Effect of elevated intracellular cAMP on MYPT1 phosphorylation. (A) Typical Western blot showing the phosphorylation of MYPT1 at Thr853 and Thr696 and the phosphorylation of MLC in response to ET-1, Fsk, and Y-27632. (B) ET-1 (100 nM; 10 minutes) led to a significant (P < 0.0001) increase in the phosphorylation of MYPT1 at Thr853 (63% ± 4%) compared with the control (100%). This increase was opposed by Y-27632 (5 μM; 150% ± 5%) and Fsk (10 μΜ) + rolipram (50 μΜ; 120% ± 4%). These agents also reduced the basal level of Thr853 phosphorylation. (C) There was no significant change in the phosphorylation of Thr696 on treatment with ET-1, Y-27632, and Fsk. (D) Change in MLC phosphorylation was similar to the phosphorylation at Thr853. Endothelin-1 increased MLC phosphorylation (ppMLC) by 92% ± 9% over the control, which was opposed by both Y-27632 (165% ± 6.7%) and Fsk (190% ± 1.6%). The bar graphs show data from at least three independent experiments, and the results are expressed as mean ± SEM. C, control; E, endothelin-1; Y, Y-27632; Fsk, forskolin.