14,15-epoxyeicosatrienoic acid (14,15-EET), 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), internal standards (14,15-EET-d11 and 14,15-DHET-d11), 4-nitrophenyl-2S,3S-epoxy-3 phenylpropyl carbonate (S-NEPC) and 12-[[(tricyclo[18.104.22.168,7]dec-1-ylamino)carbonyl]amino]-dodecanoic acid (AUDA) were purchased from Cayman Chemical (Ann Arbor, MI). High performance liquid chromatography (HPLC) grade acetonitrile, water and ethyl acetate were purchased from EM Scientific (Gibbstawn, NJ). Acrylamide, N’N’-bis-methylene-acrylamide, β-mercaptoethanol, ammonium persulfate, glycine, and N,N,N’,N’-tetramethylethylenediamine (TEMED) were purchased from Bio-Rad Laboratories (Hercules, CA). 1-(1-methanesulfonyl-piperidin-4-yl)- 3-(4-trifluoromethoxy-phenyl)-urea (TUPS) was synthesized by Paul Jones (University of California, Davis) as described in Tsai et al (Tsai et al., 2010). Trans-4-[4-(3-Adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) was synthesized by Sung Hwang (University of California, Davis) as described in Hwang et al (Hwang, Tsai, Liu, Morisseau, & Hammock, 2007). N-[[(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl)amino]carbonyl]-L-phenylalanine methyl ester (CTK8G1143) and N-[(1S,3S)-1-[(Ethoxymethoxy)methyl]-4-(hydroxyamino)-3-methyl-4-oxobutyl]-4-phenoxybenzamide (ONO-4817) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). 4-aminophenyl mercuric acetate and doxycycline was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). The catalytic domain of human recombinant MMP-2, human recombinant pro-MMP-2, human recombinant sEH and OmniMMP® fluorogenic peptide substrate were purchased from Oxford Biomedical Research (Oxford, MI). Other chemicals were purchased from Fisher Scientific Co. (Toronto, ON, Canada), or Sigma-Aldrich Chemical Co. (St. Louis, MO).
Determination of sEH activity by spectrophotometric assay
The effect of MMP inhibitors on sEH activity was determined by using spectrophotometric substrate, S-NEPC, as previously described by Dietze et al (Dietze, Kuwano, & Hammock, 1994). The principle of assay is that the epoxide group in S-NEPC is hydrolysed by sEH, releasing 4-nitrophenol, which can be quantified spectrophotometrically. Briefly, 96-well plates were pretreated by 25 mg/mL BSA, 2% Triton X-100 and 2% Tween 20 for 2 hr. Thereafter, 10 nM of sEH was added to 0.1 mM ethylenediaminetetraacetate and 2.5 mg/mL of BSA in 76 mM phosphate buffer (pH=6.4) in the absence or presence of 0.5 µM of sEH inhibitor, t-AUCB, or 100 µM of MMP inhibitors, CTK8G1143, ONO-4817 or doxycycline. After the initiation of the reaction by the addition of S-NEPC (30 µM final concentration), test and blank (without sEH) samples were monitored at 405 nm every 10 sec for 5 min at 25°C using BioTek Synergy H1 Hybrid Reader (BioTek Instruments, Inc., VT, USA). The initial rate of product formation in each well was determined by linear regression of absorbance-time data using GraphPad Prism (version 5.0, GraphPad software, San Diego, CA).
The effect of MMP inhibitors on sEH activity of immortalized human cardiomyocytes cell line, RL14, (American Type Culture Collection, Manassas, VA) was determined. Cells were maintained in Dulbecco’s modified Eagle’s medium F-12 medium supplemented with 12.5% dialysed Fetal Bovine serum,and grown in 75 cm2 tissue culture flasks at 37°C in a 5% CO2 humidified incubator. Cells were grown at a density of 5 X 105 cells per well in a 24-well tissue culture plate. On ~80% confluence (2 days), the media was replaced by 300 µL phosphate buffered saline in the absence or presence of 0.1 µM of sEH inhibitor, t-AUCB, or 10 µM of MMP inhibitors, CTK8G1143 or ONO-4817. Thereafter, S-NEPC (30 µM final concentration) was added to the cells. After 10 min incubation at 37°C, the formation of 4-nitrophenol in test and blank (without cells) samples were measured at 405 nm in 96-well plate using BioTek Synergy H1 Hybrid Reader (BioTek Instruments, Inc., VT, USA).
Determination of sEH activity by liquid chromatography-mass spectrometry (LC-MS)
The most important endogenous substrate of sEH is 14,15-EET, which is hydrolyzed by sEH to 14,15-DHET. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) (Waters Micromass ZQ 4000 spectrometer) was used to quantify the formation of 14,15-DHET as described previously by Morisseau and Hammock (Morisseau & Hammock, 2007). Briefly, 5 nM of sEH was added to 0.1 mg/mL BSA in 100 mM phosphate buffer (pH=7.4) in the absence or presence of 0.5 µM of sEH inhibitor, t-AUCB, or 100 µM of MMP inhibitors, CTK8G1143, ONO-4817 or doxycycline. The reaction was initiated by the addition of 14,15-EET (50 µM final concentration) and terminated after 7 min at 30°C by the addition of 600 ml ice-cold acetonitrile followed by the internal standards. 14,15-EET and its corresponding 14,15-DHET in test and blank (without sEH) samples were extracted by 1ml ethyl acetate twice and dried using speed vacuum (Savant, Farmingdale, NY). Extracted 14,15-EET and 14,15-DHET were analyzed using LC-MS (Waters Micromass ZQ 4000 spectrometer) method as described previously (El-Sherbeni, Aboutabl, Zordoky, Anwar-Mohamed, & El-Kadi, 2013). To determine the kinetics of sEH inhibition, concentration range of 0.05-500 nM for TUPS or t-AUCB or 0.01-100 µM for CTK8G1143 or 0.004-100 µM for ONO-4817 were tested using the same procedure as described above. Inhibitor concentrations required for 50% inhibition (IC50) and maximal inhibition (Imax) were determined by Enzyme Kinetics module from GraphPad Prism (version 5.0, GraphPad software, San Diego, CA).
Determination of MMP-2 activity by fluorescence assay
The effect of sEH inhibitors on MMP-2 activity was determined by assessing MMP-dependent hydrolysis of Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 AcOH (OmniMMP) fluorogenic substrate as previously described by Castro et al (Castro et al., 2013). 96-well black polystyrene half-area plates were pretreated by 25 mg/mL BSA, 2% Triton X-100 and 2% Tween 20 for 2 hr. Thereafter, 0.5 nM of MMP-2 catalytic domain was added to 10 mM CaCl2, 0.05% Brij 35 and 10 mM ZnSO4 in 50 mM Tris–HCl (pH=7.0) in the absence or presence of 100 µM of MMP inhibitor, ONO-4817, or 50 µM of sEH inhibitors, AUDA, TUPS or t-AUCB. The reaction was initiated by the addition of OmniMMP substrate (30 µM final concentration). The fluorescence associated with the breakdown of OmniMMP in test and blank (without MMP-2) samples were measured at 30 s intervals (excitation and emission wavelengths of 328 nm and 393 nm) for 1 hr at 37°c using a BioTek Synergy H1 Hybrid Reader (BioTek Instruments, Inc., VT, USA). The initial rate of product formation in each well was determined by linear regression of fluorescence-time data using GraphPad Prism (version 5.0, GraphPad software, San Diego, CA).
Determination of MMP activity by zymography
The effect of sEH inhibitors on the gelatinolytic activity of MMP was also determined using substrate-gel electrophoresis (zymography) as previously described by Hawkes et al (Hawkes, Li, & Taniguchi, 2010). Briefly, pro-MMP-2 was activated by 1 mM 4-aminophenyl mercuric acetate in 150 mM NaCl, 5 mM CaCl2, 50mM Tris HCl (pH=7.5). Then, pro-MMP-2 and MMP-2 were electrophoresed on an 8% polyacrylamide gel containing 1 mg/ml gelatin for 90 min (125 V, ambient temperature). Thereafter, the gels were cut into individual strips and then washed in 2.5% Triton X-100 and 5 mM CaCl2 in 50 mM Tris-HCl buffer (pH=8.0). These strips were incubated separately overnight at 37°c in development buffer (50 mM Tris–HCl, 10 mM CaCl2, 1 µM ZnSO4, pH 7.6) in the absence or presence of 100 µM of MMP inhibitor, ONO-4817, or 50 µM of sEH inhibitors, AUDA, TUPS or t-AUCB. Gels were stained with 1 mg/mL Coomassie blue, followed by destaining and, finally, gelatinolytic activity appeared as a clear band on a blue background. Moreover, gelatinolytic activity of MMP-2 and MMP-9 in the conditioned medium of cultured RL14 was determined by zymography, where 8 µL aliquots of conditioned RL14 medium were electrophoresed as described above. After washing, gel strips were incubated separately overnight at 37°c in development buffer, as described above, in the absence or presence of 10 µM CTK8G1143 or ONO-4817.