Elevated hydrostatic pressure stimulates ATP release which mediates activation of the NLRP3 inflammasome via P2X4 in rat urothelial cells
Partial bladder outlet obstruction (pBOO) is a prevalent urological condition commonly accompanied by increased intravesical pressure, inflammation, and fibrosis. Studies have demonstrated that pBOO results in increased NLRP3 inflammasome and caspase-1 activation and that ATP is released from urothelial cells in response to elevated pressure. In the present study, we investigated the role of elevated pressure in triggering caspase-1 activation via purinergic receptors activation in urothelial cells. Rat urothelial cell line, MYP3 cells, was subjected to hydrostatic pressures of 15 cmH2O for 60 min, or 40 cmH2O for 1 min to simulate elevated storage and voiding pressure conditions, respectively. ATP concentration in the supernatant media and intracellular caspase-1 activity in cell lysates were measured. Pressure experiments were repeated in the presence of antagonists for purinergic receptors to determine the mechanism for pressure-induced caspase-1 activation. Exposure of MYP3 cells to both pressure conditions resulted in an increase in extracellular ATP levels and intracellular caspase-1 activity. Treatment with P2X7 antagonist led to a decrease in pressure-induced ATP release by MYP3 cells, while P2X4 antagonist had no effect but both antagonists inhibited pressure-induced caspase-1 activation. Moreover, when MYP3 cells were treated with extracellular ATP (500 µM), P2X4 antagonist inhibited ATP-induced caspase-1 activation, but not P2X7 antagonist. We concluded that pressure-induced extracellular ATP in urothelial cells is amplified by P2X7 receptor activation and ATP-induced-ATP release. The amplified ATP signal then activates P2X4 receptors, which mediate activation of the caspase-1 inflammatory response.
KeywordsHydrostatic pressure NLRP3 Inflammasome Inflammation Purinergic receptors P2X4 P2X7 Pressure mechanobiology
The authors would like to thank Julie Fuller and the Substrate Services Core and Research Support Services (SCRSS) in the Department of Surgery for their histological embedding and sectioning. We also thank the Light Microscopy Care Facility and Yasheng Gao for help imaging. Both core facilities are at Duke University.
JTP, FMH, and JN conceived the project; CLD, JTP, FMH, and JN designed experiments; CLD, FMH, and HJ performed experiments; CLD analyzed data; CLD, JTP, FMH, and JN interpreted results of experiments; CLD and FMH prepared figures; CLD. drafted manuscript; CLD, JTP, FMH, and JN edited and revised manuscript. JTP, FMH, and JN approved final version of manuscript.
Funding was provided by National Institute of Diabetes and Digestive and Kidney Diseases (Grant No. R01DK103534), National Institutes of Health (Grant No. P20GM103444), and National Science Foundation (Grant No. 1264579).
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