Effect of Nitrous Oxide on Intracellular Events: METHODS

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17 Dec 2009

Culture of GT1-7 Cell Line
GT1-7 cells (gift of Dr P.M. Mellon, University of California, San Diego) were maintained in DMEM-4.5 g/L glucose (Life Technologies, Inc, Rockville, MD) supplemented with 10% fetal calf serum, 100 units/mL, 100 mg/mL streptomycin, and 0.0025 mg/mL amphotericin В at 37°C in a humidified atmosphere of 5% C02, 95% air or 60% N20, 35% 02, and 5% C02. For performing various tests, cells were plated in 100mm tissue culture dishes. Experiments were typically performed at day 3 of plating cells onto the tissue culture dishes.

Cell Viability Test by Tryphan Blue Exclusion
GT1-7 cells were cultured and plated in 100-mm tissue culture dishes as described above. Cells were incubated in a humidified atmosphere of 60% N20, 35% 02, and 5% C02, or 5% C02 and 95% air as control. Cells were harvested in 2 mL of trypsin/EDTA and counted under a microscope. Cell death was evaluated with the tryphan blue (0.4% tryphan blue in HBSS) exclusion method. Under microscopic examination, nonviable cells exhibit blue cytoplasm, whereas live cells exhibit clear cytoplasm. The percentage of viable cells was calculated as discussed below:

  • viable cells (%) number of viable cells X 100
  • total number of cells (dead and viable)

Nitrous Oxide Effect on GnRH mRNA Levels
GT1-7 cells were plated into filter-topped T-75 cm2 flasks at a density of 3 X 106 in 10 mL of media. Before treatment, the culture flasks were left overnight to allow cells to adhere. The flasks were then placed at 37°C in a humidified atmosphere of 60% N20, 35% 02, and 5% C02, or 95% air and 5% C02 for a set period of time (24 hours). In this series of studies, we chose 60% N20 on the basis of the results of our previous studies and reported concentrations used in people to achieve dissociation sedation and total analgesia. At the end of exposure, the cells were washed with ice-cold phosphate-buffered saline and then isolated using trypsin/ EDTA. Total RNA was then extracted from the cellular pellet using Trizol (Gibco Invitrogen Corporation, Carlsbad, Calif). Briefly, cells were added to 1 mL Trizol reagent, vortexed, and allowed to sit for 10 minutes. The tubes were centrifuged at 14,000g, and the supernatant was removed to an RNAse-free Eppendorf tube, vortex ed in 200 \xL chloroform, and allowed to sit for 10 minutes. The tubes were centrifuged at 14,000g, the top layer was carefully removed to another RNAse-free tube, and 500 |xL of isopropanol was added, agitated, and allowed to sit for 10 minutes. The tubes were centrifuged as described above. The RNA pellet was washed in 75% ethanol, centrifuged to remove excess ethanol, air-dried, and resuspended in 25 |ULL of DEPC-treated water. The RNA was dissolved by heating to 60°C briefly. Total RNA was fractionated on an 8% agarose and formaldehyde gel. The separated RNA was then blotted to nylon membranes by capillary transfer and fixed by ultraviolet cross-linking. cDNA probes were isolated, using standard protocols, from plasmids purchased through ATCC (Manassas, Va). The probes were labeled by the method of random hexagon priming (Prime-A-gene, Promega, NJ). Labeled probes were then purified and used to probe Northern blots. Hybridization was performed in aqueous solution using PerfectHyb system (Sigma Chemical Co, St Louis, Mo). The hybridized blots were then washed and exposed to x-ray film overnight. The same blots were reprobed with housekeeping gene G3PDH to normalize the amount of RNA loaded onto the gel. The intensity of Northern blot signals was determined using the program Sigma Gel (SPSS Science, Chicago, 111). Signal intensity was corrected for differences in RNA concentration by normalizing them with respect to the housekeeping gene G3PDH. The results were then plotted using Sigma Plot (SPSS Science). eriacta tablets

Western Blot Analyses of Extracellular Signal-Regulated Kinase (p44/42 or ERK1/2), p63 Tyrosine, Mitogen-Activated Protein Kinase (p38) Phosphorylation, Transcription Factor C-fos and Inhibitor of Nuclear Factor Kappa В (IKB) GT1-7 cells were cultured and plated in 100-mm tissue culture dishes as described above.

Effect of N20 Exposure on p44/42 Phosphorylation. Cells were deprived of serum for 2 hours to induce quiescence and exposed to N20 or air. Cells were collected at various time points. Phosphorylated p44/42 and total p44/42 were probed by anti-phos-pho-p44/42 antibody and anti-p44/42 antibody, respectively, and visualized by enhanced chemilumines-cence (ECL).

Effect of NzO Exposure on p63 Phosphorylation. Monolayers of GT1-7 cells in 100-mm dishes were exposed to either N20 or air for various time periods. The cells were collected in RIPA buffer. Phosphorylated p63 and actin were probed by anti-tyrosine antibody (PY99) and anti-actin antibody, respectively, and visualized by ECL.

Effect of N20 Exposure on p38 Phosphorylation. Cells were deprived of serum for 5 hours and treated and exposed to 60% N20 or air. Cells were collected at various time points. Phosphorylated p38 and total p38 were probed with anti-p38 antibody and anti-total p38, respectively, and visualized by ECL.

Effect ofN20 Exposure on Transcription Factor C-fos. Cells with or without serum deprivation were exposed to N20 or air for 3 or 12 hours. C-fos was probed by anti-c-fos antibody and visualized by ECL.

Effect of N20 Exposure on IKB. Cells were deprived of serum for 5 hours and treated and exposed to N20 or air. Cells were collected at various time points. IKB was probed by anti-kB antibody and visualized by ECL. cialis canadian pharmacy

After exposure to N20 or air for varying periods, cells were harvested in 0.4 mL of RIPA buffer (150 mmol/ L NaCL, 1 mmol/L EDTA, 1 mmol/L EGTA, 0.1% Tri-ton-100, 0.1% Nonidet P-40, 1% glycerol, 1 mmol/L dithiotreitol, 10 mmol/L Tris-Cl, pH 7.5) supplemented with complete protease inhibitor cocktail (Roche Diagnostics Corporation, Roche Molecular Biochemicals, Indianapolis, Ind), 2 mmol/L sodium vanadate and 1 mmol/L PMSF. After the addition of RIPA buffer, the cells were scraped into 1.5-mL Ependorf tubes on ice and rotated 30 minutes at 4°C. Cell debris was removed by centrifugation at 14,000g for 10 minutes at 4°C. The protein concentration of the supernatant was determined by Lowry protein assay. The extracts were mixed with equal volume of sample buffer and boiled for 10 minutes. Twenty micrograms of protein were resolved by SDS-PAGE on 10% gels and transferred to nitrocellulose membranes, which were then blocked in 5% nonfat milk in TBST (137 mmol/L NaCl, 0.1% Tween 20, 20 mmol/L Tris-HCl, pH 7.6) for 1 hour at room temperature. Membranes were incubated with antibodies (each at 1:1000 dilution) against phosphorylated and activated forms of ERK1/2, p38 (New England Biolabs, Beverly, Mass), PY99, c-fos, and IKB (Santa Cruz Biotechnology, Santa Cruz, Calif). Actin antibody was procured from New England Biolabs. The membranes were incubated in horseradish peroxidase-linked secondary antibody for 60 minutes, washed 3 times in TBST for 15 minutes and visualized by ECL (Amersham Pharmacia Biotech, Piscataway, NJ).

Effect of NzO Exposure on KCl-Stimulated GnRH Release From GT1-7 Cells

GT1-7 cells were cultured and plated on 100-mm tissue culture dishes as described above. Each experiment was conducted on 3 plates of cells. Cells were deprived of serum for 5 hours. Cells were incubated in a humidified atmosphere of 60% N20, 35% 02, and 5% C02, or 5% C02 and 95% air (control) for 6 or 24 hours. Sixty mmol/L KC1 was added to the media, which were collected at 20 minutes and pooled. Cells were pelleted for intracellular GnRH estimation after lysing the cells. Cell lysates were also prepared as described above. Cell ly-sates and media were extracted for peptides as described before. Briefly, 0.5 mL of 2N acetic acid was added to each sample and mixed well. The samples were centrifuged at 14,000g for 15 minutes. The super natants were collected, freeze-dried, and reconstituted in 0.5 mL of assay buffer provided with the assay kit (Peninsula Laboratories Inc, San Carlos, Calif). GnRH was estimated by using the ELISA kit (Peninsula Laboratories).
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Statistics and Data Presentation
Comparisons between only two means that a 2-tailed t test was used to calculate significant differences between N20-exposed and control groups. Comparisons among multiple means were achieved by analysis of variance (ANOVA) with Scheffe’s post-hoc comparisons, using computer software (Stat View, V 4.0, Abacus Concepts, Berkeley, Calif). The minimum significance level for all tests was P < .05.


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