Cell experiment:

Purified microglia and preOLs are co-cultured using a Transwell culture system. Co-cultured cells are divided into three groups: control, lipopolysaccharide (LPS)-activated, and LPS plus Diphenyleneiodonium chloride. Microglia are cultured in Transwells over established preOL layers and exposed to either LPS (100 ng/mL), LPS+Diphenyleneiodonium chloride (10 μM) or untreated. The medium supernatants and cellular protein fractions from the co-cultures are then collected for further analysis after 48 h incubation[2].

Animal experiment:

The ddy mice (6 to 7 wk of age) are individually placed in transparent cages for 30 min before experiments. An intraplantar injection of 10 μL Diphenyleneiodonium chloride (2 mM, solvent: Kolliphor EL with 20% DMSO) is then injected into the right hindpaw with or without intraperitoneal administration with HC030031 (300 mg/kg at 0.5 h prior to injection of Diphenyleneiodonium chloride; solvent: saline with 0.5% methyl cellulose). The time spent licking or biting the injected paw is recorded for 45 min after injection[1].

Diphenyleneiodonium chloride (DPI) is an agonist of GPR3 with EC50 value of 1μM [1]. Also, diphenyleneiodonium chloride inhibits NADH oxidase (NOX), NO synthase and cytochrome P450 reductase [2] [3] [4].

G protein-coupled receptor 3 (GPR3) is an orphan G protein-coupled receptor (GPCR) with constitutive G(s) signaling activity that activates cyclic AMP and is mainly expressed in mammalian brain and oocytes [1].

DPI is a GPR3 agonist. In HEK293 cells stably expressed GPR3, DPI increased the level of intracellular cAMP, which was independent of NOX inhibition. DPI activated adenylate cyclase in cells expressing GPR3. In HeLa cells coexpressed β-arrestin2 and GPR3, DPI increased Ca2+ mobilization, GPR3 receptor desensitization and membrane localization of β-arrestin2 [1]. In HeLa cells, DPI inhibited NADPH oxidation by NOX with EC50 value of 0.1 μM [2]. In mouse macrophages, DPI irreversibly and completely inhibited NO synthase, which was blocked by NADP+, NADPH or 2’ 5’ -ADP. Also, DPI inhibited acetylcholine-induced relaxation of norepinephrine-preconstricted rabbit aortic rings with IC50 value of 300 nM [3]. DPI irreversibly inhibited NADPH cytochrome P450 oxidoreductase with Ki value of 2.8 μM [4].

In rats, DPI (10-5 mol/kg) inhibited the depressor response to acetylcholine [5].

References:
[1].  Ye C, Zhang Z, Wang Z, et al. Identification of a novel small-molecule agonist for human G protein-coupled receptor 3. J Pharmacol Exp Ther, 2014, 349(3): 437-443.
[2].  Morré DJ. Preferential inhibition of the plasma membrane NADH oxidase (NOX) activity by diphenyleneiodonium chloride with NADPH as donor. Antioxid Redox Signal, 2002, 4(1): 207-212.
[3].  Stuehr DJ, Fasehun OA, Kwon NS, et al. Inhibition of macrophage and endothelial cell nitric oxide synthase by diphenyleneiodonium and its analogs. FASEB J, 1991, 5(1): 98-103.
[4].  Tew DG. Inhibition of cytochrome P450 reductase by the diphenyliodonium cation. Kinetic analysis and covalent modifications. Biochemistry, 1993, 32(38): 10209-10215.
[5].  Wang YX, Poon CI, Poon KS, et al. Inhibitory actions of diphenyleneiodonium on endothelium-dependent vasodilatations in vitro and in vivo. Br J Pharmacol, 1993, 110(3): 1232-1238.