The Role Of T-type Calcium Channel Ca_v3.1in Proliferation Of Vascular Smooth Muscle Cells

Objective:Investigated the role of voltage-gated calcium channel Cav3.1in proliferation of vascular smooth muscle cells and its underlying mechanisms.Methods:1. Identified the primary culture cell isolated from tunica media of rat thoracic aorta by Western blot for the expression of a-smooth actin.2. Identified the purity of vascular smooth muscle cells (VSMCs) isolated from rat thoracic aorta by immunofluorescence.3. Constructed α1G-adenovirus vector, infected VSMCs from rat thoracic aorta with α1G-adenovirus. The effect of α1G-adenovirus on the proliferation of VSMCs was assayed by MTS method.4. Incubated VSMCs overexpressed α1G with T-type channel blocker. The effect of T-type channel blocker on the proliferation of VSMCs overexpressed α1G was assayed by MTS method.5. Incubated VSMCs overexpressed α1G with PKC inhibitor and Pyk2inhibitor, seperately. The effects of inhibitors on the proliferation of VSMCs were assayed by MTS method.6. Use Western blot method to detect the effect of overexpressed α1G on phosphorylation of Pyk2in rat thoracic aorta VSMCs.Results:1. Western blot showed that primary culture cells which were isolated from tunica media of rat thoracic aorta expressed high level a-smooth actin, whereas the a-smooth actin expression was not detected in the skeletal muscle and cardiac muscle tissues which were isolated from the same species, suggesting the isolated primary culture is the vascular smooth muscle cell.2. Immunofluorescence showed that more than95%of isolated cells were positive stained for a-smooth actin.3. Compared with GFP control group and5%FBS control group, over expressing Cav3.1increased the proliferation of VSMCs significantly(P<0.05), with the relative proliferation rate145.44%. 4. MTS aasay showed that T-type calcium channel blocker inhibited the proliferation of the Cav3.1overexpressed VSCMs. And the T-type channel blocker can decrease the growth of Cav3.1overexpressed VSMCs to the basal level, whereas the same concentraion of T-type channel blocker showed no obvious effect on the GFP control group and5%FBS control group.5. MTS assay showed that PKC inhibitor had no obvious effect on VSMCs proliferation. Pyk2inhibitor inhibited the proliferation of the Cav3.1overexpressed VSMCs, whereas the same concentraion of Pyk2inhibitor had no obvious effect on the GFP and5%FBS control groups.6. Western blot showed that overexpressed Cav3.1in rat thoracic aorta VSMCs increased the phosphorylation level of Pyk2.Conclusion:Overexpression of Cav3.1in the VSMCs from rat thoracic aorta induced cell proliferation, the mechanism of proliferation may involved in the signal pathways which were regulated by Pyk2.