Charybdotoxin

Drugs were kept in 500X-1,000X stock solutions and diluted in SFM prior the experiments. (-)-Bicuculline methochloride (BIC; GABA_A receptor antagonist), d(-)-2-amino-5-phosphonopentanoic acid (d-AP5; NMDA receptor antagonist), 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX; AMPA/kainate receptor antagonist) and bisindolylmaleimide X (BIM-X; protein kinase C blocker) were purchased from Tocris (Bristol, UK) or Cayman Chemical (Ann Arbor, MI). SQ 25236 (adenylyl cyclase blocker) and UCL2077 (slow afterhyperpolarization blocker) were obtained from Tocris. TRAM-34 (KCa3.1 blocker) was obtained by MedChemExpress (Monmouth Juntion, NJ, USA). Rp-cAMPS triethylammonium salt (protein kinase A blocker), charybdotoxin (ChTx; fast afterhyperpolarization blocker) and apamin (medium afterhyperpolarization blocker) were obtained from Sigma (Saint-Louis, MO, USA). PG97-269 and PG99-465 (VPAC1 and VPAC2 blockers, respectively) were obtained from Bachem (Torrance, CA, USA). Cholera toxin (CTX) and wortmannin were obtained from Cayman Chemical. Vasoactive intestinal peptide (VIP) was obtained from Phoenix Pharmaceuticals (Burlingame, CA, USA).

Spadin, PE 22-28 and PE 22-28 analogs were purchased from GeneCust Europe, Luxembourg. G418, Arachidonic acid (AA), 4-AP (4-aminopyridine), TEA (tetraethylammonium), Apamin and Charybdotoxin were purchased from Sigma-Aldrich, France, Glibenclamide was purchased from ICN Biomedicals (USA).

NaCl, KCL, d‐glucose anhydrites, NaOH, and KOH were purchased from Fisher Scientific UK Ltd (Leicestershire, UK); MgCl₂, CaCl₂, ethylene‐glycol‐tetra‐acetic acid (EGTA), and Charybdotoxin (ChTX) were purchased from Sigma–Aldrich Co (Southampton, UK); N‐2‐hydroxyethylpiperazine‐N′‐2‐ethanesulfonic acid (HEPES) was purchased from VWR International (Leicestershire, UK).

Tissue samples were minced into small (> 2 mm³) pieces and incubated with Dispase/Collagenase IV (Stemcell, Vancouver, Canada) in DMEM/F12 (Sigma-Aldrich) with 5% FCS (Thermo Fisher Scientific, Waltham, MA, USA). On the next day, cells were centrifuged, and pellets were washed with DPBS (Sigma-Aldrich). Afterwards, tissue samples were digested in 5 × Trypsin/DPBS (Sigma-Aldrich) for 1 h at 4 °C followed by 1 U/mL Dispase (Stemcell) in DMEM/F-12 incubation for 2 min. Cells were passed through a 100 μm cell strainer and flow through was resuspended in 3 ml PCPM (DMEM/F12, 5% FCS, 200 mM L-glutamine, Penicillin-Streptamycin, 1 μg/ml Charybdotoxin, 200 μM Hydrocortisone, 20 mg/ml Adenine (all Sigma-Aldrich), 10 μM Y-27632 (Miltenyi Biotec, Bergisch Gladbach, Germany), 12.5 mg/ml insulin (PAN Biotech, Aidenbach, Germany), 1 mg/ml hEGF (Sigma-Aldrich)) and seeded into 12-well plates.

α-Bisabolol, diclofenac, formaldehyde, naloxone (non-selective opioid receptors antagonist), metformin (a drug hypoglycemic biguanide), NG-L-nitro-arginine methyl ester (L-NAME; a NO synthase inhibitor), 1 H-(1,2,4)-oxadiazolo (4,2-a) quinoxalin-1-one (ODQ; a NO-sensitive soluble guanylyl cyclase inhibitor), glibenclamide and glipizide (both ATP-sensitive K⁺ channel blockers; K_ir6.1-2), 4-aminopyridine (4-AP) and tetraethylammonium chloride (TEA) (both voltage-gated K⁺ channel blockers; K_V), apamin (a small conductance Ca²⁺-activated K⁺ channel blocker; K_Ca2.1-2.3), and charybdotoxin (a big conductance Ca²⁺-activated K⁺ channel blocker; K_Ca1.1), were purchased from Sigma-Aldrich (Toluca, Mexico). α-Bisabolol was diluted in 1% Tween 80. diclofenac, naloxone, metformin, L-NAME, TEA, 4-AP, apamin, and charybdotoxin were dissolved in saline solution. glibenclamide, glipizide and ODQ were dissolved in a 20% DMSO solution.

Phenylephrine hydrochloride (Sigma, cod. P6126), Acetylcholine chloride (Sigma, cod. A6625), Nω-Nitro-L-arginine methyl ester hydrochloride (Sigma, cod. N5751), charybdotoxin (Sigma, cod. C7802), indomethacin (Sigma, cod. I7378), apamin (Sigma, cod. A1289), Dihydroethidium (Sigma, cod. D7008), 2,2-Diphenyl-1-picrylhydrazyl (Sigma, cod. D9132), Folin-Ciocalteu’s (Sigma, cod. 47,641), ascorbic acid, gallic acid, from Sigma-Aldrich®, 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) from Invitrogen Molecular Probes™ (cod. D1306) and Dako Fluorescence Mounting Medium (cod. S3023) Na₂CO₃, NaCl, KCl, KH₂PO₄, NaHCO₃, C₆H₁₂O₆, CaCl₂, MgSO₄, ethylenediaminetetraacetic Acid (EDTA), ethanol, methanol, from Vetec®.