Figure 5

AlF _x ^-(not NaF) generates the LPA ₁ receptor-mediated response; AlF _x ^-also blocks the LPA phosphatase activity. (a) Functional autoradiography using horizontal brain sections was performed using a three-step protocol as detailed in Methods. AlF_x^- (NaF 10 mM + AlCl₃ 50 μM) was included during the 40 min pre-incubation step (step 1), NaF (10 mM) was present in step 2 whereas deferoxamine mesylate (DFOM, 50 μM) was present throughout steps 1–3 and in addition during a 10 min pre-incubation prior to AlF_x^- treatment. The buffer in step 3 additionally contained 0.1% BSA. Both NaF and AlF_x^- induce [³⁵ S]GTPγS binding in the LPA₁ receptor enriched white matter areas. The responses to AlF_x^- and NaF are totally abolished with the aluminium and iron(III) chelator DFOM (cc, corpus callosum; fi, fimbria of the hippocampus). Scale bar = 5 mm. (b) Slides with two horizontal brain sections underwent the three-step autoradiography mimicking protocol as detailed in Methods. AlF_x^- (NaF 10 mM + AlCl₃ 50 μM) and NaF (10 mM) were present during the 90 min incubation step (step 3). DFOM (50 μM) was present in steps 2 and 3. The buffer in step 3 additionally contained 0.1% BSA. After the final incubation step, the assay buffer was quantitatively collected and the P_i content was determined as described in Methods. Note that NaF partially and AlF_x^- totally inhibit LPA-derived P_i formation and that DFOM reverses these actions. The data are expressed as nmol P_i generated per slide (mean + SEM) and are derived from three independent experiments performed in triplicate (n = 3). Significance level: ***p < 0.001 and **p < 0.01 compared to the treatment with LPA alone.