Transient receptor potential melastatin 8 channel involvement in the regulation of vascular tone.

The transient receptor potential melastatin 8 (TRPM8) channel has been characterized as a cold and menthol receptor expressed in a subpopulation of sensory neurons but was recently identified in other tissues, including the respiratory tract, urinary system, and vasculature. Thus TRPM8 may play multiple functional roles, likely to be in a tissue- and activation state-dependent manner. We examined the TRPM8 channel presence in large arteries from rats and the functional consequences of their activation. We also aimed to examine whether these channels contribute to control of conscious human skin blood flow. TRPM8 mRNA and protein were detected in rat tail, femoral and mesenteric arteries, and thoracic aorta. This was confirmed in single isolated vascular myocytes by immunocytochemistry. Isometric contraction studies on endothelium-denuded relaxed rat vessels found small contractions on application of the TRPM8-specific agonist menthol (300 microM). However, both menthol and another agonist icilin (50 microM) caused relaxation of vessels precontracted with KCl (60 mM) or the alpha-adrenoceptor agonist phenylephrine (2 microM) and a reduction in sympathetic nerve-mediated contraction. These effects were antagonized by bromoenol lactone treatment, suggesting the involvement of Ca(2+)-independent phospholipase A(2) activation in TRPM8-mediated vasodilatation. In thoracic aorta with intact endothelium, menthol-induced inhibition of KCl-induced contraction was enhanced. This was unaltered by preincubation with either N(omega)-nitro-l-arginine methyl ester (l-NAME; 100 nM), a nitric oxide synthase inhibitor, or the ACh receptor antagonist atropine (1 microM). Application of menthol (3% solution, topical application) to skin caused increased blood flow in conscious humans, as measured by laser Doppler fluximetry. Vasodilatation was markedly reduced or abolished by prior application of l-NAME (passive application, 10 mM) or atropine (iontophoretic application, 100 nM, 30 s at 70 microA). We conclude that TRPM8 channels are present in rat artery vascular smooth muscle and on activation cause vasoconstriction or vasodilatation, dependent on previous vasomotor tone. TRPM8 channels may also contribute to human cutaneous vasculature control, likely with the involvement of additional neuronal mechanisms.

Chemotherapy-induced epidermal growth factor receptor activation determines response to combined gefitinib/chemotherapy treatment in non-small cell lung cancer cells.

Activating epidermal growth factor receptor (EGFR) mutations have been linked with sensitivity to gefitinib and erlotinib; however, there are no established predictive markers for response to the combination of EGFR inhibitors with standard chemotherapy in non-small cell lung cancer (NSCLC) patients. In this study, we characterized a panel of human EGFR wild-type and mutant NSCLC cells for their sensitivity to gefitinib alone and in combination with cisplatin or Taxol. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and crystal violet cell viability assays. Cell cycle distribution was measured by flow cytometry. EGFR expression was measured by flow cytometry, real-time PCR, and Western blotting. EGFR/Her2/Akt and extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation were measured by Western blotting. Two of nine EGFR wild type and one of two EGFR mutant NSCLC cells were sensitive to gefitinib, and this was associated with a decrease in phospho (p)-Akt and pErk1/2 following gefitinib exposure. There was no correlation between constitutive EGFR expression or activity and sensitivity to gefitinib nor was there a correlation between Her2/Akt and Erk1/2 activity and gefitinib sensitivity. However, in cells displaying a synergistic interaction between gefitinib and chemotherapy (cisplatin or Taxol), a dose-dependent increase in pEGFR was observed following chemotherapy exposure. In contrast, in cells where no change or a decrease in pEGFR following drug treatment was observed, we found an antagonistic or (at best) an additive interaction between the two compounds. Furthermore, the nature of this interaction was not dependent on the presence of a mutant EGFR. These novel findings suggest that modulation of EGFR activity following drug treatment determines response to gefitinib in combination with chemotherapy in NSCLC cells.