Amg9810

Reagents and the concentrations used were selected from previous TPMD literature [3 (link), 11 (link)–14 (link)] and from the TRP channel literature [15 (link)]. Dulbecco’s modified Eagle’s medium (DMEM) and Ca⁺⁺-free Keratinocyte-SFM medium (K-SFM), and alexa fluor 488 dextran (FDx) were purchased from ThermoFisher Scientific, Waltham, MA. Cal-520-AM dye was purchased from AAT Bioquest, Sunnyvale, CA. Cyclopiazonic acid (CPA), 18-α glycyrrhetinic acid (18α-GA), apyrase, AMG 9810, x-methyltryptophan (AMTB) and ryanodine were purchased from Sigma-Aldrich, St. Louis, MO. Thapsigargin was purchased from Abcam, Cambridge, United Kingdom, and N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl) tetrahydropyrazine-1(2H)-carbox-amide (BCTC) was purchased from Focus Biomolecules, Plymouth Meeting, PA.

Gold nanocrystals, which were grown on a KCl (100) surface via epitaxial growth under a 10⁻⁴ Pa vacuum, were dissolved in double-distilled water, and conjugated with anti (His)₆ monoclonal antibody (clone 9C11, FUJIFILM-Wako, Osaka, Japan) or anti FLAG monoclonal antibody (clone M2, Sigma-Aldrich) under borate buffer, pH 9.0. Buffer component was substituted with recording buffer (PBS (pH 7.4) with 5 mM n-decyl-β-D-maltoside (Sigma-Aldrich)) before applying to TRPV1. For the C-terminal analysis of the 12.5 ms/frame recording, ZnO crystal was used for labeling antibodies.
To immobilize biotinylated TRPV1 proteins to the basement surface, 12.5 μm thick polyimide film (Du Pont-Toray, Tokyo, Japan) was coated with cadmium and chromium (Cd/Cr) by evaporation, then biotin-functionalized self-assembled monolayers (Biotin-SAM) were formed using Biotin-SAM Formation Reagent (Dojindo, Kumamoto, Japan). After binding streptavidin on the Biotin-SAM membrane, twenty microliters of biotinylated TRPV1 (N-terminally (His)₆-tagged and C-terminally FLAG tagged, ~0.1 mg/mL) were applied and incubated at 4 °C for 6 h. Excess protein was washed out with buffer, and the gold- or ZnO-conjugated antibodies (dispersed in a recording buffer) were applied. After incubation for 20 min at 4 °C, excess antibodies were washed out. Ten microliter of recording buffer containing 10 μM capsaicin with or without 10 μM AMG9810 was applied. The sample chamber was covered with another layer of polyimide film, sandwiched by stainless steel frames and screw clamped. Capsaicin and AMG9810 were purchased from Sigma-Aldrich.

The TRPA1 antagonist HC030031 (2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-purin-7-yl)-N-(4-isopropyl-phenyl)-acetamide 1) (Tocris, UK) was either dissolved in 10% DMSO in saline and administered i.p. at a dose of 100 mg kg⁻¹ 30 min53 (link) before local cold water immersion or in 8% DMSO, 2% Tween-80 in saline and administered i.v. at a dose of 6 mg kg⁻¹ (ref. 54 (link)) at the peak vasoconstriction phase (0–2 min) following local cold water immersion. The TRPM8 antagonist AMTB (N-(3-aminopropyl)-2-[(3-methylphenyl) methyl]oxy-N-(2-thienylmethyl)benzamide hydrochloride salt) was dissolved in 10% DMSO in saline, TRPV1 antagonist SB366791 (4′-Chloro-3-methoxycinnamanilide) in 2% DMSO in saline or AMG9810 ((2E)-N-(2,3-Dihydro-1,4-benzodioxin-6-yl)-3-[4-(1,1-dimethylethyl)phenyl]-2-propenamide) (Sigma, UK) in 2% DMSO and 5% Tween-80 in saline and administered at a dose of 10 (ref. 55 (link)), 25 (ref. 56 (link)) and 50 mg kg⁻¹ (ref. 57 (link)), respectively. The CGRP receptor antagonists were dissolved as follows: CGRP_8–37 was dissolved in 0.01% bovine serum albumin (BSA) and administered i.v. at 400 nmol kg⁻¹ (ref. 58 (link)) (Tocris,UK). BIBN4096 ((1-piperidinecarboxamide,N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-,[R-(R*,S*)]) (Tocris, UK) was dissolved in a minimum volume of 1M HCl (0.5 mg per 50 μl) and the solution was made up to the required final volume with saline at a final stock concentration of 2 mg ml⁻¹, and then titrated with 1 M NaOH to return the pH to neutral. BIBN4096 was administered i.v. at 0.3 mg kg⁻¹ (refs 59 (link), 60 (link)). The NK₁ receptor antagonist SR140333 ((S)1-(2-[3-(3,4-dichlorophnyl)-1-(3-isopropoxyphenylacetyl)piperidin-3-yl]ethyl)-4-phenyl-1-azoniabicyclo[2.2.2]octane chloride)60 (link), a gift from Dr X. Emonds-Alt, Sanofi, Toulouse, France, was dissolved in saline and administered at a dose of 480 nmol kg⁻¹ i.v. The non-selective NOS inhibitor L-NAME60 (link) or the selective nNOS inhibitor SMTC (Sigma, UK) was dissolved in saline and administered i.v. at 15 mg kg⁻¹ (ref. 61 (link)). The cyclooxygenase inhibitor indomethacin (Sigma, UK) was dissolved in 5% NaHCO₃ in saline and administered i.v. at 20 mg kg⁻¹ (ref. 60 (link)). The non-selective α-adrenoceptor antagonist phentolamine62 (link) was dissolved in saline and administered i.v. at a dose of 10 mg kg⁻¹, whilst the sympathetic nerve blocker guanethidine63 (link) (Sigma, UK) was dissolved in saline and administered subcutaneously (s.c.), at a dose of 30 mg kg⁻¹ daily for four consecutive days. The α₂-adrenoceptor antagonist yohimbine hydrochloride, the selective α_2C-adrenoceptor antagonist JP1302 dihydrochloride and the selective Rho-associated protein kinase (ROCK) inhibitor Y27632 (Tocris, UK) were dissolved in saline and administered at a dose of 10 mg kg⁻¹ (i.p.)64 (link), 3 μg kg⁻¹ (s.c.)65 (link) or 5 mg kg⁻¹ (i.p.)66 (link), respectively. The SOD mimetic TEMPOL was dissolved in saline and administered i.v. at a dose of 30 mg kg⁻¹ (ref. 60 (link)), whilst the mitochondria-targeted superoxide scavenger mito-TEMPO was dissolved in saline and administered i.p. at a dose of 10 mg kg⁻¹ (ref. 67 (link)). The capsaicin analogue resiniferatoxin was dissolved in 10% ethanol, 10% Tween-80 in saline and administered s.c. at a dose of 0.3 mg kg⁻¹ for three consecutive days68 (link). These drugs were administered i.v. 5 min, i.p. 30 min and s.c. 60 min before baseline blood flow recording. The doses used were based on previous and preliminary studies.

Active reagents and the concentrations used were selected from previous TPMD literature^{7 (link),35 (link)–37 (link)} and from the TRP channel literature^{38 (link)}. AMG 9810, AMTB, ryanodine, BAPTA-AM, 18-α glycyrrhetinic acid (18α-GA), apyrase and DAPI were purchased from Sigma-Aldrich, St. Louis, MO; thapsigargin, BCTC, and Cal-520-AM dye were purchased from Abcam, Cambridge, United Kingdom, Focus Biomolecules, Plymouth Meeting, PA, and AAT Bioquest, Sunnyvale, CA, respectively. Polyclonal connexin 43 antibody was purchased from Cell Signaling, Danvers, MA; Alexa Fluor 488 was purchased from Fisher, Pittsburgh, PA. Ca⁺⁺-free Keratinocyte-SFM medium (K-SFM) was purchased from ThermoFisher Scientific (Cat. No.: 10725–018), Waltham, MA.