The embryonic rat aortic smooth muscle A7r5 cells (ATCC, Manassas, Virginia, USA) were grown, as recommended, in DMEM-high glucose (HG: 4.5 g/l equivalent to 25 mM), supplemented with 1% penicillin/streptomycin, 1% sodium pyruvate, and 10% FBS.
The proliferation of A7r5 cells was determined using trypan blue dye exclusion counting using TC10 automated cell counter (Bio-Rad, CA, USA).
To test whether the concentration of glucose affects the metabolic activity of the cells, A7r5 cells from HG, NG, and OC streams were plated, at equal density, in 96-well plates and shifted to HG or NG according to the followings: (NG:NG, NG: shifted to HG); (HG:HG, HG: shifted to NG), and (OC:OC, OC: shifted to HG).
A7r5 cells were cultured on 35 mm poly-d-lysine coated glass coverslips (MatTek corp, MA) and incubated in their respective media at 37[degrees]C.
Assessment of cell contraction was performed on A7r5 cells cultured on glass coverslips.
RNA was extracted from A7r5 cells cultured in HG, NG, and OC using Qiagen RNAeasy extraction kit (Hilden, Germany) and reversed transcribed using High-Capacity cDNA reverse transcription kit (Applied Biosystems (AB)), all following the manufacturer's instructions.
A7r5 cells were cultured in 35 mm culture dish for confocal microscopy (ibidi, Germany).
Because oxidative stress is associated with cell death, the effect of Q7 on the viability of HUVEC and the vascular smooth muscle cell line A7r5 was assessed (Figure 2).
Effect of Q7 on the Calcium Homeostasis in A7r5 Cells.
In fact, Q7 blunted the release of calcium from intracellular stores in response to PE on vascular smooth muscle cell (A7r5 cells) in a free-calcium medium.
Human umbilical vein endothelial cells (HUVEC) (a) and A7r5 vascular smooth cell line (b) were incubated in the absence or in the presence of Q7 ([10.sup.-7] to [10.sup.-4] M) for 48 h.
Figure 6: Quinone effect on intracellular Ca levels in A7r5 cells.