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Petr Svoboda - One of the best experts on this subject based on the ideXlab platform.
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Dose-response curves of DADLE-stimulated [35S]GTPγS binding in LDM and 0.025% Brij-58-treated LDM prepared from PTX-treated δ-OR-Gi1α cells.
2015Co-Authors: Lenka Roubalova, Miroslava Vosahlikova, Jana Brejchova, Jan Sykora, Vladimir Rudajev, Petr SvobodaAbstract:Sucrose gradient fractions 1–6 were combined together, mixed and used for determination of the dose-response curves of DADLE-stimulated [35S]GTPγS binding as described in Methods. (A). DADLE concentration inducing the half-maximum response [EC50 (DADLE)] was determined by GraphPad Prism4. (B) The ratio between the DADLE-stimulated and basal level of [35S]GTPγS binding was expressed as % of agonist stimulation over the basal level; the basal level represented 100%. Results represent the average of 3 experiments ± SEM. LDM, fractions collected from gradient containing no detergent; 0.025% Brij-58-treated LDM, fractions collected from gradient containing 0.025% Brij-58.
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DADLE- stimulated [35S]GTPγS binding in membranes prepared from PTX-untreated cells; dose-response curves.
2015Co-Authors: Lenka Roubalova, Miroslava Vosahlikova, Jana Brejchova, Jan Sykora, Vladimir Rudajev, Petr SvobodaAbstract:ΔDADLE (pmol × mg-1), net increment of agonist stimulation at saturating concentration of DADLE; ΔDADLE/Bmax ratio, ΔDADLE normalized to receptor number; EC50(nM), DADLE concentration inducing half-maximum stimulation of [35S]GTPγS binding.* (p
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Net increment of DADLE-stimulated [35S]GTPγS binding and [3H]DADLE binding in sucrose fractions collected from PTX-treated δ-OR-Gi1α cells.
2015Co-Authors: Lenka Roubalova, Miroslava Vosahlikova, Jana Brejchova, Jan Sykora, Vladimir Rudajev, Petr SvobodaAbstract:ΔDADLE (pmol × mg-1), net increment of agonist stimulated [35S]GTPγS binding at 10 μM DADLE; B (pmol × mg-1), [3H]DADLE binding at saturating 10 nM concentration; ΔDADLE/B, ratio between ΔDADLE and B.* (p
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Subcellular fractionation of PTX-treated δ-OR-Gi1α cells; density gradient profiles of basal and DADLE-stimulated [35S]GTPγS binding.
2015Co-Authors: Lenka Roubalova, Miroslava Vosahlikova, Jana Brejchova, Jan Sykora, Vladimir Rudajev, Petr SvobodaAbstract:Pertussis toxin-treated δ-OR-Gi1α cells were homogenized and fractionated in the absence (no detergent) or presence of 0.025% Brij-58 in sucrose gradient as in studies of PTX-untreated cells (see Methods). (A) Basal (○) and DADLE-stimulated (●) [35S]GTPγS binding was measured in fractions collected from the top to bottom of the centrifuge tube and expressed as total (pmol × ml-1) or specific binding (pmol × mg-1) in a given fraction. (B) Net increment of DADLE stimulation (ΔDADLE) was calculated as the difference between specific DADLE-stimulated and basal level of [35S]GTPγS binding and expressed as pmol × mg-1. Results represent the average of 3 experiments ± SEM. The significance of difference between the specific DADLE-stimulated and basal [35S]GTPγS binding in fractions was determined by Student´s t-test (see S8 Table).
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Basal and DADLE-stimulated [32P]GTPase activity in PNS, LDM and 0.025% Brij-58-treated LDM; determination of Vmax and Km values.
2015Co-Authors: Lenka Roubalova, Miroslava Vosahlikova, Jana Brejchova, Jan Sykora, Vladimir Rudajev, Petr SvobodaAbstract:Sucrose gradient fractions 1–6 prepared by fractionation of cell homogenate prepared from δ-OR-Gi1α cells in the absence (LDM) or presence of 0.025% Brij-58 (0.025% Brij-58-treated LDM) were combined together, mixed and used for determination of basal and DADLE-stimulated [32P]GTPase as described in Methods. Upper panels. [32P]GTPase activity (pmol × min-1 × mg-1) was measured in the absence (○) or presence (●) of 100 μM DADLE at increasing concentrations of GTP. Lower panels. The data were expressed as Eadie-Hofstee plots and Vmax (GTP) and Michaelis-Menten constant Km (GTP) calculated by GraphPad Prism4. Results represent the average of 3 experiments ± SEM. The significance of difference of Vmax and Km values (between the basal and DADLE-stimulated [32P]GTPase) in PNS, LDM and 0.025% Brij-58-treated LDM was determined by Student´s t-test. The significance of difference of Vmax and Km values (for both basal and DADLE-stimulated [32P]GTPase) in PNS versus LDM versus 0.025% Brij-58-treated LDM was determined by one-way ANOVA followed by Bonferroni´s multiple comparison test using GraphPad Prism4 (see S3 Table).
Yulong L. Chen - One of the best experts on this subject based on the ideXlab platform.
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Crosstalk between Delta Opioid Receptor and Nerve Growth Factor Signaling Modulates Neuroprotection and Differentiation in Rodent Cell Models
International Journal of Molecular Sciences, 2013Co-Authors: Michael Huchital, Yulong L. ChenAbstract:Both opioid signaling and neurotrophic factor signaling have played an important role in neuroprotection and differentiation in the nervous system. Little is known about whether the crosstalk between these two signaling pathways will affect neuroprotection and differentiation. Previously, we found that nerve growth factor (NGF) could induce expression of the delta opioid receptor gene (Oprd1, dor), mainly through PI3K/Akt/NF-κB signaling in PC12h cells. In this study, using two NGF-responsive rodent cell model systems, PC12h cells and F11 cells, we found the delta opioid neuropeptide [d-Ala2, d-Leu5] enkephalin (DADLE)-mediated neuroprotective effect could be blocked by pharmacological reagents: the delta opioid antagonist naltrindole, PI3K inhibitor LY294002, MAPK inhibitor PD98059, and Trk inhibitor K252a, respectively. Western blot analysis revealed that DADLE activated both the PI3K/Akt and MAPK pathways in the two cell lines. siRNA Oprd1 gene knockdown experiment showed that the upregulation of NGF mRNA level was inhibited with concomitant inhibition of the survival effects of DADLE in the both cell models. siRNA Oprd1 gene knockdown also attenuated the DADLE-mediated neurite outgrowth in PC12h cells as well as phosphorylation of MAPK and Akt in PC12h and F11 cells, respectively. These data together strongly suggest that delta opioid peptide DADLE acts through the NGF-induced functional G protein-coupled Oprd1 to provide its neuroprotective and differentiating effects at least in part by regulating survival and differentiating MAPK and PI3K/Akt signaling pathways in NGF-responsive rodent neuronal cells.
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Article Crosstalk between Delta Opioid Receptor and Nerve Growth Factor Signaling Modulates Neuroprotection and Differentiation in Rodent Cell Models
2013Co-Authors: Dwaipayan Sen, Michael Huchital, Yulong L. ChenAbstract:Abstract: Both opioid signaling and neurotrophic factor signaling have played an important role in neuroprotection and differentiation in the nervous system. Little is known about whether the crosstalk between these two signaling pathways will affect neuroprotection and differentiation. Previously, we found that nerve growth factor (NGF) could induce expression of the delta opioid receptor gene (Oprd1, dor), mainly through PI3K/Akt/NF-κB signaling in PC12h cells. In this study, using two NGF-responsive rodent cell model systems, PC12h cells and F11 cells, we found the delta opioid neuropeptide [D-Ala2, D-Leu5] enkephalin (DADLE)-mediated neuroprotective effect could be blocked by pharmacological reagents: the delta opioid antagonist naltrindole, PI3K inhibitor LY294002, MAPK inhibitor PD98059, and Trk inhibitor K252a, respectively. Western blot analysis revealed that DADLE activated both the PI3K/Akt and MAPK pathways in the two cell lines. siRNA Oprd1 gene knockdown experiment showed that the upregulation of NGF mRNA level was inhibited with concomitant inhibition of the survival effects of DADLE in the both cell models. siRNA Oprd1 gene knockdown also attenuated th
Lenka Roubalova - One of the best experts on this subject based on the ideXlab platform.
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Dose-response curves of DADLE-stimulated [35S]GTPγS binding in LDM and 0.025% Brij-58-treated LDM prepared from PTX-treated δ-OR-Gi1α cells.
2015Co-Authors: Lenka Roubalova, Miroslava Vosahlikova, Jana Brejchova, Jan Sykora, Vladimir Rudajev, Petr SvobodaAbstract:Sucrose gradient fractions 1–6 were combined together, mixed and used for determination of the dose-response curves of DADLE-stimulated [35S]GTPγS binding as described in Methods. (A). DADLE concentration inducing the half-maximum response [EC50 (DADLE)] was determined by GraphPad Prism4. (B) The ratio between the DADLE-stimulated and basal level of [35S]GTPγS binding was expressed as % of agonist stimulation over the basal level; the basal level represented 100%. Results represent the average of 3 experiments ± SEM. LDM, fractions collected from gradient containing no detergent; 0.025% Brij-58-treated LDM, fractions collected from gradient containing 0.025% Brij-58.
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DADLE- stimulated [35S]GTPγS binding in membranes prepared from PTX-untreated cells; dose-response curves.
2015Co-Authors: Lenka Roubalova, Miroslava Vosahlikova, Jana Brejchova, Jan Sykora, Vladimir Rudajev, Petr SvobodaAbstract:ΔDADLE (pmol × mg-1), net increment of agonist stimulation at saturating concentration of DADLE; ΔDADLE/Bmax ratio, ΔDADLE normalized to receptor number; EC50(nM), DADLE concentration inducing half-maximum stimulation of [35S]GTPγS binding.* (p
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Net increment of DADLE-stimulated [35S]GTPγS binding and [3H]DADLE binding in sucrose fractions collected from PTX-treated δ-OR-Gi1α cells.
2015Co-Authors: Lenka Roubalova, Miroslava Vosahlikova, Jana Brejchova, Jan Sykora, Vladimir Rudajev, Petr SvobodaAbstract:ΔDADLE (pmol × mg-1), net increment of agonist stimulated [35S]GTPγS binding at 10 μM DADLE; B (pmol × mg-1), [3H]DADLE binding at saturating 10 nM concentration; ΔDADLE/B, ratio between ΔDADLE and B.* (p
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Subcellular fractionation of PTX-treated δ-OR-Gi1α cells; density gradient profiles of basal and DADLE-stimulated [35S]GTPγS binding.
2015Co-Authors: Lenka Roubalova, Miroslava Vosahlikova, Jana Brejchova, Jan Sykora, Vladimir Rudajev, Petr SvobodaAbstract:Pertussis toxin-treated δ-OR-Gi1α cells were homogenized and fractionated in the absence (no detergent) or presence of 0.025% Brij-58 in sucrose gradient as in studies of PTX-untreated cells (see Methods). (A) Basal (○) and DADLE-stimulated (●) [35S]GTPγS binding was measured in fractions collected from the top to bottom of the centrifuge tube and expressed as total (pmol × ml-1) or specific binding (pmol × mg-1) in a given fraction. (B) Net increment of DADLE stimulation (ΔDADLE) was calculated as the difference between specific DADLE-stimulated and basal level of [35S]GTPγS binding and expressed as pmol × mg-1. Results represent the average of 3 experiments ± SEM. The significance of difference between the specific DADLE-stimulated and basal [35S]GTPγS binding in fractions was determined by Student´s t-test (see S8 Table).
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Basal and DADLE-stimulated [32P]GTPase activity in PNS, LDM and 0.025% Brij-58-treated LDM; determination of Vmax and Km values.
2015Co-Authors: Lenka Roubalova, Miroslava Vosahlikova, Jana Brejchova, Jan Sykora, Vladimir Rudajev, Petr SvobodaAbstract:Sucrose gradient fractions 1–6 prepared by fractionation of cell homogenate prepared from δ-OR-Gi1α cells in the absence (LDM) or presence of 0.025% Brij-58 (0.025% Brij-58-treated LDM) were combined together, mixed and used for determination of basal and DADLE-stimulated [32P]GTPase as described in Methods. Upper panels. [32P]GTPase activity (pmol × min-1 × mg-1) was measured in the absence (○) or presence (●) of 100 μM DADLE at increasing concentrations of GTP. Lower panels. The data were expressed as Eadie-Hofstee plots and Vmax (GTP) and Michaelis-Menten constant Km (GTP) calculated by GraphPad Prism4. Results represent the average of 3 experiments ± SEM. The significance of difference of Vmax and Km values (between the basal and DADLE-stimulated [32P]GTPase) in PNS, LDM and 0.025% Brij-58-treated LDM was determined by Student´s t-test. The significance of difference of Vmax and Km values (for both basal and DADLE-stimulated [32P]GTPase) in PNS versus LDM versus 0.025% Brij-58-treated LDM was determined by one-way ANOVA followed by Bonferroni´s multiple comparison test using GraphPad Prism4 (see S3 Table).
Michael Huchital - One of the best experts on this subject based on the ideXlab platform.
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Crosstalk between Delta Opioid Receptor and Nerve Growth Factor Signaling Modulates Neuroprotection and Differentiation in Rodent Cell Models
International Journal of Molecular Sciences, 2013Co-Authors: Michael Huchital, Yulong L. ChenAbstract:Both opioid signaling and neurotrophic factor signaling have played an important role in neuroprotection and differentiation in the nervous system. Little is known about whether the crosstalk between these two signaling pathways will affect neuroprotection and differentiation. Previously, we found that nerve growth factor (NGF) could induce expression of the delta opioid receptor gene (Oprd1, dor), mainly through PI3K/Akt/NF-κB signaling in PC12h cells. In this study, using two NGF-responsive rodent cell model systems, PC12h cells and F11 cells, we found the delta opioid neuropeptide [d-Ala2, d-Leu5] enkephalin (DADLE)-mediated neuroprotective effect could be blocked by pharmacological reagents: the delta opioid antagonist naltrindole, PI3K inhibitor LY294002, MAPK inhibitor PD98059, and Trk inhibitor K252a, respectively. Western blot analysis revealed that DADLE activated both the PI3K/Akt and MAPK pathways in the two cell lines. siRNA Oprd1 gene knockdown experiment showed that the upregulation of NGF mRNA level was inhibited with concomitant inhibition of the survival effects of DADLE in the both cell models. siRNA Oprd1 gene knockdown also attenuated the DADLE-mediated neurite outgrowth in PC12h cells as well as phosphorylation of MAPK and Akt in PC12h and F11 cells, respectively. These data together strongly suggest that delta opioid peptide DADLE acts through the NGF-induced functional G protein-coupled Oprd1 to provide its neuroprotective and differentiating effects at least in part by regulating survival and differentiating MAPK and PI3K/Akt signaling pathways in NGF-responsive rodent neuronal cells.
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Article Crosstalk between Delta Opioid Receptor and Nerve Growth Factor Signaling Modulates Neuroprotection and Differentiation in Rodent Cell Models
2013Co-Authors: Dwaipayan Sen, Michael Huchital, Yulong L. ChenAbstract:Abstract: Both opioid signaling and neurotrophic factor signaling have played an important role in neuroprotection and differentiation in the nervous system. Little is known about whether the crosstalk between these two signaling pathways will affect neuroprotection and differentiation. Previously, we found that nerve growth factor (NGF) could induce expression of the delta opioid receptor gene (Oprd1, dor), mainly through PI3K/Akt/NF-κB signaling in PC12h cells. In this study, using two NGF-responsive rodent cell model systems, PC12h cells and F11 cells, we found the delta opioid neuropeptide [D-Ala2, D-Leu5] enkephalin (DADLE)-mediated neuroprotective effect could be blocked by pharmacological reagents: the delta opioid antagonist naltrindole, PI3K inhibitor LY294002, MAPK inhibitor PD98059, and Trk inhibitor K252a, respectively. Western blot analysis revealed that DADLE activated both the PI3K/Akt and MAPK pathways in the two cell lines. siRNA Oprd1 gene knockdown experiment showed that the upregulation of NGF mRNA level was inhibited with concomitant inhibition of the survival effects of DADLE in the both cell models. siRNA Oprd1 gene knockdown also attenuated th
Jana Brejchova - One of the best experts on this subject based on the ideXlab platform.
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Dose-response curves of DADLE-stimulated [35S]GTPγS binding in LDM and 0.025% Brij-58-treated LDM prepared from PTX-treated δ-OR-Gi1α cells.
2015Co-Authors: Lenka Roubalova, Miroslava Vosahlikova, Jana Brejchova, Jan Sykora, Vladimir Rudajev, Petr SvobodaAbstract:Sucrose gradient fractions 1–6 were combined together, mixed and used for determination of the dose-response curves of DADLE-stimulated [35S]GTPγS binding as described in Methods. (A). DADLE concentration inducing the half-maximum response [EC50 (DADLE)] was determined by GraphPad Prism4. (B) The ratio between the DADLE-stimulated and basal level of [35S]GTPγS binding was expressed as % of agonist stimulation over the basal level; the basal level represented 100%. Results represent the average of 3 experiments ± SEM. LDM, fractions collected from gradient containing no detergent; 0.025% Brij-58-treated LDM, fractions collected from gradient containing 0.025% Brij-58.
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DADLE- stimulated [35S]GTPγS binding in membranes prepared from PTX-untreated cells; dose-response curves.
2015Co-Authors: Lenka Roubalova, Miroslava Vosahlikova, Jana Brejchova, Jan Sykora, Vladimir Rudajev, Petr SvobodaAbstract:ΔDADLE (pmol × mg-1), net increment of agonist stimulation at saturating concentration of DADLE; ΔDADLE/Bmax ratio, ΔDADLE normalized to receptor number; EC50(nM), DADLE concentration inducing half-maximum stimulation of [35S]GTPγS binding.* (p
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Net increment of DADLE-stimulated [35S]GTPγS binding and [3H]DADLE binding in sucrose fractions collected from PTX-treated δ-OR-Gi1α cells.
2015Co-Authors: Lenka Roubalova, Miroslava Vosahlikova, Jana Brejchova, Jan Sykora, Vladimir Rudajev, Petr SvobodaAbstract:ΔDADLE (pmol × mg-1), net increment of agonist stimulated [35S]GTPγS binding at 10 μM DADLE; B (pmol × mg-1), [3H]DADLE binding at saturating 10 nM concentration; ΔDADLE/B, ratio between ΔDADLE and B.* (p
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Subcellular fractionation of PTX-treated δ-OR-Gi1α cells; density gradient profiles of basal and DADLE-stimulated [35S]GTPγS binding.
2015Co-Authors: Lenka Roubalova, Miroslava Vosahlikova, Jana Brejchova, Jan Sykora, Vladimir Rudajev, Petr SvobodaAbstract:Pertussis toxin-treated δ-OR-Gi1α cells were homogenized and fractionated in the absence (no detergent) or presence of 0.025% Brij-58 in sucrose gradient as in studies of PTX-untreated cells (see Methods). (A) Basal (○) and DADLE-stimulated (●) [35S]GTPγS binding was measured in fractions collected from the top to bottom of the centrifuge tube and expressed as total (pmol × ml-1) or specific binding (pmol × mg-1) in a given fraction. (B) Net increment of DADLE stimulation (ΔDADLE) was calculated as the difference between specific DADLE-stimulated and basal level of [35S]GTPγS binding and expressed as pmol × mg-1. Results represent the average of 3 experiments ± SEM. The significance of difference between the specific DADLE-stimulated and basal [35S]GTPγS binding in fractions was determined by Student´s t-test (see S8 Table).
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Basal and DADLE-stimulated [32P]GTPase activity in PNS, LDM and 0.025% Brij-58-treated LDM; determination of Vmax and Km values.
2015Co-Authors: Lenka Roubalova, Miroslava Vosahlikova, Jana Brejchova, Jan Sykora, Vladimir Rudajev, Petr SvobodaAbstract:Sucrose gradient fractions 1–6 prepared by fractionation of cell homogenate prepared from δ-OR-Gi1α cells in the absence (LDM) or presence of 0.025% Brij-58 (0.025% Brij-58-treated LDM) were combined together, mixed and used for determination of basal and DADLE-stimulated [32P]GTPase as described in Methods. Upper panels. [32P]GTPase activity (pmol × min-1 × mg-1) was measured in the absence (○) or presence (●) of 100 μM DADLE at increasing concentrations of GTP. Lower panels. The data were expressed as Eadie-Hofstee plots and Vmax (GTP) and Michaelis-Menten constant Km (GTP) calculated by GraphPad Prism4. Results represent the average of 3 experiments ± SEM. The significance of difference of Vmax and Km values (between the basal and DADLE-stimulated [32P]GTPase) in PNS, LDM and 0.025% Brij-58-treated LDM was determined by Student´s t-test. The significance of difference of Vmax and Km values (for both basal and DADLE-stimulated [32P]GTPase) in PNS versus LDM versus 0.025% Brij-58-treated LDM was determined by one-way ANOVA followed by Bonferroni´s multiple comparison test using GraphPad Prism4 (see S3 Table).
