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Fig. 4. Stimulation of phosphoinositide hydrolysis by LPA₁ receptors is inhibited by cholesterol extraction with methyl-ß-cyclodextrin. (A) Either native HeLa cells or stably transfected LPA₁/HeLa cells were labeled overnight with [³H] myo-inositol and then either left untreated (HeLa and lane 1) or pre-incubated with 5 mM MßCD for 1 hour (lane 2), 50 µg/ml nystatin for 1 hour (lane 4), 5 mM MßCD for 1 hour followed by 10 mM cholesterol/MßCD complexes for 60 minutes (lane 3), or 50 µg/ml nystatin for 1 hour followed by 10 mM cholesterol/MßCD complexes for 60 minutes (lane 5) prior to an additional 1 hour treatment with 10 µM LPA. Cells were then solubilized and the total accumulation of labeled inositol phosphates was determined. The radioactivity recovered in the different samples was normalized to total cellular protein and the data are presented as the mean±s.e.m. of triplicate measurements from a representative experiment that was repeated four times. ^**a, P<0.01, comparison of LPA-stimulated phosphoinositide hydrolysis in MßCD-treated LPA₁/HeLa cells to that observed in non-MßCD-treated LPA₁/HeLa cells. ^**b, P<0.01, comparing phosphoinositide hydrolysis in MßCD-treated or nystatin-treated LPA₁/HeLa cells that were incubated with water-soluble cholesterol to that observed in unstimulated LPA₁/HeLa cells. (B) HeLa cells were transiently transfected with plasmids encoding either vector alone (lanes 1 and 2), LPA₁ (lanes 3 and 4), or M₁ mAChRs (lanes 5 and 6). The cells were incubated in the absence (–) or presence (+) of 5 mM MßCD for 1 hour prior to a subsequent 1 hour incubation with agonist (10 µM LPA or 1 mM carbachol). After solubilization, the radioactively-labeled inositol phosphates were isolated as described. The radioactivity recovered in the different samples was normalized to total cellular protein and the data are presented as the mean±s.e.m. of triplicate measurements from a representative experiment that was repeated three times. ^**P<0.01, comparison of LPA-stimulated phosphoinositide hydrolysis in MßCD-treated LPA₁-transfected HeLa cells to that observed in non-MßCD-treated cells.

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