Fig 3. TRPV4-mediated increase in cytosolic Ca²⁺ ([Ca²⁺]_i) in mouse primary colonic epithelial cells and CCD 841 cells.

(A) [Ca²⁺]_i changes in response to the TRPV4 specific agonist, GSK101 (GSK, 30 nM), in WT or TRPV4-KO (V4KO) primary colonic epithelial cells (mean ± SEM). Ionomycin (iono) was used as a positive control. Bars indicate the period of chemical application. Significant increases in [Ca²⁺]_i at 90 seconds were observed in WT than V4KO (p<0.05). (B) GSK101 (300 nM) significantly increased [Ca²⁺]_i in CCD 841 cells (mean ± SEM, n = 17) compared to basal conditions (# p < 0.05 vs control). Clodronate-liposomes did not affect the [Ca²⁺]_i responses. (C) Dose-response [Ca²⁺]_i increase curve in CCD 841 cells responding to GSK101 determined EC50 value of 231.3 nM. (D) Dose-response [Ca²⁺]_i inhibition curve with a TRPV4 specific inhibitor, RN1734 in CCD 841 cells responding to GSK101 (3 μM) determined IC50 value of 702.6 pM. (E) Dose-response [Ca²⁺]_i increase curve in CCD 841 cells responding to an endogenous TRPV4 agonist, 8,9-EET determined EC50 value of 111.1 nM. (F) 8,9-EET (1.5 μM) significantly increased [Ca²⁺]_i in CCD 841 cells (mean ± SEM, n = 11) compared to basal conditions (# p < 0.05 vs control). RN1734 (10 μM) significantly inhibited the [Ca²⁺]_i responses, but clodronate-liposomes did not.