Cytodifferentiation and expression of alpha-smooth muscle actin mRNA and protein during primary culture of aortic smooth muscle cells. Correlation with cell density and proliferative state.
To investigate a possible correlation between cytodifferentiation, proliferation, and actin expression, smooth muscle cells from the 9-week-old rabbit aortic media were enzyme-dispersed into single cells and were plated in primary culture at different initial seeding densities. The volume fraction of myofilaments (Vv myo) in cells seeded moderately densely fell from 39.5% +/- 1.2% in the intact aortic media to 11.5% +/- 1.6% on Day 5, one day before the onset of logarithmic growth. The Vv myo remained low over the next 3 days, then began to rise as the density of cells increased, returning almost to the original levels after confluency and 1.84 cumulative population doublings (CPD). The expression of alpha-smooth muscle actin mRNA followed a similar time course of change, falling from 84.7% +/- 1.2% of total actin mRNA in freshly isolated cells to 54.0% +/- 6.5% on Day 5, returning to 87.5% +/- 0.5% after confluency. In these cultures, the alpha-smooth muscle actin protein content was 93.7% +/- 2.9% of total actin in freshly isolated cells, 68.7% +/- 3.1% on Day 5, and 73.3% +/- 2.5% 3 days after confluency. In densely seeded cultures, the Vv myo and expression of alpha-smooth muscle actin mRNA fell only slightly on Day 5 and rose to original levels upon confluency after 0.33 CPD. However, at the protein level, alpha-smooth muscle actin decreased on Day 5 and remained low on Day 12. The Vv myo, alpha-smooth muscle actin mRNA, and actin protein of sparsely seeded cells fell on Day 5 and then remained low throughout the culture period, including 5 days after confluency (Day 24), when the cells had undergone 5.37 CPD. Cells that were maintained subconfluent but quiescent on Day 7 in culture had the same low Vv myo, low alpha-actin mRNA expression, and low alpha-actin protein content as actively proliferating cells. The results show that Vv myo and alpha-smooth muscle actin mRNA undergo parallel changes during primary culture according to seeding density, but not to replication, and that alpha-smooth muscle actin protein decreases in culture then remains low irrespective of culture conditions.
Campbell
J H
JH
Cell Biology Laboratory, Baker Medical Research Institute, Prahran, Melbourne, Australia.
Kocher
O
O
Skalli
O
O
Gabbiani
G
G
Campbell
G R
GR
D000199:Actins / Q000235:genetics*; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D002452:Cell Count; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D003584:Cytological Techniques; D009124:Muscle Proteins / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*
D000199:Actins; D009124:Muscle Proteins; D012333:RNA, Messenger