Antiproliferative effect of brief exposure to cholera toxin in vascular smooth muscle cells: role of cAMP and protein kinase A

The effect of cholera toxin (CTX), an activator of the adenylate cyclase-co upled G protein alpha(S) subunit, was studied on cultured vascular smooth m uscle cell (VSMC) proliferation. Continuous exposure (48 h) to CTX as well as 2-min pretreatment of VSMC with CTX led to the same level of cAMP produc tion, inhibition of DNA synthesis, and arrest in the G(1) phase without ind uction of necrosis or apoptosis in VSMC. Protein kinase A (PKA) activity in CTX-pretreated cells was transiently elevated by 3-fold after 3 h of incub ation, whereas after 48 h it was reduced by 2-fold compared with baseline v alues without modulation of the expression of its catalytic alpha subunit. The PKA inhibitors H89 and KT 5720 did not protect VSMC from the antiprolif erative effect of CTX. Two-dimensional electrophoresis was used to analyze the influence of CTX on protein phosphorylation. After 3 h of incubation of CTX-pretreated cells, we observed both newly-phosphorylated and dephosphor ylated proteins (77 and 50 protein species, respectively). After 24 h of in cubation, the number of phosphorylated proteins in CTX-treated cells was de creased to 39, whereas the number of dephosphorylated proteins was increase d to 106. In conclusion, brief exposure to CTX leads to full-scale activati on of cAMP signaling and evokes VSMC arrest in the G(1) phase.