Table 1.
Test compounds.
Fig 1.
Saturation binding of [125I]-NKA to human recombinant NK2 receptors.
[125I]-NKA binding to human NK2 receptors at increasing radioligand concentrations. Saturation data were subjected to nonlinear regression analysis. Values are means ± SD of triplicate determinations of TB and duplicates of NSB at each radioligand concentration. TB: total binding; SB: specific binding; NSB: nonspecific binding.
Fig 2.
Representative curves showing displacement of [125I]-NKA binding to human recombinant NK2 receptors by NKA, [Lys5,MeLeu9,Nle10]-NKA(4–10), [Lys5,MeLeu9]-NKA(4–10) and [Arg5,MeLeu9]-NKA(4–10).
Displacement curves were fitted using a one-site model. Values are means ± SD from a representative experiment performed in duplicate.
Table 2.
Displacement of [125I]-NKA binding, intracellular calcium response and stimulation of cyclic AMP in CHO cells expressing human recombinant NK2 receptors.
Fig 3.
Concentration-response curves for NKA and substance P on intracellular calcium response using human cloned NK2 receptors.
Data are expressed as % of maximal response to NKA (30 nM). Each data point is the mean ± SD of data from an individual experiment performed in duplicate.
Fig 4.
Concentration-response curves for individual test compounds on intracellular calcium levels using human cloned NK2 and NK1 receptors.
Data are expressed as % of maximal response to 30 nM NKA (for NK2 receptors) or 30 nM substance P (for NK1 receptors). Triangles are NK2 receptors; circles are NK1 receptors. Each data point is the mean ± SD of data from an individual experiment performed in duplicate.
Fig 5.
Representative calcium traces for NKA, substance P, [Lys5,MeLeu9,Nle10]-NKA(4–10) and [β-Ala8]-NKA(4–10) using human recombinant NK2 and NK1 receptors.
Compounds were tested at the EC50 concentration. Data are expressed as relative fluorescence units (RFU) measured over a period of 200 seconds. A: NK2 receptors, B: NK1 receptors.
Fig 6.
Correlation between displacement of [125I]-NKA binding (pKi) and intracellular calcium response (pEC50) at human recombinant NK2 receptors expressed in CHO cells.
Scatterplot and regression analysis of pKi versus pEC50 values at human recombinant NK2 receptors for a series of NK2 agonists. Solid line: regression analysis; dotted lines: 95% confidence intervals. For [Arg5,β-Ala8]-NKA(4–10) and [β-Ala8]-NKA(4–10), pIC50 values were used.
Fig 7.
Concentration-response curves for stimulation of cyclic AMP by selected compounds using human cloned NK2 receptors.
Data are expressed as % of maximal response to 30 nM NKA. Triangles are [Lys5,MeLeu9,Nle10]-NKA(4–10); inverted triangles are [β-Ala8]-NKA(4–10); squares are NKA; circles are substance P. Data are the mean ± SD of one representative experiment performed in duplicate.
Fig 8.
Saturation binding of [3H]-Septide to human recombinant NK1 receptors.
[3H]-Septide binding to human NK1 receptors expressed in CHO membranes with increasing radioligand concentrations. Saturation data were subjected to nonlinear regression analysis. Values are means ± SD of triplicate determinations of TB and duplicates of NSB at each radioligand concentration. TB: total binding; SB: specific binding; NSB: nonspecific binding.
Fig 9.
Representative curves showing displacement of [3H]-septide binding to human recombinant NK1 receptors by substance P, NKA, septide, [Lys5,MeLeu9,Nle10]-NKA(4–10), [Lys5,MeLeu9]-NKA(4–10) and [Arg5,MeLeu9]-NKA(4–10).
Displacement curves were fitted using a one-site model. Values are means ± SD from a representative experiment performed in duplicate.
Table 3.
Displacement of [3H]-Septide binding, intracellular calcium response and stimulation of cyclic AMP in CHO cells expressing human recombinant NK1 receptors.
Fig 10.
Concentration-response curves for substance P and NKA on intracellular calcium levels using human cloned NK1 receptors.
Data are expressed as % of maximal response to substance P (30 nM). Each data point is the mean ± SD of data from an individual experiment performed in duplicate.
Fig 11.
Correlation between displacement of [3H]-Septide binding (pKi) and intracellular calcium response (pEC50) at human recombinant NK1 receptors expressed in CHO cells.
Scatterplot and regression analysis of pKi versus pEC50 values at human recombinant NK1 receptors for a series of NK2 agonists. Solid line: regression analysis; dotted lines: 95% confidence intervals.
Fig 12.
Concentration-response curves for stimulation of cyclic AMP by selected compounds using human cloned NK1 receptors.
Data are expressed as % of maximal response to 30 nM substance P. Triangles are [Lys5,MeLeu9,Nle10]-NKA(4–10); inverted triangles are [β-Ala8]-NKA(4-10); squares are NKA; circles are substance P. Data are the mean ± SD of one representative experiment performed in duplicate.
Table 4.
Ratio of Ki values (in nM) for displacement of [125I]-NKA and [3H]-Septide binding.
Table 5.
Ratio of EC50s (in nM) for intracellular calcium response.
Table 6.
Ratio of EC50s (in nM) for cAMP stimulation.