Tyrphostin inhibition of ATP-stimulated DNA synthesis, cell proliferation and fos-protein expression in vascular smooth muscle cells.

1. We and others have shown that extracellular ATP (adenosine triphosphate), released from sympathetic nerves and platelets, stimulates growth of vascular smooth muscle cells (SMC). To study the importance of tyrosine kinases for ATP-mediated proliferation in vascular smooth muscle cells we used tyrphostins, a recently developed group of highly specific inhibitors of tyrosine kinases. 2. ATP induced a powerful concentration-dependent increase in DNA synthesis measured by [3H]-thymidine incorporation in rat aorta SMC (RASMC) and an increase in total cell number after 72 h of incubation as measured by an enzymatic cell proliferation assay. Tyrphostin 25 (10(-5) M) had no effect per se on basal DNA synthesis but reduced ATP-stimulated DNA synthesis and increase in cell number in a dose-dependent manner. Higher concentrations of ATP could not reverse the inhibitory effect of tyrphostin 25. The potency of several (six) other tyrphostins was also examined and found to be slightly greater than tyrphostin 25 with equal efficacy. 3. When RASMC were incubated with 10(-5) M ATP for 2 h, nearly all of the cells (87 +/- 5%) were intensely stained with an antibody to the Fos protein while in the controls only 1 +/- 2% of the cells were weakly stained. Tyrphostin 25 greatly reduced the Fos-protein staining (14 +/- 2%). 4. ATP induced a concentration-dependent increase in 45Ca(2+)-influx and formation of inositol phosphates (IPtotal) in RASMC. These effects were not inhibited by tyrphostin 25. 5. Tyrphostin 25 did not alter ATP-induced contraction in ring segments of rat aorta. 6. In conclusion, tyrphostin 25 inhibited ATP-induced DNA synthesis, cell proliferation and Fos-protein expression, but not ATP-induced 45Ca(2+)-influx, inositolphosphate-production or vasoconstriction. This indicates that the mitogenic effect of ATP on vascular smooth muscle cells is dependent on tyrosine kinases in contrast to the contractile effect of ATP in blood vessels.