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Alterations in 3‐hydroxy‐3‐methylglutaryl‐CoA reductase mRNA concentration in cultured chick aortic smooth muscle cells
Author(s) -
Carazo Angel,
Alejandre M José,
Suarez M Dolores,
Linares Ana
Publication year - 2000
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/s11745-000-0560-8
Subject(s) - reductase , hydroxymethylglutaryl coa reductase , biology , messenger rna , hmg coa reductase , microbiology and biotechnology , enzyme , complementary dna , biochemistry , gene
We observed and compared alterations in 3‐hydroxy‐3‐methylglutaryl (HMG)‐CoA reductase at the transcriptional level in unsynchronized, three‐passage cultures of smooth‐muscle cells from the aorta of chicks fed on a control diet (C‐SMC) and those of chicks fed on a similar diet plus cholesterol (Ch‐SMC). Alterations in reductase mRNA concentrations in senescent cultures were much lower. We used a modification of the competitive (c) reverse transcription polymerase chain reaction method, using a Thermus thermophilus DNA polymerase (Tth pol) to quantify the very scarce species of HMG‐CoA reductase mRNA in samples of cytoplasmic SMC mRNA. We cloned and sequenced a 199 bp cDNA fragment of chicken HMG‐CoA reductase, which encoded a region of 66 amino acids belonging to the catalytic domain of the enzyme. HMG‐CoA reductase mRNA concentrations from young C‐SMC cultures rose 3.89‐fold 4 h after the change of medium and returned to base levels between 8 to 12 h afterward. Concentrations in Ch‐SMC cultures increased less (2.36‐fold) 8 h after the change to fresh medium. Increases in reductase mRNA in senescent cultures of Ch‐SMC and C‐SMC measured under similar conditions were only 1.28‐ and 1.39‐fold, respectively.