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.     

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.     

Regulation of the stimulant actions of neurokinin a and human hemokinin-1 on the human uterus: a comparison with histamine

Regulation of the contractile effects of tachykinins and histamine on the human uterus was investigated with biopsy sections of the outer myometrial layer. The effects of neurokinin A (NKA) and human hemokinin-1 (hHK-1) in tissues from pregnant but not from nonpregnant women were enhanced by the inhibition of neprilysin. The effects of NKA and eledoisin were blocked by the NK2 receptor antagonist SR 48968 but not by the NK1 receptor antagonist SR 140333 in tissues from both groups of women. Human HK-1 acted as a partial agonist blocked by SR 48968 and, to a lesser extent, by SR 140333; endokinin D was inactive. In tissues from pregnant women, responses to high potassium-containing Krebs solution were 2-3-fold higher than those from nonpregnant women. Mepyramine-sensitive maximal responses to histamine were similarly enhanced. The absolute maximum responses to NKA and its stable NK2 receptor-selective analogue, [Lys5MeLeu9Nle10]NKA(4-10), were increased in pregnancy, but their efficacies relative to potassium responses were decreased. Tachykinin potencies were lower in tissues from pregnant women than in those from nonpregnant women. These data 1) show for the first time that hHK-1 is a uterine stimulant in the human, 2) confirm that the NK2 receptor is predominant in mediating tachykinin actions on the human myometrium, and 3) indicate that mammalian tachykinin effects are tightly regulated during pregnancy in a manner that would negate an inappropriate uterotonic effect. The potencies of these peptides in tissues from nonpregnant women undergoing hysterectomy are consistent with their possible role in menstrual and menopausal disorders.     

Receptor subtypes involved in tachykinin-mediated edema formation.

Intradermal (ID) injection of the natural tachykinins substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) (0.3-30 nmol) resulted in a marked and dose-related rat paw edema, with mean ED50 values of 2.68 nmol, 1.17 nmol, and 0.80 nmol, respectively. The ID injection of the selective NK1, SP methyl-ester (1-30 nmol), NK2, [beta-Ala8]-neurokinin A4-10) (beta-Ala, 0.3-30 nmol), or NK3, senktide (1-10 nmol) agonists, caused extensive edema formation with mean ED50s of 0.48 nmol, 0.41 nmol, and 0.18 nmol, respectively. The ID injection of the selective NK1 antagonist FK888 (0.1-3 nmol) produced marked inhibition (94%, 52%, and 66%, respectively) of rat paw edema induced by SP, NKA, or SP methyl-ester. The ID co-injection of the NK2 receptor antagonist SR 48968 elicited a graded inhibition (52%, 67%, and 35%, respectively) of rat paw edema induced by NKA, beta-Ala and, to a lesser extent, the edema caused by SP. Finally, the ID co-injection of the NK, receptor antagonist SR 142801 significantly inhibited (53%, 76%, 53%, and 100%, respectively) the edema formation caused by NKB and NKA or by SP and senktide. Together, the data of the present study suggest that tachykinin-mediated rat paw edema depends on the activation of NK1, NK2 and NK3 receptor subtypes, with apparent major involvement of NK1 receptors subtypes.     

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.     

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.     

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.     

Developmental expression of neurokinin A and functional neurokinin-2 receptors in lung

Peribronchial smooth muscle constriction causes airway stretch, an important mechanical force in developing lung. Little is known about factors influencing these spontaneously active muscle elements. We measured contractile activity of neurokinin (NK) receptors on fetal intrapulmonary smooth muscle by tracheal perfusion assay (n = 11). Injecting either capsaicin or the NK(2) receptor agonist [NLE(10)]NKA resulted in significant (P < 0.05) bronchoconstriction. A specific NK(2) receptor antagonist inhibited constriction caused by endogenous tachykinins released by capsaicin. We then examined NK(2) receptor (n = 44) and NKA (n = 23) ontogeny in human lung. NKA immunostaining was identified in peribronchial nerves in samples with gestational age >12 wk. NK(2) receptor protein was identified in peribronchial and perivascular smooth muscle. These results indicate that endogenous tachykinins released by the developing lung act via NK(2) receptors to cause smooth muscle constriction. We speculate that tachykinins could modulate lung development.     

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.     

Pharmacological profile of hemokinin 1: a novel member of the tachykinin family

Recently, the cloning of a novel preprotachykinin gene (PPT-C) has been reported. This gene codes for a novel peptide named hemokinin 1 (HK-1). In contrast with the known tachykinins, which are exclusively expressed in neuronal tissues, PPT-C mRNA was detected primarily in hematopoietic cells. In this study, we pharmacologically characterised the effects of HK-1 using three tachykinin monoreceptor systems, namely the rabbit jugular vein (rbJV) for NK(1), the rabbit pulmonary artery (rbPA) for NK(2), and rat portal vein (rPV) for NK(3) receptors. In all these preparations substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) elicited concentration dependent contractions showing similar maximal effects and the following rank order of potency: SP > NKA = NKB in the rbJV, NKA > NKB > SP in the rbPA, and NKB > NKA > SP in the rPV. In those vessels HK-1 behaved as a full agonist displaying potencies similar (rbPA and rPV) or slightly higher (rbJV) than those of SP. In the rbJV, SR 140333, a selective NK(1) receptor antagonist, antagonised the effects of HK-1 and SP with similar high potencies (pK(B) 9.3 and 9.5, respectively). Similar results were obtained with the pseudopeptide NK(1) antagonist, MEN 11467 (pK(B) 8.8 and 8.6, respectively). Taken together, these data indicate that HK-1 behaves as a NK(1) preferring receptor agonist.     

Functional characterisation of tachykinin receptors in the circular muscle layer of the mouse ileum

OBJECTIVES: Tachykinins are important mediators in neuromuscular signalling but have not been thoroughly characterised in the mouse gut. We investigated the participation of tachykinin receptors in contractility of circular muscle strips of the mouse ileum. RESULTS: Electrical field stimulation (EFS) of excitatory nonadrenergic noncholinergic (NANC) nerves induced frequency-dependent contractions which were mimicked by substance P (SP). Desensitisation of SP and NK(1), NK(2) or NK(3) receptors significantly reduced contractions to EFS. The NK(1) receptor blocker RP67580 significantly inhibited NANC contractions to EFS. The NK(2) and NK(3) receptor blockers nepadutant and SR142801 did not affect NANC contractions per se but increased the RP67580-induced inhibition of NANC contractions to EFS. Contractions to SP were significantly reduced by RP67580 but not affected by nepadutant or SR142801. The NK(1) and NK(2) receptor agonists, septide and [beta-ala(8)]-NKA 4-10 (beta-A-NKA), respectively, but not the NK(3) receptor agonist senktide-induced dose-dependent contractions. Atropine inhibited and l-NNA augmented contractions to septide. Contractions to beta-A-NKA were insensitive to atropine but augmented by l-NNA. CONCLUSIONS: Tachykinins mediate NANC contractions to EFS in the mouse small intestine. Endogenously released tachykinins activate mainly NK(1) receptors, located on cholinergic nerves and smooth muscle cells and, to a lesser degree, NK(2) and NK(3) receptors, most likely located presynaptically.     

Tachykinin receptor subtype that mediates the increase in vascular permeability in guinea pig skin

To determine the tachykinin receptor subtype that mediates the increase in vascular permeability, we examined the rank order of potency of tachykinins for inducing plasma extravasation in guinea pig skin and the specificity of tachykinin-induced tachyphylaxis of the responses. Plasma extravasation of the skin induced by tachykinins was NK-1 (SP-P)-type response from the rank order of potency of mammalian and nonmammalian tachykinins. Tachyphylaxis of the vascular response was induced by intradermal preinjection of mammalian tachykinins and was tachykinin-specific. In substance P (SP) tachyphylaxis (where SP was preinjected), the response to SP, not to neurokinin A (NKA) or neurokinin B (NKB), was decreased. In NKA tachyphylaxis and NKB tachyphylaxis, the response to NKA, not to SP or NKB, and the response to NKB, not to SP or NKA, were decreased, respectively. Thus, we conclude that the apparent NK-1-type response is mediated through three mammalian tachykinin receptors, NK-1, NK-2, and NK-3, which are specifically stimulated by their preferred agonist, SP, NKA, and NKB, respectively.     

Expression and coupling of neurokinin receptor subtypes to inositol phosphate and calcium signaling pathways in human airway smooth muscle cells

Neuropeptide tachykinins (substance P, neurokinin A, and neurokinin B) are present in peripheral terminals of sensory nerve fibers within the respiratory tract and cause airway contractile responses and hyperresponsiveness in humans and most mammalian species. Three subtypes of neurokinin receptors (NK1R, NK2R, and NK3R) classically couple to Gq protein-mediated inositol 1,4,5-trisphosphate (IP3) synthesis and liberation of intracellular Ca2+, which initiates contraction, but their expression and calcium signaling mechanisms are incompletely understood in airway smooth muscle. All three subtypes were identified in native and cultured human airway smooth muscle (HASM) and were subsequently overexpressed in HASM cells using a human immunodeficiency virus-1-based lentivirus transduction system. Specific NKR agonists {NK1R, [Sar9,Met(O2)11]-substance P; NK2R, [beta-Ala8]-neurokinin A(4-10); NK3R, senktide} stimulated inositol phosphate synthesis and increased intracellular Ca2+ concentration ([Ca2+]i) in native HASM cells and in HASM cells transfected with each NKR subtype. These effects were blocked by NKR-selective antagonists (NK1R, L-732138; NK2R, GR-159897; NK3R, SB-222200). The initial transient and sustained phases of increased [Ca2+]i were predominantly inhibited by the IP3 receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) or the store-operated Ca2+ channel antagonist SKF-96365, respectively. These results show that all three subtypes of NKRs are expressed in native HASM cells and that IP3 levels are the primary mediators of NKR-stimulated initial [Ca2+]i increases, whereas store-operated Ca2+ channels mediate the sustained phase of the [Ca2+]i increase.     

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.     

Activation of neurokinin 1 receptors on interstitial cells of Cajal of the guinea-pig small intestine by substance P

 The aims of this work were to determine whether cells that are similar to the interstitial cells of Cajal (ICC) and have immunoreactivity for the neurokinin 1 (NK1) receptor are indeed ICC; to determine whether the agonist, substance P, binds to and activates the receptor on presumptive ICC; and to investigate the relationship between substance P-immunoreactive nerve fibres and ICC. ICC at the level of the myenteric plexus and in the deep muscular plexus in the duodenum and ileum of the guinea-pig were investigated. Immunoreactivities for the ICC marker, Kit, and the NK1 receptor were colocalised in ICC of the myenteric and deep muscular plexuses. In tissue fixed immediately after its removal from the animal, NK1 receptor-immunoreactive ICC were found at the level of the myenteric plexus in the duodenum, but not in the ileum, and in the deep muscular plexus in the duodenum and ileum. The majority of receptor immunoreactivity was on the cell surface. ICC were exposed to substance P (10^–7 M), initially at 4°C for 1 h to allow the agonist to bind, followed by incubation at 37°C to allow receptor internalisation to proceed. Exposure to substance P caused the NK1 receptor immunoreactivity to aggregate in clumps in the cytoplasm of ICC of the myenteric and deep muscular plexuses, including the ICC of the myenteric plexus of the ileum, where NK1 receptor immunoreactivity was not seen if tissue was not exposed to substance P. Substance P, to which the fluorescent label, cyanine 3.18 (Cy-3), was coupled, bound to the ICC. The Cy-3-substance P was internalised with the receptor following warming to 37°C. Many, but not all, ICC were closely apposed by nerve fibres with immunoreactivity for substance P. It is concluded that the NK1 receptor immunoreactivity on ICC represents receptor that is functional in the sense that it binds the natural agonist substance P and undergoes agonist-induced internalisation. ICC are likely to receive excitatory innervation from the close approaches of tachykinin-containing nerve fibres. Accepted: 10 March 1998     

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.