Inhibition of intracellular Ca2+ mobilisation by low antiproliferative concentrations of thapsigargin in human vascular smooth-muscle cells

Low nanomolar concentrations of thapsigargin, a modulator of intracellular Ca2+ ([Ca2+](i)) pools, inhibit vascular smooth-muscle cell (VSMC) prolifer ation. Because the mechanisms underlying this effect have not been defined, the effect of antiproliferative concentrations of thapsigargin on [Ca2+](i ) in fura-2-loaded VSMCs was studied by using dynamic video imaging of [Ca2 +](i). After seeding on coverslips, human VSMCs were incubated for 1-48 h w ith thapsigargin before loading with fura-2 or during imaging. Mobilisation of [Ca-2+](i) was stimulated with 1 mu M ionomycin in Ca2+-free medium and the increase in [Ca2+](i) detected by using Ca2+ imaging techniques. Conti nuous exposure of cells to low concentrations of thapsigargin (which failed measurably to increase in [Ca2+](i)) reduced the ionomycin response in a t ime- and dose-dependent manner (100% inhibition at 10 nM thapsigargin after 1 h exposure). After exposure of cells to 10 nM thapsigargin for 1 h follo wed by washing and further incubation for less than or equal to 72 h, there was a time-dependent recovery of the ionomycin response. Because the conce ntrations of thapsigargin and exposure times are identical to those that in hibit replication in VSMCs, it is proposed that depletion of [Ca2+](i) pool s mediates the inhibitory effect of thapsigargin on VSMC proliferation.