Journal: Oncology Letters

Article Title: Potential role of melastatin-related transient receptor potential cation channel subfamily M gene expression in the pathogenesis of urinary bladder cancer

doi: 10.3892/ol.2016.5359

Figure Lengend Snippet: Primers used for reverse transcription-quantitative polymerase chain reaction.

Article Snippet: TRPM8 , Hs00375481_m1.

Techniques:

Journal: Oncology Letters

Article Title: Potential role of melastatin-related transient receptor potential cation channel subfamily M gene expression in the pathogenesis of urinary bladder cancer

doi: 10.3892/ol.2016.5359

Figure Lengend Snippet: Fold changes in the mRNA levels of the TRPM genes. A comparison of the mRNA levels of TRPM2, TRPM4, TRPM5, TRPM7 and TRPM8 ion channel genes in bladder cancer patients and controls is shown. Gene expression levels were normalized according to the expression of glyceraldehyde 3-phosphate dehydrogenase. Results are presented as the mean ± standard deviation for all groups. ***P<0.05 vs. bladder control tissues. TRPM, transient receptor potential cation channel subfamily M.

Article Snippet: TRPM8 , Hs00375481_m1.

Techniques: Comparison, Gene Expression, Expressing, Standard Deviation, Control

Journal: Experimental Animals

Article Title: Effects of β-estradiol on cold-sensitive receptor channel TRPM8 in ovariectomized rats

doi: 10.1538/expanim.17-0028

Figure Lengend Snippet: Sequences of primers used for real-time RT-PCR assays

Article Snippet: After rinsing with PBS, the sections were incubated with an anti-TRPM8 antibody (1:500, rabbit polyclonal, Novus Biologicals, Littleton, CO, USA) and anti-PGP9.5 antibody (1:500, mouse polyclonal, Abcam, Tokyo, Japan) overnight at 4°C.

Techniques: Quantitative RT-PCR

Journal: Experimental Animals

Article Title: Effects of β-estradiol on cold-sensitive receptor channel TRPM8 in ovariectomized rats

doi: 10.1538/expanim.17-0028

Figure Lengend Snippet: Relative expression levels of TRPM8 mRNA. NON-OPE: non-operated rats. OVX: ovariectomized rats. OVX + E2: ovariectomized rats treated for 28 days with 17β-estradiol. The OVX + E2 group showed a trend for decreased expression of TRPM8 channel mRNA in lumbar skin in comparison with the OVX group, although the difference between the two groups did not reach statistical significance ( P =0.0873>0.05).

Article Snippet: After rinsing with PBS, the sections were incubated with an anti-TRPM8 antibody (1:500, rabbit polyclonal, Novus Biologicals, Littleton, CO, USA) and anti-PGP9.5 antibody (1:500, mouse polyclonal, Abcam, Tokyo, Japan) overnight at 4°C.

Techniques: Expressing, Comparison

Journal: Experimental Animals

Article Title: Effects of β-estradiol on cold-sensitive receptor channel TRPM8 in ovariectomized rats

doi: 10.1538/expanim.17-0028

Figure Lengend Snippet: TRPM8 protein levels. NON-OPE: non-operated rats. OVX: ovariectomized rats. OVX + E2: ovariectomized rats treated for 28 days with 17β-estradiol. There was no statistical difference between mean TRPM8 protein levels in lumbar skin of the OVX and OVX + E2 groups ( P =0.3095>0.05).

Article Snippet: After rinsing with PBS, the sections were incubated with an anti-TRPM8 antibody (1:500, rabbit polyclonal, Novus Biologicals, Littleton, CO, USA) and anti-PGP9.5 antibody (1:500, mouse polyclonal, Abcam, Tokyo, Japan) overnight at 4°C.

Techniques:

Journal: Experimental Animals

Article Title: Effects of β-estradiol on cold-sensitive receptor channel TRPM8 in ovariectomized rats

doi: 10.1538/expanim.17-0028

Figure Lengend Snippet: Immunohistochemistry. A. NON-OPE: non-operated rats. B. OVX: ovariectomized rats. C. OVX + E2: ovariectomized rats treated for 28 days with 17β-estradiol. TRPM8 channels and PGP9.5-positive nerve fibers in lumbar skin of NON-OPE, OVX, and OVX + E2 rats were visualized by immunohistochemistry. TRPM8 channels were stained red, and PGP9.5-positive nerve fibers were stained green. Cell nuclei are shown in blue. PGP9.5-positive nerve fibers were scattered in a line at the external side of the epidermal tissues (arrows). Scale bar, 10 µ m. Expression of TRPM8 in the OVX rat was greater than in the NON-OPE rat (A, B), whereas expression of TRPM8 in the OVX + E2 rat was lesser than in the OVX rat (B, C).

Article Snippet: After rinsing with PBS, the sections were incubated with an anti-TRPM8 antibody (1:500, rabbit polyclonal, Novus Biologicals, Littleton, CO, USA) and anti-PGP9.5 antibody (1:500, mouse polyclonal, Abcam, Tokyo, Japan) overnight at 4°C.

Techniques: Immunohistochemistry, Staining, Expressing

Journal: Scientific Reports

Article Title: Control of motor coordination by transient receptor potential melastatin 8 through γ-aminobutyric acidergic circuit modulation in the male mouse cerebellum

doi: 10.1038/s41598-025-98837-9

Figure Lengend Snippet: Effects of transient receptor potential melastatin 8 (TRPM8) antagonist (RQ-00203078) administration on motor coordination. ( A ) Latency to fall off the rotarod in mice that were intraperitoneally treated with vehicle (control) or RQ-00203078. Data are presented as the mean ± standard error (SE) for 8–10 mice per group. * P < 0.05 compared with vehicle. ( B ) Latency to fall off the rotarod in mice that were treated with vehicle or RQ-00203078 via intracerebellar administration. Data are presented as the mean ± SE for 8 mice per group. * P < 0.05 compared with vehicle. ( C ) Double labeling of TRPM8 with c-Fos and γ-aminobutyric acid (GABA) in the cerebellum of a TRPM8-EGFP mouse. Scale bars: 20 μm.

Article Snippet: Animals received an intraperitoneal or intracerebellar administration of the TRPM8 selective antagonist RQ-00203078 (MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: Control, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

doi: 10.3390/ijms232416164

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: TRPM8 , ECM3 , ECM biosciences , TM5721 , Mouse , Primary , Human TRPM8 extracellular domain. Monoclonal , 1:200 (ICC, ICH) 1:500 (WB, ICC, ICH).

Techniques: Isolation

Journal: Journal of the American Heart Association

Article Title: Activation of Transient Receptor Potential Melastatin Subtype 8 Attenuates Cold‐Induced Hypertension Through Ameliorating Vascular Mitochondrial Dysfunction

doi: 10.1161/jaha.117.005495

Figure Lengend Snippet: Figure 1. Transient receptor potential melastatin subtype 8 (TRPM8) is a functional Ca2+ channel in the sarcoplasmic reticulum (SR) coupling Ca2+ release to mitochondria in vascular smooth muscle cells (VSMCs). A, TRPM8 immunoreactivity was detected by the monoclonal anti- TRPM8 antibody (125 kDa) in homogenates (h) and SR fractions of VSMCs from wild-type (WT) but not Trpm8/ mice. IP3R served as an SR marker, voltage-dependent anion channel as a mitochondrial marker, and tubulin as a cytosolic marker. Menthol (300 lmol/L)-induced Ca2+

Article Snippet: Fifty-microgram portions of the sample protein were separated on 10% SDS polyacrylamide gels and Western blotting performed as we previously described.23 The primary antibodies used included TRPM8 (NB200-145, Novus Biologicals), RhoA (sc-418), MYPT1 (sc-514261), p-MYPT1 (sc377531), tubulin (sc-134237), IP3R (sc-271197), b-actin (sc47778), and AT1R (sc-515884) and were purchased from Santa Cruz; MLC (#3672), p-MLC (#3675), and PDH (#2784) were purchased from Cell Signaling; and p-PDH (ab92696) and VDAC (ab15895) were purchased from Abcam.

Techniques: Functional Assay, Marker

Journal: Journal of the American Heart Association

Article Title: Activation of Transient Receptor Potential Melastatin Subtype 8 Attenuates Cold‐Induced Hypertension Through Ameliorating Vascular Mitochondrial Dysfunction

doi: 10.1161/jaha.117.005495

Figure Lengend Snippet: Figure 2. Transient receptor potential melastatin subtype 8 (TRPM8) activation by menthol inhibits Angiotensin II (Ang II)–induced cytosolic Ca2+ influx and mitochondrial Ca2+ elevation. A and B, Change in Fura-2 fluorescence intensity showing that menthol pretreatment blunted Ang II–induced Ca2+ influx in primary cultured vascular smooth muscle cells (VSMCs) from wild-type (WT) but not Trpm8/ mice in Ca2+-free 60 mmol/L K+-containing solution. MitoTEMPO (Mito T, 20 lmol/L) treatment hindered Ang II–induced Ca2+ influx. Mitochondrial Ca2+

Article Snippet: Fifty-microgram portions of the sample protein were separated on 10% SDS polyacrylamide gels and Western blotting performed as we previously described.23 The primary antibodies used included TRPM8 (NB200-145, Novus Biologicals), RhoA (sc-418), MYPT1 (sc-514261), p-MYPT1 (sc377531), tubulin (sc-134237), IP3R (sc-271197), b-actin (sc47778), and AT1R (sc-515884) and were purchased from Santa Cruz; MLC (#3672), p-MLC (#3675), and PDH (#2784) were purchased from Cell Signaling; and p-PDH (ab92696) and VDAC (ab15895) were purchased from Abcam.

Techniques: Activation Assay, Cell Culture

Journal: Journal of the American Heart Association

Article Title: Activation of Transient Receptor Potential Melastatin Subtype 8 Attenuates Cold‐Induced Hypertension Through Ameliorating Vascular Mitochondrial Dysfunction

doi: 10.1161/jaha.117.005495

Figure Lengend Snippet: 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-E1a (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 lmol/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 lmol/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 meanSEM. 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.

Article Snippet: Fifty-microgram portions of the sample protein were separated on 10% SDS polyacrylamide gels and Western blotting performed as we previously described.23 The primary antibodies used included TRPM8 (NB200-145, Novus Biologicals), RhoA (sc-418), MYPT1 (sc-514261), p-MYPT1 (sc377531), tubulin (sc-134237), IP3R (sc-271197), b-actin (sc47778), and AT1R (sc-515884) and were purchased from Santa Cruz; MLC (#3672), p-MLC (#3675), and PDH (#2784) were purchased from Cell Signaling; and p-PDH (ab92696) and VDAC (ab15895) were purchased from Abcam.

Techniques: Activation Assay, Western Blot, Cell Culture, Saline, Activity Assay, Phospho-proteomics

Journal: Journal of the American Heart Association

Article Title: Activation of Transient Receptor Potential Melastatin Subtype 8 Attenuates Cold‐Induced Hypertension Through Ameliorating Vascular Mitochondrial Dysfunction

doi: 10.1161/jaha.117.005495

Figure Lengend Snippet: Figure 4. Transient receptor potential melastatin subtype 8 (TRPM8) activation by dietary menthol attenuates cold-induced hypertension. A and B, Dietary menthol (0.5%) attenuated cold stimulation (CS)–induced elevation of systolic blood pressure (SBP) in wild-type (WT) mice but not Trpm8/ mice. n=6 in mice fed a normal diet (ND), n=8 in mice fed on menthol in normal temperature, and n=10 in CS-ND and CS-menthol group. C through F, After 6 months of treatment, the results of telemetric ambulatory blood pressure showed that dietary menthol supplementation lowered SBP and diastolic BP in WT mice in a TRPM8-dependent manner. n=6. *P<0.05, **P<0.01 vs mice fed an ND; #P<0.05 vs mice with CS and ND; &P<0.05, &&P<0.01 vs baseline (0 month) using repeated-measures ANOVA. Data are expressed as meanSEM. Con-Men indicates mice fed on menthol in normal temperature; Con-ND, mice fed an ND; CS-Men, mice fed on menthol treated by CS.

Article Snippet: Fifty-microgram portions of the sample protein were separated on 10% SDS polyacrylamide gels and Western blotting performed as we previously described.23 The primary antibodies used included TRPM8 (NB200-145, Novus Biologicals), RhoA (sc-418), MYPT1 (sc-514261), p-MYPT1 (sc377531), tubulin (sc-134237), IP3R (sc-271197), b-actin (sc47778), and AT1R (sc-515884) and were purchased from Santa Cruz; MLC (#3672), p-MLC (#3675), and PDH (#2784) were purchased from Cell Signaling; and p-PDH (ab92696) and VDAC (ab15895) were purchased from Abcam.

Techniques: Activation Assay

Journal: Journal of the American Heart Association

Article Title: Activation of Transient Receptor Potential Melastatin Subtype 8 Attenuates Cold‐Induced Hypertension Through Ameliorating Vascular Mitochondrial Dysfunction

doi: 10.1161/jaha.117.005495

Figure Lengend Snippet: Figure 5. Transient receptor potential melastatin subtype 8 (TRPM8) activation by dietary menthol attenuates vasoconstriction in cold- induced hypertension. A and B, Dietary menthol (normal diet [ND] plus menthol supplementation [0.5%]) attenuated cold stimulation (CS)– induced vasoconstriction of mesenteric arterial rings in wild-type (WT) but not Trpm8/ mice. C and D, Phenylephrine (PE)-induced vasoconstriction in mesenteric arterial rings pretreated by the endothelial nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester (L- NAME). n=5 in WT and Trpm8/ normal temperature (Con)-ND group, and n=6 per group for the rest. *P<0.05 vs Con-ND; #P<0.05 vs CS-ND. Data are expressed as meanSEM. Men indicates menthol.

Article Snippet: Fifty-microgram portions of the sample protein were separated on 10% SDS polyacrylamide gels and Western blotting performed as we previously described.23 The primary antibodies used included TRPM8 (NB200-145, Novus Biologicals), RhoA (sc-418), MYPT1 (sc-514261), p-MYPT1 (sc377531), tubulin (sc-134237), IP3R (sc-271197), b-actin (sc47778), and AT1R (sc-515884) and were purchased from Santa Cruz; MLC (#3672), p-MLC (#3675), and PDH (#2784) were purchased from Cell Signaling; and p-PDH (ab92696) and VDAC (ab15895) were purchased from Abcam.

Techniques: Activation Assay

Journal: Journal of the American Heart Association

Article Title: Activation of Transient Receptor Potential Melastatin Subtype 8 Attenuates Cold‐Induced Hypertension Through Ameliorating Vascular Mitochondrial Dysfunction

doi: 10.1161/jaha.117.005495

Figure Lengend Snippet: 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 lm. 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 lmol/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 lmol/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 meanSEM. Men indicates ND plus menthol supplementation (0.5%); Con, vehicle (saline or ethanol as appropriate).

Article Snippet: Fifty-microgram portions of the sample protein were separated on 10% SDS polyacrylamide gels and Western blotting performed as we previously described.23 The primary antibodies used included TRPM8 (NB200-145, Novus Biologicals), RhoA (sc-418), MYPT1 (sc-514261), p-MYPT1 (sc377531), tubulin (sc-134237), IP3R (sc-271197), b-actin (sc47778), and AT1R (sc-515884) and were purchased from Santa Cruz; MLC (#3672), p-MLC (#3675), and PDH (#2784) were purchased from Cell Signaling; and p-PDH (ab92696) and VDAC (ab15895) were purchased from Abcam.

Techniques: Activation Assay, Western Blot, Expressing, Saline

Journal: Journal of the American Heart Association

Article Title: Activation of Transient Receptor Potential Melastatin Subtype 8 Attenuates Cold‐Induced Hypertension Through Ameliorating Vascular Mitochondrial Dysfunction

doi: 10.1161/jaha.117.005495

Figure Lengend Snippet: Figure 7. Transient receptor potential melastatin subtype 8 (TRPM8) activation by dietary menthol attenuates vascular dysfunction in Angiotensin II (Ang II)–induced hypertensive mice. A and B, Dietary menthol (0.5%) for 2 weeks before Ang II infusion (0.7 mg/kg per day) partially normalized systolic blood pressure in wild-type (WT) but not Trpm8/ mice. n=6 for Saline-normal diet (ND) group, and n=8 for Ang II groups. C and D, Dietary menthol ameliorated mesenteric artery constriction in Ang II–treated WT mice but not Trpm8/ mice. Preincubation of mitoTEMPO (Mito T, 50 lmol/L) for 2 hours blunted the enhanced vasoconstriction from Ang II–induced hypertensive mice. n=6. *P<0.05 vs saline-ND; #P<0.05 vs Ang II-ND; &&P<0.01 vs baseline (0 day) using repeated-measures ANOVA. Data are expressed as meanSEM. Men indicates ND plus menthol supplementation (0.5%).

Article Snippet: Fifty-microgram portions of the sample protein were separated on 10% SDS polyacrylamide gels and Western blotting performed as we previously described.23 The primary antibodies used included TRPM8 (NB200-145, Novus Biologicals), RhoA (sc-418), MYPT1 (sc-514261), p-MYPT1 (sc377531), tubulin (sc-134237), IP3R (sc-271197), b-actin (sc47778), and AT1R (sc-515884) and were purchased from Santa Cruz; MLC (#3672), p-MLC (#3675), and PDH (#2784) were purchased from Cell Signaling; and p-PDH (ab92696) and VDAC (ab15895) were purchased from Abcam.

Techniques: Activation Assay, Saline

Journal: Journal of the American Heart Association

Article Title: Activation of Transient Receptor Potential Melastatin Subtype 8 Attenuates Cold‐Induced Hypertension Through Ameliorating Vascular Mitochondrial Dysfunction

doi: 10.1161/jaha.117.005495

Figure Lengend Snippet: Figure 8. Transient receptor potential melastatin subtype 8 (TRPM8) activation by dietary menthol inhibits reactive oxygen species (ROS) production and vasoconstriction in Angiotensin II (Ang II)–induced hypertensive mice. 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. Bar, 200 lm. n=6. 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. n=4. *P<0.05, **P<0.01 vs saline-normal diet (ND); #P<0.05, ##P<0.01 vs Ang II-ND. Data are expressed as meanSEM. Men indicates ND plus menthol supplementation (0.5%).

Article Snippet: Fifty-microgram portions of the sample protein were separated on 10% SDS polyacrylamide gels and Western blotting performed as we previously described.23 The primary antibodies used included TRPM8 (NB200-145, Novus Biologicals), RhoA (sc-418), MYPT1 (sc-514261), p-MYPT1 (sc377531), tubulin (sc-134237), IP3R (sc-271197), b-actin (sc47778), and AT1R (sc-515884) and were purchased from Santa Cruz; MLC (#3672), p-MLC (#3675), and PDH (#2784) were purchased from Cell Signaling; and p-PDH (ab92696) and VDAC (ab15895) were purchased from Abcam.

Techniques: Activation Assay, Western Blot, Saline

Journal: Journal of the American Heart Association

Article Title: Activation of Transient Receptor Potential Melastatin Subtype 8 Attenuates Cold‐Induced Hypertension Through Ameliorating Vascular Mitochondrial Dysfunction

doi: 10.1161/jaha.117.005495

Figure Lengend Snippet: Figure 9. Schematic diagram of the protective effect of transient receptor potential melastatin subtype 8 (TRPM8) activation by menthol in vascular smooth muscle cell function. On angiotensin II (Ang II) stimulation, the Ca2+ concentration in mitochondria [Ca2+]m is increased and the activity of pyruvate dehydrogenase (PDH) is impaired, leading to the dysfunction of mitochondrial oxidative phosphorylation (OXPHOS) and the excessive generation of reactive oxygen species (ROS) in vascular smooth muscle cells. The activation of TRPM8 by menthol couples sarcoplasmic reticulum Ca2+ release to mitochondrial Ca2+ uptake, which promotes the activity of PDH and OXPHOS, and inhibited ROS production. Preserving mitochondrial function by menthol blunts Ang II–induced [Ca2+]m overload and mitochondrial dysfunction through inhibiting ROS-triggered Ca2+ influx through the L-type Ca2+ channel.

Article Snippet: Fifty-microgram portions of the sample protein were separated on 10% SDS polyacrylamide gels and Western blotting performed as we previously described.23 The primary antibodies used included TRPM8 (NB200-145, Novus Biologicals), RhoA (sc-418), MYPT1 (sc-514261), p-MYPT1 (sc377531), tubulin (sc-134237), IP3R (sc-271197), b-actin (sc47778), and AT1R (sc-515884) and were purchased from Santa Cruz; MLC (#3672), p-MLC (#3675), and PDH (#2784) were purchased from Cell Signaling; and p-PDH (ab92696) and VDAC (ab15895) were purchased from Abcam.

Techniques: Activation Assay, Cell Function Assay, Concentration Assay, Activity Assay, Phospho-proteomics, Preserving