Michael R. Hanley

The possible existence of multiple receptors for substance P

Summary1.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.

Tissue selectivity of substance P alkyl esters: Suggesting multiple receptors

Previous studies from this laboratory suggested that two subtypes of substance P receptor may exist, based on the observations that substance P and related peptides did not exhibit complete cross-desensitisation on guinea-pig ileum, and that two distinct rank orders of potency of tachykinins were observed in various test systems. The present study has added support to this hypothesis by extending the screening of tachykinins to further bioassays and by testing novel analogues. In particular, C-terminal alkyl esters of substance P were found to exhibit a high degree of selectivity to one putative receptor subtype. The synthesis of the alkyl esters by esterification of substance P free acid is described.

Specific binding of 3H-substance P to rat brain membranes.

The undecapeptide substance P is a putative neurotransmitter in the mammalian central nervous system (CNS), and may be associated with pain fibres in the spinal cord1,2. Radiolabelled derivatives of other neuropeptides havd been used to demonstrate specific interactions with receptor sites on brain membranes3–6, and this approach has now been explored with substance P. We have now prepared [4-3H-Phe8]-substance P and we find that it binds reversibly to a saturable population of sites in rat brain particulate fractions. Scatchard analysis of concentration-dependent saturation of binding indicates a single population of non-interacting sites with a high affinity (Kd = 0.38 nM) and a low density (Bmax = 27.2 fmol per mg protein). Kinetic analyses indicate an apparent dissociation equilibrium constant of 0.46 nM. A variety of neurotransmitter amines and amino acids, and other peptides do not compete at the substance P sites, but structurally related peptides or shorter C-terininal fragments of substance P are active. The rank order of potency of these substance P-related peptides agrees with that reported for their effects in depolarizing spinal cord neurones7. The regional distribution of the specific binding sites for 3H-substance P parallels that of substance P immunoreactivity, being high in the hypothalamus and low in the cerebellum and cerebral cortex. The characteristics of the 3H-substance P binding sites are consistent with those expected for substance P receptors.

Pharmacological characterization of a receptor for calcitonin gene-related peptide on rat, L6 myocytes

1 The L6 myocyte cell line expresses high affinity receptors for calcitonin gene‐related peptide (CGRP) which are coupled to activation of adenylyl cyclase. The biochemical pharmacology of these receptors has been examined by radioligand binding or adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) accumulation. 2 In intact cells at 37°C, human and rat α‐ and β‐CGRP all activated adenylyl cyclase with EC50s of about 1.5 nm. A number of CGRP analogues containing up to five amino acid substitutions showed similar potencies. In membrane binding studies at 22°C in 1 mm Mg2+, the above all bound to a single site with IC50s of 0.1–0.4 nm. 3 The fragment CGRP(8–37) acted as a competitive antagonist of CGRP stimulation of adenylyl cyclase with a calculated Kd of 5 nm. The Kd determined in membrane binding assays was lower (0.5 nm). 4 The N‐terminal extended human α‐CGRP analogue Tyro‐CGRP activated adenylyl cyclase and inhibited [125I]‐iodohistidyl‐CGRP binding less potently than human α‐CGRP (EC50 for cyclase = 12 nm, IC50 for binding = 4 nm). 5 The pharmacological profile of the L6 CGRP receptor suggests that it most closely resembles sites on skeletal muscle, cardiac myocytes and hepatocytes. The L6 cell line should be a stable homogeneous model system in which to study CGRP mechanisms and pharmacology.

Substance P Receptors

Substance P (SP) is an undecapeptide with powerful spasmogenic activity on smooth muscle originally identified in extracts of equine intestine by von Euler and Gaddum (1931). The peptide was later isolated from hypothalamus and characterized by Chang and Leeman (1970) and Chang et al. (1971), and from intestine by Studer et al. (1973). It belongs to a family of related peptides with rapid stimulant actions on vascular and extravascular smooth muscle, known as the tachykinins (Erspamer and Anastasi, 1966) (Table 3.1). SP is the only such mammalian peptide, the others being of amphibian origin, except for eledoisin which occurs in octopus salivary gland.

THE EFFECTS OF SUBSTANCE P AND RELATED PEPTIDES ON α‐AMYLASE RELEASE FROM RAT PAROTID GLAND SLICES

1 The effects of substance P and related peptides on amylase release from rat parotid gland slices have been investigated. 2 Supramaximal concentrations (1 μ) of substance P caused enhancement of amylase release over the basal level within 1 min; this lasted for at least 40 min at 30°C. 3 Substance P‐stimulated amylase release was partially dependent on extracellular calcium and could be inhibited by 50% upon removal of extracellular calcium. 4 Substance P stimulated amylase release in a dose‐dependent manner with an ED50 of 18 nM. 5 All C‐terminal fragments of substance P were less potent than substance P in stimulating amylase release. The C‐terminal hexapeptide of substance P was the minimum structure for potent activity in this system, having 1/3 to 1/8 the potency of substance P. There was a dramatic drop in potency for the C‐terminal pentapeptide of substance P or substance P free acid. Physalaemin was more potent than substance P (ED50 = 7 nM), eledoisin was about equipotent with substance P (ED50 = 17 nM), and kassinin less potent than substance P (ED50 = 150 nM). 6 The structure‐activity profile observed is very similar to that for stimulation of salivation in vivo, indicating that the same receptors are involved in mediating these responses. 7 All the fragments of substance P tested were capable of eliciting a full amylase release response. This indicates that the apparent partial agonist action of the C‐terminal nonapeptide fragment on in vivo salivation is not explicable at the receptor level.

Biochemical and Pharmacological Studies of Substance P Receptors

Abstract Receptors for the biologically-active peptide, substance P, have been studied in the nervous system and in peripheral tissues using radio-ligand binding, stimulation of phospholipid turnover, and pharmacological bioassays. The binding of 3 H-substance P to rat brain membranes indicates the presence of a single saturable population of sites. They are ecognize zed by a high affinity (K d = 0.4 nM) and a low density (B max = 27 fmol/mg protein). The structural requirements for this site have been determined by measuring the potencies of shorter fragments and residue-substituted analogues in competing for 3 H-substance P binding. The CNS site appears to differ from peripheral sites in that it appears to ecognize N-terminal fragments with a C-terminal amide as well as C-terminal fragments, which are active in peripheral tissues. The substance P-induced stimulation of 3 H-inositol incorporation into rat parotid lipids is an accurate model of substance P receptor events and the phosphatidylinositol effect may be an event closely coupled to receptor occupancy. The rank order of potency of substance P, and the naturally occurring structural homologues, kassinin, eledoisin and physalaemin in peripheral bioassays has revealed two classes of responses: eledoisin-≃kassinin≫substance P≃physalaemin and physalaemin≤substance P≤eledoisin≤kassinin. We propose that these correspond to two categories of substance P receptor “SPP”, preferring physalaemin, and “SPE”, preferring eledoisin.

Substance P-Stimulated Phosphatidylinositol Breakdown in Guinea Pig Ileum

The breakdown of phosphatidylinositol lipids in cell membranes may have a key role in controlling the rise in cytosolic Ca2+ which occurs on stimulation of a variety of cell-surface receptors. Pre-iously this phenomenon has been studied by monitoring the rate of incorporation or loss of radioactivity from isotopically labelled membrane phospholipids. However, these approaches suffer from the drawback that changes in the levels of the substrates and not the enzymatic products are measured. We have recently found that lithium can be used to inhibit the breakdown of a metabolite of phosphatidylinositol hydrolysis, inositol-1-phosphate, and that the receptor-stimulated accumulation of inositol-1-phosphate after lithium treatment provides a model for the study of drug action [1]. We have now applied this approach to the characterization of substance P receptors on smooth muscle of guinea-pig ileum.