The study of voltage-dependent calcium entry in A7r5 vascular smooth muscle cell proliferation
Description
A7r5 cells were employed to delineate the relationship between cell proliferation and voltage-gated Ca$\sp{2+}$ entry in vascular smooth muscle cells of the noncontractile, synthetic phenotype. The main hypothesis tested was that Ca$\sp{2+}$ entry through the voltage-gated Ca$\sp{2+}$ channels (L-type) is differentially regulated during various growth ages of the A7r5 cells as they switch from a rapidly proliferative state to a confluent state in culture Under resting conditions, binding of the Ca$\sp{2+}$ channel blocker isradipine as a function of membrane potential (E$\sb{m})$ indicated that proliferating 3 day cells contained active L-type Ca$\sp{2+}$ channels compared to 5 and 10 day confluent cultures. Both tetraphenylphosphonium distribution and patch-clamp studies showed that 3 day cells are more depolarized and closer to the threshold for L-type Ca$\sp{2+}$ channel activation than cells at either 5 or 10 days. Tetraphenylphosphonium assays demonstrated that the depolarized E$\sb{m}$ of proliferating cells was not entirely due to either Na$\sp{+}$ or Ca$\sp{2+}$ entry. Determination of intracellular $\rm\lbrack$Ca$\sp{2+}\rbrack$ with a Ca$\sp{2+}$-sensitive dye showed that proliferating cells had a significantly higher basal cytosolic Ca$\sp{2+}$ than either of the confluent cultures. The $\sp{45}$Ca$\sp{2+}$ influx rates were also measured under resting conditions, and both 3 and 5 day cells displayed a similar rate which was lower than the mature 10 day cultures. Under depolarizing conditions uptake rates were similar. Measurement of maximal $\rm Na\sp{+},K\sp{+}$-ATPase activity revealed that the pump's activity increased as the cells matured in culture Thymidine incorporation assays showed that as cells reached confluence, DNA synthesis decreased significantly. The addition of Ca$\sp{2+}$ channel blockers to the growth medium of all 3 cultures decreased DNA synthesis in a dose-dependent manner, with nifedipine exhibiting an increase in IC$\sb{50}$ as the cells matured Application of cyclical stretch to 10 day cultures increased Ca$\sp{2+}$ uptake via a dihydropyridine-sensitive, Na$\sp{+}$-dependent pathway. The converse was observed in 7 day cultures, where stretch caused a significant reduction in Ca$\sp{2+}$ uptake These studies suggest that Ca$\sp{2+}$ entry through L-type Ca$\sp{2+}$ channels plays an important role in early events associated with proliferation of A7r5 cells. The regulation of these channels appears to be modified through gradual hyperpolarization of the membrane as the cells reach confluence