We previously demonstrated that thapsigargin (TG), an inhibitor of Ca²⁺ ATPase pumps in the sarcoplasmic reticulum (SR) is a potent inhibitor of vascular smooth muscle cell (VSMC) replication at low nM concentrations, an effect that persisted after TG removal (Shukla et al., 1997; George et al., 1997; Birkett et al., 1999). The precise role of SR-Ca²⁺ in mediating VSMC proliferation, however, has not been elucidated. Therefore, the effect of pre-treatment with thapsigargin (TG) on extracellular signal regulated kinase (ERK1/2) activation, its translocation to the nucleus, cyclin D1 expression, the onset of S phase and cytosolic Ca²⁺ levels were studied in human VSMCs (hVSMC)

hVSMCs, derived from saphenous veins, were incubated with 10 nM TG at 37 °C for 24 hr. Cells were then thoroughly washed and incubated with 10% fetal calf serum (FCS; elicits cell replication). At varying times after stimulation with FCS, the activation of ERK1/2 and cyclin D1 were assessed by western blotting, translocation of ERK1/2 to the nucleus using indirect immunofluorescence, the onset of S-phase with the incorporation of bromodeoxyuridine (BrdU) and cytosolic Ca²⁺ levels using FURA-2 (Shukla et al.,1997; George et al.,1997; Birkett et al., 1999).

FCS elicited an immediate increase in the activation (phosphorylation) of ERK1/2 (table 1). TG had a marginal inhibitory effect on ERK1/2 activation at only 5 and 10 min after FCS stimulation (table 1). By contrast, the rapid translocation of ERK1/2 to the nucleus was completely blocked by pre-treatment with 10 nM TG (table 1). S phase, as determined with BrDu incorporation was delayed by 8 hours by TG which coincided with the recovery of Ca²⁺release and cyclin D1 expression (8 hr). Data were analysed using one-way ANOVA.

Table 1. Effect of pre-incubation of hVSMCs with 10 nM TG for 24 hours compared to controls (con) on: A) phosphorylation of ERK1/2 (% maximal) and B) the translocation of pERK1/2 to the nucleus (% positive nuclear immunofluorescence) after stimulation with 10 % fetal calf serum. Data = mean ± SEM, n = 6. * p < 0.05 compared to controls.

The inhibitory effect of TG (depletion of Ca²⁺pools) on hVSMC replication is mediated principally through the inhibition of translocation of phosphorylated ERK1/2 to the nucleus, which in turn prevents cyclin D1 expression and thus progression of the cell cycle. These observations have implications for treating diseases that involve VSMC proliferation which include atherogenesis, vein graft failure and restenosis.

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George, S., et al., (1997) ATVB, 17 , 2500-2506.
Shukla , N., et al., (1997) Br J Surg, 84 , 325-330.