Characterization of the substance P receptor in guinea pig lung tissues.
The biologic activity of substance P has been demonstrated to be limited both in in vivo and in vitro by a membrane-bound protease, neutral endopeptidase (EC 3.4.24.11). The interaction of substance P with its receptor on guinea pig lung tissues was studied in the presence of an inhibitor of neutral endopeptidase under conditions that protect the peptide from degradation. Uptake of 0.1 nM [125I]-BH-substance P in lung membrane preparations was rapid at 4 degrees C, reaching equilibrium in 30 to 40 min, and binding was stable for at least 30 min thereafter. Binding was reversible and saturable. Scatchard analyses of saturation binding data are consistent with a single class of receptor molecules in both lung parenchymal and airway membranes, with a Kd of 2 to 3 nM and a receptor density of 4,000 to 5,000 fmol/g wet wt of tissue. In competitive binding experiments, neurokinin A and substance P methyl ester were equipotent and required approximately 100-fold higher concentrations to effect equivalent displacement than unlabeled substance P. Eledoisin also competed for [125I]-BH-substance P binding, but was less effective than the other analogs. The spasmogenically inactive derivative, substance P 1-9, did not compete for substance P binding at concentrations as high as 1 microM. Binding of [125I]-BH-substance P was rapidly and completely reversed by addition of 0.1 mM GTP, suggesting that association with a GTP binding protein is required for high affinity binding of substance P to its receptor in lung. The substance P receptor molecule was further characterized by covalently crosslinking [125I]-BH-substance P to membrane preparations followed by SDS-PAGE of the solubilized material.(ABSTRACT TRUNCATED AT 250 WORDS)
Coats
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SR
Department of Medicine, Harvard Medical School, Beth Israel Hospital, Boston, Massachusetts 02215.
Gerard
N P
NP
D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D006168:Guinea Pigs; D007700:Kinetics; D008168:Lung / Q000378:metabolism*; D008297:Male; D011869:Radioligand Assay; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D013373:Substance P / Q000378:metabolism*; D013388:Succinimides / Q000378:metabolism
C043194:Bolton Hunter reagent-substance P conjugate; D003432:Cross-Linking Reagents; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter; D013388:Succinimides; D013373:Substance P; C019358:disuccinimidyl suberate