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Department of Biology, Marquette University, Milwaukee, Wisconsin 5320l-1881
These experiments were performed to test the hypotheses that myosin light chain 17 (MLC17) a and b isoform expression varies between individual vascular smooth muscle (SM) cells and that their expression correlates with cell unloaded shortening velocity. Single SM cells isolated from rabbit aorta and carotid arteries were used to measure unloaded shortening velocity and subsequently were analyzed via RT-PCR for MLC17 a and b mRNA ratio. The MLC17b/a mRNA and protein ratios from adjacent tissue sections correlate very well (R2 = 0.68), allowing use of the mRNA ratio to predict the protein ratio. The rabbit MLC17 isoform protein sequence was found to be similar to, but unique from, the swine, mouse, and chicken sequences. Isolated single SM cells from the aorta and carotid have resting lengths of 70-280 µm and shorten to 33-88 µm after contraction. Isolated cell maximum unloaded shortening velocity is highly variable (0.5-7.5 µm/s) but becomes more uniform when normalized to initial cell length (0.01-0.05 cell lengths/s). Carotid cells activated in the presence of okadaic acid (1 µm) have mean maximal unloaded shortening velocities not significantly different from carotid cells activated without okadaic acid (0.016 vs. 0.019 cell lengths/s). Resting cell length before activation is significantly correlated with final cell length after unloaded shortening. Neither initial cell length, final cell length, total cell length change, nor maximum unloaded shortening velocity (absolute or normalized) was significantly correlated with single-cell MLC17b/a mRNA ratio. These studies were performed in isolated single SM cells where unloaded shortening velocity and MLC17b/a mRNA ratios were measured in the same cell. In this preparation, the three-dimensional organization and milieu of the cell is kept intact, but without the intercellular heterogeneity concerns of multicellular preparations. These results suggest the MLC17b/a ratio is variable between individual SM cells from the same tissue, but it is not a determinant of unloaded shortening velocity in single SM cells.
arterial muscle; aorta; carotid; reverse transcription-polymerase chain reaction; muscle mechanics
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