Striatal neurons cultivated for 7 days in culture were incubated for 4 h (A) or 72 h (B) with dynorphin A (1C17) (10 M) in the presence or absence of CNQX (10 M), MK (+)801 (10 M), and/or CX546 [1-(1,4-Benzodioxan-6-ylcarbonyl)piperidine]. and cultivated on poly-D-lysine (0.1 mg/ml) coated 24-well plates at 35 C in 5% Rabbit Polyclonal to GANP CO2/ 95% air flow. Neuronal viability MK 8742 (elbasvir) was assessed as explained before (Goody et al., 2003). Briefly, coverslips were securely attached into 24-well Costar plates using Syl-Gard (Dow Corning, MI, USA) and the underside of wells obtained to aid in locating the same neurons repeatedly. Time-lapse digital microscopic images of individual neurons were collected using a Spot 2 digital camera (Diagnostic Tools, MK 8742 (elbasvir) Sterling Heights, MI, USA) and a Nikon Diaphot inverted microscope with phase contrast optics and a 20X objective (Goody et al., 2003). Z-DEVD-FMK was dissolved in dimethyl sulfoxide to the final volume 0.05% v/v dimethyl sulfoxide in all treatment groups. Cells were pretreated with the cell-permeable caspase-3 inhibitor Z-DEVD-FMK for 4 h prior to exposure to dynorphin A (1C17). Caspase-3 Activity Caspase-3 protease activity was measured as previously explained (Rigamonti et al., 2000). Embryonic mouse striatal neurons were continually treated with dynorphin A (1C17) (10 M) in the presence and absence of numerous antagonists and harvested in ice-cold harvesting buffer (25 mM HEPES, pH 7.5, 5 mM EDTA, 1 mM EGTA, 5 mM magnesium chloride, 10 mM sucrose, 5 mM dithiothreitol, 1% 3-[-(3-chloramidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), 10 g/ml pepstatin, 10 g/ml leupeptin and 1 mM PMSF). After freezing and thawing 3 times, the cell lysates were centrifuged for 10 min at 5000 rpm, and the supernatants were centrifuged at 10,000g for 60 min. The cell lysates therefore acquired were stored at ?80 C. Lysates were incubated at 37 C inside a buffer comprising 25 mM HEPES, pH 7.5, 10% sucrose, 0.1% CHAPS, and 1 MK 8742 (elbasvir) mM dithiothreitol supplemented with 50 M Ac-DEVD-7- amino-4-methylcoumarin (AMC) in 96-well Costar plates. As a negative control, Ac-DEVD-CHO, a caspase-3 inhibitor, was added to the cell lysates 30 min before incubation with caspase-3 substrate, Ac-DEVD-AMC. The increase in fluorescence after the cleavage of the fluorogenic AMC moiety was monitored using a Cytofluor 4000 fluorimeter (Perspective Biosystems, Framingham, MA, USA) at 360 nm excitation and 460 nm emission wavelengths. Caspase-3 activity was normalized to protein concentration and indicated as fluorescence devices or devices per milligram of total cytosolic protein. Mitochondrial Cytochrome c Launch Launch of cytochrome c from mitochondria was measured as previously explained (Yang et al., 1997). After incubation of the striatal neurons with dynorphin A in the presence and absence of numerous antagonists, cells were harvested with ice-cold buffer A comprising 20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and 250 mM sucrose. Cell homogenates were centrifuged 1st at 750 g for 10 min at 4 C and the supernatant was centrifuged at 12,000g for 30 min at 4 C. The supernatant portion and the pellet comprising mitochondria resuspended in buffer A were freezing at ?80 C until assayed. The supernatant (cytosolic) portion and the mitochondrial pellet portion were utilized for the detection MK 8742 (elbasvir) of cytochrome c. Equivalent amounts of cytosolic and mitochondrial proteins from control and treated cultures were immunoprecipitated with 1 g of purified mouse anti-cytochrome c monoclonal antibody (clone.6H2.B4 mouse IgG from BD PharMingen) for 3 h at 4 C and incubated 10 l of a 50% slurry of protein G-agarose beads in PBS overnight at 4 C. Immunoprecipitates were then washed three times with PBS and resuspended in sodium dodecyl sulfate (SDS)-sample buffer and electrophoresed on SDS-polyacrylamide gel (SDS-PAGE). Proteins were electrophoresed to blotting PVDF membranes and then incubated with main monoclonal antibodies against cytochrome c (7H8.2C12 mouse IgG, PharMingen, 1:333 dilution) at space temp for 3 h. Finally, proteins were visualized using a peroxidase-conjugated antibody to mouse IgG and a chemiluminescence detection system. After immunoblotting, the bands on the.