Pd146176

Exogenous K⁺ was added as an isotonic physiological salt solution in which all the NaCl was replaced with an equivalent amount of KCl. Concentrations of K⁺ used are expressed as final bath concentration. Ebselen (2-Phenyl-1,2-benzisoselenazol-3(2H)-one), L-NAME (N^G-nitro-L-arginine methyl ester), Nordihydroguaiaretic acid (NDGA), papaverine HCl, PD 146176 (6,11-Dihydro[1]benzothiopyrano[4,3-b]indole), tempol (4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl), TRAM-34 (1-[(2-Chlorophenyl)diphenylmethyl]-1H-pyrazole) and U46619 (9,11-Dideoxy-11α,9α-epoxymethanoprostaglandin F_2α) were all obtained from Sigma (Poole, U.K.). Apamin and iberotoxin, from Latoxan (Valence, France). SLIGRL-NH₂ (serine, leucine, isoleucine, glycine, arginine, leucine) from Auspep (Parkville, Australia). 8-iso PGF_2α from Cayman-Europe (Tallinn, Estonia). tAUCB (trans-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid) was a generous gift from Professor Bruce D. Hammock (University of California, Davis). All stock solutions (100 mM) were prepared in dimethylsulfoxide (DMSO) except L-NAME, apamin, iberiotoxin, papaverine and SLIGRL that were dissolved in 0.9% NaCl and tempol which was dissolved in ultrapure water. Vehicle controls were performed when necessary.

Following tail transection at 3 dpf, zebrafish larvae were screened at 4 hpi for neutrophil recruitment. Selected larvae were immersed in 1 μM PGE₂ (Sigma-Aldrich) at 8 hpi with counts performed at 12 hpi to look for accelerated inflammation resolution and 24 hpi for correction of phenotype assays. Concentrations of PGE₂ ranged from 0.01 to 10 μM for dose-response assays. The EP4 antagonist AH23848 (Cayman Chemicals) was used at a final concentration of 1 μM. Fish were incubated at 2 dpf, and tail transection was performed at 3 dpf. PD146176, a 15-LO inhibitor (Sigma-Aldrich), was injected at 6 hpi into the Duct of Cuvier to a final concentration of 27 nM in 2 dpf larvae. LXA₄ (Cayman Chemicals) was injected into the Duct of Cuvier to a final concentration of 27 nM at 2 dpf, followed by tail transection and 6 hpi counts.