AB276. SPR-03 In vitro evaluation of hydrostatic pressure on ATP release and Caspase-1 activation in rat urothelial cells

Abstract

ObjectiveBladder outlet obstruction (BOO) is projected to affect approximately 1.1 billion men and women by 2018. While BOO results from a multitude of etiologies, they are all characterized by chronically elevated storage and voiding pressures, along with inflammation in the bladder tissue, which can lead to bladder fibrosis and overactive bladder symptoms. Previous research in our lab demonstrated that ATP release by rat primary urothelial cells increased in response to elevated pressure (10–15 cmH2O) in vitro. Thus, we hypothesize that the release of ATP by urothelial cells upon exposure to pathological pressures in BOO initiates a cascade of events that includes the formation of the NLRP3 inflammasome and caspase-1 activation. In the present study, we examined the effects of elevated pressure on ATP-purinergic signaling and caspase-1 activation in rat urothelial cells in vitro.MethodsUsing a custom built pressure system, rat urothelial cell line MYP3 cells (1.2×106 cells/well in F-12 K medium) were exposed to pressure conditions that represent both pathological storage and voiding pressures: 15 cmH2O 60 min, 15, 40 and 75 cmH2O 1 min. Cells that were prepared in a similar manner but maintained under atmospheric pressure served as a control. In addition, MYP3 cells that were exposed to a hypotonic condition (240 mOsm) and cells treated with 1.25 mM ATP served as two positive controls. After exposure to pressure, the supernatant media was collected and the extracellular ATP concentration was measured using a luciferin-luciferase assay kit (Life Technologies). Cells were then lysed and intracellular caspase-1 activity was measured using an established method. The pressure experiments (40 cmH2O 1 min) were repeated in the presence of a P2X7 antagonist [100 µM A-438079 (R&D Systems)] to determine the mechanism for pressure-induced caspase-1 activation.ResultsExposure of MYP3 cells to hydrostatic pressure for 1 min at 15, 40 and 75 cmH2O resulted in a 3-, 3.5- and 4.5-fold increase in extracellular ATP levels, respectively, compared to the 0 cmH2O control. When these cells were exposed to a hypotonic solution, ATP release increased by 6-fold compared to the isotonic control. Exposure of MYP3 cells to pressure also resulted in up to 1.3-fold increases in caspase-1 levels, which were similar to the positive controls, indicating caspase-1 levels are maximized after exposure to pathologic pressure conditions.ConclusionsThe significant increase in ATP release indicates that hydrostatic pressure is a good mechanical stimulus to model BOO in vitro. The acute caspase-1 responses after 1-minute exposure to pressure suggest that high-pressure voiding may be an important trigger of the NLRP3 inflammasome in BOO. The results that demonstrate exposure to pressure and treatment with ATP both result in caspase-1 activation implicate autocrine purinergic signaling as a mediator of NLRP inflammasome formation.Funding Source(s)NIH (R01DK103534, P20GM103444), NSF (1264579)