A rapid, PPAR- -dependent effect of pioglitazone on the phosphorylation of MYPT

Atkins KB, Irey B, Xiang N, Brosius FC 3rd. A rapid, PPAR-dependent effect of pioglitazone on the phosphorylation of MYPT. Am J Physiol Cell Physiol 296: C1151–C1161, 2009. First published March 4, 2009; doi:10.1152/ajpcell.00343.2008.—Peroxisome proliferator-activated receptor (PPAR)ligands, thiazolidinediones, have been demonstrated to regulate vascular reactivity. We examined the effect of pioglitazone (PIO; 20 M) in rat primary cultured aortic smooth muscle cells on constitutive phosphorylation of the regulatory subunit of myosin phosphatase (MYPT). PIO decreased the phosphorylation of Thr on MYPT within 15 min, and the inhibition was maintained up to 6 h. The PPARantagonist GW-9662 (5 M) abrogated the inhibition of Thr phosphorylation mediated by PIO. Because longer-term PIO treatment inhibits RhoA/Rho kinase (ROCK) signaling and Thr phosphorylation, we tested the effect of the ROCK inhibitor Y-27632 (10 M) on the inhibition of Thr phosphorylation by PIO. Y-27632 alone inhibited Thr phosphorylation, and there was an additive effect with PIO. In addition, up to 1 h of PIO treatment did not affect RhoA localization or decrease ROCKdependent phosphorylation of Thr. These results suggest that the effect of PIO is independent of inhibition of RhoA/ROCK. PIO increased the phosphorylation of Ser in the same time course as its effect on Thr. Ser has been shown to be phosphorylated by PKA and PKG. PKA inhibitor H-89 (10 M) and PKG inhibitor KT-5823 (0.5 M) abrogated the effect of PIO on both Thr and Ser phosphorylation. The constitutive turnover of phosphorylation of Thr is rapid, suggesting that the decreased phosphorylation of Thr by PIO is due to enhanced phosphorylation of Ser. This is supported by the finding that PIO blocks ANG II-stimulated phosphorylation of Thr but not ANG II-stimulated RhoA translocation. Therefore, the effect of shorter-term PIO apparently is to increase myosin light chain phosphatase activity, thereby desensitizing the vascular smooth muscle to agonist signaling.