Figure 4.

c48/80- and LPA-stimulated G protein activity in brain cryostat sections is mediated by LPA receptors, likely LPA₁. (a) [³⁵S]GTPγS autoradiography was conducted using a three-step protocol with DPCPX (1 μM) present throughout steps 2 and 3, as detailed in the Methods section. c48/80 (100 μg ml⁻¹; MP Biomedicals) or LPA (5 μM in 0.1% fatty acid-free BSA) was included in the [³⁵S]GTPγS labeling step of developing (4-week-old) rat brain sections along with or without the LPA₁/LPA₃ receptor-selective antagonist Ki16425 (5 μM). Note that Ki16425 clearly abolishes [³⁵S]GTPγS binding responses to c48/80 and LPA throughout the white matter tracts. Like 1-butanol, Ki16425 also suppresses basal G protein activity in the white matter regions, indicating tonic LPA receptor activity in the developing rat brain. Abbreviations: cca, corpus callosum. Scale bar=5 mm. (b) Quantitative autoradiography data on the corpus callosum, selected to represent the white matter regions. Autoradiography images were digitized and bound radioactivity values were obtained from the digitized images for statistical analysis. Ki16425 dose-dependently decreases the basal [³⁵S]GTPγS binding as well as that evoked by c48/80 (100 μg ml⁻¹; MP Biomedicals) or LPA (5 μM) with IC₅₀ values of 35±9, 59±59 and 87±19 nM, respectively. Values are mean±s.e.m. (or IC₅₀±s.e.m.) representing six sections from six developing (4-week-old) animals.