Autoradiographic distribution of tachykinin NK2 binding sites in the rat brain: comparison with NK1 and NK3 binding sites.

The autoradiographic distribution of tachykinin NK(2) binding sites was determined in the adult rat brain using [(125)I]neurokinin A in the presence of either senktide (NK(3) agonist) and [Pro(9)]substance P (NK(1) agonist) or senktide and SR 140333 (NK(1) antagonist). Indeed, this radioligand labels two subtypes of NK(1) binding sites (which present a high affinity not only for SP but also for neurokinin A, neuropeptide K and neuropeptide gamma) as well as NK(3) binding sites. The distribution of NK(2) binding sites was also compared with those of NK(1) and NK(3) binding sites, these sites being labeled with [(125)I]Bolton and Hunter substance P and [(125)I]Bolton and Hunter eledoisin, respectively. In agreement with our results obtained with membranes from various brain structures, NK(2)-sensitive [(125)I]neurokinin A labeling was mainly observed in few structures including the dorsal and ventral hippocampus, the septum, the thalamus and the prefrontal cortex. The density of NK(2) binding sites was weak when compared with those of NK(1) and NK(3) binding sites. Marked differences were observed in the distributions of NK(1), NK(2) and NK(3) binding sites. These results are discussed taking into consideration differences or similarities between the distributions of NK(2)-sensitive [(125)I]neurokinin A binding sites and of their endogenous ligands (neurokinin A, neuropeptide K and neuropeptide gamma) but also local NK(2) agonist responses blocked by NK(2) antagonists. Insights on the roles of endogenous tachykinins in several brain functions are also discussed on the basis of the respective distributions of different neurokinin binding sites.

Presence of NK2 binding sites in the rat brain.

Attempts were made to label tachykinin NK2 binding sites in the adult rat brain using [125I]neurokinin A (NKA) as ligand in the presence of NK1 and NK3 agonist or antagonist to avoid labelling of NK1 and NK3 binding sites, respectively. A high-affinity, specifically NK2-sensitive, [125I]NKA-binding, temperature-dependent, reversible, sensitive to GTPgammaS and correspondence to a single population of binding sites (K(D) and B(max) values: 2.2 nM and 7.3 fmol/mg protein) was demonstrated on hippocampal membranes. Competition studies performed with tachykinins and tachykinin-related compounds indicated that the pharmacological properties of these NK2-sensitive [125I]NKA binding sites were identical to those identified in the rat urinary bladder and duodenum. NKA, neuropeptide K, and neuropeptide gamma, as well as the potent and selective NK2 antagonists SR 144190, SR 48968 and MEN 10627, presented a nanomolar affinity for these sites. The regional distribution of these NK2-sensitive [125I]NKA binding sites differs markedly from those of NK1 and NK3 binding sites, with the largest labeling being found in the hippocampus, the thalamus and the septum. Binding in other brain structures was low or negligible. A preliminary autoradiographic analysis confirmed [125I]NKA selective binding in hippocampal CA1 and CA3 areas, particularly, and in several thalamic nuclei.

Different subtypes of tachykinin NK(1) receptor binding sites are present in the rat brain.

(2-[(125)I]iodohistidyl(1))Neurokinin A ([(125)I]NKA), which labels "septide-sensitive" but not classic NK(1) binding sites in peripheral tissues, was used to determine whether septide-sensitive binding sites are also present in the rat brain. Binding studies were performed in the presence of SR 48968 (NK(2) antagonist) and senktide (NK(3) agonist) because [(125)I]NKA also labels peripheral NK(2) binding sites and, as shown in this study, central NK(3) binding sites. [(125)I]NKA was found to label not only septide-sensitive binding sites but also a new subtype of NK(1) binding site distinct from classic NK(1) binding sites. Both subtypes of [(125)I]NKA binding sites were sensitive to tachykinin NK(1) antagonists and agonists but also to the endogenous tachykinins NKA, neuropeptide K (NPK), and neuropeptide gamma (NPgamma). However, compounds of the septide family such as substance P(6-11) [SP(6-11)] and propionyl-[Met(O(2))(11)]SP(7-11) and some NK(1) antagonists, GR 82334, RP 67580, and CP 96345, had a much lower affinity for the new NK(1)-sensitive sites than for the septide-sensitive sites. The hypothalamus and colliculi possess only this new subtype of NK(1) site, whereas both types of [(125)I]NKA binding sites were found in the amygdala and some other brain structures. These results not only explain the central effects of septide or SP(6-11), but also those of NKA, NPK, and NPgamma, which can be selectively blocked by NK(1) receptor antagonists.

Further evidence for the presence of "septide-sensitive" tachykinin binding sites in tissues possessing solely NK(1) tachykinin receptors.

Binding experiments performed with [(125)I]-NKA allowed us to demonstrate the presence of "septide-sensitive" specific binding sites on membranes from rat CHO cells transfected with the NK(1) receptor cDNA (CHO-rat-NK1 cells), human astrocytoma U373 MG, or mouse cortical astrocytes, cells which express NK(1) but neither NK(2) nor NK(3) receptors. In all cases, [(125)I]-NKA was specifically bound with high affinity (2 to 5 nM) to a single population of sites. In the three preparations, pharmacological characteristics of [(125)I]-NKA binding sites were notably different from those of classical NK(1) binding sites selectively labelled with [(125)I]-BHSP. Indeed, the endogenous tachykinins NKA, NPK, and NKB and the septide-like compounds such as septide, SP(6-11), ALIE-124, [Apa(9-10)]SP, or [Lys(5)]NKA(4-10) had a much higher affinity for [(125)I]-NKA than [(125)I]-BHSP binding sites. Interestingly, differences were also found in the ratio of B(max) values for [(125)I]-NKA and [(125)I]-BHSP specific bindings from one tissue to another. These latter observations suggest that these two types of NK(1) binding sites are present on distinct NK(1) receptor isoforms (or conformers). Finally, while several tachykinins and tachykinin-related compounds stimulated cAMP formation or increased inositol phosphate accumulation in CHO-rat-NK1 cells, these compounds only increased the accumulation of inositol phosphates in the two other preparations.

Pharmacological characterization of tachykinin septide-sensitive binding sites in the rat submaxillary gland.

Binding studies have shown that [125I]NKA is a selective ligand of tachykinin septide-sensitive binding sites from membranes of the rat submaxillary gland. Indeed, this ligand bound with high affinity to a single population of sites. In addition, competition studies indicated that natural tachykinins and tachykinin-related compounds had a similar affinity for these sites than for those labeled with [3H]ALIE-124, a selective ligand of septide-sensitive binding sites. Moreover, selective tachykinin NK2, or NK3 agonists or antagonists exhibited weak or no affinity for [125I]NKA binding sites. As indicated by Ki values of several compounds, the pharmacological characteristics of the septide-sensitive binding sites (labeled with [125I]NKA) largely differ from those of classic NK1 binding sites, as determined on crude synaptosomes from the rat brain using [125I]Bolton-Hunter substance P (SP) as ligand. Indeed, several tachykinins including neurokinin A (NKA), neuropeptide K (NPK), neuropeptide gamma (NKgamma), and neurokinin B, as well as some SP and NKA analogues or C-terminal fragments such as septide, ALIE-124, SP(6-11), NKA(4-10), which have a weak affinity for classic tachykinin NK1 binding sites exhibited a high affinity for the septide-sensitive binding sites. In contrast, SP, classic selective NK1 agonists, and antagonists had a high affinity for both types of binding sites. The presence of a large population of tachykinin septide-sensitive binding sites in the rat submaxillary gland may thus explain why NPK and NPgamma induce salivary secretion and may potentiate the SP-evoked response in spite of the absence of tachykinin NK2 receptors in this tissue.

Functional coupling of the NK1 tachykinin receptor to phospholipase D in chinese hamster ovary cells and astrocytoma cells.

In [3H]myristic acid-prelabeled Chinese hamster ovary cells stably expressing the rat NK1 tachykinin receptor, the selective NK1 agonist [Pro9]substance P ([Pro9]SP) time and concentration dependently stimulated the formation of [3H]phosphatidylethanol in the presence of ethanol. This [Pro9]SP-induced activation of phospholipase D (PLD) was blocked by NK1 receptor antagonists and poorly or not mimicked by NK2 and NK3 agonists, respectively. In confirmation of previous observations, [Pro9]SP also stimulated the hydrolysis of phosphoinositides, the release of arachidonic acid, and the formation of cyclic AMP (cAMP). All these [Pro9]SP-evoked responses could be mimicked by aluminum fluoride, but they remained unaffected in cells pretreated with pertussis toxin, suggesting that a Gi/Go protein is not involved in these different signaling pathways. The activation of PLD by [Pro9]SP was sensitive to external calcium and required an active protein kinase C because the inhibition of this kinase (Ro 31-8220) or its down-regulation (long-term treatment with a phorbol ester) abolished the response. In contrast, a cAMP-dependent process was not involved in the activation of PLD because the [Pro9]SP-evoked response was neither affected by Rp-8-bromoadenosine 3',5'-cyclic monophosphorothioate nor mimicked by cAMP-generating compounds (cholera toxin or forskolin) or by 8-bromo-cyclic AMP. A functional coupling of NK1 receptors to PLD was also demonstrated in the human astrocytoma cell line U 373 MG stimulated by SP or [Pro9]SP. These results suggest that PLD activation could be an additional signaling pathway involved in the mechanism of action of SP in target cells expressing NK1 receptors.

High affinity binding of [3H]propionyl-[Met(O2)11]substance P(7-11), a tritiated septide-like peptide, in Chinese hamster ovary cells expressing human neurokinin-1 receptors and in rat submandibular glands.

Propionyl-[Met(O2)11]substance P(7-11) [ALIE-124 or propionyl-[Met(O2)11]SP(7-11)] has been designed as a septide-like ligand adequate for tritiation and, therefore, adequate for binding studies. In Chinese hamster ovary (CHO) cells expressing human tachykinin neurokinin (NK)-1 receptors, ALIE-124 displaced [3H][Pro9]substance P (SP) from its binding site at micromolar concentrations. However, ALIE-124 stimulated phosphatidylinositol hydrolysis, as previously shown for septide-like peptides. With [3H]ALIE-124 (95 Ci/mmol), we have been able to reveal a high affinity binding site in CHO cells (Kd = 6.6 +/- 1.0 nM), with a low maximal binding capacity. [3H]ALIE-124 specific maximal binding represented only 15-20% of that observed with [3H][Pro9]SP in CHO cells. Septide-like peptides, including septide and NKA, were potent competitors (in the nanomolar range) of [3H]ALIE-124 specific binding site. Interestingly, SP and [Pro9]SP were also potent competitors, with 10-fold greater potency for sites labeled with [3H]ALIE-124 than for sites labeled with [3H][Pro9]SP. The NK-1 antagonist RP 67580 also showed a higher potency for [3H]ALIE-124 than for [3H][Pro9]SP-specific binding sites. NKB and [Lys5,methyl-Leu9,Nle10]NKA(4-10) displaced [3H]ALIE-124 binding but with lower potency, whereas senktide had no affinity. The existence of [3H]ALIE-124 specific binding sites was also demonstrated in rat submandibular gland. In this tissue, [3H]ALIE-124 specific maximal binding was higher, reaching 40-50% of that achieved with [3H][Pro9]SP.

Substance P(6-11) and natural tachykinins interact with septide-sensitive tachykinin receptors coupled to a phospholipase C in the rat urinary bladder.

The rat urinary bladder possesses NK1, NK2 (but not NK3) and 'septide-sensitive' tachykinin receptors coupled to a phospholipase C. The present study performed with SR48968 (10(-6) M) to avoid any interaction of the tested peptides with NK2 receptors, indicates that substance P(6-11) (with a high potency), neurokinin A, neurokinin B and to a lesser extent neuropeptide K (with a lower potency) stimulate [3H]-inositol monophosphate ([3H]-IP1) formation in this tissue by acting on the 'septide-sensitive' tachykinin receptors. Substance P(6-11) had little affinity for NK1 binding sites and stimulated [3H]-IP1 formation with an EC50 value and a maximal amplitude similar to those of septide. As previously observed with septide, this maximal response of substance P(6-11) (insensitive to 10(-6) M SR48968) which was about three-fold that of substance P, was blocked by the NK1 receptor antagonist RP67580 and prevented by [Pro9]substance P (NK1 receptor agonist). Similarly, substance P and several substance P C-terminal fragments prevented the substance P(6-11)-evoked response. In addition, neurokinin A, neuropeptide K and neurokinin B induced SR48968-resistant responses which exhibited a maximal amplitude similar to that of substance P (6-11) and were blocked by RP67580 and totally or partially (neuropeptide K) prevented by [Pro9]substance P.

Potency and selectivity of the tachykinin NK3 receptor antagonist SR 142801.

Binding studies indicated that tachykinin NK3 binding sites in peripheral (ileum) and central (cerebral cortex) tissues of the guinea pig exhibit similar pharmacological properties. They also confirmed that the tachykinin NK3 receptor antagonist (S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl) propyl)-4-phenylpiperidin-4-yl)-N-methylacetamide (SR 142801) has a higher affinity for tachykinin NK3 binding sites in the guinea pig than in the rat. SR 142801 exhibited a much lower affinity for tachykinin NK2 and NK1 binding sites. SR 142801 was shown to be a potent uncompetitive antagonist of the senktide-induced formation of [3H]inositol monophosphate in slices from the guinea-pig ileum (apparent KB = 3.2 nM, 51% reduction of the maximal response), a functional test for tachykinin NK3 receptors. In agreement with results of binding studies, the effect of SR 142801 was stereoselective since its enantiomer SR 142806 was much less potent. In the rat urinary bladder, a tissue devoid of tachykinin NK3 receptors, SR 142801 was without effect on the [Pro9]substance P- or the septide-induced formation of [3H]inositol monophosphate but it slightly reduced the response of the tachykinin NK2 receptor agonist [Lys5,MeLeu9,Nle10]neurokinin A-(4-10) (KB = 339 nM). Altogether, these data indicate that SR 142801 is a highly selective tachykinin NK3 receptor antagonist which is more potent in the guinea pig than in the rat.

Involvement of septide-sensitive tachykinin receptors in inositol phospholipid hydrolysis in the rat urinary bladder.

The selective NK2 agonist [Lys5-MeLeu9,Nle10]NKA(4-10) markedly stimulated [3H]inositol monophosphate (PI1) formation in prisms from the rat urinary bladder. This response was blocked by the NK2 antagonist SR 48968. Senktide (NK3 agonist) was inactive. Septide, a short SP analogue, and the NK1 agonists [Pro9]SP and [Sar9,Met(O2)11]SP also stimulated [3H]IP1 formation and several NK1 tachykinin antagonists (RP 67580, CP 96345, GR 82334, and [D-Pro9,t beta-BPr10,Trp11]SP) were more potent in blocking the septide than the [Pro9]SP response. GR 82334 was the most discriminative. SR 48968 (10(-6) M shifted the [Pro9]SP dose-response curve but did not modify the septide dose-response curve. Septide had a low affinity for [3H][Pro9]SP binding sites, suggesting further that septide and NK1 agonists act on different receptors. Finally, both [Pro9]SP and [Sar9,Met(O2)11]SP blocked the septide-evoked response, acting as partial agonists at the septide-sensitive tachykinin receptors.

Design and synthesis of side-chain conformationally restricted phenylalanines and their use for structure-activity studies on tachykinin NK-1 receptor.

Constrained analogues of phenylalanine have been conceptually designed for analyzing the binding pockets of Phe7 (S7) and Phe8 (S8), two aromatic residues important for the pharmacological properties of SP, i.e., L-tetrahydroisoquinoleic acid, L-diphenylalanine, L-9-fluorenylglycine (Flg), 2-indanylglycine, the diastereomers of L-1-indanylglycine (Ing) and L-1-benz[f]indanylglycine (Bfi), and the Z and E isomers of dehydrophenylalanine (delta ZPhe, delta EPhe). Binding studies were performed with appropriate ligands and tissue preparations allowing the discrimination of the three tachykinin binding sites, NK-1, NK-2, and NK-3. The potencies of these agonists were evaluated in the guinea pig ileum bioassay. According to the binding data, we can conclude that the S7 subsite is small, only the gauche (-) probe [(2S,3S)-Ing7]SP presents a high affinity for specific NK-1 binding sites. Surprisingly, the [delta EPhe7]SP analogue, which projects the aromatic ring toward the trans orientation, is over 40-fold more potent than the Z isomer, [delta ZPhe7]SP. A plausible explanation of these conflictual results is that either the binding protein quenches the minor trans rotamer of [(2S,3S)-Ing7]SP in solution or this constrained amino acid side chain rotates when inserted in the protein. In position 8, the high binding affinities of [Flg8]SP and [(2S,3S)-Bfi8]SP suggest that the S8 subsite is large enough to accept two aromatic rings in the gauche (-) and one aromatic ring in the trans direction. Peptides bearing two conformational probes in positions 7, 8, or 9 led to postulate that S7, S8, and S9 subsites are independent from each other. The volumes available for side chains 7 and 8 can be estimated to be close to 110 and 240 A3, respectively. The large volume of the S8 subsite raises question on the localization of the SP-binding site in the NK-1 receptor. If SP were to bind in the transmembrane domains, the cleft defined by the seven transmembrane segments must rearrange during the binding process in order to bind a peptide in an alpha-helical structure and at least one large binding subsite in position 8. Thus, indirect topographical analysis with constrained amino acids might contribute to the analysis of the receptor/ligand dynamics. Finally, this study demonstrates that a good knowledge of the peptidic backbone structure and a combination of constrained amino acids are prerequisites to confidently attribute the preferred orientation(s) of an amino acid side chain.

Differential localization of 3H-[Pro9]SP binding sites in the guinea pig and rat brain.

Due to the existence of differences in the pharmacological properties of tachykinin NK-1 receptors in the rat and the guinea pig, the autoradiographic distribution of NK-1 binding sites was compared in the brain of the two species using the selective NK-1 ligand 3H-[Pro9]SP. If a good similarity in the distribution of NK-1 binding sites could be seen in basal ganglia, a relative absence of correlation was observed between the estimated optical densities in other brain structures of the two species. For instance, the interpeduncular nucleus, the lateral habenular nucleus and the deep layers of the cerebral cortex were labeled in the guinea pig but not in the rat while the reverse was observed for the columns of the vermis lobules 9-10, the dorsal raphe nucleus, the medial habenular nucleus, the superficial cortical layers and the dorsal hippocampus. Furthermore, the high similarity found in the localization of 125I-BHSP (a non selective ligand) and 3H-[Pro9]SP binding sites, does not suggest the existence of NK-1 binding site subtypes in the guinea pig brain.

A new selective bioassay for tachykinin NK3 receptors based on inositol monophosphate accumulation in the guinea pig ileum.

The selective agonists of tachykinin NK1, NK2 and NK3 receptors, respectively [Pro9]substance P, [Lys5,MeLeu9,Nle10]neurokinin A-(4-10) and senktide, stimulated phosphoinositide breakdown in slices of the guinea pig ileum. This was also the case with septide which has recently been found to act on a new type of tachykinin receptors in this tissue. The NK1, NK2 and septide-evoked responses were completely antagonized in the combined presence of (+/-)-CP-96,345 and MEN 10,376 which are potent and selective antagonists of tachykinin NK1 and NK2 receptors respectively in the guinea pig ileum. Like senktide, other available NK3 receptor agonists, such as [MePhe7]neurokinin B, [MeVal7]neurokinin B, [Pro7]neurokinin B and DiMe-C7, stimulated phosphoinositide hydrolysis in either the absence or combined presence of (+/-)-CP-96,345 and MEN 10,376, although senktide was the most potent. Therefore, following the blockade of tachykinin NK1, NK2 and septide-sensitive receptors, the accumulation of inositol monophosphate appears to be a valuable, rapid and sensitive bioassay for determining the activity of NK3 receptor agonists and putative NK3 receptor antagonists.

NK-1 tachykinin receptor in rat and guinea pig brains: pharmacological and autoradiographical evidence for a species difference.

The affinities of a large variety of peptide or nonpeptide tachykinin analogues were determined on membranes from rat and guinea pig brains using the selective NK-1 radioligand 3H-[Pro9]SP. Nonpeptide antagonists clearly revealed a species difference; (+/-)CP-96,345 was more potent in the guinea pig, while RP 67580 was found to be a better competitor of 3H-[Pro9]SP binding to rat brain membranes. This was confirmed on brain slices by autoradiography. Numerous brain structures were analyzed by optical densitometry. From these data, a heterogeneity of NK-1 binding sites among different structures can be excluded in both rat and guinea pig brains.

Higher potency of RP 67580, in the mouse and the rat compared with other nonpeptide and peptide tachykinin NK1 antagonists.

1. This study was undertaken to compare the potency and selectivity of the nonpeptide (RP 67580, (+/-)-CP-96,345 and its chloro-derivative [(+/-)-cis-3-(2-chlorobenzylamino)-2-benzhydrylquinuclidine] (CP-C1)) and peptide (GR 71,251 and spantide) neurokinin1 (NK1) antagonists in mouse and rat preparations. 2. Among the NK1 antagonists tested, RP 67580 was the most potent in inhibiting the specific binding of [125I]-Bolton Hunter substance P ([125I]-BHSP) to crude synaptosomes from the rat brain (Ki: 2.9 nM). (+/-)-CP-96,345 was about ten fold less potent (Ki: 31 nM) than RP 67580 while other compounds exhibited even less affinity. 3. All NK1 antagonists inhibit competitively the activation of phospholipase C by [Pro9]substance P ([Pro9]SP) in cultured cortical astrocytes from the newborn mouse, a preparation rich in NK1 receptors but devoid of NK2 and NK3 receptors. pA2 values for the most potent compounds, RP 67580 and (+/-)-CP-96,345, were 8.28 and 7.08 respectively. When used alone, all antagonists showed some agonist activity at 10(-5) M, except spantide which was already effective at 10(-6) M. 4. An excellent correlation was found between the potency of the NK1 antagonists in blocking the stimulation by [Pro9]SP of phosphoinositide breakdown in cortical astrocytes and in inhibiting [125I]-BHSP specific binding to rat brain synaptosomes. 5. As shown on single cells by use of the Indo-1 microfluorometric method, RP 67580 (10(-7) M) prevented reversibly the elevation of cytosolic calcium concentration induced by [Pro9]SP (10(-8) M) in cultured cortical astrocytes. 6. Several experiments indicated that the antagonists were highly selective for NK1 receptors. RP 67580 did not modify the noradrenaline-evoked activation of phospholipase C in cortical astrocytes; when used at 10-5 M all antagonists had no or only little affinity for NK2 or NK3 binding sites and did not block the NKA (10-8 M)-induced activation of phospholipase C in the hamster urinary bladder (a selectiveNK2 test).7. In conclusion, RP 67580 appears to be a potent NK1 antagonist in the mouse and the rat. Results obtained with (+/-)-CP-96,345 confirm the lower potency of this compound in these two species when compared with reported data obtained in the guinea-pig or man.

The nonpeptide NK-2 antagonist SR 48968 is also a NK-3 antagonist in the guinea but not in the rat.

SR 48968 was first described as a NK-2 nonpeptide receptor antagonist; we report here that SR 48968 interacts also with guinea pig but not rat NK-3 cortical binding sites. Furthermore, SR 48968 is shown to inhibit the senktide- (a NK-3 selective agonist) evoked stimulation of phosphoinositide turnover in guinea pig ileum slices. The species difference observed for the NK-3 receptor with SR 48968 was confirmed by the determination of the affinities of NK-3 peptide agonists. [Pro7]neurokinin B particularly was found to have a greater affinity for cortical NK-3 binding sites in the rat than in the guinea pig.

[Functional properties of tachykinin receptors in the central nervous system]. / Propriétés fonctionnelles des récepteurs des tachykinines dans le système nerveux central.

Three types of tachykinin receptors (NK1, NK2 and NK3) have been identified, their endogenous ligands being substance P, neurokinin A and neurokinin B respectively. Peptide agonists and non peptide antagonists are now available allowing to study the functional properties of these receptors which are coupled to G proteins. Depending on the tissue, stimulations of phospholipase C occur with NK1, NK2 or NK3 agonists. Peptide, a C-terminal substance P analog and substance P(1-7) seem to act on tachykinin receptors distinct from those of the NK1, NK2 or NK3 type. NK2 binding sites have not been visualized in the brain. Nevertheless, biological responses of the NK2 type have been shown both in the substantia nigra and the striatum in studies on dopamine release.

[Pro9]SP and [pGlu6, Pro9]SP(6-11) interact with two different receptors in the guinea-pig ileum as demonstrated with new SP antagonists.

Structural considerations led us to postulate that the introduction of the dipeptides DPro9-Pro10 and DPro9-MeLeu10 should lock the C-terminal tetrapeptide of SP in a type II' beta-turn structure, a prerequisite for antagonist activity. Indeed, as the GR 71251, [DPro9, Pro10, Trp11]SP was more potent in inhibiting the septide, (pA2 = 6.5), than the [Pro9]SP, (pA2 < or = 5), spasmogenic activity in the guinea-pig ileum bioassay. This result confirms that septide, [pGlu6, Pro9]SP(6-11), a peptide active in the guinea-pig ileum bioassay and practically devoid of binding potencies for the three specific NK-1, NK-2 and NK-3 tachykinin binding sites interacts with a tachykinin receptor different from the NK-1 receptor sensitive to [Pro9]SP. Interestingly enough, the reintroduction of the leucine side-chain in position 10 yielded [DPro9, MeLeu10, Trp11]SP, an antagonist, equipotent in inhibiting both the septide- and the [Pro9]SP-evoked contractile response in the guinea-pig ileum bioassay, (pA2 = 6.6).

[A new tachykinin receptor revealed by substance P analogues in the guinea pig ileum]. / Un nouveau récepteur des tachykinines révélé par des analogues de la substance P dans l'iléon de cobaye.

[Pro9]SP and septide have been described as selective agonists for the SP receptor (NK-1 type). These two peptides contract with a great efficacy the guinea-pig ileum, but unexpectedly septide was practically devoid of affinity for the NK-1 site labelled by 3H-[Pro9]SP. Like septide, SP analogues like SP-O-CH3, [Apa9-10]SP and [Pro9,10]SP share the same peculiar properties. In addition, the contracting activity of these peptides is not explained by an interaction with NK-2 or NK-3 sites. GR 71,251, a compound which has been described as NK-1 antagonist, was more potent in inhibiting the septide- and the [Apa9-10]SP- than the [Pro9]SP-evoked contracting responses. Altogether, these results suggest that septide, SP-O-CH3, [Apa9-10]SP and [Pro9,10]SP exert their high contracting activity in the guinea-pig ileum by acting on a new type of tachykinin receptor.