Figure 3.

Transient receptor potential melastatin subtype 8 (TRPM8) activation attenuates Angiotensin II (Ang II)–induced mitochondrial dysfunction and mitochondrial reactive oxygen species (ROS) generation. A, Western blot analysis of Ser²⁹³‐phosphorylated pyruvate dehydrogenase‐E1α (p‐PDH) and total pyruvate dehydrogenase (PDH) in primary cultured vascular smooth muscle cells (VSMCs) from wild‐type (WT) or Trpm8^−/− mice pretreated with menthol (100 μmol/L) or vehicle (ethanol) for 2 hours, followed by Ang II (200 nmol/L) or vehicle (saline) treatment for another 4 hours. n=3. The effect of menthol and/or Ang II on PDH activity of VSMCs from WT (B) or Trpm8^−/− (C) mice was also determined. n=8. D through G, Total cytosolic (dihydroethidium [DHE]) and mitochondrial ROS (MitoSOX Red) were measured in primary cultured vascular smooth muscle cells (VSMCs) from WT and Trpm8^−/− mice. VSMCs were pretreated with menthol (M, 100 μmol/L) or vehicle (ethanol) for 2 hours, followed by Ang II (200 nmol/L) or vehicle (saline) treatment for another 4 hours. n=9 per group for DHE measurement, and n=10 per group for MitoSOX Red measurement. H, The representative image shows the measurement of mitochondrial respiratory function and ROS generation by Oxygraph‐2K. VSMCs mitochondrial respiratory function and ROS production were measured by a specially designed substrate‐uncoupler‐inhibitor titrations protocol. The red line indicates O₂ consumption and the blue line shows H₂O₂ generation in response to the application of substrates for complex I (CI) and complex II (CII). The values are expressed in pmol/s per 10⁶ cells. Summarized data for oxygen consumption capacity (I) and H₂O₂ generation (J) in the mitochondria of WT VSMCs. K and L, The same experiments as in (I and J) were performed with Trpm8^−/− VSMCs. n=5 for the vehicle (saline or ethanol as appropriate) group and the menthol group. n=8 for the Ang II group, and n=6 for the menthol plus Ang II treatment group in WT VSMCs. n=6 for the vehicle group and menthol group, and n=5 for the Ang II group and menthol plus Ang II treatment group in Trpm8^−/− VSMCs. *P<0.05, **P<0.01 vs vehicle; ^# P<0.05, ^## P<0.01 vs Ang II. Data are expressed as mean±SEM. A indicates angiotensin II; A+M, menthol plus angiotensin II treatment; Amp, Amplex UltraRed; Close, inserting the stoppers to seal the chambers; Con, vehicle (saline or ethanol as appropriate); Dig, digitonin; FCCP, carbonyl cyanide 4‐trifluoromethoxy phenylhydrazone; G/M, glutamine and malate; HRP, horseradish peroxidase; M, menthol; Men, menthol; Omy, oligomycin; open, pulling out the stoppers for reoxygenation; OXP, oxidative phosphorylation; Unc, uncoupler; succinate.