WNT-5A-induced G protein activation mediates decrease in cAMP and mobilization of [Ca
. (A) Isolated plasma membranes from mouse primary microglia were stimulated with WNT-5A (300 ng/ml) as described in the Methods section to assess activation of heterotrimeric G proteins. Incorporation of [γ-35 S]GTP, a measure for activation of G proteins, was quantified by scintillation counting. Data from three independent experiments are shown in the bar graph. ***, P < 0.001. Error bars give s.e.m.. (B) cDNA from mouse primary microglia was analyzed for expression of PTX-sensitive variants of the Gi/o family of Gα subunits by RT-PCR. cDNA synthesis was performed in the absence and presence of RT (+/− RT) to control for the purity of the preparation. (C) Detection of intracellular cAMP levels reveals that WNT-5A reduces forskolin-induces cAMP production in a dose-dependent manner (error bars: s.e.m.; n = 3; **, P < 0.01; ***, P < 0.001.). (D-F) Stimulation of Fluo-3-loaded primary microglia with WNT-5A induced fast and transient elevation of [Ca2+]i. ATP was used as a positive control. The [Ca2+]i trace shown in D originates from a single cell. Typically 15% to 30% of the cultured microglia responded to WNT-5A. (E) shows a representative view of Fluo-3-loaded cells at baseline and upon WNT-5A (300 ng/ml) exposure. The images are pseudocolored with warm colors presenting high [Ca2+]i and cold colors low [Ca2+]i. Size bar 20 μm. The bar graph summarizes data from five different experiments with ATP and WNT-5A in combination with PTX. Error bar gives s.e.m. n, number; PTX, pertussis toxin; s.e.m., standard error of the mean.