Methoxamine hydrochloride

Apart from the anaesthetic (diethyl ether), the compounds used in this study (obtained from the sources indicated) were: gallamine triethiodide, hexamethonium chloride, rat α-CGRP, methoxamine hydrochloride, rauwolscine hydrochloride, (Sigma Chemical Co., St Louis, MO, USA); N-[methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)[1,1-biphenyl]-4-carboxamidehydrochloride (GR127935) (gift from GlaxoSmithKline, Stevenage, Hertfordshire, UK); and DHE tartrate (gift from Novartis Pharma A.G., Basel, Switzerland). All compounds were dissolved in saline, except: (i) DHE, which was dissolved in propylene glycol and gauged with saline to have a final solution of 1% PPG; and (ii) haloperidol, which was dissolved in some drops of 5% ascorbic acid and the resulting solution was finally diluted with saline. These vehicles had no effect on baseline diastolic blood pressure or heart rate (data not shown). Fresh solutions were prepared for each experiment. The doses of agonists refer to their respective salts, whereas those of the antagonists refer to their free base.

Metabolites were derivatized using a combination of methoxamine hydrochloride (Sigma-Aldrich Co.) and N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA; Sigma-Aldrich Co.). methoxamine hydrochloride solution (20 μL; 40 mg mL⁻¹ in pyridine) was added to the dried polar and non-polar fractions and incubated at 30 °C for 90 min with shaking (1,000 rpm; Thermomixer Comfort). Then, 80 μL of MSTFA was added and incubated at 37 °C for 30 min with shaking as above. The sample was centrifuged at 12,000 × g for 3 min, the supernatant was filtered through a hydrophilic polytetrafluoroethylene membrane filter unit (Millex-LG; Merck Millipore Co., Darmstadt, Germany), and a 1-μL aliquot was used in GC/TOF-MS analysis.

The following reagents were used: 4,5-dihydro-N-[4-[[4-(1-methylethoxy)phenyl]methyl]phenyl]-1H-imadazol-2-amine (CAY10441), 1-(4-fluorobenzoyl)-3-[[(6-methoxy-2-naphthalenyl)oxy]methyl]-3-azetidinecarboxylic acid (PF-04418948), prostaglandin E₂ (PGE₂), prostaglandin I₂ (PGI₂) (Cayman Chemical Co., Ann Arbor, MI, USA), (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3; Dojindo, Kumamoto, Japan), dimethyl sulfoxide, tetrodotoxin (Nacalai Tesque, Kyoto, Japan), fluorescein sodium salt, indomethacin, methoxamine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA), pontamine sky blue 6B, pentobarbital sodium (Tokyo Chemical Industry, Tokyo, Japan), and hydroxyethyl cellulose (Scopisol 15^®; Senju Pharmaceutical, Osaka, Japan).

The volatile standards were purchased from Sigma Aldrich (Saint Louis, MO, USA) and Shanghai Yuanye Ltd. (Shanghai, China). Detailed information of volatile standards was showed in Table S2. Methanol (HPLC-grade) from Fisher Scientific (Fair lawn, NJ, USA), ribitol (99%), N-methyl-N-(trimethylsilyl) trifluoroacetamide (98%) and methoxamine hydrochloride (98%) from Sigma Aldrich were applied for primary metabolites determination. A standard n-paraffin mixture (C7-C40) from ANPEL Laboratory Technologies Inc. (Shanghai, China), methyl nonanoate (98%) from Sigma Aldrich and aldrich sodium chloride (99%) from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) were for volatile compounds profiling.

Stable isotope internal standard 5-¹³C-glutamine was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). Myristic-1,2-¹³C2 acid, methoxamine hydrochloride (purity 98%), and pyridine (≥99.8% GC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-methyl-trimethylsilyl-trifluoroacetamide (MSTFA) and 1% trimethylchlorosilane (TMCS) were provided by Pierce Chemical (Rockford, IL, USA). Methanol, acetonitrile, and n-heptane were of HPLC grade and obtained from Merck (Darmstadt, Germany). Purified water was produced by a Milli-Q system (Millipore, Bedford, MA, USA). Ammonium acetate (purity 98.0%) and ammonia solution (25%, w/w) were purchased from Aladdin (Shanghai, China) and Nanjing Chemical Reagent (Nanjing, China), respectively.

The organic chemicals of analytical grade, namely ethanol, methanol, n-hexane, ethyl acetate, and dimethyl sulfoxide (DMSO), were obtained from Merck (Darmstadt, Germany). The four inorganic salts (sodium chloride, potassium chloride, calcium chloride, and magnesium sulfate) and methylene blue were purchased from Merck (Darmstadt, Germany). Ascorbic acid, rosiglitazone, ρ-nitrophenyl-ρ-D-glucopyranosidase (PNPG), quercetin, insulin, pyridine anhydrous, methoxamine hydrochloride, N-methyl-N-(trimethylsilyl) tryfluoroacetamide (MSTFA), and phosphate-buffered saline (PBS) were bought from Sigma-Aldrich (St. Louis, MO, USA). The AG enzyme (source: yeast maltase) was purchased from Megazyme, Ireland. Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco, Life Technologies (Carlsbad, CA, USA). Furthermore, the 2-NBDG Glucose Uptake assay kit and tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were procured from Abcam (Cambridge, UK) and Life Technologies (Carlsbad, CA, USA), respectively. The 3T3-L1 cell line was obtained from American Type Culture Collection ATCC (Manassas, VA, USA).