C. Tousignant

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.

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.

Antagonists for the neurokinin NK-3 receptor evaluated in selective receptor systems

Four isolated vessels that are monoreceptor systems for neurokinins, the dog carotid artery and rabbit jugular vein (NK-1), the rabbit pulmonary artery (NK-2) and the rat portal vein (NK-3), were used to compare the activities of selective neurokinin agonists and evaluate the affinities of new NK-3 antagonists. Chemical modifications in the partial sequences NKA (4-10) and NKB (4-10), particularly the replacement of Val7 with an aromatic residue (Tyr, MePhe or Trp) and the extension of the peptide backbone in position 8, obtained with beta-Ala, led to compounds that maintain weak agonistic activities on the NK-1 and NK-2, and some of them also on NK-3 receptors but exert potent antagonism against NKB on the NK-3 receptor of the rat portal vein. Antagonistic affinity is the highest when Trp is used in position 7 of [beta-Ala8]-NKA (4-10) and MePhe in position 7 of [beta-Ala8]-NKB (4-10). Antagonism is selective for NKB or [MePhe7]-NKB, and appears to be specific, since the most active compound [Trp7, beta-Ala8]-NKA (4-10) is inactive against bradykinin on the rabbit jugular vein (B2 receptor), against SP on the rabbit jugular vein (NK-1 receptor), against desArg9-bradykinin on the rabbit aorta (B1 receptor), and against angiotensin II and histamine (AT and H receptors, respectively) in the rabbit aorta. The new NK-3 receptor antagonists described in the present study provide useful tools for neurokinin receptor characterization and for determining the roles of neurokinins in physiopathology.

Characterization of pre- and postjunctional receptors for neurokinins and kinins in the rat vas deferens

Neurokinins and kinins are potent stimulants of the rat vas deferens: they increase both the twitch response to electrical stimulation by facilitating transmitter release and the basal tone of the preparation by activating smooth muscle receptors. The two sites of action have been studied separately in the present experiments by using the prostatic for the prejunction site and the epididymal section of the vas deferens for the postjunction site. Receptors for neurokinins, characterized by the order of potency of agonists, appear to be of the NK-A type both at the pre- and postjunction level. Receptors for kinins are present also at both sites and are probably of the B2 type: they have been characterized by the use of agonists and, the B2 postjunction type has been convalidated with antagonists. These compounds could not be used for the prejunction receptor characterization, because they act as agonists. Receptor for angiotensin at the prejunction level are of the same type as in the cardiovascular and other systems.

125I-BH[Sar9, Met(O2)11]-SP, a new selective ligand for the NK-1 receptor in the central nervous system.

The selective agonist [Sar9,Met(O2)11]-SP was radioiodinated with 125I-Bolton Hunter in order to study its binding to rat brain membranes and for further comparison with 125I-BH.SP. Specific binding of 125I-BH[Sar9,Met(O2)11]-SP was temperature-dependent, saturable and reversible. In brain homogenates, 125I-BH[Sar9,Met(O2)11]-SP interacted with a single class of high affinity (kd = 1.0 nM) non-interacting binding sites (Bmax of 15 fmol/mg protein). In the central nervous system, 125I-BH-[Sar9,Met(O2)11]-SP apparently labeled the same number of binding sites as 125I-BH.SP (19 fmol/mg proteins). Competition studies with tachykinins, neurokinins and selective neurokinin agonists indicated that the pharmacological profile of the site labeled by 125I-BH[Sar9,Met(O2)11]-SP is identical with that of NK-1 receptors. In dose-displacement studies made with radiolabeled SP and [Sar9,Met(O2)11)]-SP, an excellent correlation (r = 0.96) was found for the Ki values of the different compounds tested; these findings suggest that both radioligands recognize the same receptor in rat brain. The affinity (Ki) of various neurokinin-related peptides for the brain site were compared with their biological activities on various isolated organs (dog carotid artery, guinea-pig ileum, rat portal vein). NK-1 binding sites characterized in rat brain homogenates appear to be identical with those present on the dog carotid artery, a preparation known to possess exclusively the NK-1 receptor type.

Neurokinin A. A pharmacological study

Discovered in 1983, the decapeptide neurokinin A has been shown to occur in several peripheral organs and to exert a variety of biological effects. In this article, we review the most sensitive and selective in vivo and in vitro tests which have been used in various laboratories to evaluate naturally occurring or synthetic neurokinin A. A comparison of the effects of neurokinin A and those of its mammalian homologues, substance P and neurokinin B as well as those of tachykinins and related peptides is presented in the frame of a study directed toward characterization of neurokinin receptors. Indeed, neurokinin A has been shown to be particularly active on a neurokinin receptor subtype, the NK-2. Structure-activity studies performed with neurokinin A and its fragments as well as with several analogues of both the decapeptide and the heptapeptide NKA(4-10) have brought to the identification of the minimum structure required for activation of NK-2 receptors. Selective agonists for this receptor have been identified, in particular [Nle10]-NKA(4-10) and [beta-Ala8]-NKA(4-10).

Basic pharmacology of kinins: pharmacologic receptors and other mechanisms.

Kinins are a group of peptides released in blood and tissues by the action of trypsin-like endopeptidases, the kallikreins. A variety of stimuli have been shown to transform the inactive prekallikreins into kallikreins and thus start the process of the liberation of kinins from large protein precursors of hepatic origin, the kininogens (1). Bradykinin and related kinins appear to be autacoids, released in tissues to act on a variety of cells and structures, including arterial and venous smooth muscles, endothelia, especially capillary endothelia, mast cells, fibroblasts and both sensory and motor nerve endings. Kinins are indeed the most potent endogenous stimulants of pain fibers and of a variety of sensory fibers distributed throughout the cardiovascular system to monitoring vascular reactivity, blood flow and cardiovascular reflexes (2). Kinins act also as modulators of transmitter release, especially in the sympathetic nervous system (3).

Structure-activity study of neurokinins: antagonists for the neurokinin-2 receptor.

A series of 21 peptides were synthesized and tested in a variety of isolated organs in order to determine their potential as neurokinin-2 (NK-2) antagonists. The peptides have been tested in the three monoreceptor systems, the dog carotid artery (NK-1), the rabbit pulmonary artery (NK-2) and the rat portal vein (NK-3) as well as on other preparations containing NK-2 receptors, such as the rat vas deferens, the hamster urinary bladder, the guinea-pig trachea and the human urinary bladder. Some of the compounds have also been tested on the human isolated bronchus. Three compounds, of which two are linear peptides, Ac.Leu-Asp-Gln-Trp-Phe-Gly.NH2, Thr-Asp-Tyr-D-Trp-Val-D-Trp-D-Trp-Arg.NH2 and a cyclic one, cyclo[Gln-Trp-Phe-Gly-Leu-Met] have been shown to reduce or eliminate the effects of neurokinin A (NKA) in practically all the preparations containing NK-2 receptors. The first compound was found to be selective for the NK-2 receptor and showed only agonistic or no activity on the other receptor systems, while the second compound showed some antagonistic effects not only on the NK-2 but also on the other systems. The cyclic compound was found to be fairly selective for the NK-2 receptor. The first compound (Ac.Leu-Asp-Gln-Trp-Phe-Gly.NH2) was characterized with respect to its specificity for neurokinins (NK): it was found to be inactive on receptors for acetylcholine, noradrenaline, angiotensin and des Arg9-bradykinin in the rabbit pulmonary artery. Moreover, the compound exerted a competitive type of antagonism on the rabbit pulmonary artery and on the hamster urinary bladder. Although of moderate affinity, the NK-2 receptor antagonists described in this paper provide important tools for pharmacological studies on NK.

Binding sites for [3H][Sar9, Met(O2)11]substance P in rat bain and guinea pig ileum

[3H][Sar9,Met(O2)11]substance P (SP) with high specific activity (32 Ci/mmol) was used to study neurokinin-1 (NK-1) binding sites on rat brain and smooth muscle membranes of the guinea pig ileum. The specific binding of [3H][Sar9,Met(O2)11]SP was shown to be saturable, reversible and increased in parallel with the protein concentration. Scatchard analyses of equilibrium binding experiments revealed that [3H][Sar9,Met(O2)11]SP binds to a class of non-interacting binding sites in rat brain membranes (Kd = 2 nM, Bmax = 56 fmol/mg of protein) and ileum muscle membranes (Kd = 2 nM, Bmax = 194 fmol/mg of protein). Competition of [3H][Sar9,Met(O2)11]SP, 125I-BH[Sar9,Met(O2)11]SP and 125I-BH.SP with different tachykinin-related peptides gave the following rank order of potencies: SP greater than physalaemin greater than [Sar9,Met(O2)11]SP greater than N-Ac[Arg6,Sar9,Met(O2)11]SP(6-11) greater than neurokinin A (NKA) greater than or equal to eledoisin greater than or equal to neurokinin B (NKB) greater than [MePhe7]NKB (4-10) greater than [beta-Ala8]NKA(4-10). A very similar pattern was observed on ileum muscle membranes. [3H][Sar9,Met(O2)11]SP was found to be highly selective for NK-1 binding sites in rat brain and in the intestinal tissue. Binding showed good correlation with the biological activity of tachykinins and related peptides. From these data it can be suggested that (a) the NK-1 receptor characterized in the central nervous system is identical to the one in the periphery, (b) the NK-1 binding site of the muscle membranes appears to be similar to the contractile receptor of the guinea pig ileum and (c) the functional site mediating relaxation of the dog carotid artery is similar to the contractile receptor of the guinea pig ileum.

New highly potent bradykinin B2 receptor antagonists

AbstractpA2 values of new B2 receptor antagonists ranging from 7.51 to 8.86 were measured on the rabbit jugular vein, while lower values were observed in the other preparations (for instance, the hamster urinary bladder). The most potent antagonists were those containing a hydroxyproline (Hyp) in position 3, ad-Arg at the N-terminal and a Leu instead of a Phe in position 8, with or without other chemical changes.d-Arg[Hyp3,d-Phe7, Leu8]-BK was found to be competitive, selective for B2 receptors and specific for kinins since it was without effect against substanceP and angiotensin II in the rabbit jugular vein. The essential feature for obtaining B2 receptor antagonists appears to be the replacement or reorientation of Phe8 of bradykinin. The rabbit jugular vein provides a sensitive bioassay in which the potency and specificity of B2 receptor antagonists can be adequately evaluated.