Fig. 2.

Specific agonists of transient receptor potential vanilloid member 4 (TRPV4) activate currents and increase intracellular calcium concentration ([Ca²⁺]_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 [Ca²⁺]_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 <0.05 vs. mock transfected.