"Specific" binding of [3H]-[Sar9,Met(O2)11]-substance P to tissue culture plates is found when substance P is used to define non-specific binding.
Following incubation at 4 degrees C, [3H]-[Sar9,Met(O2)11]-substance P bound to tissue culture plates in a manner displaceable by substance P (SP). The IC50 for SP was about 100 nM. Although the total binding to the wells could be reduced by increasing the assay bovine serum albumin concentration, by the presence of divalent ions and by gelatin-precoating of the wells, the remaining binding could be inhibited by about 70% by SP, and would thus appear as "specific" binding in assays. The non-peptide antagonist (+/-)CP-96345 also inhibited the binding to the wells at high concentrations (IC50 about 2 mcM), whereas physalaemin did not. Physalaemin is thus a better agent to define non-specific binding. The consequences of the "specific" binding to the culture wells observed with SP to define non-specific binding are demonstrated in two cell systems: in cultured human umbilical vein endothelial cells, an apparent receptor density of about 100,000/cell is shown to be due mainly to binding to the wells, rather than the cells. In UC11MG cells, which express 40,000-60,000 NK-1 recognition sites/cell, the use of SP to define binding gives artifactually high KD and Bmax values, and an artifactually low potency (and Hill slope) of (+/-)CP-96345 when compared with data obtained using physalaemin to define the non-specific binding.
Fowler
C J
CJ
Department of Biochemical Pharmacology, Astra Pain Control AB, Södertälje, Sweden.
Martinsson
T
T
Brännström
G
G
D001254:Astrocytoma / Q000378:metabolism; D001667:Binding, Competitive / Q000187:drug effects; D001713:Biphenyl Compounds / Q000494:pharmacology; D046508:Culture Techniques; D006801:Humans; D006993:Hypnotics and Sedatives / Q000494:pharmacology; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D010803:Physalaemin / Q000494:pharmacology; D018040:Receptors, Neurokinin-1 / Q000378:metabolism; D013373:Substance P / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D001713:Biphenyl Compounds; D006993:Hypnotics and Sedatives; D018040:Receptors, Neurokinin-1; C062618:substance P, Sar(9)-Met(O2)(11)-; D010803:Physalaemin; D013373:Substance P; C066892:CP 96345