TRPV4 channel opening mediates pressure-induced pancreatitis initiated by Piezo1 activation
Sandip M. Swain, … , Steven R. Vigna, Rodger A. Liddle
Sandip M. Swain, … , Steven R. Vigna, Rodger A. Liddle
Published January 30, 2020
Citation Information: J Clin Invest. 2020;130(5):2527-2541. https://doi.org/10.1172/JCI134111.
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Research Article Gastroenterology

TRPV4 channel opening mediates pressure-induced pancreatitis initiated by Piezo1 activation

Abstract

Elevated pressure in the pancreatic gland is the central cause of pancreatitis following abdominal trauma, surgery, endoscopic retrograde cholangiopancreatography, and gallstones. In the pancreas, excessive intracellular calcium causes mitochondrial dysfunction, premature zymogen activation, and necrosis, ultimately leading to pancreatitis. Although stimulation of the mechanically activated, calcium-permeable ion channel Piezo1 in the pancreatic acinar cell is the initial step in pressure-induced pancreatitis, activation of Piezo1 produces only transient elevation in intracellular calcium that is insufficient to cause pancreatitis. Therefore, how pressure produces a prolonged calcium elevation necessary to induce pancreatitis is unknown. We demonstrate that Piezo1 activation in pancreatic acinar cells caused a prolonged elevation in intracellular calcium levels, mitochondrial depolarization, intracellular trypsin activation, and cell death. Notably, these effects were dependent on the degree and duration of force applied to the cell. Low or transient force was insufficient to activate these pathological changes, whereas higher and prolonged application of force triggered sustained elevation in intracellular calcium, leading to enzyme activation and cell death. All of these pathological events were rescued in acinar cells treated with a Piezo1 antagonist and in acinar cells from mice with genetic deletion of Piezo1. We discovered that Piezo1 stimulation triggered transient receptor potential vanilloid subfamily 4 (TRPV4) channel opening, which was responsible for the sustained elevation in intracellular calcium that caused intracellular organelle dysfunction. Moreover, TRPV4 gene–KO mice were protected from Piezo1 agonist– and pressure-induced pancreatitis. These studies unveil a calcium signaling pathway in which a Piezo1-induced TRPV4 channel opening causes pancreatitis.

Authors

Sandip M. Swain, Joelle M.-J. Romac, Rafiq A. Shahid, Stephen J. Pandol, Wolfgang Liedtke, Steven R. Vigna, Rodger A. Liddle

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Figure 3

Mechanical pushing and fluid shear stress increase [Ca2+]i in pancreatic acini.

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Mechanical pushing and fluid shear stress increase [Ca2+]i in pancreatic...
(A) Brightfield and live-cell imaging of pancreatic acini loaded with calcium 6-QF at time 0 and at time of maximum fluorescence 1:40 (min:s) after mechanical pushing. Scale bars: 10 μm. (B) Representative [Ca2+]i fluorescence tracings from single acinar cells in an acinus during the course of mechanical pushing with a blunt pipette. (C) Peak [Ca2+]i levels following mechanical pushing of acini from WT or Piezo1aci-KO mice. (D) Blunt pushing with a micropipette increased [Ca2+]i fluorescence only in the presence of extracellular Ca2+. Results represent data from 25–39 cells in 3 independent experiments. (E) Representative traces for relative fluorescence intensity (ΔF/F0) of calcium 6-QF–loaded cells are shown in response to applied shear stress at the forces shown in the graph. The duration of fluid flow shear stress was 30 seconds, as indicated by the orange bar. (F) The average peak [Ca2+]i intensity of Fmax/F0 is shown for 41–54 cells. n = 3–4 independent experiments. (G and H) The relative fluorescence intensity (ΔF/F0) and average peak [Ca2+]i intensity of pancreatic acini in response to 12 dyne/cm2 shear stress for 1 or 5 seconds. Data in H are averaged from 14–41 cells. (I) Representative traces of relative fluorescence intensity (ΔF/F0) from calcium 6-QF–loaded WT and Piezo1-KO acinar cells before and after applying 12 dyne/cm2 shear stress for 30 seconds. (J) Average peak intensity of Fmax/F0 from the experiment depicted in I. Data represent a total of 53–67 cells and 3 independent experiments. Statistical analyses were performed using Student’s t test (D, H, and J) and 1-way ANOVA with Tukey’s multiple comparison (C and F). Data are shown as mean ± SEM. **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.