Alterations in 3-hydroxy-3-methylglutaryl-CoA reductase mRNA concentrationin cultured chick aortic smooth muscle cells

We observed and compared alterations in 3-hydroxy-3-methylglutaryl (HMG)-Co A reductase at the transcriptional level in unsynchronized, three-passage c ultures of smooth-muscle cells from the aorta of chicks fed on a control di et (C-SMC) and those of chicks fed on a similar diet plus cholesterol (Ch-S MC). Alterations in reductase mRNA concentrations in senescent cultures wer e much lower. We used a modification of the competitive (c) reverse transcr iption polymerase chain reaction method, using a Thermus thermophilus DNA p olymerase (Tth pol) to quantify the very scarce species of HMG-CoA reductas e 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 a mino acids belonging to the catalytic domain of the enzyme. HMG-CoA reducta se mRNA concentrations from young C-SMC cultures rose 3.89-fold 4 h after t he 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 th e 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 139-fold, respectively.