The new neurokinin 1-sensitive receptor mediates the facilitation by endogenous tachykinins of the NMDA-evoked release of acetylcholine after suppression of dopaminergic transmission in the matrix of the rat striatum

Using an in vitro microsuperfusion procedure, the NMDA-evoked release of [3H]ACh was studied after suppression of dopamine (DA) transmission (alpha-methyl-p-tyrosine) in striatal compartments of the rat. The effects of tachykinin neurokinin 1 (NK1) receptor antagonists and the ability of appropriate agonists to counteract the antagonist responses were investigated to determine whether tachykinin NK1 classic, septide-sensitive and/or new NK1-sensitive receptors mediate these regulations. The NK1 antagonists, SR140333, SSR240600, GR205171 but not GR82334 and RP67580 (0.1 and 1 microM) markedly reduced the NMDA (1 mm + D-serine 10 microM)-evoked release of [3H]ACh only in the matrix. These responses unchanged by coapplication with NMDA of NK2 or NK3 agonists, [Lys5,MeLeu9,Nle10]NKA(4-10) or senktide, respectively, were completely counteracted by the selective NK1 agonist, [Pro9]substance P but also by neurokinin A and neuropeptide K (1 nM each). According to the rank order of potency of agonists for counteracting the antagonist responses ([Pro9]substance P, 0.013 nM > neurokinin A, 0.15 nM > substance P(6-11) 7.7 nM = septide 8.7 nM), the new NK1-sensitive receptors mediate the facilitation by endogenous tachykinins of the NMDA-evoked release of ACh in the matrix, after suppression of DA transmission. Solely the NK1 antagonists having a high affinity for these receptors could be used as indirect anti-cholinergic agents.     

Diversity in mammalian tachykinin peptidergic neurons: multiple peptides, receptors, and regulatory mechanisms

The tachykinins comprise a family of closely related peptides that participate in the regulation of diverse biological processes. The tachykinin peptides substance P, neurokinin A, neurokinin A(3-10), neuropeptide K, and neuropeptide gamma are produced from a single preprotachykinin gene as a result of differential RNA splicing and differential posttranslational processing. Another tachykinin, neurokinin B, is produced from a separate preprotachykinin gene. These preprotachykinin mRNAs and peptide products are differentially distributed throughout the nervous system. Three distinct G protein-coupled tachykinin receptors exist for these tachykinin peptides. The three receptors interact differentially with the tachykinin peptides and are uniquely distributed throughout the nervous system. The NK-1 receptor preferentially interacts with substance P, the NK-2 receptor prefers neurokinin A, neuropeptide K, and neuropeptide gamma, and the NK-3 receptor interacts best with neurokinin B. Examples of the roles of tachykinin peptidergic neuronal systems are taken from the spinal cord sensory system and the nigrostriatal extrapyramidal motor system. Analysis of the functional significance of multiple tachykinin peptide systems, receptor-second messenger coupling mechanisms, and developmental and regulatory mechanisms underlying peptide mRNA and receptor expression represent areas of current and future investigation.     

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.     

Tachykinins and tachykinin receptors: a growing family

The peptides of the tachykinin family are widely distributed within the mammalian peripheral and central nervous systems and play a well-recognized role as excitatory neurotransmitters. Currently, the concept that tachykinins act exclusively as neuropeptides is being challenged, since the best known members of the family, substance P, neurokinin A and neurokinin B, are also present in non-neuronal cells and in non-innervated tissues. Moreover, the recently cloned mammalian tachykinins hemokinin-1 and endokinins are primarily expressed in non-neuronal cells, suggesting a widespread distribution and important role for these peptides as intercellular signaling molecules. The biological actions of tachykinins are mediated through three types of receptors denoted NK(1), NK(2) and NK(3) that belong to the family of G protein-coupled receptors. The identification of additional tachykinins has reopened the debate of whether more tachykinin receptors exist. In this review, we summarize the current knowledge of tachykinins and their receptors.     

Central administration of substance P inhibits feeding behavior in chicks

The purpose of the present study was to determine whether central administration of substance P (SP), a tachykinin neuropeptide, influenced feeding behavior in layer chicks (Gallus gallus). Intracerebroventricular (ICV) injections of 5 nmol SP decreased food intake in 5- and 6-day-old chicks under both ad libitum and 3-h fasting conditions. There are 3 major subtypes of tachykinin receptors, namely, neurokinin 1, 2 and 3 receptors. Injection of neurokinin A and neurokinin B, which are respectively endogenous agonists for neurokinin 2 and 3 receptors, did not suppress feeding behavior in chicks, suggesting that the anorexigenic effect of SP might be mediated by the neurokinin 1 receptor rather than neurokinin 2 and 3 receptors. Chicks that received 5 nmol SP did not change their locomotion, standing, sitting or drinking time, suggesting that its anorexigenic action might not be due to SP-induced hyperactivity or sedation. ICV injection of SP increased water intake, also indicating that SP likely did not affect feeding behavior through malaise. In addition, the anorexigenic effect of SP might not be related to corticotrophin-releasing hormone (CRH) because plasma corticosterone concentration was not affected by ICV injection of SP and co-administration of the CRH receptor antagonist astressin did not affect the anorexigenic effect of SP. The present study suggests that central SP acts as an anorexigenic neuropeptide in chicks.     

Functional and molecular characterization of tachykinins and tachykinin receptors in the mouse uterus

The aim of this study was to analyze the function and expression of tachykinins, tachykinin receptors, and neprilysin (NEP) in the mouse uterus. A previous study showed that the uterotonic effects of substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) in estrogen-treated mice were mainly mediated by the tachykinin NK1 receptor. In the present work, further contractility studies were undertaken to determine the nature of the receptors mediating responses to tachykinins in uteri of late pregnant mice. Endpoint and real-time quantitative RT-PCR were used to analyze the expression of the genes that encode the tachykinins SP/NKA, NKB, and hemokinin-1 (HK-1) (Tac1, Tac2, and Tac4); and the genes that encode tachykinin NK1 (Tacr1), NK2 (Tacr2), and NK3 (Tacr3) receptors in uteri from pregnant and nonpregnant mice. The data show that the mRNAs of tachykinins (particularly NKB and HK-1), tachykinin receptors, and NEP are locally expressed in the mouse uterus, and their expression changes during the estrous cycle and during pregnancy. The tachykinin NK1 receptor is the predominant tachykinin receptor in the nonpregnant and early pregnant mouse and may mediate tachykinin-induced uterine contractions in the nonpregnant mouse. The tachykinin NK2 receptor is predominant in the late pregnant mouse and is the main receptor mediating uterotonic responses to tachykinins at late pregnancy. The tachykinin NK3 receptor is expressed in considerable amounts only in uteri from nonpregnant diestrous animals, and its physiological significance remains to be clarified.     

Tachykinins Induce Secretion of Prolactin from Perifused Rat Anterior Pituitary Cells by Interactions with Two Different Binding Sites

Abstract

Substance P and the two other mammalian tachykinins, neurokinin A and B, are accepted to have direct regulating effects at the anterior pituitary level. We have examined the effects of substance P (SP) and neurokinin B (NKB), alone and in combination, on prolactin release from cultured anterior pituitary cells grown on collagen-coated micro beads and placed in a perfusion system. Prolactin (Prl) secretion was observed within 25 s after exposure to either secretagogue and reached a maximum within 60-80 s. Furthermore, the prolactin response induced by SP and NKB was dose-dependent. Prl secretion remained constant for up to 4 h when SP or NKB were perifused and then fell gradually towards basal levels. Simultaneous addition of submaximal concentrations of SP and NKB resulted in an additive response compared with the responses of either secretagogue alone. Continuous (8 h) perifusion with SP did not prevent a normal prolactin response by NKB or TRH. These results indicate that the tachykinins, substance P and neurokinin B, release Prl from perifused female rat anterior pituitary cells by interaction with two different receptors, possibly the NK1 and NK3 tachykinin receptor subtypes.     

NK1, NK2, NK3 and CGRP1 receptors identified in rat oral soft tissues,and in bone and dental hard tissue cells

The distribution of the tachykinin receptors neurokinin-1 (NK1), neurokinin-2 (NK2) and neurokinin-3 (NK3), and the calcitonin gene-related peptide-1 (CGRP1) receptor were examined in rat teeth and tooth-supporting tissues by immunohistochemical methods and light and confocal microscopy. Western blot analysis was performed to identify the NK1- and the CGRP1-receptor proteins in the dental pulp. The results showed that odontoblasts and ameloblasts, cementoblasts and cementocytes, osteoblasts and osteocytes are all supported with the tachykinin receptors NK1 and NK2, but a distinct, graded cellular labeling pattern was demonstrated. The ameloblasts were also positive for CGRP1 receptor. Blood vessels in oral tissues expressed the tachykinin receptors NK1, NK2 and NK3, and the CGRP1 receptor. Both gingival and Malassez epithelium were abundantly supplied by NK2 receptor. Pulpal and periodontal fibroblasts demonstrated NK1 and NK2 receptors. Western blot analysis identified both the NK1- and the CGRP1-receptor proteins in the dental pulp. These results clearly indicate that the neuropeptides substance P, neurokinin A, neurokinin B and CGRP, released from sensory axons upon stimulation, directly modulate the function of the different types of bone and dental hard tissue cells, and regulate functions of blood vessels, fibroblasts and epithelial cells in oral tissues.     

Tachykinin regulation of cholinergic transmission in the limbic/prefrontal territory of the rat dorsal striatum: implication of new neurokinine 1-sensitive receptor binding site and interaction with enkephalin/mu opioid receptor transmission

The tachykinin neurokinin 1 receptors (NK₁Rs) regulation of acetylcholine release and its interaction with the enkephalin/mu opioid receptors (MORs) transmission was investigated in the limbic/prefrontal (PF) territory of the dorsal striatum. Using double immunohistochemistry, we first showed that in this territory, cholinergic interneurons contain tachykinin NK₁Rs and co-express MORs in the last part of the light period (afternoon). In slices of the striatal limbic/PF territory, following suppression of the dopaminergic inhibitory control of acetylcholine release, application of the tachykinin NK₁R antagonist, SSR240600, markedly reduced the NMDA-induced acetylcholine release in the morning but not in the afternoon when the enkephalin/MOR regulation is operational. In the afternoon, the NK₁R antagonist response required the suppression of the enkephalin/MOR inhibitory control of acetylcholine release by βfunaltrexamine. The pharmacological profile of the tachykinin NK₁R regulation tested by application of the receptor agonists [[Pro⁹]substance P, neurokinin A, neuropeptide K, and substance P(6–11)] and antagonists (SSR240600, GR205171, GR82334, and RP67580) indicated that the subtype of tachykinin NK₁R implicated are the new NK₁-sensitive receptor binding site. Therefore, in the limbic/PF territory of the dorsal striatum, endogenous tachykinin facilitates acetylcholine release via a tachykinin NK₁R subtype. In the afternoon, the tachykinin/NK₁R and the enkephalin/MOR transmissions interact to control cholinergic transmission.     

Neurokinin-immunoreactivity in human neuroblastomas. Evidence for selective expression of the preprotachykinin (PPT) II gene.

Factors regulating differentiation of the peripheral nervous system (PNS) have been widely studied in neuroblastomas which are tumors of the PNS. Five neuroblastomas were investigated for their content of tachykinin neuropeptides, which arise from two distinct genes which appear differentially expressed in the PNS. Radioimmunoassay and column chromatography revealed large amounts of neurokinin B in three of these tumors and the absence of substance P, neurokinin A, neuropeptide K and neuropeptide gamma from all five tumors. This suggests that neuroblastomas can selectively express the preprotachykinin (PPT) II gene and that they may be valuable for investigating the factors involved in the regulation of these two structurally-related neuropeptide genes.     

Multiple tachykinins are produced and secreted upon post-translational processing of the three substance P precursor proteins, alpha-, beta-, and gamma-preprotachykinin. Expression of the preprotachykinins in AtT-20 cells infected with vaccinia virus recombinants

The rat preprotachykinin I gene mRNA is alternatively spliced to yield three different mRNA species differing in their protein coding regions. We have produced recombinant vaccinia viruses expressing alpha-, beta-, and gamma-preprotachykinin to examine the tachykinin-related peptides produced upon post-translational processing of each individual precursor. Infection of BSC-40 or AtT-20 cell lines with a beta-preprotachykinin-encoding vaccinia virus recombinant results in the expression of the precursor protein. The pro-form (signal peptide removed) can be immunoprecipitated from extracts of infected cells. Infected cells of both types secrete into the culture medium a product(s) which reacts in radioimmunoassay with an antiserum shown to recognize precursor as well as mature substance P. Infected AtT-20, but not BSC-40, cells secrete into the culture medium a processed form(s) of beta-preprotachykinin which reacts in radioimmunoassay with an anti-serum which recognizes the amidated carboxyl terminus of substance P. The molecular nature of the tachykinin products produced in and secreted from AtT-20 cells infected with alpha-, beta-, and gamma-preprotachykinin-encoding recombinants was analyzed by combined high performance liquid chromatography and radioimmunoassay. Peptides were identified based on comigration with synthetic standards and antisera cross-reactivity. We determined that alpha-preprotachykinin is processed to the mature undecapeptide, substance P. beta-Preprotachykinin was processed into multiple products, including substance P, neurokinin A, neurokinin A(3-10), and neuropeptide K. gamma-Preprotachykinin was processed into substance P, neurokinin A, neurokinin A(3-10), and neuropeptide gamma. These five tachykinin peptide products were all routed through the regulated secretory pathway and were secreted into the medium in a cAMP-stimulatable fashion. Since all of these peptides have been shown to be biologically active, it is important to consider the biological consequences of their co-secretion in vivo.     

Tachykinin receptors mediating airway macromolecular secretion.

Three tachykinin receptor types, termed NK1, NK2, and NK3, can be distinguished by the relative potency of various peptides in eliciting tissue responses. Airway macromolecular secretion is stimulated by the tachykinin substance P (SP). The purposes of this study were to determine the tachykinin receptor subtype responsible for this stimulation, and to examine the possible involvement of other neurotransmitters in mediating this effect. Ferret tracheal explants maintained in organ culture were labeled with 3H-glucosamine, a precursor of high molecular weight glycoconjugates (HMWG) which are released by airway secretory cells. Secretion of labeled HMWG then was determined in the absence and presence of the tachykinins SP, neurokinin A (NKA), neurokinin B (NKB), physalaemin (PHY), and eledoisin (ELE). All the tachykinins tested stimulated HMWG release to an approximately equal degree. Stimulation was concentration-related, with log concentrations giving half-maximal effects (EC50) as follows: SP -9.47, NKA -7.37, NKB -5.98, PHY -8.08, and ELE -7.68. This rank order of potency (SP greater than PHY greater than or equal to ELE greater than or equal to NKA greater than NKB) is most consistent with NK1 receptors. To evaluate the possible contribution of other mediators, tachykinin stimulation was examined in the presence of several receptor blockers. The potency of SP was not diminished by pretreatment with atropine, propranolol, or chlorpheniramine, and atropine actually increased the magnitude of the secretory response. The SP receptor antagonist [D-Arg1,D-Phe5, D-Trp7,9, Leu11]-SP blocked SP-induced secretion. These findings indicate that SP is a potent stimulus of airway macromolecular secretion. This effect occurs through the action of NK1 receptors, and is not dependent upon cholinergic, beta-adrenergic, or H-1 histamine receptors. The facilitation by atropine of SP stimulation suggests the existence of a mechanism of cholinergic inhibition of SP-induced stimulation.     

Stable expression of high affinity NK1 (substance P) and NK2 (neurokinin A) receptors but low affinity NK3 (neurokinin B) receptors in transfected CHO cells.

Stable CHO cell clones which selectively express all three rat tachykinin receptors were established by transfection. The binding of radiolabled substance P and neurokinin A (substance K) to CHO clones expressing the NK1 and NK2 receptors, respectively, were saturatable and of high affinity (Kd = 0.17 nM (NK1); 3.4 nM (NK2)). Scatchard analysis of the binding data indicated for both receptors binding to a single population of binding sites, and competition binding studies showed that the binding specificities of the receptors corresponded to those of classical NK1 and NK2 receptors. In contrast, the binding of eledoisin to the NK3 receptor expressed in the transfected CHO cells was of low affinity (IC50 = 240 nM) compared to the high affinity of the receptor found when it was transiently expressed in COS-7 cells (IC50 = 8 nM). However, in both cases the receptor exhibited the specificity of a classical NK3 receptor. The established cell clones may provide an important tool for further analysis of the molecular mechanisms involved in binding, activation, and coupling of receptors for tachykinin peptides.     

Binding sites for tachykinin peptides in the brain and stomach of the dogfish, Scyliorhinus canicula.

In membranes of dogfish brain and stomach, two binding sites for tachykinins were identified. One site specifically bound [125I]-Bolton-Hunter substance P (BH-SP) and the rank potency of tachykinins to compete for BH-SP binding revealed similarities with the rank potency of an NK1 receptor. The pharmacology of the other site, which specifically bound [125I]-Bolton-Hunter scyliorhinin II (BH-Scy II), did not resemble any of the mammalian tachykinin receptors. The rank potency to inhibit BH-Scy II binding to this second site was: scyliorhinin II approximately scyliorhinin I greater than eledoisin approximately substance P approximately neurokinin A greater than phyllomedusin approximately physalaemin greater than [Sar9Met(O2)11]substance P. Neurokinin B and senktide did not displace BH-Scy II binding. In addition, nucleotide analogues inhibited BH-SP binding but not BH-Scy II binding. Our binding data suggest the existence of a mammalian-like NK1 receptor and of a nonmammalian tachykinin receptor in the dogfish.     

Effects of supraspinal administration of PG-SPI and PG-KII, two amphibian tachykinin peptides, on nociception in the rat

We investigated and compared the effects of two amphibian tachykinins, the NK1 receptor agonist PG-SPI and the NK3 receptor agonist PG-KII, and the mammalian tachykinins substance P, neurokinin A and neurokinin B on the reaction time to a painful radiant heat stimulus (tail-flick test in rats) after intracerebroventricular injection. PG-SPI (1, 10 and 20 microg) and PG-KII (1, 5 and 10 microg) significantly increased the reaction time. Substance P (10 microg) injected intracerebroventricularly induced antinociception, whereas neurokinin A and neurokinin B did not. Like analgesia evoked by exogenous substance P, PG-SPI-evoked analgesia was blocked by pretreatment with naloxone. Naloxone left PG-KII antinociception unchanged, but the NK3 receptor selective antagonist markedly reduced it. These findings suggest NK1 and NK3 tachykinin receptor system involvement in supraspinal analgesia in rats.     

Role of tachykinins in non-adrenergic non-cholinergic excitation in smooth muscles of the gastrointestinal tract

Two peptides from the tachykinin family, substance P (SP) and neurokinin A (NKA), were identified as neurotransmitters (co-transmitters) of non-adrenergic non-cholinergic (NANCh) excitation in the gastrointestinal tract. The contraction of smooth muscles produced by tachykinins released from the excitatory enteric motoneurons is mediated by the NK1 and/or the NK2 tachykinin receptors. The differing contribution of these receptors in mediating the NANCh excitatory responses has been demonstrated in various regions of the intestine. The NK3 tachykinin receptors are confined only to the enteric neurons; they mediate release of different excitatory and inhibitory transmitters. The main secondary messenger pathway for all three tachykinin receptors is phosphoinositide breakdown that results in an increase of intracellular Ca²⁺ concentration. Signal transduction mechanisms are still not adequately known for tahykinin receptors. A multiple ionic mechanism has been proposed to mediate excitatory action of SP; it comprises activation of non-selective cationic channels, or activation of maxi Cl^– channels, and/or inhibition of K⁺ channels. Data about the ionic mechanism underlying the NK2 receptor activation are still missing. In conclusion, SP and NKA play a physiological role as NANCh neurotransmitters in smooth muscles of the gastrointestinal tract and, therefore, tachykinins may have a significant pathophysiological relevance in humans.Neirofiziologiya/Neurophysiology, Vol. 27, No. 5/6, pp. 425–432, September–December, 1995.     

Differential alterations in tachykinin NK2 receptors in isolated colonic circular smooth muscle in inflammatory bowel disease and idiopathic chronic constipation

Inflammatory bowel disease (IBD) and idiopathic chronic constipation (ICC) are intestinal disorders which disrupt normal colonic motility. Enteric tachykinins are well-recognised to play a role in the motor control of the gut, and increased colonic levels of substance P are seen in IBD, whereas decreased levels have been reported in ICC. In this investigation, we have characterised the tachykinin receptor population of normal human colonic circular smooth muscle and examined any changes that occur in IBD and ICC. The selective tachykinin NK2 receptor agonist, [beta-Ala8]neurokinin A(4-10), caused concentration-dependent contractions in healthy tissues; neither NK1 receptor-selective nor NK3 receptor-selective agonists were contractile. In diseased preparations also, only [beta-Ala8]neurokinin A(4-10) caused contractions with EC50 values similar to health. The maximum contractile responses (Emax), however, were significantly decreased in both forms of IBD but significantly increased in ICC. The muscarinic acetylcholine receptor agonist, carbachol, also caused contractions in diseased tissues, but EC50 and Emax values were not significantly different from health. The differential changes in contractility found in IBD and ICC are specific to NK2 receptors, and may reflect the altered levels of substance P or other tachykinins found in these intestinal disorders.     

Determination of ligand binding affinities for endogenous seven-transmembrane receptors using fluorometric microvolume assay technology.

We have developed a fluorescence-based mix and read method for the quantitative determination of receptor-ligand binding interactions. This method was used to determine IC(50) values for peptide ligands of two endogenous seven-transmembrane receptors that are expressed in cultured human cancer cells. Substance P, neurokinin A, and galanin were labeled with Cy5 and were shown to retain their native binding affinities. The cell-associated fluorescence was quantified using a fluorometric microvolume assay technology (FMAT) scanner that was designed to perform high-throughput screening assays in multiwell plates with no wash steps. The binding of fluorescently labeled substance P and neurokinin A was tested on the human astrocytoma cell line UC11 that expresses endogenous NK(1) receptor. Galanin binding was measured on endogenous galanin type 1 receptors in the Bowes neuroblastoma cell line. IC(50) values were determined for substance P, neurokinin A, and galanin and were found to correspond well with reported values from radioligand binding determinations. To demonstrate FMAT as instrumentation for high-throughput screening, it was utilized to successfully identify individual wells in a 96-well plate in which Cy5-substance P binding in UC11 cells was competed with unlabeled substance P. In addition, we developed a two-color multiplex assay in which cells individually expressing neuropeptide Y and substance P receptors were mixed in the same well. In this assay, the fluorescent ligands substance P and neuropeptide Y bound only to their respective cell types and binding was specifically competed. Therefore, two different seven-transmembrane receptor targets can be tested in one screen to minimize reagent consumption and increase throughput.