Guy Drapeau

New selective agonists for neurokinin receptors: pharmacological tools for receptor characterization

Abstract The neurokinin system is replete with a multiplicity of endogenous ligands (substance P, neurokinin A, neurokinin B) and their receptors (NK 1 , NK 2 and NK 3 ). Neurokinin receptors have previously been studied almost exclusively with natural — but non-selective — agonists. However, new selective agonists have now been developed . Domenico Regoli and colleagues describe how such compounds have been successfully used in vitro to characterize the responses of peripheral organs to neurokinins, and in vivo to elucidate possible physiological roles of the neurokinin system .

Pharmacological receptors for substance P and neurokinins.

The three neurokinins identified in mammals, substance P, neurokinin A and neurokinin B, as well as their C-terminal biologically active fragments, have been used to characterize the responses of a variety of isolated organs. Three preparations selective either for substance P (the dog carotid artery), or for neurokinin A (the rabbit pulmonary artery) or for neurokinin B (the rat portal vein) are described. A neurokinin receptor classification is attempted using the neurokinins and their fragments to determine the order of potency of agonists. Three receptor subtypes have been identified: the NK-P, on which substance P (SP) is more active than neurokinin A (NKA) and neurokinin B (NKB), and the neurokinins are more active than their respective fragments; the NK-A on which NKA greater than NKB greater than SP, and some NKA fragments are more discriminative than their precursor; the NK-B on which NKB greater than NKA greater than SP, and fragments of NKB are less active than their precursor. Among the peptides studied, some potent compounds have been identified that could provide selective receptor ligands.

Selective agonists for substance P and neurokinin receptors

A series of neurokinin analogues and fragments have been prepared in an attempt to identify selective agonists for NK-P, NK-A and NK-B receptors. The compounds have been tested on the dog carotid artery (NK-P receptor system), the rabbit pulmonary artery (NK-A) and the rat portal vein (NK-B). C-terminal substituted analogues of the three neurokinins have provided indication that NK-P receptor selectivity is improved by the oxidation of methionine to Met(O2), while selectivity for NK-A is favoured by replacing Met with NIe. Selectivity for NK-P receptors is further improved by the replacement of Gly9 with Sar. Selectivity and affinity for NK-B receptors is markedly increased when Val7 is replaced with MePhe in both the fragment NKB (4-10) and NKB. The results of the present study indicate that a) [Sar9,Met(O2)11]SP is a potent and selective agonist for the NK-P receptors of the dog carotid artery; b) [MePhe7]NKB is a very potent and selective stimulant of receptors for neurokinin B and c) [Nle10]NKA (4-10) is a promising compound, showing some selectivity for NK-A receptor; further modifications are however needed to improve its affinity.

Receptors for Substance P and Related Neurokinins

The most widely used smooth muscle preparations for neurokinin bioassays have been critically analyzed in order to determine whether neurokinins act directly or by the intermediary of other natural agents. Indeed, part of the contraction of the GPI in response to neurokinins appears to be mediated by acetylcholine and possibly prostaglandins. Active metabolites of the arachidonic acid cascade also intervene in the response of the HUB. Neurokinins produce relaxation of the DCA by stimulating the release of a vascular smooth muscle relaxing factor from the endothelium. In the other preparations (the RD, the RPA without endothelium and the RPV) neurokinins may act directly on the smooth muscle fibers. Neurokinins produce their biological effects by activating specific receptors. Three different receptor types, one for each mammalian neurokinin, have been identified by using four groups of natural peptide sequences and some selective agonists. The receptor for SP is particularly sensitive to SP and physalaemin and shows higher affinity for the whole natural peptides (SP, NKA) than for their C-terminal fragments. The receptor for neurokinin A is highly sensitive to NKA and eledoisin: it shows high affinity for heptapeptide fragments such as NKA4-10 and SP5-11. The receptor for NKB is sensitive to NKB and kassinin more than to the other natural peptides and their fragments. The natural peptides show however little selectivity. Synthetic analogues active on a single receptor type (selective agonists) have been used to find out whether the responses of the isolated organs are due to the activation of one or more than one receptor. It has been found that the GPI, the RD and the HUB contain all three or at least two receptors, while the DCA has only the NK1, the RPA has only the NK2 and the RPV only the NK3 type. Binding sites specific for each neurokinin have been identified in brain and peripheral organs with accurate biochemical assays, using labeled neurokinins. Competitive displacement assays have been performed with a variety of neurokinin-related peptides, and their Ki have been determined. By plotting Ki values against the ED50, estimated from biological assays, positive significant correlations have been found for the monoreceptor (DCA, RPA, RPV) but not for the multiple receptor systems (GPI, RD, HUB). This suggests that pharmacological receptors may be identical with the recognition sites which bind the labeled neurokinins. The availability of monoreceptor systems and of selective agonists opens the way for the identification of potential antagonists and accurate estimation of their affinities.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of neurokinin receptors in various isolated organs by the use of selective agonists

The three mammalian neurokinins, substance P, neurokinin A and neurokinin B, as well as some agonists selective for their respective receptors, NK-P, NK-A and NK-B, were tested in a variety of pharmacological preparations in order to evaluate if the biological responses of the various tissues were mediated by single or multiple receptor types. Previous observations that the dog carotid artery, the rabbit pulmonary artery and the rat portal vein are selective preparations respectively for SP, NKA and NKB were confirmed in the present study by showing that only the respective selective agonists were active on these tissues. Multiple functional sites were demonstrated in intestinal tissues (guinea pig ileum, rat duodenum), which apparently contain the three neurokinin receptors. A large number of NK-P, together with some NK-A receptor sites were found in the guinea pig and rat urinary bladder. Similarly, the guinea pig trachea and the rabbit mesenteric vein contain NK-A and NK-P functional sites. Rat and rabbit vas deferens stimulated electrically respond as typical NK-A preparations, since they are almost insensitive to SP or NKB selective agonists. A mixture of NK-A and NK-B receptor sites has been shown to be present in the hamster urinary bladder: dog and human urinary bladder definitely contain NK-A receptors and the dog bladder also some NK-P functional sites.

New selective bradykinin receptor antagonists and bradykinin B2 receptor characterization

Substantial progress has been made recently in the field of kinin pharmacology with the identification of sensitive bioassay organs and the discovery of bradykinin B2 receptor antagonists. Data obtained with such compounds in various laboratories support the hypothesis that kinins act on multiple (at least two) receptor types. Domenico Regoli and colleagues review here the basic criteria of receptor characterization as they apply to kinins and present a critical analysis of the bioassay organs and B2 receptor antagonists currently used in kinin pharmacology.

Comparison of the effects of epithelium removal and of an enkephalinase inhibitor on the neurokinin‐induced contractions of guinea‐pig isolated trachea

1 The influence of epithelium removal and/or thiorphan on the effects of neurokinins (substance P (SP), neurokinin A (NKA), neurokinin B (NKB)) and related peptides on airway contractility was investigated on the guinea‐pig isolated trachea. 2 Removing the tracheal epithelium significantly enhanced the sensitivity but not the maximum contractile responses to the peptides. 3 After removal of the epithelial layer, the shifts to the left of the log concentration response curves were greater for SP and SP‐OMe (1.62 and 1.94 log units, respectively) than for two SP analogues substituted in position 9 namely [Pro9]SP sulfone and [β‐Ala,4 Sar9]SP(4–11)sulfone (0.66 and 0.68 log units, respectively). The leftward shifts for compounds related to NKA or NKB lay between 0.58 and 0.73 log units. 4 The leftward shifts of the log concentration‐response curves for SP, SP‐OMe, [Pro9]SP sulfone, [β‐Ala4, Sar9]SP(4–11) sulfone and NKA were of similar magnitude after removal of the epithelium or after pretreatment with thiorphan (10−5 m), an enkephalinase inhibitor, in the presence of epithelium. No significant additional shift of the curves to the left was observed with thiorphan plus epithelium removal. 5 The results obtained with the selective agonists for each of the three classes of neurokinin receptor (i.e NK1, NK2, NK3) suggest that the guinea‐pig trachea contains receptors for SP and NKA but few if any for NKB. 6 It was concluded that neurokinins and related peptides (especially SP and analogues not substituted in position 9) are degraded by enkephalinase mainly located in the tracheal epithelium and that the addition of thiorphan or epithelium removal results in an inhibition or loss of enkephalinase activity, thereby increasing similarly the potencies of these peptides. It was, therefore, suggested that the supersensitivity to neurokinins produced by epithelium removal was due neither to the elimination of a permeability barrier nor to reduced production of a relaxant factor, but mainly to reduced peptide degradation.

Different receptors are involved in the endothelium-mediated relaxation and the smooth muscle contraction of the rabbit pulmonary artery in response to substance and related neurokinins

Four neurokinins, substance P (SP), neurokinin A (NKA) neurokinin B (NKB) and kassinin (Kass) were used in the present study together with other peptides and nonpeptide agents to demonstrate the existence of two different neurokinin receptor types in the rabbit isolated pulmonary artery. Similar to other arterial vessels, the endothelium-dependent relaxation of the pulmonary artery in response to neurokinins is due to the activation of a SP-P receptor more sensitive to SP than to the other neurokinins. The endothelium-dependent relaxation is an indirect phenomenon, mediated by an unknown endothelial agent, similar to that released by acetylcholine. The contraction of the pulmonary artery in response to neurokinins is due to receptors of the NK-A type, particularly sensitive to NKA and NKB, and much less sensitive to SP. The contraction is a direct phenomenon, apparently not involving any of the known endogenous autacoids and neurotransmitters or metabolites of arachidonic acid. Contraction appears to be due to stimulation by the neurokinins of receptors located in the arterial smooth muscle. The results presented in this paper indicate that NK-A receptors for neurokinins (which are present in the tracheo-bronchial tree) are also to be found in pulmonary vessels and mediate contraction of arterial vascular smooth muscle, an interesting property of neurokinins.

Relative potencies of neurokinins in guinea pig trachea and human bronchus.

The three endogenous neurokinins, substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), as well as NKA-(4-10), carbachol, acetylcholine and histamine, were tested in guinea pig tracheae and human bronchi in order to compare the activities of peptides and non-peptide agents and to characterize the neurokinin receptors by means of agonists. Neurokinin A and NKA-(4-10) were potent stimulants of the two preparations: pD2 values for NKA-(4-10) averaged 8.62 in the guinea pig trachea and 7.50 in the human bronchus. The rank order of potency of neurokinins was NKA-(4-10) greater than NKA greater than SP greater than NKB in the human bronchus and NKA-(4-10) greater than NKA greater than NKB greater than SP in the guinea pig trachea. SP was 2-3 orders of magnitude less active than NKA and appears to be a partial agonist. NKB is inactive on the human bronchus. Compared to non-peptide agents, NKA had an affinity 2-3 orders of magnitude greater than acetylcholine and histamine but produced only 75-80% of the maximal effect of acetylcholine. The present results indicate that neurokinins contract the human bronchus by activating a NK-A receptor type which is more sensitive to NKA than to SP and is insensitive to NKB. The guinea pig trachea appears to be a complex preparation containing not only NK-A but also other neurokinin receptors.

Structure-activity studies of neurokinin A

A structure-activity study on neurokinin A and its C-terminal fragment NKA (4-10) has been performed in order to find selective agonists for the NK-2 receptor and identify chemical modifications suitable for protecting the peptides from degradation, while maintaining activity. Five series of compounds have been prepared and tested: 1. the complete series of the L-Ala monosubstituted analogues of NKA; 2. a series of NKA fragments from the C- or N-terminal; 3. the complete series of NKA (4-10) analogues monosubstituted with beta-Ala; 4. a series of NKA (4-10) analogues with monosubstitutions in pos. 4, 8, 10 or multisubstitutions in two or more of the same positions; and 5. a series of 6 NKA (4-10) analogues monosubstituted with 1-amino,1-cyclohexane carboxylic acid residue. It has been found that the most selective agonists for the NK-2 receptor system are [beta Ala8]NKA (4-10) and [Nle10]NKA (4-10). Protection from aminopeptidase may be obtained by acetylation of the N-terminal amide of NKA (4-10), while partial protection from endopeptidases should be expected from the presence of beta-Ala in position 8. Conformational constraints induced with 1,amino,1-cyclohexane carboxylic acid residue gave weakly active compounds. Multiple substitutions reduce rather than potentiating the favorable effects of the corresponding monosubstituted compounds.