NK-1 Binding Sites Research Articles

The alternative protein isoform NK2B, encoded by the vnd/NK-2 proneural gene, directly activates transcription and is expressed following the start of cells differentiation

NK-2 is a homeodomain protein essential for the development of the central nervous system in the Drosophila embryo. Here, we show that the vnd/NK-2 gene encodes an additional protein isoform (NK-2B) that differs from the known one (NK-2A) in its N-terminal domain. While NK-2A is a transcription repressor, NK-2B directly activates transcription from promoters containing NK-2 binding sites, with its N-terminal domain possessing a strong transcription activation potency. The transcription of NK-2B starts at the onset of metamorphosis. Its expression is observed in precursors of differentiating photoreceptors and in photoreceptors of the adult eye. Both NK-2B and NK-2A are expressed in the lamina. However, the expression of NK-2A is mostly associated with the undifferentiated state of nervous cells.

Effects of intra-hippocampal injections of the NK2 receptor antagonist saredutant on the elevated plus maze, and the mouse defense test battery

Intracerebroventricular (i.c.v.) or intraperitoneal (IP) administration of saredutant (SR48968), an NK2 receptor antagonist, produces anxiolytic-like effects in rodents in a number of animal models of anxiety. NK2 binding sites are present in several limbic structures in rats, including the hippocampus, thalamus, septum and prefrontal cortex, suggesting involvement in the modulation of emotional processes. The current study investigated the behavioral effects of saredutant infused into the ventral hippocampus (VH), a structure associated with cognitive and emotional processes, to clarify the neural substrate underlying the anxiolytic-like effect of the compound. Saredutant (10, 100 or 500 pmol/0.2 μL) was injected bilaterally into the VH of male CD-1 mice tested in the elevated plus-maze and mouse defense test battery (MDTB). Results from the EPM showed that microinjections of 10 pmol/0.2 μL of saredutant increased entries and time spent in the open arms and enhanced end-arm exploration. In the MDTB, saredutant (500 pmol/0.2 μL) decreased vocalizations and increased escape attempts in mice confronted with a rat. Taken together, these results suggest that hippocampal tachykinin mechanisms are involved in the modulation of anxiety and defensive behaviors.

The two NK-1 binding sites correspond to distinct, independent, and non-interconvertible receptor conformational states as confirmed by plasmon-waveguide resonance spectroscopy.

Two nonstoichiometric ligand binding sites have been previously reported for the NK-1 receptor, with the use of classical methods (radioligand binding and second messenger assays). The most populated (major, NK-1M) binding site binds substance P (SP) and is related to the adenylyl cyclase pathway. The less populated (minor, NK-1m) binding site binds substance P, C-terminal hexa- and heptapeptide analogues of SP, and the NK-2 endogenous ligand, neurokinin A, and is coupled to the phospholipase C pathway. Here, we have examined these two binding sites with plasmon-waveguide resonance (PWR) spectroscopy that allows the thermodynamics and kinetics of ligand-receptor binding processes and the accompanying structural changes of the receptor to be monitored, through measurements of the anisotropic optical properties of lipid bilayers into which the receptor is incorporated. The binding of the three peptides, substance P, neurokinin A, and propionyl[Met(O(2))(11)]SP(7-11), to the partially purified NK-1 receptor has been analyzed by this method. Substance P and neurokinin A bind to the reconstituted receptor in a biphasic manner with two affinities (K(d1) = 0.14 +/- 0.02 nM and K(d2) = 1.4 +/- 0.18 nM, and K(d1) = 5.5 +/- 0.7 nM and K(d2) = 620 +/- 117 nM, respectively), whereas only one binding affinity (K(d) = 5.5 +/- 0.4 nM) could be observed for propionyl[Met(O(2))(11)]SP(7-11). Moreover, binding experiments in which one ligand was added after another one has been bound to the receptor have shown that the binding of these ligands to each binding site was unaffected by the fact that the other site was already occupied. These data strongly suggest that these two binding sites are independent and non-interconvertible on the time scale of these experiments (1-2 h).

Conformational Analysis of the C-Terminal Gly-Leu-Met-NH 2 Tripeptide of Substance P Bound to the NK-1 Receptor

We examined the effect of simultaneously incorporating proline or proline-amino acid chimeras in positions 9, 10, and/or 11 of substance P, on the affinity for the two NK-1 binding sites and on second-messenger activation. Because these 3-substituted prolines constrain not only the (phi,psi) values of the peptide backbone, but also the chi space of the amino acid side chain, we were able to gather data on the structural requirements for high-affinity binding to the NK-1 receptor. We were able to confirm that this C-terminal component is crucial and that it should adopt an extended conformation close to a polyproline II structure when bound to the receptor. The partial additivity of these constraints, more specifically, for the NK-1M site, suggests that the peptide backbone flexibility around the hinge-point residue Gly9 is essential to subtly position crucial side chains.

Membrane-Induced Structure of Scyliorhinin I: A Dual NK1/NK2 Agonist

Scyliorhinin I, a linear decapeptide, is the only known tachykinin that shows high affinity for both NK-1 and NK-2 binding sites and low affinity for NK-3 binding sites. As a first step to understand the structure-activity relationship, we report the membrane-induced structure of scyliorhinin I with the aid of circular dichroism and 2D-(1)H NMR spectroscopy. Sequence specific resonance assignments of protons have been made from correlation spectroscopy (TOCSY, DQF-COSY) and NOESY spectroscopy. The interproton distance constraints and dihedral angle constraints have been utilized to generate a family of structures using DYANA. The superimposition of 20 final structures has been reported with backbone pairwise root mean-square deviation of 0.38 +/- 0.19 A. The results show that scyliorhinin I exists in a random coil state in aqueous environments, whereas helical conformation is induced toward the C-terminal region of the peptide (D4-M10) in the presence of dodecyl phosphocholine micelles. Analysis of NMR data is suggestive of the presence of a 3(10)-helix that is in equilibrium with an alpha-helix in this region from residue 4 to 10. An extended highly flexible N-terminus of scyliorhinin I displays some degree of order and a possible turn structure. Observed conformational features have been compared with respect to that of substance P and neurokinin A, which are endogenous agonists of NK-1 and NK-2 receptors, respectively.

Control of foot differentiation in Hydra: in vitro evidence that the NK-2 homeobox factor CnNK-2 autoregulates its own expression and uses pedibin as target gene

The foot of the simple metazoan Hydra is a highly dynamic body region of constant tissue movement, cell proliferation, determination and differentiation. Previously, two genes have been shown to participate in the development and differentiation of this body region: homeodomain factor CnNK-2 and signal peptide pedibin [Dev. Biol. 180 (1996) 473; Development 126 (1999) 517; Development 122 (1996) 1941; Mech. Dev. 106 (2001) 37]. CnNk-2 functions as transcriptional regulator and is responsive to changes in the positional value while the secreted peptide pedibin serves as ‘extrinsic’ positional signal. Exposure of polyps to pedibin increases the spatial domain of CnNK-2 expression towards the gastric region, indicating that positional signals are integrated at the cis-regulatory region of CnNK-2. In the present study, to elucidate the molecular basis of the interaction of CnNK-2 and pedibin, we characterized the 5′ regulatory regions of both genes. Within the CnNK-2 5′ upstream region, electrophoretic mobility shift assays showed that putative NK-2 binding motifs are specifically bound by both nuclear protein from Hydra foot and by recombinant CnNK-2, suggesting that CnNK-2 might autoregulate its own expression. This is the first indication for an autoregulatory circuit in Hydra. In addition, we also identified NK-2 binding sites in the cis-regulatory region of the pedibin gene, indicating that this gene is one of the targets of the transcription factor CnNK-2. On the basis of these results, we present a model for the regulatory interactions required for patterning the basal end of the single axis in Hydra which postulates that CnNK-2 together with pedibin orchestrates foot specific differentiation.

The two NK-1 binding sites are distinguished by one radiolabelled substance P analogue

Two non-stoichiometric binding sites had previously been characterized for the NK-1 receptor using two different types of radiolabelled analogues of substance P. However, the question remained on their eventual conformational interconversion induced or not by the ligand. In this study, kinetic, saturation, and competition studies using [ 3H]propionyl[Pro 9]SP demonstrate the existence of two independent binding components in CHO cells transfected with the human NK-1 receptor, with K d values of 0.040 nM (≈20% of total sites) and 5.9 nM (≈80% of total sites) that correspond to those of the two previously described binding sites. These two binding sites do not seem to interconvert since the minor one can be selectively extinguished in saturation studies in the presence of a SP analogue specific of this binding site.

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.

EVIDENCE OF A PERIPHERAL ROLE OF NEUROKININS IN DETRUSOR HYPERREFLEXIA: A FURTHER STUDY OF SELECTIVE TACHYKININ ANTAGONISTS IN CHRONIC SPINAL INJURED RATS

Spinal cord injury above the sacral micturition center usually leads to detrusor hyperreflexia, increased intravesical pressure and post-void residual urine. Detrusor hyperreflexia is believed to be mediated by afferent C fibers with tachykinins as neurotransmitters. We investigated the selective peptide tachykinin antagonists MEN 11420 and GR 82334 of NK-2 and NK-1 receptors, respectively, in a chronic rat model of detrusor hyperreflexia after suprasacral spinal cord injury. Adult female Sprague-Dawley rats weighing 200 to 250 gm. were used. The spinal cord was transected at the T10 level. The bladder was evacuated by the Credé maneuver 3 times daily. After 6 weeks the rats were implanted with femoral vein and bladder dome catheters 2 days before filling cystometry. The 5 rats in group 1 received 100 nmol./kg. of the NK-2 antagonist MEN 11420 intravenously. The 5 rats in group 2 received 100 nmol./kg. of the NK-1 antagonist GR 82334 intravenously. The 5 rats in group 3 received a combination of the same dose of each antagonist. Three repetitive micturition cycles were recorded before injection. Three micturition cycles were done 20 minutes after the injection of each antagonist. Mean cystometric parameters were reported, including bladder capacity, micturition pressure, baseline pressure, post-void residual urine and micturition volume, and the number and amplitude of hyperreflexic contractions greater than 15 cm. water. MEN 11420 significantly reduced the frequency of hyperreflexic contractions and baseline bladder pressure (p <0.05). There was no statistically significant effect on the other cystometric parameters. GR 82334 reduced the amplitude of hyperreflexic contractions but not statistically significant. A combination of MEN 11420 and GR 82334 significantly reduced the frequency and amplitude of hyperreflexic contractions (p <0.05) with no significant effects on other cystometric parameters, although there was a tendency toward increased micturition volume and bladder capacity. These results suggest that at the peripheral level there is an efferent role of tachykinins in detrusor hyperreflexia after spinal cord injury. NK-1 and NK-2 receptor selective antagonists reduced the frequency and amplitude of hyperreflexic contractions as well as baseline bladder pressure. This finding may lead to potential new therapeutic modalities using selective tachykinins antagonists with other pharmacological agents to combat detrusor hyperreflexia.

Further Evidence for the Presence of “Septide-Sensitive” Tachykinin Binding Sites in Tissues Possessing Solely NK1 Tachykinin Receptors

Binding experiments performed with [125I]-NKA allowed us to demonstrate the presence of “septide-sensitive” specific binding sites on membranes from rat CHO cells transfected with the NK1 receptor cDNA (CHO-rat-NK1 cells), human astrocytoma U373 MG, or mouse cortical astrocytes, cells which express NK1 but neither NK2 nor NK3 receptors. In all cases, [125I]-NKA was specifically bound with high affinity (2 to 5 nM) to a single population of sites. In the three preparations, pharmacological characteristics of [125I]-NKA binding sites were notably different from those of classical NK1 binding sites selectively labelled with [125I]-BHSP. Indeed, the endogenous tachykinins NKA, NPK, and NKB and the septide-like compounds such as septide, SP(6–11), ALIE-124, [Apa9–10]SP, or [Lys5]NKA(4–10) had a much higher affinity for [125I]-NKA than [125I]-BHSP binding sites. Interestingly, differences were also found in the ratio of Bmax values for [125I]-NKA and [125I]-BHSP specific bindings from one tissue to another. These latter observations suggest that these two types of NK1 binding sites are present on distinct NK1 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.

NK1, NMDA, 5HT1a, and 5HT2 receptor binding sites in the rat lumbar spinal cord: modulation following sciatic nerve crush.

Quantitative receptor binding autoradiography was used to study the NK1, NMDA, 5HT1a, and 5HT2 receptor binding densities in the adult rat lumbar spinal cord from 3 days to 20 weeks following a unilateral crush lesion of the sciatic nerve. NK1 binding density increased unilaterally in the superficial dorsal horn on the side of the sciatic crush to reach levels 60% above controls by 4 weeks following the lesion and returned to control values by 12 weeks. NMDA binding density increased bilaterally and equally in both the dorsal and ventral horns to reach 300% of control values at 2 weeks following the crush and returned to near control values by 20 weeks following the lesion. Serotonergic receptor binding did not change. The changes in NK1 receptor binding density on postsynaptic dorsal horn cells are consistent with a response to the decrease and recovery in the synthesis and transport of tachykinins by the dorsal root ganglion cells following peripheral nerve injury. the bilateral changes in NMDA receptor binding are more likely mediated by polysynaptic pathways in the spinal cord that respond to the changes in metabolic events of the dorsal root ganglion cells evoked by axotomy and regeneration.

Expression and presence of septal neurokinin-2 receptors controlling hippocampal acetylcholine release during sensory stimulation in rat.

We examined the expression and presence of NK2 receptors in the septal area of rat brain, and investigated their functional role in the regulation of the septohippocampal cholinergic system. Using reverse transcription-polymerase chain reaction (RT-PCR) analysis, we showed the presence of NK2 receptor mRNA expression in the septal area, and detected septal NK2 binding sites by using a fluorescent-tagged neurokinin A (NKA) derivative. In vivo microdialysis was employed to explore the functional role of NK2 receptors in the release of hippocampal acetylcholine evoked by tactile stimulation in freely moving rats. Two sessions of stroking of the neck and back of the rat for 30 min, at 90 min intervals, produced a marked and reproducible increase in hippocampal acetylcholine release. This effect was dose-dependently prevented by intraperitoneal administration of the two selective non-peptide tachykinin NK2 receptor antagonists SR144190 (0.03-0.3 mg/kg, i.p.) and SR48968 (0.3 and 1 mg/kg, i.p.), but not by the inactive enantiomer of SR48968 (SR48965, 1 mg/kg) nor by the two non-peptide NK1 receptor antagonists SR140333 (3 mg/kg, i.p.) and GR205171 (1 mg/kg, i.p.). Furthermore, the intraseptal application of SR144190 (10(-8) M) reduced the sensory response. Finally, intraseptal perfusion of neurokinin A (0.01-10 microM) in anaesthetized rats produced a concentration-dependent increase in hippocampal acetylcholine release. The response to neurokinin A (0.1 microM) was prevented by SR144190 (0.03-0.3 mg/kg, i.p.) and SR48968 (0.3-1 mg/kg, i.p.). In conclusion, this study provides direct evidence for the role of endogenous NKA/substance P, through the activation of NK2 receptors, in regulating the septohippocampal cholinergic function.

Heterogeneity of tachykinin receptors in the rabbit lung.

The tachykinins, substance P (SP) and neurokinin A (NKA), are agonists for the NK(1) and NK(2) receptors, respectively. Tachykinins have various respiratory effects, including bronchoconstriction. This study characterizes tachykinin binding sites in the rabbit lung. We hypothesize that (2-[(125)I]iodohistidyl(1))Neurokinin A ([(125)I]NKA) interacts with NK1 and NK2 binding sites in the rabbit lung. The K d determined from saturation isotherms was 0.69 times/divided by 1.14 nM (geometric mean times/divided by SEM) and the B max was 4.15 + or - 0.22 femtomole/mg protein (arithmetic mean + or - SEM). Competitive inhibition studies with NKA, SP and various selective tachykinin agonists showed the rank order of potency; [beta-Ala(8)]-Neurokinin A 4-10 = SP >> NKA >> [Sar(9),Met(02)11]-Substance P. [beta-Ala(8)]-Neurokinin A 4-10, a selective NK(2) agonist, and SP inhibition of [(125)I]NKA binding were best described using a two-site model. Competitive inhibition studies using the selective nonpeptide NK(2) antagonist (SR 48968) and the selective nonpeptide NK(1) antagonist (CP 96,345) revealed Ki's of 5.5 nM and 8.1 nM, respectively. Our data therefore suggest that [(125)I]NKA binds to both the NK(1) and NK(2) receptors in the lung.

Chapter 6 Tachykinin receptors in the spinal cord

In summary, all three tachykinin receptors appear to be important modulators of physiological systems in the spinal cord. However, although there is a good deal of data concerning binding characteristics in peripheral tissues, work done in the spinal cord is scanty, leading to a number of unanswered questions. Firstly, Lui et al. (1993) have suggested a discrepancy between the location of SP binding sites and SP containing terminals. This might explain the conflicting evidence on the role of NK1 receptors in the dorsal horn. Furthermore, evidence that NK2 receptors are involved in nociception is increasing, however binding sites for these receptors in the spinal cord have not been demonstrated. This appears to be due to the difficulty in locating an ideal receptor specific ligand. The role of NK2 receptors in autonomic function is also unclear, perhaps for the same reason. Finally, there is evidence indicating that NK3 binding sites are increased following transection of the LIV-VI dorsal roots, however, studies on the effects of inflammation have not been done, as they have with the NK1 and NK2 receptors. All of these and many more unanswered questions require further investigation.

Differential ability of tachykinin NK-1 and NK-2 agonists to produce scratching and grooming behaviours in mice

Potent and selective NK-1 and NK-2 agonists as well as compounds with lower selectivity and affinity for NK-1 binding sites were compared in their ability to produce scratching and grooming behaviours when injected intracerebroventricularly in mice. Septide, an agonist with a low affinity for NK-1 binding sites, [Sar 9, Met(O 2) 11]SP and to a lesser extent [Pro 9]SP, two potent and selective NK-1 agonists were the most effective drugs in stimulating these behaviours. Only high doses of [Apa 9,10]SP and [Lys 5, Tyr 7, Pro 8]NKA(4–10), two agonists with low affinity for NK-1 binding sites, produced scratching and grooming responses. Similarly, only high doses of [Lys 5, MeLeu 9, NLe 10]NKA(4–10), a potent NK-2 agonist, produced grooming behaviour. When coinjected with the endopeptidase enzyme inhibitor phosphoramidon, the effects of [Apa 9,10]SP, [Lys 5, Tyr 7, Pro 8]NKA(4–10) and [Pro 9]SP were markedly enhanced. Analyses of the potency of the different agents to displace 3H-SP binding in mouse subcortical structures revealed that the affinities of the agonists for NK-1 receptors are similar to those previously reported in rat brain. The efficacy of the agonists at producing behavioural responses was not equivalent to their potency to bind to central NK-1 receptors. These findings therefore suggest that a stimulation of NK-1 but also non classical NK-1 receptors are involved in the induction of scratching and grooming behaviours.

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.

Evidence for tachykinin NK-2 receptors in guinea-pig airways from binding and functional studies, using [ 125I]-[Lys 5,Tyr(I 2) 7,MeLeu 9,Nle 10]-NKA(4–10)

The potent contractile responses of guinea-pig airways to neurokinin A (NKA) and neuropeptide γ (NPγ) are thought to be mediated by NK-2 receptors. However, NK-2 binding sites are not detectable using the radioligand [ 125I]-iodohistidyl-NKA. Here, a novel, highly selective iodinated-radioligand, [ 125I]-[Lys 5,Tyr(I 2) 7,MeLeu 9,Nle 10]-NKA(4–10), and a number of related peptides have been used to characterize NK-2 receptors on guinea-pig airways, using binding and functional studies. Specific binding of [ 125I]-[Lys 5,Tyr(I 2) 7,MeLeu 9,Nle 10]-NKA(4–10), was saturable and to a single high affinity site, with K D 1.29 ± 0.36 nM (n = 4). The rank order of potency for tachykinins and analogues as competitors for the binding was: [Lys 5,Tyr(I 2) 7,MeLeu 9,Nle 10]-NKA(4–10) ≥ NPγ ≥ [Lys 5,MeLeu 9,Nle 10]-NKA(4–10) > NKA ≥ SR 48968 ⪢ MDL 29913 ≥ substance P (SP) = [ 127I]-Bolton-Hunter NKA (BHNKA) ≥ MEN 10207 > neurokinin B (NKB). Septide, [DPro 9,Pro 10,Trp 11]-SP, the NK-1 selective ligands [Sar 9,Met(O 2) 11]-SP, [Pro 9]-SP and CP 96345, the NK-3 selective senktide, and calcitonin gene-related peptide (CGRP) were weak or ineffective. On guinea-pig isolated bronchi, the potency order of contractile agonists was: [Lys 5,MeLeu 9,Nle 10]-NKA(4–10) > NKA ≥ NPγ ≥ [Lys 5,Tyr 7,MeLeu 9,Nle 10]-NKA(4–10) ≥ septide = BHNKA ≥ [Lys 5,Tyr(I 2) 7,MeLeu 9,Nle 10]-NKA(4–10) ≥ [Sar 9,Met(O 2) 11]-SP ≥ NKB = [Pro 9]-SP ≥ SP ⪢ senktide. All NK-2 receptor preferring agonists, except BHNKA, had similar potencies in both systems. In conclusion, binding sites for [ 125I]-[Lys 5,Tyr(I 2) 7,MeLeu 9,Nle 10]-NKA(4–10) probably represent the NK-2-like receptors mediating contractions to NK-2 preferring tachykinins. NK-1 receptors also mediate contractile responses but NK-3 receptors appear to be negligible in this tissue.

Differential localization of 3H-[Pro 9]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-[Pro 9]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-[Pro 9]SP binding sites, does not suggest the existence of NK-1 binding site subtypes in the guinea pig brain.

Distinct presynaptic control of dopamine release in striosomal- and matrix-enriched areas of the rat striatum by selective agonists of NK1, NK2 and NK3 tachykinin receptors

Distinct presynaptic control of dopamine release in striosomal- and matrix-enriched areas of the rat striatum by selective agonists of NK1, NK2 and NK3 tachykinin receptors

Receptor binding profile of neuropeptide γ and its fragments: Comparison with the nonmammalian peptides carassin and ranakinin at three mammalian tachykinin receptors

The tachykinin binding site preferences of neuropeptide γ (NPγ), its C- terminal fragments AcNPγ(3–21), AcNPγ(5–21), AcNPγ(7–21), and AcNPγ(9–21), other mammalian tachykinins, and the nonmammalian tachykinins ranakinin and carassin were examined in membrane binding competition studies. [ 125I]-Bolton-Hunter [Sar 9,Met(O 2) 11]SP (BHSarSP), [ 125I]-neurokinin A (INKA) and [ 125I]-Bolton-Hunter scyliorhinin II (BHScyII) were used to investigate NK-1, NK-2, and NK-3 sites, in rat submandibular gland, gastric fundus, and brain, respectively. Elongation of the neurokinin A molecule does not appear to influence binding to rat tachykinin NK-1 and NK-2 binding sites. Ranakinin has affinity for the NK-1 and NK-2 site similar to that of substance P and neurokinin A, respectively, but has low affinity for the NK-3 site. Despite its structural similarities to neuropeptide γ, carassin has only moderate affinity for rat tachykinin binding sites. Possession of an acidic residue at position 4 appears critical for binding to rat NK-2 sites.