Heptanol

The CON+H group was the same as the CON group, except that 2.5 μmol/L of the gap junction uncoupler heptanol (Sigma-Aldrich) was added to the growth media.

The standards of volatile compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA), including isobutanol, 3-methyl-1-butanol, cis-3-hexen-1-ol, 1-hexanol, heptanol, octanol, phenethyl alcohol, 2,6-nonadien-1-ol, dodecanol, ethyl acetate, isoamyl acetate, ethyl lactate, ethyl hexanoate, ethyl octanoate, diethyl succinate, phenethyl acetate, ethyl decanoate, butanoic acid, hexanoic acid, octanoic acid, dodecanoic acid, octanal, (2e,6z)-2,6-nonadienal, benzaldehyde, furfural, citronellol, linalool, rose oxide, geraniol, nerol, β-damascenone, β-ionone, nerolidol, hexanal, guaiacol, 4-Ethylphenol, and 2-octanol which were used as the internal standard.
Folin–Ciocalteu reagent and sodium chloride were purchased from Beijing Chemical Works (Beijing, China). Potassium hydrogen tartrate were purchased at Kemiou Chemical Reagent Co. (Tianjin, China). Bovine serum albumin was obtained from Asahi Kasei Corporation (Tokyo, Japan). Deionized water (<18 MW resistance) was purified by using a Milli-Q purification system (Molecular, Chongqing, China). Bentonite, soybean protein and potassium metabisulfite were purchased from Lallemand Company (Lallemand, Toulouse, France).

Twelve thousand primary astrocytes were seeded in 48-well plates. After 24 h, cells were incubated in serum-free media containing 100 μM of the exonuclease inhibitor ARL-67156 (Santa Cruz Biotechnologies) for 30 min at 37 °C. Following this, cells were stimulated with Thy-1-Fc:Protein A for 15 min. Where indicated, cells were incubated with a combination of Heptanol (Sigma-Aldrich Co.) (500 μM) and Probenecid (Sigma-Aldrich Co.) (1 mM). Next, culture medium was recovered and centrifuged for 5 min at 1000×g. The supernatants were incubated in the dark with 50 μl CellTiter-Glo® reaction mix (Promega, Madison, WI). Luminescence intensity was determined in a Synergy2 multi-mode reader (Biotek Instruments, Inc., Vermont), and the values were interpolated using a calibration curve obtained from different ATP concentrations (0.01, 0.1, 1, and 10 μM).

The present experiments used Krebs-Henseleit solution [119 mM NaCl, 25 mM NaHCO₃, 4 mM KCl, 1.2 mM KH₂PO₄, 1 mM MgCl₂, 1.8 mM CaCl₂, 10 mM glucose and 2 mM sodium pyruvate (pH 7.4)] that had been bicarbonate-buffered and bubbled with 95% O₂ and 5% CO₂ (7 (link)). Hypokalaemic solution was generated by decreasing the amount of KCl added to the Krebs-Henseleit solution to result in a total of 3 mM [K⁺]. Heptanol (Sigma-Aldrich, Dorset, UK; density, 0.82 g ml⁻¹) was added directly to the hypokalaemic solution to produce a final concentration of 0.1 mM.

Commercially available food additive products were used in the human sensory test. Food-grade NaCl was purchased from Naikaisyoji (Tokyo, Japan), and monosodium glutamate (MSG) was purchased from Ajinomoto Co., Inc. (Tokyo, Japan). All flavor compounds (of food additive grade), including propionaldehyde, butanal, isobutylaldehyde, 2-methylbutylaldehyde, pentanal, IVA, hexanal, heptanal, octanal, propionic acid, 2-methylbutyric acid, isovaleric acid, hexanoic acid, heptanoic acid, propyl alcohol, 2-methyl-1-butanol, hexanol, heptanol, methional, methionol, and 3-(methiylthiol) propionic acid, were purchased from Sigma-Aldrich (St. Louis. MO, USA). Deionized water for test solutions and mouth rinsing was supplied by a Mill-Q water purification system (Millipore, Bedford, MA, USA). γ-Glu-Val-Gly was obtained from Ajinomoto Co., Inc. (Tokyo, Japan). NPS-2143, a CaSR inhibitor, was synthesized, as described by Rybczynska et al. [41 (link)]. Chemicals for cell based-assays, such as CaCl₂, ethylenediaminetetraacetic acid (EDTA), and probenecid, were purchased from Sigma-Aldrich (St. Louis. MO, USA).

Gap junction blockers heptanol (500 μM, Sigma-Aldrich) (Guan et al., 1997 (link); Johnston et al., 1980 (link); Weingart and Bukauskas, 1998 (link)) or propofol (10 μM, Sigma-Aldrich) (Mantz et al., 1993 (link); Wentlandt et al., 2006 (link)) were added to the bath for at least 30 min to allow adequate diffusion before imaging acquisition. Additional putative gap junction blockers were also tested in preliminary studies but were difficult to dissolve in embryo media (carbenoxolone) or toxic (mefloquine) and not included here. For all drugs, toxicity tests were performed wherein multiple concentrations of each drug were bath applied to 3 agar-embedded larvae for 1.5 hours, then heart rate was monitored to identify maximum non-toxic concentration of each drug to be used. During all imaging studies, heart rate was continuously monitored as a means to confirm vitality, and fish with no or barely observable heart activity after imaging data acquistions were excluded from analysis.

NRK-52E cells, a cell line of rat kidney tubular epithelial cell origin, were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM/F-12 supplemented with 10% fetal bovine serum. Cells were grown at 37 °C in an atmosphere of 5% CO₂ in air^{48 (link)}, and then pretreated with connexin channel inhibitors heptanol, 2 mM, for 1 h (Sigma-Aldrich, a Cx43 uncoupler)^{49 (link)} or Gap26, 300 μM, for 1 h (Sigma-Aldrich, a connexin mimetic peptide)^{50 (link)}; a Cx43 expression enhancer, retinoic acid (RA), 10 μM, for 24 h (Sigma-Aldrich)^{4 (link)}; NAC (Sigma-Aldrich, a kind of ROS scavenger), 10 mM, for 1 h before H/R or/and LPS exposure. “Parachute” dye-coupling assay was performed as described below. The solvents of heptanol and RA were DMSO.