Animals: induction of chronic inflammation, drug treatment and cell isolation
Pathogen free New Zealand White male rabbits (Charles River Laboratories International Inc.) were used for the study. Four cohorts of animals, 4 animals in each cohort, were used: normal, inflamed, normal + ketotifen and inflamed + ketotifen. Chronic intestinal inflammation was induced in rabbits by intragastrically inoculating them with Eimeria magna oocytes as previously reported [18,20]. Normal and inflamed rabbits that were injected intramuscularly with saline were used as untreated controls. For drug treatment, normal and inflamed rabbits were intramuscularly injected with ketotifen (10 mg/kg body weight), a noncompetitive H1-antihistamine and mast cell stabilizer, 12 and 13 days post coccidia inoculation and the animals were euthanized on day 14. All animal handling, treatments and euthanization were carried out according to the protocol approved by the Institutional Animal Care and Use Committee of West Virginia University (ACUC protocol # 12–0102).
Villus and crypt cells were isolated from the rabbit ileum by a calcium chelation technique as previously described [18]. Briefly, a 3-ft section of ileum was filled and incubated with cell isolation buffer (0.15 mM EDTA, 112 mM NaCl, 25 mM NaHCO3, 2.4 mM K2HPO4, 0.4 mM KH2PO4, 2.5 mM L-glutamine, 0.5 mM β-hydroxybutyrate, and 0.5 mM dithiothreitol; gassed with 95% O2 and 5% CO2, pH 7.4, at 37°C) for 3 min and gently palpitated for another 3 min to facilitate cell dispersion. The buffer was then drained out from the ileal section, phenylmethylsulfonyl fluoride was added, and the suspension was centrifuged at 100 g for 3 min. Cells to be used for BBM vesicle (BBMV) preparation were frozen immediately in liquid nitrogen and stored at −80°C until required.
β-Hexosaminidase assay
When activated mast cells undergo degranulation they release a substantial amount of enzymes that mediate several inflammatory pathways. One such enzyme that is released is β-Hexosaminidase, the levels of which are estimated as an index of mast cell degranulation during inflammation. In the present study, β-Hexosaminidase assay was performed as previously reported [21], to detect mast cell degranulation in vivo. Briefly, enterocytes lysed with 1% Triton X-100 were incubated with the substrate solution (P-nitrophenyl-Nacetyl-β-D-glucosaminide from Sigma, St Louis, MO) in 0.1 M citrate buffer (pH 4.5) for 60 min at 37°C. The reaction was terminated by the addition of 0.2 M NaOH/0.2 M glycine. Using an enzyme-linked immunosorbent assay reader, absorbance was acquired at 405 nm and the results were expressed as percentage β-Hexosaminidase activity relative to control.
Na-K-ATPase measurement
Na-K-ATPase was measured as Pi liberated [22] in cellular homogenates from the same amount of cells from all experimental conditions as previously described [9,18,22]. Enzyme-specific activity was expressed as nanomoles of Pi released per milligram protein per minute.
Uptake studies in villus and crypt cells
Intact villus and crypt cell uptakes were done using previously described protocol [9,18]. Briefly, villus or crypt cells (100 mg wet wt.) were washed and resuspended in HEPES buffer containing 0.2 mM glutamine, 4.5 mM KCl, 1.2 mM KH2PO4, 1.0 mM MgSO4, 1.25 mM CaCl2, 20 mM HEPES, and either 130 mM sodium chloride or choline chloride and was gassed with 100% O2 (pH 7.4 at 37°C). Ten μCi of 3H-glutamine was added to 1 mL of cell suspension in HEPES buffer and 100 μL aliquots were removed at 2 minutes and mixed with 1 mL ice-cold stop solution (choline-HEPES buffer) to stop the uptake. The mixture was then filtered on 0.65 μm Millipore (Bedford, MA; HAWP) filters and washed twice with ice cold-stop solution. The filter was dissolved in 5 mL Ecoscint and the radioactivity was determined in a Beckman Coulter LS6500 Scintillation counter.
BBMV preparation and uptake studies
BBMV from rabbit ileal villus and crypt cells were prepared by CaCl2 precipitation and differential centrifugation as previously described [18]. Uptake studies were performed by rapid filtration technique [18,20]. Briefly, 5 μL of BBMV resuspended in vesicle medium (100 mM choline chloride, 0.10 mM MgSO4, 50 mM HEPES-Tris (pH 7.5), 50 mM mannitol, 50 mM KCl) were voltage clamped with 10 μM valinomycin and 100 mM carbonyl cyanide p-(tri-fluoromethoxy) phenyl-hydrozone. The vesicles were then incubated in 95 μL of reaction medium (50 mM HEPES-Tris buffer (pH 7.5), 0.2 mM glutamine, 20 μCi 3H-glutamine, 0.10 mM MgSO4, 50 mM KCl, 50 mM mannitol, 100 mM of either NaCl or choline chloride) and at desired time points uptake was arrested by mixing with ice-cold stop solution of 50 mM HEPES-Tris buffer (pH 7.5), 0.10 mM MgSO4, 75 mM KCl, and 100 mM choline chloride. The stopped reaction mixture was filtered on a 0.45 μm Millipore (HAWP) filter and washed with 10 mL ice-cold stop solution, twice. Filters were then dissolved in Ecoscint solution and radioactivity was determined in a Beckman Coulter LS6500 Scintillation counter.
Kinetic studies
Na-dependent glutamine uptake was performed as described above in BBMV from all experimental conditions. Kinetic parameters were derived from Na-dependent glutamine uptake at 6 seconds performed at varying concentrations of extra vesicular glutamine (0.2, 0.5, 1, 5, 10, 25, 50, 75 and 100 mM). Uptake values were analyzed for simple Michaelis-Menten kinetics using a non-linear regression data analysis using GraphPad Prism 4 (San Diego, CA).
Western blot analysis
BBM protein was used for all Western blot experiments. BBM was solubilized in a buffer consisting of 50 mM Tris–HCl (pH 7.4), 1% Igepal, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, supplemented with a mixture of a protease inhibitor (Sigma, St.Louis, MO). Equal amounts of protein were denatured in a sodium dodecyl sulfate (SDS)/sample buffer (20 mM Tris pH 7, 12% glycerol, 2% SDS, 0.01% bromophenol blue, and freshly added 1 mM Dithiothreitol) and separated by electrophoresis on a 4%–20% gradient Gel (Bio-Rad Laboratories, Hercules, CA). Proteins on the gel were transferred to a polyvinylidene membrane which was blocked with 5% dry milk in TBS (20 mM Tris, pH 7.5, 150 mM NaCl) with 0.1% Tween-20 and then incubated with a goat polyclonal antibody against B0AT1, overnight at 4°C followed by incubation with an anti-goat IgG conjugated to horseradish peroxidase (Jackson Immunoresearch Laboratories, West Grove, PA) for an hour at room temperature. For SN2/SNAT5, a chicken polyclonal antibody against SN2/SNAT5 was used as the primary antibody and an anti-chicken IgG was used as the secondary. Both the primary antibodies were obtained through the custom antibody services provided by Invitrogen. ECL Western blotting detection reagent (GE Healthcare Bio-Sciences, Piscataway, NJ) was used to detect the immobilized protein. The resultant chemiluminescence was detected using biomax film (Kodak, Rochester, NY) and the intensity of the bands was analyzed by a FluorChem™ instrument (Alpha Innotech, San Leandro, CA).
Statistical analysis
Data are shown as mean ± SEM in all figures. All individual uptakes were done in triplicate. The ‘n’ number for any set of uptake experiments or Western blot analysis refers to vesicle or isolated cell preparations from different animals. Data were analyzed using one-way analysis of variance (ANOVA) to assess the significance between control and experimental groups by using GraphPad Instat 4 (San Diego, CA) for statistical analysis and a p value of less than 0.01 was considered significant.