
Scientific Podium Short Oral Session 24
Thursday 30th August 2018
14:07 - 14:15
Hall C
Basic Science Voiding Dysfunction Biochemistry Pathophysiology
1. University of Pittsburgh

Jonathan Beckel



Hypothesis / aims of study
The importance of ATP release from the bladder epithelium (urothelium) in bladder physiology/pathophysiology has been a hotly debated topic in the field of urology.  Previous research has revealed that urinary ATP levels are elevated in patients suffering from conditions such as overactive bladder (OAB), Painful Bladder Syndrome/Interstitial Cystitis (PBS/IC), or bacterial cystitis, suggesting a role for ATP in bladder pathology.  It has also been demonstrated that alteration of basal levels of urinary ATP, either by inhibiting endogenous ecto-ATPases in the bladder lumen or by introducing an exogenous ATPase into the bladder lumen, can modulate reflex bladder activity in the rat. This suggests that urinary ATP also plays an important role in the physiological control of micturition. It has become clear, then, that differences must exist in urinary purinergic signaling that account for ATP’s dual role in bladder physiology/pathophysiology.
It has been recently confirmed that the urothelium releases ATP from both vesicular and non-vesicular (pannexin channels) mechanisms.  It is unclear, however, if these two release mechanisms mediate different downstream events.  Research involving other cell types that express both ATP release mechanisms, such as the retinal epithelium, suggests that vesicular and non-vesicular ATP release mechanisms are activated by separate stimuli, with physical stimuli such as stretch activating ATP release through pannexin channels and simulation of Toll-like receptors (TLRs) activating vesicular (lysosomal) release.  With this information in mind, we hypothesized that the same separation exists in the urothelium and that distinct ATP release mechanisms may mediate different downstream events. To this end we examined two pathways known to cause ATP release from the urothelium: the stimulation of urothelial α3* nicotinic receptors (nAChRs) with cytisine and the activation of TLR4 with bacterial lipopolysaccharides (LPS). Both of these stimuli have been previously shown to excite bladder reflexes in vivo in addition to causing ATP release, however LPS also induces bladder inflammation, while α3* nAChR stimulation does not.
Study design, materials and methods
The in vitro portion of our study used immortalized normal human urothelial cells (TRT-HU1) at passage number 25-35, plated in 96-well, white walled, clear bottomed culture plates at a density of approximately 1x106/ml. Cells were used for experiments after 1-2 days when they had reached 50-70% confluency. 
To measure extracellular ATP concentrations, the media supporting the cells was first replaced with 50µl of Krebs solution alone or antagonists (2X final concentration) and incubated at 37°C for 20 minutes.  50µl of Krebs (for non-stimulated controls) or agonists (2X final concentration) was then added and the plate incubated again for 20 minutes (cytisine) or 1 hour (LPS) at 37°C.  50µl of the luciferin/luciferase assay mix (Sigma-Aldrich) was then added and the luminescence measured using a plate-based luminometer (Victor3).  Luminescence readings were converted to ATP concentrations using known concentrations of ATP dissolved in solutions containing the same concentrations of the drugs used in the study to correct for any interference they might have on the assay.
To measure extracellular acid phosphatase activity (as a measure of lysosomal exocytosis), cells were incubated in agonists/antagonists as described above. After incubation, 50µl samples of the extracellular fluid were taken from each well and tested for acid phosphatase activity using the commercially available colorimetric kit from Sigma-Aldrich.  At the end of each experiment, cells in each well were lysed using RIPA buffer and protein concentrations determined by BCA assay as a measure of cell number with which to normalize the acid phosphatase measurements.  
To determine the influence of each ATP release mechanism on bladder function, we measured contractile activity in an in vitro rat bladder strip setup.  Bladders were removed from female Sprague Dawley rats, cut into strips longitudinally, and attached to a force displacement transducer in a tissue bath containing oxygenated Krebs solution at 37°C.  Agonists and antagonists were bath applied and changes in basal tone and spontaneous contraction amplitude recorded.
Stimulation of TRT-HU1 cells with the α3* nicotinic agonist cytisine (100µM) increased extracellular ATP concentrations 26.1 ± 3.1% after 20 minutes. This increase was significantly diminished after pretreatment with the pannexin channel antagonist Brilliant Blue FCF (BB-FCF, 100µM) but not by glycyl-L-phenylalanine 2-naphthylamide (GPN, 20µM), which destroys lysosomes. Stimulation of TRT-HU1 cells with the TLR4 agonist LPS (from O111:B4 E. coli, 100µg/ml) also increased extracellular ATP by 16.6 ± 1.3% after 1 hour.  This increase blocked by pretreatment with GPN (1.2 ± 1.9% increase), but was enhanced by pretreatment with BB-FCF (30.8 ± 2.1% increase) (Figure 1).
Stimulation of TRT-HU1 cells with LPS also increased extracellular concentrations of acid phosphatase by 63.5 ± 25.2%.  This release was blocked after pre-incubation of the cells with GPN.  Cytisine, conversely, did not increase extracellular acid phosphatase concentrations. 
Rat bladder strips exhibited increased basal tone and increased amplitude of spontaneous contractions following stimulation with LPS (AUC: 112.1 ± 17.6% increase).  This response was blocked by pre-incubation with GPN, but potentiated after Brilliant Blue FCF. LPS-induced increases were also blocked by apyrase (2U/ml), an ATPase, indomethacin (10µM), a COX inhibitor or removal of the urothelium. Cytisine had no effect on detrusor contractions, even at twice the dose that elicits ATP release (200µM).
Interpretation of results
Stimulation of urothelial cell cultures with either cytisine or LPS significantly increased extracellular ATP, however cytisine-induced ATP release was blocked using a pannexin channel antagonist, while LPS-induced release was not. LPS-induced ATP release was prevented following destruction of lysosomes, indicating that LPS causes ATP release through lysosomal exocytosis. This was confirmed by measuring extracellular acid phosphatase concentrations; LPS increased the release of lysosomally-stored acid phosphatase, while cytisine did not.  LPS stimulation of bladder strips induced increases in basal tone and bladder contraction amplitude which were blocked by incubation with apyrase or removal of the urothelium, indicating that release of urothelial ATP mediated this effect.  This effect was also blocked by indomethacin, suggesting that LPS-induced ATP release induces prostaglandin synthesis.  Cytisine did not alter detrusor activity, even at concentrations higher than those that caused ATP release.  Because ATP release in response to intravesical cytisine has been previously shown to cause bladder hyperactivity in vivo, we conclude that pannexin-mediated ATP release mediates its effects by either directly or indirectly stimulating afferent nerves, but does not activate the same pro-inflammatory pathways stimulated by LPS.
Concluding message
This study provides the first evidence that urothelial ATP release can mediate separate downstream effects on bladder function depending on the mechanism of its release. These exciting results raise the possibility that the physiological and pathophysiological roles of urinary ATP can be separated based on the mechanism of release. Although the complete cellular pathways involved in these phenomena are unclear, we believe this research is an excellent first step in developing of treatments for bladder dysfunction that specifically target the pathological role of urinary ATP, while sparing any physiological role ATP may play in normal micturition.
Figure 1
Funding K01DK106115 & R03DK114492 (JMB) Clinical Trial No Subjects Animal Species Rat Ethics Committee Institutional Care and Use Committee University of Pittsburgh