Identification and characterization of the substance P receptor in sheep intestinal smooth muscle membranes
Keefer, J.F.; Mong, S.
Journal of Pharmacology and Experimental Therapeutics 255(1): 120-127
1990
ISSN/ISBN: 0022-3565 PMID: 1698967 Document Number: 356945
Substance P (SP) is one of the endogenous tachykinin peptides implicated in neurogenic inflammation and may be critically involved in diseases as diverse as asthma, arthritis and inflammatory bowel disease. The current study was initiated to identify a rich source of SP receptor that would be amenable for studying the regulatory mechanism of the receptor. By using a radioligand receptor binding technique, sheep ileal smooth muscle membranes showed a much higher density of [3H]SP specific binding than other non-neural rat or sheep tissues and organs surveyed. Of the protease inhibitors tested, only phosphoramidon, a specific and potent enkephalinase inhibitor, prevented the degradation of [3H]SP and enhanced [3H]SP binding to the membrane. [3H]SP binding to the specific binding sites in the membranes was time-dependent and reached a steady state after 60 min at 22.degree.C in 25 mM Tris.NH3 (pH 7.4). Calcium and magnesium ions enhanced [3H]SP specific binding. Saturation binding studies showed that the dissociation constant (Kd) and the density of maximum binding sites for [3H]SP binding were 0.54 nM and 83 fmol/mg of protein, respectively. The specificity of the [3H]SP labeled sites was SP > (4-11) SP > eledoisin > spantide > neurokinin-A > D-Pro2D-Phe7D-Trp9-SP. Neurokinin-B and senktide showed no inhibition of [3H]SP binding. These unlabeled ligands completed with 2 nM (2H]SP binding resulting in IC50 values at 6, 35, 1200, 3000, 5000 and 20,000 nM, respectively. Binding of [3H]SP to the receptors in sheep ileal smooth muscle membranes was inhibited by guanosine-5-O-3-thiotriphosphate and guanyl-5-yl-imidophosphate in a concentration-dependent manner. Sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis of receptor cross-linked to [125I]SP and solubilized, yielded a single band with a molecular weight of 44,000. These results indicate that [3H]SP binds to a highly selective, G proteins coupled, 44 KDa, neurokinin-1 receptor to sheep ileal smooth membranes.