Journal: ACS Pharmacology & Translational Science

Article Title: Toward Defining the Pharmacophore for Positive Allosteric Modulation of PTH1 Receptor Signaling by Extracellular Nucleotides

doi: 10.1021/acsptsci.8b00053

Figure Lengend Snippet: Extracellular ribose-5-phosphate (R5P) enhances PTH-induced activation of adenylyl cyclase. (A) Two-dimensional structures of protonated forms of ATP, CMP, R5P and ribose. (B) UMR-106 osteoblastic cells were transfected with GloSensor cAMP biosensor plasmid. At time 0, cells were stimulated with PTH (0.1 nM) in the presence of ATP (1.5 mM, red), R5P (1.5 mM, green), or vehicle (Veh2, blue). Luminescence intensity, which corresponds to the level of cytosolic cAMP, was measured from live cells every 1.5 min. Values are means of triplicate determinations from an individual experiment, representative of five independent experiments. (C) Cells were stimulated with PTH (0.1 nM) in the presence of vehicle (Veh2, negative control) or the indicated test substance (1.5 mM): ATP, CMP, R5P, or ribose (Ri). The maximal rate of cAMP accumulation was determined from the greatest slope of the cAMP vs time curve (e.g., panel B). In the presence of the cyclic nucleotide phosphodiesterase inhibitor IBMX, the rate of cAMP accumulation reflects the relative activity of adenylyl cyclase. In the absence of PTH, none of the test substances altered adenylyl cyclase activity. Data were normalized to the average value within each individual experiment. Vertical bars illustrate means ± SEM, data points represent values from each independent experiment (n = 5 independent experiments, each performed in duplicate or triplicate). The asterisk (∗) indicates significant difference from PTH + Veh2 (p < 0.05, based on one-way ANOVA and Bonferroni test). (D) Cells were stimulated with indicated concentrations of PTH or its vehicle in the presence of R5P (1.5 mM). Note that R5P had no detectable effect on cAMP levels in the absence of PTH. Values are means of duplicate determinations from an individual experiment, representative of four independent experiments. (E) The maximal rate of cAMP accumulation was determined for the indicated concentrations of PTH (or its vehicle, Veh1) in the presence of R5P (1.5 mM, green solid line), ATP (1.5 mM, red dashed line), or vehicle (Veh2, blue solid line). Data were normalized to the maximal rate of cAMP accumulation induced by PTH alone (1 μM). Values are means ± SEM (n = 4 independent experiments, each performed in duplicate). pEC50 values for PTH in the presence of R5P and in the presence of ATP were both significantly greater than the pEC50 for PTH alone (based on extra sum-of-squares F-test, Table 1). The maximum response to PTH was also enhanced significantly by R5P and ATP.

Article Snippet: Cells and Culture UMR-106 rat osteoblast-like cells 22 were obtained from the American Type Culture Collection (Rockville, MD).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Negative Control, Activity Assay

Journal: ACS Pharmacology & Translational Science

Article Title: Toward Defining the Pharmacophore for Positive Allosteric Modulation of PTH1 Receptor Signaling by Extracellular Nucleotides

doi: 10.1021/acsptsci.8b00053

Figure Lengend Snippet: Effect of extracellular sugar phosphates on PTH-induced adenylyl cyclase activity. (A) Two-dimensional structures of the protonated forms of glucose, glucose-1-phosphate (G1P), glucose-6-phosphate (G6P), fructose, fructose-6-phosphate (F6P), and fructose-1,6-bisphosphate (F16bP). (B) UMR-106 cells were transfected with GloSensor cAMP biosensor plasmid. Parallel samples of cells were stimulated with PTH (0.1 nM) in the presence of vehicle (Veh2, negative control) or the indicated test substance (1.5 mM): ATP (positive control), glucose (Glu), G1P, G6P, fructose (Fru), F6P, or F16bP. Data are the maximal rate of cAMP accumulation (maximal adenylyl cyclase activity) under each condition, normalized to the average value within each independent experiment. Vertical bars illustrate means ± SEM, data points represent values from each independent experiment (n = 4 independent experiments, each performed in triplicate). The asterisk (∗) indicates significant difference from PTH + Veh2 (p < 0.05, based on one-way ANOVA and Bonferroni test).

Article Snippet: Cells and Culture UMR-106 rat osteoblast-like cells 22 were obtained from the American Type Culture Collection (Rockville, MD).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Negative Control, Positive Control

Journal: ACS Pharmacology & Translational Science

Article Title: Toward Defining the Pharmacophore for Positive Allosteric Modulation of PTH1 Receptor Signaling by Extracellular Nucleotides

doi: 10.1021/acsptsci.8b00053

Figure Lengend Snippet: Potentiation of PTH-induced adenylyl cyclase activity—dependence on the concentration of glucose phosphates and PTH. UMR-106 osteoblastic cells were transfected with GloSensor cAMP biosensor plasmid. (A) Parallel samples of cells were stimulated with PTH (0.1 nM) in the presence of vehicle (Veh2) or the indicated concentrations of glucose-1-phosphate (G1P, orange symbols), or glucose-6-phosphate (G6P, brown symbols). Data are the maximal rate of cAMP accumulation under each condition, normalized to the maximal rate of cAMP accumulation induced by PTH in the presence of G1P at 3 mM. Values are means ± SEM (n = 3 independent experiments, each performed in duplicate). The asterisk (∗) indicates significant difference between equivalent concentrations of G1P and G6P (p < 0.05, based on one-way ANOVA and Bonferroni test). (B–E) The maximal rate of cAMP accumulation was determined for the indicated concentrations of PTH (or its vehicle, Veh1) in the presence of the indicated concentrations of G1P (orange lines), G6P (brown lines), ATP (red dashed lines), or their vehicle (Veh2, blue lines). Data were normalized to the maximal rate of cAMP accumulation induced by PTH alone (1 μM). (B,C) Values shown are means ± SEM (n = 3 independent experiments with duplicate determinations, performed separately for graphs B and C). (B) The pEC50 values for PTH in the presence of G1P (316 μM, 1 mM and 10 mM) were significantly greater than the pEC50 for PTH alone (based on extra sum-of-squares F-test, Table 1). Maximal PTH activity was also significantly enhanced by G1P. (C) With G6P, maximal PTH activity was not significantly increased, while pEC50 values for PTH in the presence of G6P (1 and 10 mM) were significantly greater than the corresponding pEC50 for PTH alone. (D,E) Values shown are means ± SEM (n = 3–6 independent experiments with duplicate determinations, performed separately for graphs D and E). In graph D, the pEC50 values for PTH in the presence of 1.5 mM ATP and 1.5 mM G6P were significantly greater than the pEC50 for PTH alone. In graph E, the pEC50 values for PTH in the presence of 1.5 mM ATP, 6 mM G1P, and 6 mM G6P were significantly greater than the corresponding pEC50 for PTH alone. As well, the maximum response to PTH was significantly enhanced by 1.5 mM ATP, 6 mM G1P, and 6 mM G6P.

Article Snippet: Cells and Culture UMR-106 rat osteoblast-like cells 22 were obtained from the American Type Culture Collection (Rockville, MD).

Techniques: Activity Assay, Concentration Assay, Transfection, Plasmid Preparation

Journal: ACS Pharmacology & Translational Science

Article Title: Toward Defining the Pharmacophore for Positive Allosteric Modulation of PTH1 Receptor Signaling by Extracellular Nucleotides

doi: 10.1021/acsptsci.8b00053

Figure Lengend Snippet: Potentiation of PTH-induced adenylyl cyclase activity—dependence on the concentration of fructose phosphates and PTH. UMR-106 osteoblastic cells were transfected with GloSensor cAMP biosensor plasmid. (A) Parallel samples of cells were stimulated with PTH (0.1 nM) in the presence of vehicle (Veh2) or the indicated concentration of fructose-1,6-bisphosphate (F16bP, dark purple symbols), fructose-6-phosphate (F6P, light purple symbols), or fructose (black symbols). Data are the maximal rate of cAMP accumulation under each condition, normalized to the maximal rate of cAMP accumulation induced by PTH in the presence of F16bP at 3 mM. Values shown are means ± SEM (n = 6 independent experiments, each performed in duplicate). (∗) Significantly greater effect than that induced by corresponding concentration of fructose; (†) significantly greater effect than that induced by corresponding concentration of F6P; at concentrations ≥ 3 mM, fructose significantly enhanced PTH-stimulated adenylyl cyclase activity; p < 0.05, based on one-way ANOVA and Bonferroni test. (B–E) The maximal rate of cAMP accumulation was determined for the indicated concentrations of PTH (or its vehicle, Veh1) in the presence of the indicated concentrations of F6P (light purple lines), F16bP (dark purple lines), ATP (red dashed lines), or their vehicle (Veh2, blue lines). Data were normalized to the maximal rate of cAMP accumulation induced by PTH alone (1 μM). Values plotted are means ± SEM (n = 3 independent experiments with duplicate determinations, performed separately for each panel). (B,C) The pEC50 values for PTH in the presence of F6P (316 μM, 1 mM, and 10 mM) and F16bP (316 μM, 1 mM, and 10 mM) were significantly greater than the corresponding pEC50 for PTH alone, based on extra sum-of-squares F-test (Table 1). As well, the maximum response to PTH was significantly enhanced by F6P (1 and 10 mM) and F16bP (316 μM, 1 mM, and 10 mM). In graph D, the pEC50 values for PTH in the presence of 1.5 mM ATP, 1.5 mM F6P, and F16bP were significantly greater than the pEC50 for PTH alone. In graph E, the pEC50 values for PTH in the presence of 1.5 mM ATP, 6 mM F6P, and 6 mM F16bP were significantly greater than the corresponding pEC50 for PTH alone. As well, in graphs D and E, the maximum response to PTH was significantly enhanced at all concentrations of test substances.

Article Snippet: Cells and Culture UMR-106 rat osteoblast-like cells 22 were obtained from the American Type Culture Collection (Rockville, MD).

Techniques: Activity Assay, Concentration Assay, Transfection, Plasmid Preparation

Journal: ACS Pharmacology & Translational Science

Article Title: Toward Defining the Pharmacophore for Positive Allosteric Modulation of PTH1 Receptor Signaling by Extracellular Nucleotides

doi: 10.1021/acsptsci.8b00053

Figure Lengend Snippet: Fructose does not alter potentiation of PTH-stimulated adenylyl cyclase activity by fructose-1,6-bisphosphate (F16bP) or ATP. UMR-106 cells were transfected with GloSensor cAMP biosensor plasmid. Parallel samples of cells were treated with PTH (0.1 nM) or its vehicle in the presence or absence of the indicated concentrations of fructose, F16bP and ATP. Data are the maximal rate of cAMP accumulation (maximal adenylyl cyclase activity) under each condition, normalized to the average value within each independent experiment. Vertical bars illustrate means ± SEM; data points represent values from each independent experiment (n = 7 independent experiments, each performed in quadruplicate). The asterisk (∗) indicates significant difference from PTH alone (p < 0.05, based on one-way ANOVA and Bonferroni test). Fructose had no significant effect on the ability of F16bP or ATP to potentiate signaling (as indicated by the horizontal bar labeled n.s.).

Article Snippet: Cells and Culture UMR-106 rat osteoblast-like cells 22 were obtained from the American Type Culture Collection (Rockville, MD).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Labeling