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The P2Y2 receptor mediates uptake of matrix‐retained and aggregated low‐density lipoprotein in primary smooth muscle cells
Author(s) -
Dissmore Tixieanna,
Mamedova Laman,
Medeiros Denis,
Weisman Gary A
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.373.6
Subject(s) - transfection , endocytosis , internalization , microbiology and biotechnology , chemistry , colocalization , scavenger receptor , p2y receptor , lipoprotein , receptor , biochemistry , biology , cholesterol , purinergic receptor , gene
The internalization of matrix retained and aggregated low‐density lipoprotein (agLDL) may involve the actin cytoskeleton in a manner that distinguishes this process from the endocytosis of soluble LDL. We have previously shown that UTP‐induced phosphorylation of filamin A (FLN‐A) is associated with actin polymerization in 1321N1 cells expressing the hemagglutinin (HA)‐tagged P2Y 2 receptor (P2Y 2 R). Aortic smooth muscle cells (SMCs) were isolated from wild type (WT) and P2Y 2 R−/− mice to investigate the role of P2Y 2 R/FLN‐A interaction in the uptake of matrix‐bound agLDL. Cells were transiently transfected with cDNA encoding HA‐tagged WT P2Y 2 R or a mutant P2Y 2 R that does not bind FLN‐A, then treated with 10 μM UTP and plated on top of the matrix. We produced the SMC‐derived matrix by extracting the lipids with 3:2 hexane:propanol (v/v). DiI‐LDL was aggregated by incubation with 50‐milliunits/ml bacterial sphingomyelinase (SMase) in the presence of 5mM MgCl 2 for 4 h at 37°C under argon. In preparation for LDL‐matrix binding 1 μg of lipoprotein lipase was added to the lipid‐extracted matrix for 1 h followed by 100 μl Smase‐treated DiI‐LDL. Confocal microscopy results showed no uptake of agLDL in P2Y 2 R−/− cells transfected with mutant P2Y 2 R. However, there was agLDL uptake in P2Y 2 R −/− cells transfected with the WT P2Y 2 R, indicating that an intact P2Y 2 R is necessary for agLDL uptake in mouse vascular SMCs. Grant Funding Source : NHLBI Ruth L Kirschstein pre‐doctoral NRSA