1. The possible existence of multiple receptors for substance P (SP) was investigated by examining the relative pharmacological potencies of SP and related peptides in contracting guinea pig ileum, in potentiating electrically evoked contractions of rat vas deferens preparations and in competing for 3H-SP receptor binding in rat brain membranes, and by comparing the extent of cross-tachyphylaxis of various analogues with SP in the guinea pig ileum. 2. Different rank orders of potencies were observed among SP, its C-terminal fragments, analogues and related tachykinins in the different test systems, and these could not be explained by differential access to the target organ receptors. 3. In contrast to SP and physalaemin, both eledoisin and a metabolically stable SP analogue, [pGlu5, MePhe8, Sar9]-SP5-11 exhibited differential recovery from SP tachyphylaxis in the guinea pig ileum, and part of their spasmogenic action in this preparation was atropine-sensitive. 4. The results suggest the possible existence of multiple SP receptors, and the specificity of those in the brain may be different from those in the gut. The structural and pharmacological basis for subdividing tachykinins into SP-physalaemin and eledoisin-kassinin families is also discussed.

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A membrane-bound enzyme which degrades substance P (an undecapeptide) has been purified from human brain. The properties of this enzyme suggest that it may be involved in the physiological inactivation of the peptide by neural tissues. Enzyme activity was extracted from a membrane fraction of human diencephalon with a non-ionic detergent, Brij 35, and activity was monitored by measuring the disappearance of added substance P using radioimmunoassay, bioassay or radiochemical assay. The enzyme was purified about 1000-fold by chromatography on DEAE-cellulose, hydroxyapatite and Sephadex gel filtration columns. To identify the cleavage sites in substance P, the peptide was incubated with the purified enzyme and the breakdown products were separated by reverse-phase high-performance liquid chromatography and identified by amino acid analysis. The results suggested that the enzyme preparation was functionally homogeneous and it cleaved substance P between Gln6-Phe', Phe7-Phe' and Phe'-Gly', with no exopeptidase action. The enzyme had a pH optimum in the range 7-9 and was strongly inhibited by metal-chelating agents, but not affected by most other peptidase inhibitors; it can thus be classified as a neutral metallo-endopeptidase. The enzyme was thermolabile and had a molecular weight of 40000 -50000 as estimated by gel filtration, density-gradient ultracentrifugation and sodium dodecylsulphate gel electrophoresis. The highly purified substance-P-degrading enzyme could be distinguished from previously described peptidases for which substance P is a substrate. An important feature was that substance P was the preferred substrate among various other neuropeptides tested.Substance P, an undecapeptide (Fig.l) The mechanism involved in the inactivation of substance P after its synaptic release is unknown, although a number of enzymes capable of degrading this peptide have been reported to exist in brain [I0 -121. These include both cytosolic [12-151 and membrane-bound [13,14,16-181 substance-Pdegrading enzyme activities. A cytosolic enzyme has been partially purified from rat brain and shown to release phenylalanine and leucine preferentially from substance P, suggesting cleavage at two or more internal peptide bonds (Gln6-Phe7 or Phe7-Phe' and Glyg-Leu") [19]. Akopyan and coworkers [20] purified a neutral endopeptidase from bovine hypothalamus which degraded luteinizing-hormone-releasing hormone (luliberin) and somatostatin and also cleaved substance P, probably between Gln6-Phe' and Phe7-Phe'. In addition, a prolyl endopeptidase has been purified from rabbit brain and shown to cleave substance P between Abbreviations. Substance P, the undecapeptide Arg-Pro-Lys-ProGln-Gln-Phe-Phe-Gly-Leu-MetNHz; [3H]substance P, [PheK-3H]substance P ; Ala-Ala-Phe-Nan, alanyl-alanyl-phenylalanyl p-nitroanilide; Fao-Gly-LeuNHZ, 3-(2-furylacryloyl)-glycyl-~-leucine amide; Hepes, 4-(2-hydroxyethyl)-l -piperazineethanesulphonic acid.Enzymes. Substance-P-degrading enzyme (EC 3.4.24.-) ; angiotensinconverting enzyme or dipeptidyl carboxypeptidase...