Figure 6.

Transient receptor potential melastatin subtype 8 (TRPM8) activation by menthol blunts reactive oxygen species (ROS)–induced Ras homolog gene family, member A (RhoA) activation in cold‐induced hypertension. A, Cytosolic ROS measured by dihydroethidium (DHE) and (B) mitochondrial ROS measured by MitoSOX Red in the aortas from wild‐type (WT) and Trpm8^−/− mice fed a normal diet (ND) or menthol treated by normal temperature (Con) or cold stimulation (CS). Bar, 200 μm. n=5. C through F, Western blot analysis of RhoA activation, myosin phosphatase targeting subunit 1 (MYPT1), Ser⁶⁹⁵‐phosphorylated MYPT1 (p‐MYPT1), myosin light chain (MLC), and Ser¹⁹‐phosphorylated MLC (p‐MLC) in aortas form WT and Trpm8^−/− mice treated by Con‐ND, Con‐menthol, CS‐ND, or CS‐menthol. n=3. *P<0.05, **P<0.01 vs normal temperature‐ND; ^# P<0.05, ^## P<0.01 vs CS‐ND; ^&& P<0.01 vs normal temperature‐menthol. G through J, Western blot analysis of RhoA activation, MYPT1, p‐MYPT1, MLC, and p‐MLC in vascular smooth muscle cells (VSMCs). The activation of TRPM8 by menthol pretreatment (100 μmol/L) inhibited the expression ratios of RhoA‐GTP/RhoA, p‐MYPT1/MYPT1, and p‐MLC/MLC in VSMCs treated by Angiotensin II (Ang II; 200 nmol/L). This effect of menthol was similar with mitoTEMPO (Mito T, 20 μmol/L). n=3. *P<0.05, **P<0.01 vs normal temperature; ^# P<0.05, ^## P<0.01 vs Ang II. Data are expressed as mean±SEM. Men indicates ND plus menthol supplementation (0.5%); Con, vehicle (saline or ethanol as appropriate).