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Amyloid β (Aβ) protein is responsible for Alzheimer's disease, and one of its important fragments, Aβ(25-35), is found in the brain and has been shown to be neurotoxic. Tachykinin neuropeptides, including Neuromedin K (NK), Kassinin, and Substance P, have been reported to reduce Aβ(25-35)'s toxicity in cells even though they share similar primary structures with Aβ(25-35). Here, we seek to understand the molecular mechanisms of how these peptides interact with Aβ(25-35) and to shed light on why some peptides with similar primary structures are toxic and others nontoxic. We use both experimental and computational methods, including ion mobility mass spectrometry and enhanced-sampling replica-exchange molecular dynamics simulations, to study the aggregation pathways of Aβ(25-35), NK, Kassinin, Substance P, and mixtures of the latter three with Aβ(25-35). NK and Substance P were observed to remove the higher-order oligomers (i.e., hexamers and dodecamers) of Aβ(25-35), which are related to its toxicity, although Substance P did so more slowly. In contrast, Kassinin was found to promote the formation of these higher-order oligomers. This result conflicts with what is expected and is elaborated on in the text. We also observe that even though they have significant structural homology with Aβ(25-35), NK, Kassinin, and Substance P do not form hexamers with a β-sheet structure like Aβ(25-35). The hexamer structure of Aβ(25-35) has been identified as a cylindrin, and this structure has been strongly correlated to toxic species. The reasons why the three tachykinin peptides behave so differently when mixed with Aβ(25-35) are discussed.

The neurokinin-1 receptor plays a profound role in inflammatory processes and is involved in immune cell differentiation, cytokine release, and mast cell activation. Due to their similar peptide structures, the neurokinin-1 receptor does not discriminate between the endogenous ligands substance P (SP) and human hemokinin-1 (hHK-1), which both demonstrate biological receptor affinity. In addition, due to cross-reactivity, the current bioanalytical method of choice-immunoassays-also displays limitations in differentiating between these peptides. Thus, a recently developed mass spectrometric assay was utilized for the selective quantification of SP and hHK-1 in various biofluids and tissue. By applying the sample processing protocols developed, SP was quantified in porcine brain tissue (4.49 ± 0.53 nM), human saliva (113.3 ± 67.0 pM), and human seminal fluid (0.52 ± 0.15 nM) by mass spectrometric analysis. As previously reported, neither SP nor hHK-1 could be detected in human plasma by mass spectrometry. Comparison with analysis using a commercial immunoassay of the same plasma sample revealed SP like-immunoreactivity concentrations of 37.1-178.0 pM. The previously reported carboxylic acid of SP, whose identity was confirmed by high-resolution mass spectrometric analysis, did not show cross-reactivity in the applied immunoassay and did not contribute to SP-like immunoreactivity results. Subsequent compound discovery of the immunocaptured substance indicated the presence of a precursor of SP as possible cross-reactor in human plasma samples. The found cross-reactivity might be the cause for the high variance of SP plasma levels in former determinations., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Ligand-receptor binding kinetics (i.e. association and dissociation rates) are emerging as important parameters for drug efficacy in vivo. Awareness of the kinetic behavior of endogenous ligands is pivotal, as drugs often have to compete with those. The binding kinetics of neurokinin 1 (NK1) receptor antagonists have been widely investigated while binding kinetics of endogenous tachykinins have hardly been reported, if at all. Therefore, the aim of this research was to investigate the binding kinetics of endogenous tachykinins and derivatives thereof and their role in the activation of the NK1 receptor. We determined the binding kinetics of seven tachykinins targeting the NK1 receptor. Dissociation rate constants (k off ) ranged from 0.026±0.0029min -1 (Sar 9 ,Met(O 2 ) 11 -SP) to 0.21±0.015min -1 (septide). Association rate constants (k on ) were more diverse: substance P (SP) associated the fastest with a k on value of 0.24±0.046nM -1 min -1 while neurokinin A (NKA) had the slowest association rate constant of 0.001±0.0002nM -1 min -1 . Kinetic binding parameters were highly correlated with potency and maximal response values determined in label-free impedance-based experiments on U-251 MG cells. Our research demonstrates large variations in binding kinetics of tachykinins which correlate to receptor activation. These findings provide new insights into the ligand-receptor interactions of tachykinins and underline the importance of measuring binding kinetics of both drug candidates and competing endogenous ligands., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Uperolein, a physalaemin-like endecapeptide, has been shown to be selective for Neurokinin 1 receptor. As a first step towards understanding the structure-activity relationship, we report the membrane-induced structure of Uperolein with the aid of circular dichroism and 2D (1)H NMR spectroscopy. Sequence-specific resonance assignments of protons have been made using 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 torsion angle molecular dynamics within program DYANA. The conformational range of the peptide revealed by NMR and CD studies has been analysed in terms of characteristic secondary features. Analysis of NMR data indicates that the global fold of Uperolein can be explained in terms of equilibrium between 3(10)-helix and alpha-helix from residues 5 to 11. An extended highly flexible N-terminus displays some degree of order and a possible turn structure. A comparison between the structures of Uperolein and Substance P, a prototype and endogenous Neurokinin 1 receptor agonist, indicates several common features in the distribution of hydrophobic and hydrophilic residues. Both the peptides show an amphiphilic character towards the middle region. The similarities suggest that the molecules interact with the receptor in an analogous manner.

Opioid and tachykinin systems are involved in modulation of pain transmission in the spinal cord. Regulation of surface opioid receptors on nociceptive afferents is critical for opioid analgesia. Plasma-membrane insertion of delta-opioid receptors (DORs) is induced by stimulus-triggered exocytosis of DOR-containing large dense-core vesicles (LDCVs), but how DORs become sorted into the regulated secretory pathway is unknown. Here we report that direct interaction between protachykinin and DOR is responsible for sorting of DORs into LDCVs, allowing stimulus-induced surface insertion of DORs and DOR-mediated spinal analgesia. This interaction is mediated by the substance P domain of protachykinin and the third luminal domain of DOR. Furthermore, deletion of the preprotachykinin A gene reduced stimulus-induced surface insertion of DORs and abolished DOR-mediated spinal analgesia and morphine tolerance. Thus, protachykinin is essential for modulation of the sensitivity of nociceptive afferents to opioids, and the opioid and tachykinin systems are directly linked by protachykinin/DOR interaction.

Two peptides with limited structural similarity to mammalian substance P (SP) and neurokinin A (NKA) have been isolated from extracts of the intestine of the African clawed frog (Xenopus laevis). The primary structure of an SP-like peptide was established as: Lys-Pro-Arg-Pro-Asp-Gln-Phe-Tyr-Gly-Leu-Met.NH(2), which is identical to the previously characterized peptide, bufokinin isolated from the toad Bufo marinus. The primary structure of an NKA-related peptide was established as Thr-Leu-Thr-Thr-Gly-Lys-Asp-Phe-Val-Gly-Leu-Met.NH(2). Only the five amino acids at the C-terminal region of the peptide are identical to mammalian NKA whereas the N-terminal region shows no structural similarity to previously characterized tachykinins. Immunohistochemical investigations of the gut wall revealed a dense network of nerve fibres and nerve cell bodies containing SP/NKA-like substances. The myotropic effects of the Xenopus tachykinins were compared with the contractile effect of mammalian SP and NKA on isolated strips of circular smooth muscle from Xenopus stomach. No significant differences in potencies (-log EC(50)) or in intrinsic activities were observed between the Xenopus and mammalian peptides. The potencies for the Xenopus SP-like (8.49+/-0.15) and the NKA-like peptide (8.12+/-0.06) were similar suggesting that the amino acid sequence at the N-terminal region of the tachykinins is not important in activating the tachykinin receptors in Xenopus gastric smooth muscle. The maximum response to Xenopus SP (alpha=0.59+/-0.06) was significantly lower than to the NKA-like peptide (alpha=1.0) suggesting a more effective interaction of the NKA-like peptide with the tachykinin receptor(s) in Xenopus stomach.

The conformation of the C-terminal octapeptide fragment of Substance P (SP4-11, Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) has been investigated by 2D-NMR and MD methods. The octapeptide exists in a blend of conformations. The molecule seems to shuttle between conformations with gamma-bends either at Phe5 or Gly6 or Gln3 or Leu7 and between a nearly extended structure.

Peptides with substance P-like immunoreactivity, neurokinin A-like immunoreactivity and neurotensin-like immunoreactivity were isolated in pure form from an extract of the intestine of the Burmese python (Python molurus). The primary structure of python substance P (Arg-Pro-Arg-Pro-Gln-Gln-Phe-Tyr-Gly-Leu- Met-NH2) shows one amino acid substitution (Phe8-->Tyr) compared with chicken/alligator substance P and an additional substitution (Lys3-->Arg) as compared with mammalian substance P. The neurokinin A-like immunoreactivity was separated into two components. Python neuropeptide gamma (Asp-Ala-Gly-Tyr- Ser-Pro-Leu-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 shows three substitutions (Gly5-->Ser, Gln6-->Pro and Ile7-->Leu) compared with alligator neuropeptide gamma and an additional substitution (His4-->Tyr) compared with mammalian neuropeptide gamma. Python neurokinin A (His-Lys-Thr-Asp-Ser-Phe-Val-Gly- Leu-Met.NH2) is identical to human/chicken/alligator neurokinin A. Python neurotensin (pGlu-Leu-Val-His-Asn-Lys-Ala-Arg-Pro-Tyr-Ile-Leu) is identical to chicken/alligator neurotensin. The data are indicative of differential evolutionary pressure to conserve the amino acid sequences of reptilian gastrointestinal peptides.

The bowfin is an extant representative of an ancient group of ray-finned fish with evolutionary connections to modern teleosts. A peptide with substance P-like immunoreactivity was isolated from an extract of bowfin stomach and its primary structure was established as Ser-Lys-Ser-His-Gln-Phe-Tyr-Gly-Leu-Met-NH2. This amino acid sequence resembles mammalian substance P only in the COOH-terminal region of the peptide. A second tachykinin with neurokinin A-like immunoreactivity isolated from the extract comprises 23 amino acid residues and shows limited structural similarity to mammalian neuropeptide-gamma. A randomly distributed population of cells in the gastric glands of the bowfin were immunostained with an antiserum raised against substance P, but no immunopositive structures were identified in the surface epithelium, lamina propria, or the nerve plexuses of the submucosa. Bolus injections of synthetic bowfin substance P (0.1-10 nmol/kg) into the bulbus arteriosus of unanesthetized bowfin resulted in a significant and dose-dependent rise in vascular resistance and arterial blood pressure (P < 0.01) and a fall in cardiac output (P < 0.05) without change in heart rate. After 5-10 min, arterial pressure and vascular resistance returned to preinjection levels, but cardiac output significantly (P < 0.05) increased over baseline values. The response to the peptide was unaffected by pretreatment of the animals with phentolamine. The study has shown that the stomach of the bowfin synthesizes tachykinins with novel structural features that display cardiovascular activity in this species.

The rat preprotachykinin-A (rPPT-A) gene encodes the precursor of several tachykinin neuropeptides including substance P. Previous studies have demonstrated the presence of multiple DNA sequences important for directing expression of the rPPT-A gene in dorsal root ganglion neurons within a region of the promoter spanning nucleotides -865 and -47. In order to identify potential cis acting elements, we have carried out DNase 1 footprinting analysis using a series of constructs containing fragments from this region of the promoter. This study has defined three potential AP-1 complex interactions, two E box binding protein interactions and two dG rich elements, which are potentially bound by complexes related to AP-2 or Sp1 in this region of the promoter.

Methods for extraction and isolation of intact RNA are often laborious, time consuming, and preclude the direct analysis of peptides. Similarly, the conditions for extraction and isolation of peptides are unsuitable for the isolation of intact RNA. Thus, to study changes in the levels of neuropeptides and gene expression of the corresponding mRNAs, separate procedures are required. A simple and rapid method for the simultaneous extraction of RNA and peptides from tissues is described. RNA and peptides are extracted with guanidinium isothiocyanate, followed by delipidation, and peptides are isolated by a simple solid-phase extraction procedure. RNA is isolated by differentially partitioning DNA into an organic phase, followed by precipitation with ethanol. The RNA and peptides isolated by this method are of high yield and quality. Furthermore, this method for RNA isolation is successful and efficient, even with tissues that proved recalcitrant with other procedures, and allows the simultaneous processing of multiple samples. We describe the successful application of this procedure for measuring tachykinins and the corresponding preprotachykinin A mRNA from tissues. Extraction of neuropeptide K, a 36-mer tachykinin, was dramatically more efficient with the present method than other methods in common use.

We have studied the structure-activity relationship of a series of tachykinin receptor antagonists based on spantide II. Fifteen novel peptides were tested for their ability to antagonize the electrically evoked tachykinin receptor-mediated response in the isolated rabbit iris sphincter muscle. Substitution or deletion of one to three amino acids in the spantide II sequence caused significant changes in biological activity. Eight of the novel analogues were found to be as potent as or more potent than spantide II and some were found to have better water solubility. We tested the selectivity for different tachykinin receptors of spantide II and two of the eight most potent analogues. They all interacted with tachykinin NK1 (rabbit jugular vein) and tachykinin NK2 (rabbit pulmonary artery) receptors with pA2 values of about 6.5-7.5 at the NK1 receptor and of 5.9-7.2 at the NK2 receptor, while being inactive at the tachykinin NK3 receptor (rat portal vein). Spantide II and the novel analogues were without effect on electrically evoked cholinergic responses of the isolated rabbit iris sphincter and on electrically evoked sympathetic responses of the guinea-pig vas deferens; moreover, they were without local anaesthetic-like effects on action potentials of the frog sciatic nerve, which suggests that they do not produce a general neurosuppressive effect. They were as effective as or slightly less effective than spantide II in causing histamine release from rat peritoneal mast cells.

Various procedures for extraction at acid, neutral and alkaline pH were compared with regard to the yield of different tachykinins and tachykinin-like substances from rat spinal cord. Reverse phase high performance liquid chromatography (RP-HPLC) and radioimmunoassay with various C-terminally directed tachykinin antisera and a newly developed N-terminally directed substance P (SP)-antiserum (SPN 1) were used. Antiserum SPN 1 fully reacts with SP-analogues modified at the C-terminal end (SP free acid and SP-Gly-Lys) and also (77%) with SP(1-9) but not with C-terminal SP-fragments lacking 2 or more N-terminal amino acids. The highest levels of SP-like immunoreactivity (LI) and neurokinin A (NKA)-LI were measured after combined water and acetic acid extraction procedures. Also when measuring cholecystokinin-like immunoreactivity the highest level was obtained following this extraction procedure. RP-HPLC revealed a major component of SP-LI at the position of synthetic SP irrespectively of the extraction method and if the C- or N-terminally directed antiserum was used. Neutral water extracts contained a late eluting component detected with the C-terminally, but not with the N-terminally, directed antiserum. Acid and alkaline extracts, in contrast, contained components which could be detected with the N-terminally, but not with the C-terminally, directed SP-antiserum. Immunoreactive components eluting at the position of NKA and NKB were found in all types of extracts with NKA-, kassinin- and eledoisin-antisera. The NKB- and neuropeptide K (NPK)-components were more prominent in acid than in neutral and alkaline extracts. In conclusion, the present results indicate that rat spinal cord may contain molecular forms of tachykinin-like immunoreactivity in addition to those previously described and illustrate the importance of the choice of extraction method in immunochemical studies. Combined extraction in water and acetic acid appears to be a suitable method when the content of peptides with different chemical properties are to be measured in a tissue sample.

An extract of the whole brain of the alligator (Alligator mississipiensis) contained very high concentrations of substance P-like immunoreactivity (405 pmol/g wet tissue) and neurokinin A-like immunoreactivity (514 pmol/g), as measured with antisera raised against the mammalian peptides. The primary structure of alligator substance P was established as: Arg-Pro-Arg-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2. This sequence is the same as that of chicken substance P and shows one substitution (Arg for Lys3) as compared with mammalian substance P. The neurokinin A-like immunoreactivity was separated into two components. Neuropeptide gamma was the most abundant peptide and its primary structure was established as Asp-Ala-Gly-Tyr-Gly-Gln-Ile-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser- Phe-Val-Gly-Leu-Met-NH2. This sequence shows one substitution (Tyr for His4) compared with mammalian neuropeptide gamma. The second component was identical to mammalian neurokinin A. A peptide with the chromatographic properties of mammalian neuropeptide K was not identified in the extract.

The purpose of this study was to determine whether substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) induce the release of neutrophil chemotactic activity (NCA) from bovine bronchial epithelial cells (BBEC) and whether neutral endopeptidase (NEP), a membrane-bound metalloenzyme that hydrolyzes tachykinins, modulates these effects. BBEC monolayers were exposed to SP, NKA, and NKB in the absence or presence of phosphoramidon (10(-6) M), a selective NEP inhibitor, for 72 h. Using a modified blind-well in vitro neutrophil chemotaxis assay, we found that tachykinin-exposed BBEC culture supernatant fluids induced significant neutrophil chemotaxis compared with supernatants obtained from unstimulated BBEC. Maximal effect was observed after 48 h of incubation and at SP concentration of 10(-13) M [92 +/- 3 (SP) vs. 64 +/- 2 (media) cells/high-power field (HPF), mean +/- SE, n = 7, P less than 0.05]. Release of NCA was mediated by the COOH-terminal of the SP molecule. The rank order of potency of tachykinins in inducing release of NCA was SP greater than NKA = NKB. SP-induced response was significantly potentiated by phosphoramidon (109 +/- 3 vs. 92 +/- 3 cells/HPF, n = 7, P less than 0.05), whereas other proteinase inhibitors had no effect. The released NCA was composed of protein and lipid-soluble components. These data indicate that mammalian tachykinins induce the release of NCA from BBEC and that NEP modulates these effects. We suggest that tachykinins regulate neutrophil recruitment into the lower respiratory tract, in part, by inducing the release of NCA from airway epithelial cells.

The aim of the study was to assess which tachykinin receptors mediate the contractile response in the guinea-pig isolated bronchi. Experiments with natural tachykinins and receptor-selective tachykinin agonists were performed in the absence or presence of peptidase inhibitors and in bronchi pretreated with phenoxybenzamine. Both NK-1 (substance P, substance P methylester and septide) and NK-2 (neurokinin A, [beta-Ala8]neurokinin A-(4-10) and MDL 28,564) receptor agonists produced concentration-dependent contraction. NK-3 agonists (senktide and [MePhe7]neurokinin B) were active only at high concentrations. Phenoxybenzamine pretreatment reduced the maximal response to NK-1 agonists and produced a rightward shift of the curve to NK-2 agonists, without depression of the maximum. Five tachykinin antagonists selective for the NK-1 (L 668,169) or the NK-2 (MEN 10,207, MEN 10,376, L 659,877 and R 396) receptor were tested against substance P methylester and [beta-Ala8]neurokinin A-(4-10). The results indicated that these receptor-selective antagonists maintain their characteristic even when tested in a multireceptor assay such as the guinea-pig bronchus. The rank order of potency of NK-2 antagonists against [beta-Ala8]neurokinin A-(4-10) was MEN 10,207 = MEN 10,376 greater than L 659,877 much greater than R 396. This pattern, with the observation of the full agonist activity of MDL 28,564, indicates that in addition to NK-1 receptors, NK-2 receptors also are present in the guinea-pig bronchi and belong to the same subtype (NK-2A) as present in the rabbit pulmonary artery.

Theoretical conformational analysis of N-terminal fragments of the title peptides has been carried out using the potential energy calculations. The number of conformational states for each fragment is very limited, and they are easily interconverted. Since these fragments cannot form alpha-helises, it is unlikely that upon binding of tachikinins to their receptors, their N-terminal fragments could overcome the hydrophobic barrier of the cell membrane's lipid belayer.