Pregs

The following drugs were used: PregS (Sigma‐Aldrich), nifedipine (Nif, Sigma‐Aldrich), Dic (Wako), aspirin (Asp, Wako), ibuprofen (Ibu, Wako), loxoprofen (Lox, Wako), Ind (Wako), etodolac (Etd, Wako), piloxicam (Pil, Wako), niflumic acid (Nfm, Sigma‐Aldrich), aceclofenac (Afc, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), GSK1016790A (GSK; Sigma‐Aldrich), hydrogen peroxide (H₂O₂, Sigma‐Aldrich), and menthol (Men, Wako). Each drug was dissolved in the vehicle recommended by the manufacturer.

The TRPM3 agonist PregS and TRPM8 agonist menthol were purchased from Sigma-Aldrich. ATP was purchased from Roche. The nonhydrolyzable ATP analogue adenosine-5-[(β,γ)-methyleno]triphosphate (AMPPCP) was obtained from Jena Bioscience and adenosine-5-[(β,γ)-imido]triphosphate (AMPPNP) was purchased from Sigma-Aldrich. Water-soluble diC8 PIPs and diC16 PI(4,5)P₂ were purchased from Echelon, IP₃ was obtained from Sigma-Aldrich, and brain-derived natural PI(4,5)P₂ was obtained from Avanti. Stock solutions of PIPs were reconstituted in H₂O and stored at −80°C, and they were intensively sonicated before use. Estimated mole fractions in the inner leaflet of the membrane for diC8- PI(4,5)P₂ and diC8- PI(4)P were calculated based on the polynomial functions provided in Collins and Gordon (2013) (link), which provide a relationship between PIP concentrations in solution and their distribution in artificial liposomes. To scavenge PIPs, poly-L-lysine (PLL) and neomycin were used (both from Sigma-Aldrich). The phosphatidylinositol-3 kinase (PI-3K) inhibitors wortmannin and LY294,002, as well as rapamycin were from LC Laboratories. The mAChR receptors were activated by oxotremorine-M (N,N,N-trimethyl-4-(2-oxo-1-pyrolidinyl)-2-butyn-1-ammonium iodide; Oxo-M) from BioTrend Chemicals.