In this regard, in order to eliminate the effects potentially mediated by TLRs, in our study we used cultured SH-SY5Y cells, wherein the expression of TLR 7/8 and 9 is not reported in literature. Conclusion Targeting NO-cGMP-PKG-ERK signaling pathway may represent an interesting approach for the development of new drugs in the treatment of memory dysfunctions occurring in neurodegenerative and psychiatric diseases, Cefsulodin sodium among others Alzheimers disease and dementia. a model of SH-SY5Y neuroblastoma cell line, the signal transduction pathway activated by extracellular GUA. Altogether, our results showed that: (i) in addition to an enhanced phosphorylation of ASK1, p38 and JNK, likely linked to a non-massive and transient ROS production, the PKB/NO/sGC/cGMP/PKG/ERK cascade seems to be the main signaling pathway elicited by extracellular GUA; (ii) the activation of this pathway occurs in a pertussis-toxin sensitive manner, thus suggesting the involvement of a putative G protein coupled receptor; (iii) the GUA-induced NO production, strongly reduced by cell pre-treatment with L-NAME, is usually negatively modulated by the EPAC-cAMP-CaMKII pathway, which causes the over-expression of GDA that, in ATF1 turn, reduces the levels of GUA. These molecular mechanisms activated by GUA may be useful to support our previous observation showing that GUA improves learning and memory functions through the stimulation of NO signaling pathway, and underscore the therapeutic potential of oral administration of guanine for treating memory-related disorders. for 20 min, 4C. Before performing Immunoblot, a sample buffer (5 Laemmli buffer with 10% mercaptoethanol) was added to melted lysates 1:4. Protein concentrations were obtained using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, United States) based on the Bradford method. An equal amount of 50C70 g of protein was resolved by 10% sodium dodecyl sulfateCpolyacrylamide gel electrophoresis (SDS-PAGE). The resolved proteins were transferred onto a nitrocellulose membrane and then incubated with blocking buffer 1 TBS made up of 0.1% Tween-20 (TBST) and 3% BSA or 5% non-fat dry milk for 2 h, RT, and subsequently probed with specific primary antibody at 4C, overnight. After washing with TBST, the membrane was further probed with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies at RT for 1 h. Membranes were finally washed, before subjecting them to ECL Plus Immunoblot Detection Reagent (Amersham, GE Healthcare). The immunoreactive bands were visualized under a chemiluminescence detection system (UVItec, Cambridge, United Kingdom). Band intensity data were obtained using Quantity One software (Bio-Rad Laboratories). Blotting membranes were stripped and re-probed with anti-actin antibody as equal loading control. Estimates of phosphorylated proteins are presented as densitometric ratios, normalized to the corresponding total protein content. Apart from PNP antibody (1:500), all primary antibodies [Phospho-ASK1 (Ser83), Phospho-p38 MAPK (Thr180/Tyr182), Phospho-SAPK/JNK (Thr183/Tyr185), Phospho-PKC (Ser660), Phospho-Akt (Thr450), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), Guanase Deaminase, -Actin] were diluted 1:1000 in 3% BSA/1 TBS/0.1% Tween 20 or 2.5% non-fat dry milk/1 TBS/0.1% Tween 20. The secondary antibody was used at 1:2500 dilution in 3% BSA/1 TBS/0.1% Tween 20 or 2.5% non-fat dry milk/1 TBS/0.1% Tween 20. Measurement of Cellular Reactive Oxygen Species (ROS) The amount of intracellular reactive oxygen species (ROS) was measured by using the probe H2DCF-DA (Ha et al., 1997), which diffuses into the cells and is oxidized to the green fluorescent compound 2,7-dichlorofluorescein (DCF) upon reaction with intracellular hydrogen peroxide or low-molecular-weight hydroperoxides. Cells were seeded at 1 106 cells/well in 6-well culture plates and incubated overnight. After exposure to different concentration of GUA for 30 min, cells were incubated with 5 M H2DCF-DA for 30 min, in the dark, at 37C. At the end of incubation, the cells were washed with PBS and fluorescence was measured at an excitation wavelength of 480 nm and an emission wavelength Cefsulodin sodium of 540 nm in a fluorescence microplate reader Cefsulodin sodium (Thermo Fischer Scientific, Monza, Italy). ROS production was determined by analyzing DCF fluorescence normalized for total protein content. The fluorescence intensity was proportional to the amount of ROS produced by cells. Determination of Nitric Oxide Synthase (NOS) Activity Nitric oxide synthase activity was measured from the conversion of L-[3 H]-arginine to L-[3 H]-citrulline based on the method of Bredt et al. (1991) with modifications. Cefsulodin sodium SH-SY5Y cells were grown overnight in 6-well plates. After 24-h starvation, cells were uncovered for 30 min to 50 M GUA, 5 M L-NAME or 2 M Ionomycin, the latter used as positive control. When used in combination, L-NAME was administered 15 min before cell exposure to GUA or Ionomycin. Thereafter, cells were washed three times with ice-cold 1X PBS, scraped in 1X PBS made up of 1 mM EDTA, and centrifuged for 10 min at 1200 0.05. All experiments were performed at least three times. Results The Levels of Guanine-Based Purines in SH-SY5Y Culture Media Are Controlled by Specific Nucleobase and Nucleoside Transporters and by the Presence of.Membranes were finally washed, before subjecting them to ECL Plus Immunoblot Detection Reagent (Amersham, GE Healthcare). involvement of a putative G protein coupled receptor; (iii) the GUA-induced NO production, strongly reduced by cell pre-treatment with L-NAME, is usually negatively modulated by the EPAC-cAMP-CaMKII pathway, which causes the over-expression of GDA that, in turn, reduces the levels of GUA. These molecular mechanisms activated by GUA may be useful to support our previous observation showing that GUA improves learning and memory functions through the stimulation of NO signaling pathway, and underscore the therapeutic potential of oral administration of guanine for treating memory-related disorders. for 20 min, 4C. Before performing Immunoblot, a sample buffer (5 Laemmli buffer with 10% mercaptoethanol) was added to melted lysates 1:4. Protein concentrations were obtained using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, United States) based on the Bradford method. An equal amount of 50C70 g of protein was resolved by 10% sodium dodecyl sulfateCpolyacrylamide gel electrophoresis (SDS-PAGE). The resolved proteins were transferred onto a nitrocellulose membrane and then incubated with blocking buffer 1 TBS made up of 0.1% Tween-20 (TBST) and 3% BSA or 5% non-fat dry milk for 2 h, RT, and subsequently probed with specific primary antibody at 4C, overnight. After washing with TBST, the membrane was further probed with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies at RT for 1 h. Membranes were finally washed, before subjecting them to ECL Plus Immunoblot Detection Reagent (Amersham, GE Healthcare). The immunoreactive bands were visualized under a chemiluminescence detection system (UVItec, Cambridge, United Kingdom). Band intensity data were obtained using Quantity One software (Bio-Rad Laboratories). Blotting membranes were stripped and re-probed with anti-actin antibody as equal loading control. Estimates of phosphorylated proteins are presented as densitometric ratios, normalized to the corresponding total protein content. Apart from PNP antibody (1:500), all primary antibodies [Phospho-ASK1 (Ser83), Phospho-p38 MAPK (Thr180/Tyr182), Phospho-SAPK/JNK (Thr183/Tyr185), Phospho-PKC (Ser660), Phospho-Akt (Thr450), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), Guanase Deaminase, -Actin] were diluted 1:1000 in 3% BSA/1 TBS/0.1% Tween 20 or 2.5% non-fat dry milk/1 TBS/0.1% Tween 20. The secondary antibody was used at 1:2500 dilution in 3% BSA/1 TBS/0.1% Tween 20 or 2.5% non-fat dry milk/1 TBS/0.1% Tween 20. Measurement of Cellular Reactive Oxygen Species (ROS) The amount of intracellular reactive oxygen species (ROS) was measured by using the probe H2DCF-DA (Ha et al., 1997), which diffuses into the cells and is oxidized to the green fluorescent compound 2,7-dichlorofluorescein (DCF) upon reaction with intracellular hydrogen peroxide or low-molecular-weight hydroperoxides. Cells were seeded at 1 106 cells/well in 6-well culture plates and incubated overnight. After exposure to different concentration of GUA for 30 min, cells were incubated with 5 M H2DCF-DA for 30 min, in the dark, at 37C. At the end of incubation, the cells were washed with PBS and fluorescence was measured at an excitation wavelength of 480 nm and an emission wavelength of 540 nm in a fluorescence microplate reader (Thermo Fischer Scientific, Monza, Italy). ROS production was determined by analyzing DCF fluorescence normalized for total protein content. The fluorescence intensity was proportional to the amount of ROS produced by cells. Determination of Nitric Oxide Synthase (NOS) Activity Nitric oxide synthase activity was measured from the conversion of L-[3 H]-arginine to L-[3 H]-citrulline based on the method of Bredt et al. (1991) with modifications. SH-SY5Y cells were grown overnight in 6-well plates. After 24-h starvation, cells were uncovered for 30 min to 50 M GUA, 5 M L-NAME or 2 M Ionomycin, the latter used as positive control. When used in combination, L-NAME was administered 15 min before cell exposure to GUA or Ionomycin. Thereafter, cells were washed three times with ice-cold 1X PBS, scraped.