Figure 4: Wnt5a does not activate cAMP and PKA.

(a) No significant elevation of cAMP concentration in response to Wnt5a. The mpkCCD cells were treated with Wnt5a (500 ng ml⁻¹) or dDAVP (1 nM) for 1 or 4 h. Error bars are mean values±s.d. from three experiments. Asterisk indicates a significant difference compared with control. Tukey, **P<0.01. (b) No significant elevation of PKA kinase activity in response to Wnt5a. The mpkCCD cells were treated with Wnt5a (500 ng ml⁻¹) or dDAVP (1 nM) for 1 or 4 h. Error bars are mean values±s.d. from three experiments. Asterisk indicates a significant difference compared with control. Tukey, **P<0.01. (c–e) The effects of CyA and H89 on AQP2 phosphorylation. Wnt5a (500 ng ml⁻¹) or dDAVP (1 nM) was added in the presence or absence of CyA (10 μM) or H89 (50 μM) for 1 h. The mpkCCD cells were pretreated with each inhibitor for 45 min before Wnt5a or dDAVP stimulation. Non-glycosylated AQP2 bands (arrow) were quantified by densitometric analysis, and the results are presented in the bar graphs as fold change compared with the value in the control cells. Mean values±s.d. were determined from three experiments. Tukey, *P<0.05, **P<0.01. (f) The effects of CyA and H89 on AQP2 trafficking. (Left) Wnt5a (500 ng ml⁻¹) or dDAVP (1 nM) was added in the presence or absence of CyA (10 μM), or H89 (50 μM) for 1 h. The mpkCCD cells were pretreated with each inhibitor for 45 min before Wnt5a or dDAVP stimulation. Immunofluorescence staining of AQP2 was analyzed as in Fig. 2. Scale bars, 10 μm. (Right) Fluorescence intensities of apical AQP2 were quantified, and the results are presented in the bar graphs. Error bars are mean values±s.d. from three experiments. Tukey, *P<0.05, **P<0.01.