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.
View: Text | PDF
Research Article Gastroenterology Article has an altmetric score of 12

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

×

Figure 5

TRPV4 is expressed in pancreatic acini, and Piezo1 induces TRPV4 activation.

Options: View larger image (or click on image) Download as PowerPoint
TRPV4 is expressed in pancreatic acini, and Piezo1 induces TRPV4 activat...
(A) mRNA (fold expression) of TRPV4 and Piezo1 in pancreatic acini relative to the housekeeping gene actb (n = 3–5 experiments). (B) Immunostaining of mouse pancreatic acini with a TRPV4 antibody (red). Nuclei (blue) were stained with Nunc blue. Scale bar: 10 μm. (C and D) Effects of the TRPV4 agonist GSK101 (50 nM) and GSK101 + the TRPV4 antagonist HC067 (100 nM) or RN1734 (30 μM) on [Ca2+]i are shown. (C) A representative experiment shows the relative fluorescence intensity (ΔF/F0) of calcium dye over time and (D) the average peak [Ca2+]i intensity of pancreatic acini from 43 to 63 cells. (E and F) Arachidonic acid (20 μM) and 5′,6′-EET (5 μM) increased [Ca2+]i. The effects of 5′,6′-EET (5 μM) were blocked by HC067 (100 nM). (E) The relative fluorescence intensity of calcium dye and (F) average peak [Ca2+]i intensity of pancreatic acini from 40 to 54 cells are shown. (G, H, I, and J) The TRPV4 antagonist HC067 (1 μM) blocked the effects of shear stress (12 dyne/cm2) and Yoda1 (25 μM) on the sustained [Ca2+]i responses. (G and I) Representative tracings of the relative fluorescence intensity of calcium dye over time with the different stimuli. (H and J) The average peak [Ca2+]i responses were calculated from 35 to 86 cells. (K and L) The shear stress– and Yoda-induced sustained increases in [Ca2+]i were not seen in pancreatic acini isolated from TRPV4-KO mice. (K) Representative traces demonstrate the effects of shear stress or Yoda1 on [Ca2+]i and (L) show the average peak [Ca2+]i intensity at different time intervals (from 45 to 47 cells). Statistical analyses were performed using Student’s t test. Data are shown as the mean ± SEM. *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001.