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

Abstract BackgroundPrevious studies have demonstrated that purinergic receptors could be therapeutic targets to modulate the inflammatory response in multiple brain disease models. However, tools for the selective and efficient targeting of these receptors are scarce. The new development of P2X7-specific nanobodies (nbs) enables us to effectively block the P2X7-channel.MethodsTemporary middle cerebral artery occlusion (tMCAO) in wildtype and P2X7-transgenic mice was used as a model for ischemic stroke. ATP release was assessed in transgenic ATP sensor mice. Stroke size was measured without treatment and after injection of P2X7-specific nbs i.v. and i.c.v. directly before tMCAO-surgery. P2X7-GFP expressing transgenic mice were used to show immunhistochemically P2X7 distribution in the brain. In vitro cultured microglia were used to investigate calcium-influx, pore-formation via DAPI uptake, caspase 1 activation and IL-1b release after incubation with P2X7-specific nbs. ResultsATP sensor mice showed an increase of ATP-release in the ischemic hemisphere compared to the contralateral hemisphere or sham mice up to 24 h after stroke. We could further verify the role of the ATP-P2X7 axis in P2X7-overexpressing mice, which showed significantly greater stroke volumes after 24 h. In vitro experiments with primary microglia cells showed that P2X7-specific nanobodies were capable of dampening the ATP-trigged calcium-influx and formation of membrane pores measured by Fluo4 fluorescence or DAPI uptake. We found a lower caspase 1 activity and a subsequently lower IL-1b release. However, the intravenous (i.v.) injection of P2X7-specific nanobodies compared to isotype controls before the tMCAO-surgery did not result in smaller stroke size compared to isotype controls. As demonstrated by FACS, nbs had only reached brain infiltrating macrophages but not microglia. To reach microglia, we injected the P2X7-spezific nbs or the isotype directly intraventricularly (icv). 30 mg of P2X7-specific nbs proved efficient for microglial targeting, reducing post-stroke microglia activation and stroke size significantly.ConclusionHere, we demonstrate the importance of locally produced ATP for the tissue damage observed in ischemic stroke and we show the potential of icv injected P2X7-specific nbs to reduce ischemic tissue damage.

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Elevated hydrostatic pressure stimulates ATP release which mediates activation of the NLRP3 inflammasome via P2X4 in rat urothelial cells

International Urology and Nephrology ◽

10.1007/s11255-018-1948-0 ◽

2018 ◽

Vol 50 (9) ◽

pp. 1607-1617 ◽

Cited By ~ 7

Author(s):

Cody L. Dunton ◽

J. Todd Purves ◽

Francis M. Hughes ◽

Huixia Jin ◽

Jiro Nagatomi

Keyword(s):

Hydrostatic Pressure ◽

Nlrp3 Inflammasome ◽

Atp Release ◽

Urothelial Cells ◽

Elevated Hydrostatic Pressure

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Electrophysiological Investigation of Hydrostatic Pressure Mechanotransduction by Urothelial Cell Lines

ASME 2011 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2011-53518 ◽

2011 ◽

Author(s):

Kevin D. Champaigne ◽

Sarette N. Jenderny ◽

Jiro Nagatomi

Keyword(s):

Hydrostatic Pressure ◽

Transient Receptor Potential ◽

Trp Channels ◽

Primary Cultures ◽

Receptor Potential ◽

Ruthenium Red ◽

Mechanical Stimuli ◽

Urothelial Cells ◽

Membrane Stretch

The urothelium is the epithelial lining of the ureters, urinary bladder, and urethra. Recent discoveries have suggested that in addition to providing a barrier function to urine, the urothelium actively participates in sensory functions related to thermal, chemical, and mechanical stimuli, and releases chemical signals in response[1]. In addition to a sensitivity to cell membrane stretch caused by wall tension upon bladder filling, in vitro studies by our group have shown that urothelial cells may be sensitive to hydrostatic pressure directly without requiring membrane stretching [2]. Specifically, primary cultures of rat bladder urothelial cells exposed to 10 cmH2O pressure on rigid substrates released significantly greater amounts of ATP compared to the baseline control without exposure to pressure. Moreover, this ATP response by rat urothelial cells to pressure was inhibited by pre-treatment of cells with ruthenium red, a non-specific antagonist of transient receptor potential (TRP) channels, suggesting a potential involvement of these channels in pressure mechanotransduction. Further understanding of the mechanisms, however, is needed to improve treatment of bladder dysfunction such as overactive bladder.

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An Optical Approach for Studying the Cellular Mechanotransduction of Hydrostatic Pressure by Bladder Urothelial Cells

ASME 2011 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2011-53872 ◽

2011 ◽

Author(s):

Shawn Olsen ◽

Jiro Nagatomi

Keyword(s):

Ion Channels ◽

Hydrostatic Pressure ◽

Cell Volume ◽

Transient Receptor Potential ◽

Trp Channels ◽

Receptor Potential ◽

Fluorescent Imaging ◽

Urothelial Cells ◽

Bladder Urothelium

Recent studies have suggested that the bladder urothelium is sensitive to both stretch and hydrostatic pressure during bladder filling, and is considered to play a mechanosensory role in sensing bladder fullness [1, 2]. In a previous study [3], our group demonstrated that compared to the control, rat bladder urothelial cells (UCs) exposed to hydrostatic pressure (10–15 cmH2O for 5 minutes) in vitro released significantly higher levels of ATP and that this response was attenuated by pharmacologically blocking transient receptor potential (TRP) channels, as well as epithelial sodium channels (ENACs). While blocking these ion channels inhibited the ATP response by UCs to hydrostatic pressure, it remains unclear whether these ion channels are being activated directly by hydrostatic pressure or by membrane deformation. Our current hypothesis is that a change in cell volume may occur due to the application of hydrostatic pressure and subsequent changes in cellular osmolality, which, in turn, activate the membrane-bound mechanosensitive channels. Using real-time fluorescent imaging and a custom experimental setup, the present study sought to quantify the UC cell volume changes during exposure to hydrostatic pressure and to better understand the mechanisms by which UCs sense hydrostatic pressure.

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