-(not NaF) generates the LPA
receptor-mediated response; AlF
-also blocks the LPA phosphatase activity. (a) Functional autoradiography using horizontal brain sections was performed using a three-step protocol as detailed in Methods. AlFx- (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 AlFx- treatment. The buffer in step 3 additionally contained 0.1% BSA. Both NaF and AlFx- induce [35 S]GTPγS binding in the LPA1 receptor enriched white matter areas. The responses to AlFx- 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. AlFx- (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 Pi content was determined as described in Methods. Note that NaF partially and AlFx- totally inhibit LPA-derived Pi formation and that DFOM reverses these actions. The data are expressed as nmol Pi 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.