Fig. 5.

Hypotonicity-induced Ca²⁺ response is reconstituted in AQP4 (M1 or M23)-transfected but not in AQP1-transfected DI TNC1 cells. (A) Traces representative of typical variations in [Ca²⁺]_i observed in fura-2–loaded DI TNC1 nontransfected (NT) cells exposed to hypotonic solution (black bar; ΔOsm = 60 mOsm) in the presence or absence of 5 mM [Ca²⁺]_o (hatched bar) and stimulated with 5 μM 4αPDD (gray line). (B–D) Representative traces depicting [Ca²⁺]_i DI TNC1 cells transfected with EGFP (B), EGFP + M1 (C), or EGFP + M23 (D). The experimental design is as in A. (E) Typical Ca²⁺ dynamics of three Fluo-4-AM–loaded DI TNC1 cells transfected with AQP1 showing no [Ca²⁺]_i signal upon hypotonicity. Thrombine (10 μM; THB; gray line) was used as positive control. (Inset) Quantitative analyses of maximal variation in fluorescence ratio (ΔF/F) recorded in AQP1 (gray bar; n = 16) and AQP4-M23 (n = 6) transfected cells upon cell exposure to hypotonic solution. (F) Quantitative analysis of data represented in A–D. Mean of variation in fluorescence ratio (ΔF340/F380) recorded upon cell exposure to hypotonic solution in presence (Left) or in absence (Center) of [Ca²⁺]_o (NT, nontransfected, light gray bar, n = 36; EGFP, hatched bar, n = 32; AQP4-M1, black bar, n = 19; AQP4-M23, white bar, n = 13) (ANOVA and post hoc, independent t-test P < 0.001 for AQP4-M1/NT-EGFP and AQP4-M23/NT-EGFP).The increase is abolished in absence of [Ca²⁺]_o. The histogram also includes data obtained by adding gadolinium chloride (Gd³⁺) to the hypotonic saline to block TRPV cation channels (Right). The application of Gd³⁺ abolished the [Ca²⁺]_i signal. (paired t-test, P < 0.001 for analyses in presence and absence of [Ca²⁺ ]_o; independent t-test for experiment with Gd³⁺, P = 0.004 in AQP4-M1 cells, n = 10; P < 0.001 in AQP4-M23 cells, n = 16; P < 0.05 in EGFP cells, n = 6). P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.