Tetraethyl ammonium tea

Neurons were isolated from their surrounding synaptic environment by blocking AMPA, NMDA and GABA_A and glycine receptors with CNQX (20 μM, Tocris), AP5 (50 μM, Tocris), Gabazine (10 μM, Tocris) and Strychnine (10 μM, Sigma-Aldrich). Ca²⁺ currents were isolated by blocking voltage gated K⁺ channels with 4-Aminopyridine (4-AP, 3 mM; Sigma-Aldrich), tetraethylammonium (TEA, 0.1 mM; Sigma-Aldrich) cesium (1 mM; Sigma-Aldrich) and Na⁺ channels with tetrodotoxin (TTX, 1 μM; Alomone Labs). Drugs applied to the extracellular medium: 5-HT hydrochloride (10 μM; Sigma Aldrich); 4-Iodo-2,5-dimethoxy-α-methylbenzeneethanamine hydrochloride (DOI hydrochloride; 10–20 μM, Tocris); Mibefradil (8–16 μM, Tocris).

Radioactive isotope [³H]choline (78.3 Ci/mmol) was purchased from PerkinElmer (Boston, MA, USA). Choline chloride, acetylcholine (ACh), hemicholinium-3 (HC-3), verapamil, diphenhydramine (DPH), nifedipine, donepezil, edaravone, tetraethyl ammonium (TEA), amiloride, betaine, and L-carnitine were purchased from Sigma Aldrich (St. Louis, MO, USA). All chemicals were of reagent grade.

For the experiments, the following were used: ketamine hydrochloride and xylazine (from Syntec, São Paulo, SP, Brazil), 4-aminopyridine (4-AP), acetylcholine chloride (ACh), atropine, CaCl₂, dextrose, ethylenediaminetetraacetic acid, glibenclamide, HOE-140, KCl, KH₂PO₄, NaCl, NaHCO₃, MgSO₄, Nω-Nitro-L-arginine methyl ester (L-NAME), indomethacin, phenylephrine (Phe), sodium deoxycholate, and tetraethylammonium (TEA) were obtained from Sigma-Aldrich (St. Louis, MO, USA) and heparin from Hipolabor (São Paulo, SP, Brazil)

The drugs phenylephrine (PE), captopril, A779, PD123,319, LY-294,002, Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), Nω-Nitro-L-arginine (L-NNA), 1,400 W, 7-Nitroindazole (7-Ni), catalase and tetraethylammonium (TEA) were purchased from Sigma-Aldrich (St. Louis, MO, United States). DAF-2DA and DCF-DA fluorescent probes were obtained from Invitrogen (Carlsbad, CA, United States).

The radiolabeled labeling agent [³H]paeonol (7.2 Ci/mmol) was procured from PerkinElmer (Boston, MA, USA). Other unlabeled reagent-grade chemicals, including paeonol, carnitine, cimetidine, 1-methyl-4-phenylpyridinium ion (MPP⁺), quinidine, verapamil, edaravone, clonidine, tramadol, acetyl-l-carnitine (ALC), tetraethyl ammonium (TEA), para-amminohippuric acid (PAH), and glutamic acid, were obtained from Sigma Aldrich (St. Louis, MO, USA). Lipopolysaccharide (LPS) was acquired from List Biological Laboratories (Campbell, CA, USA).

Tetraethylammonium (TEA) a non-specific K_V channel blocker, was obtained from Sigma-Aldrich (St. Louis, MO, USA). K_V7 channel modulators, including flupirtine maleate, linopirdine dihydrochloride, and XE991 dihydrochloride, were purchased from Tocris Bioscience (Minneapolis, MN, USA). Riluzole, a glutamate release inhibitor, and CNQX and MK801, glutamate receptor antagonists, were obtained from Tocris Bioscience.
Rabbit polyclonal anti-K_V7.3 antibody was obtained from Alomone Labs (Jerusalem, Israel). Rabbit polyclonal anti-ERK1/2 and phospho-ERK1/2 antibody were purchased from Cell Signaling Technology (Danvers, MA, USA) and goat polyclonal synapsin Ia/b and goat polyclonal type 1 collagen antibody were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Horseradish peroxidase-conjugated anti-rabbit, anti-mouse antibodies were purchased from GenDEPOT (Barker, TX, USA) and the anti-goat antibody came from Santa Cruz Biotechnology, Inc.

BIMs I-XI and the PKC inhibitor LY 333531 (also known as ruboxistaurin), that contains a BIM core, were provided by Santa Cruz Biotechnology (Dallas, Texas) and Sigma-Aldrich (Saint-Quentin Fallavier, France). The chemical structures of these BIMs are shown in Fig 1. Verapamil, cyclosporin A, Gö 6983, amitriptyline, U0126, wortmannin, KN62, staurosporine, chelerythrine, tetra-ethylammonium (TEA), 4',6-diamidino-2-phenylindole (DAPI) and phorbol-12-myristate-13-acetate (PMA) were purchased by Sigma-Aldrich (Saint-Quentin Fallavier, France). [1-¹⁴C]-TEA (sp. act. 2.4 mCi/mmol) and [³H(G)] taurocholic acid (sp. act. 1.19 Ci/mmol) were from Perkin-Elmer (Boston, MA). All other chemicals were commercial products of the highest purity available.