Identification and characterization of the substance P receptor in sheep intestinal smooth muscle membranes.
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 degrees 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 specific binding were 0.54 nM and 83 fmol/mg of protein, respectively. The specificity of the [3H]SP labeled sites was SP greater than (4-11) SP greater than eledoisin greater than spantide greater than neurokinin-A greater than D-Pro2D-Phe7D-Trp9-SP. Neurokinin-B and senktide showed no inhibition of [3H]SP binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Immunology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania.
J F
JF
Keefer
S
S
Mong
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007422:Intestines / Q000737:chemistry; D007700:Kinetics; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000737:chemistry*; D011480:Protease Inhibitors / Q000494:pharmacology; D011869:Radioligand Assay; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter / Q000302:isolation & purification*; D012756:Sheep; D013373:Substance P / Q000378:metabolism; D013696:Temperature
D011480:Protease Inhibitors; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter; D013373:Substance P; D006160:Guanosine Triphosphate; D002118:Calcium