Sumatriptan

All chemical reagents were purchased and used without any additional purification. A standard stock solution of 0.01 mol L⁻¹ sumatriptan (≥98%, Sigma Aldrich, St. Louis, MO, USA) was prepared dissolution in double deionized water and stored in the fridge between measurements. Solutions with lower SUM concentration were prepared daily. Phosphate buffer (1 mol L⁻¹, pH 6.0) was prepared by mixing the proper amounts of KH₂PO₄ and K₂HPO₄ (both Merck, Darmstadt, Germany). Carbon black nanoparticles (CB) characterized by the following parameters, surface area: 100 m²g⁻¹ and average particles size: 30 nm, were purchased from 3D-nano (Poland), and dimethyloformamide (DMF) was obtained from Sigma Aldrich (St. Louis, MO, USA). Freeze dried urine (Medidrug Basis-line U) was purchased from Medichem (Steinenbronn, Germany), and human plasma was purchased from Biowest (Nuaillé, France). Double-deionized water was used to prepare all solutions.

Apart from the anaesthetic (sodium pentobarbital), the drugs used in the present study were: isometheptene racemate, (R)-isometheptene and (S)-isometheptene (Tonix Pharmaceuticals Inc., New York, N.Y., U.S.A.); sumatriptan, bradykinin, noradrenaline, capsaicin, U46619 and substance P (Sigma Chemical Co., St. Louis, MO, U.S.A); and rat/human α-CGRP (NeoMPS S.A., Strasbourg, France). capsaicin was dissolved in a mixture of tween 80, ethanol 70% and water (1:1:8), while the rest of the compounds were dissolved in either distilled water (in vitro) or physiological saline (in vivo). The doses mentioned in the text refer to the free base of substances in all cases.

The human colorectal cancer LoVo, HCT-116, HT-29 and SW403 cell lines were purchased from the Shanghai Cell Collection (Shanghai, China). Cells were grown in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA) at 37°C in a 5% CO₂-humidified atmosphere. Monoclonal antibodies against 5-HT family, Axin1, β-catenin, APC, GSK3β, CKIα, LEF1, TCF4, cyclin D1, c-Myc, MMP-2, MMP-9, MMP-7 and β-actin were products of Cell Signaling Technology (Beverly, MA, USA). 5-HT_1DR agonist (Sumatriptan) and 5-HT_1DR antagonist (GR127935) were from Sigma Chemical Co. (St. Louis, MO).

Alzet osmotic minipumps (model 1007D, Charles River, France) with a nominal flow rate of 0.5 μl/h for 6 days were used for drug infusion. The minipumps were implanted subcutaneously under anesthesia with isoflurane. The day of the pump implant was considered as day 0. Drugs administered by infusion were sumatriptan (0.6 mg/kg/day, Sigma, St. Louis USA) and its vehicle (NaCl, 0.9%). SNP was injected subcutaneously into the loose skin over the neck at 0.03 mg/kg. Infusion of sumatriptan/saline and injection of SNP/saline were made blind, e.g. the investigator was not aware of the content of the minipump, nor of the nature of the solution (SNP or vehicle) injected on day 21. Animals were randomly assigned to treatment groups.

Ketamine, xylazine, and isoflurane were purchased from VetOne (IL, United Stated). DH376 was a generous gift from Prof. Mario van der Stelt, Leiden University. LEI106 was purchased from Cayman Chemicals (Ann Arbor, MI). KT109, olcegepant, sumatriptan, and morphine were purchased from Sigma-Aldrich (St. Louis, MO). LEI106, KT109, and olcegepant were dissolved in DMSO-Tween80-saline 0.9% (1:1:8, v/v/v). LEI106 was injected intraperitoneally at doses of 10, 20, and 40 mg/kg. KT109 was interperitoneally administered at 5, 10, 20 mg/kg doses. DH376 was dissolved in ethanol-PEG-saline 0.9% (1:1:18, v/v/v). sumatriptan succinate (0.6 mg/kg, dissolved in saline) was dosed subcutaneously 30 min before the injection of DAGL inhibitor. olcegepant 0.8 mg/kg, dissolved in DMSO-Tween80-saline 0.9% (1:1:8, v/v/v) was injected intraperitoneally 30 min before the administration of DAGL inhibitor. AEA-d4 and 2-AG-d5 were purchased from Cayman Chemicals (Ann Arbor, MI).

A modified version of NTG model described by Bates et al. [14 (link)] was used. Animals received intraperitoneal (i.p.) injections of 15 mg/kg NTG (Nitro POHL®, G. Pohl-Boskamp GmbH & Co. KG, Hohenlockstedt, Germany) or vehicle (49 mg glucose monohydrate/ml) after the measurement of the baseline thermal nociceptive threshold. The dose of NTG was chosen based on literature data [14 (link)] and preliminary tests. sumatriptan succinate (Sigma-Aldrich, Hungary), dissolved in saline was used to validate our experiments: each animal was given i.p. sumatriptan at the dose of 600 μg/kg [14 (link)] or saline 5 minutes after NTG administration. The P2X7 antagonist, Brilliant Blue G (BBG, Sigma-Aldrich, Hungary) or its vehicle (0.9% saline), were applied in an identical way, or using two different prophylactic application protocols: acutely (50 mg/kg), it was applied i.p. 30 minutes prior to NTG treatment and after the measurement of the baseline thermal nociceptive threshold on the day of testing. In subacute treatment, mice were treated for 5 consecutive days with the daily doses of BBG (50 mg/kg i.p.) or saline and 30 min after the last injection were subjected to NTG. Selection of dose was based on previous studies [38 (link),39 (link)]. All drug solutions were freshly prepared on the day of use.

[³H]5-HT (5-hydroxytryptamine[³H(G)] (serotonin), spec. activity 113.2 Ci/mmol) and [³H]Glu (Glutamic acid, L-[2,3,4-3H], spec. activity 60 Ci/mmol) were obtained from American Radiolabelled Chemicals Inc. Tetrodotoxin (TTX, from Alomone labs), CNQX (disodium salt; Tocris), DL-AP-5 (Tocris), sumatriptan (Sigma), buspirone (Sigma) and AZ-10606120 (Tocris) were dissolved in distilled water, veratridine (Tocris) was dissolved in DMSO. JNJ-47965567 (Tocris) was dissolved in 30% sulfobutylether beta-cyclodextrin (SBE, from Ligand Pharmaceuticals Inc.). The maximum concentrations of vehicles used had no significant effect on the [³H]5-HT/[³H]Glu release. All solutions were freshly prepared on the day of use.