Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology
Article Title: Mechanosensor transient receptor potential vanilloid member 4 (TRPV4) regulates mouse cholangiocyte secretion and bile formation
Figure Lengend Snippet: Specific agonists of transient receptor potential vanilloid member 4 (TRPV4) activate currents and increase intracellular calcium concentration ([Ca 2+ ] i ) in large mouse cholangiocytes (MLC). A, top : representative whole cell recording. The specific TRPV4 agonist 4α -phorbol-12,13-didecanoate (4αPDD; 1 µM) activates whole cell currents. Currents measured at +100 mV (○) and at −100 mV (●) are shown. Voltage protocol −100 mV to +100 mV ramp every 5 s. Bottom : cells on coverglass were loaded with fura-2 AM, washed with PBS, and exposed to 4αPDD (1 µM); y -axis values represent the ratio of fluorescence at 340 and at 380 nm. B, top : the specific TRPV4 agonist GSK (100 nM) activates whole cell currents. Currents measured at +100 mv (○) and at −100 mV (●) are shown. Bottom : fura-2-loaded MLC cells were exposed to glycogen synthase kinase (GSK; 100 nM). The y -axis values represent the ratio of fluorescence at 340 and at 380 nm. C, top : cumulative data demonstrating maximal current density (pA/pF) measured at +100 mV in response to 4αPDD or GSK. Currents were significantly inhibited by ruthenium red (RR, 10 µM, n = 9), HC-067047 (HC, 10 µM, n = 16), and after transfection with TRPV4 small interfering (si)RNA ( n = 8). Bottom : cumulative data demonstrating the change in [Ca 2+ ] i in response to 4αPDD or GSK in control cells ( n = 78) and in the presence of RR ( n = 46), HC ( n = 54), or after transfection with TRPV4 siRNA ( n = 27). Values represent relative change in fluorescence ratio 340/380. Maximal increase vs. basal ** P ≤ 0.01, inhibitor vs. control, * P ≤ 0.05, vs. control ** P ≤ 0.01, * P
Article Snippet: After blocking, immunoblots were incubated overnight with anti-mouse TRPV4 (1:200, Alomone Laboratories) This was followed by incubation with peroxidase-conjugated goat antirabbit antibody (dilution 1:10,000, Jackson Immuno Research Laboratories) and visualized with an ECL+ detection kit (GE Healthcare).
Techniques: Concentration Assay, Fluorescence, Transfection