Alterations in 3-hydroxy-3-methylglutaryl-CoA reductase mRNA concentration in cultured chick aortic smooth muscle...

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.