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AWL3106 composed of opioid (dermorphin) and tachykinin (substance P 7-11 ) pharmacophores is a new compound with high analgesic potency and markedly reduced ability to induce tolerance and dependence. The present study aimed to determine the respiratory and cardiovascular responses evoked by this peptide in urethane-chloralose anaesthetized, spontaneously breathing rats in the presence or absence of vagal connection. Intravenous injection of AWL3106 at a dose of 0.3μmol/kg in intact rats resulted in apnoea lasting 5.1 ± 0.7s. Breathing that followed was of diminished frequency (F) and augmented tidal volume (V T ) with no significant impact on minute ventilation. AWL3106-challenge induced biphasic fall in arterial blood pressure with no effect on heart rate. Midcervical and supranodosal sectioning the vagal nerves prevented the occurrence of the apnoea and abrogated the post-AWL3106 reduction in F but failed to eliminate the increase in V T . Hypotensive response appeared to be less profound following supranodose vagotomy. NaloxoneHCl abolished solely the occurrence of apnoea. However additional blockade of tachykinin NK 1 receptors with SR140333 was required to abolish V T increase, deceleration of breathing and to markedly suppress AWL3106-induced hypotension. The present study shows that extravagally controlled stimulation of V T maintains fairly regular ventilation by levelling the bradypnoeic effects. Although the peptide showed no cardiac effects, hypotension occurring beyond the vagal loop may limit future therapeutic benefits of this chimeric compound., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Tachykinins are comprised of the family of related peptides, substance P (SP), neurokinin A (NKA), and neurokinin B (NKB). NKB has emerged as regulator of kisspeptin release in the arcuate nucleus (ARC), whereas the roles of SP and NKA in reproduction remain unknown. This work explores the roles of SP and NKA in the central regulation of GnRH release. First, central infusion of specific agonists for the receptors of SP (neurokinin receptor 1, NK1R), NKA (NK2R) and NKB (NK3R) each induced gonadotropin release in adult male and ovariectomized, estradiol-replaced female mice, which was absent in Kiss1r(-/-) mice, indicating a kisspeptin-dependent action. The NK2R agonist, however, decreased LH release in ovariectomized-sham replaced females, as documented for NK3R agonists but in contrast to the NK1R agonist, which further increased LH release. Second, Tac1 (encoding SP and NKA) expression in the ARC and ventromedial nucleus was inhibited by circulating estradiol but did not colocalize with Kiss1 mRNA. Third, about half of isolated ARC Kiss1 neurons expressed Tacr1 (NK1R) and 100% Tacr3 (NK3R); for anteroventral-periventricular Kiss1 neurons and GnRH neurons, approximately one-fourth expressed Tacr1 and one-tenth Tacr3; Tacr2 (NK2R) expression was absent in all cases. Overall, these results identify a potent regulation of gonadotropin release by the SP/NK1R and NKA/NK2R systems in the presence of kisspeptin-Kiss1r signaling, indicating that they may, along with NKB/NK3R, control GnRH release, at least in part through actions on Kiss1 neurons.

This Review deals essentially with the elucidation of structural features of Tachykinin family of neuropeptides, which are known to interact through three distinct GPCR subtypes, namely NK1 (Neurokinin 1), NK2 (Neurokinin 2) and NK3 (Neurokinin 3) receptors. In mammals, Tachykinins have been shown to elicit a wide array of activities such as powerful vasodilatation, hypertensive action and stimulation of extravascular smooth muscle and are known to be involved in a variety of clinical conditions including chronic pain, Parkinson's disease, Alzheimer's disease, depression, rheumatoid arthritis, irritable bowel syndrome and asthma. This broad spectrum of action of Tachykinins is attributed to the lack of selectivity of tachykinins to their receptors. All tachykinins interact with all the three-receptor subtypes with SP preferring NK1, NKA preferring NK2 and NKB preferring NK3. This lack of specificity can be accounted for by the conformational flexibility of these short, linear peptides. Hence, identification of structural features of the agonists important for receptor binding and biological activity is of great significance in unraveling the molecular mechanisms involved in tachykinin receptor activation and also in rational design of novel therapeutic agents. Understanding structure of the ligand-receptor complex and analysis of topography of the binding pocket of the tachykinin receptor is also crucial in rational design of drugs.

Kisspeptin, neurokinin B (NKB) and dynorphin A (Dyn) are coexpressed within KNDy neurons that project from the hypothalamic arcuate nucleus (ARC) to GnRH neurons and numerous other hypothalamic targets. Each of the KNDy neuropeptides has been implicated in regulating pulsatile GnRH/LH secretion. In isolation, kisspeptin is generally known to stimulate, and Dyn to inhibit LH secretion. However, the NKB analog, senktide, has variously been reported to inhibit, stimulate or have no effect on LH secretion. In prepubertal mice, rats and monkeys, senktide stimulates LH secretion. Furthermore, in the monkey this effect is dependent on kisspeptin signaling through its receptor, GPR54. The present study tested the hypotheses that the stimulatory effects of NKB on LH secretion in intact rats are mediated by kisspeptin/GPR54 signaling and are independent of a Dyn tone. To test this, ovarian-intact prepubertal rats were subjected to frequent automated blood sampling before and after intracerebroventricular injections of KNDy neuropeptide analogs. Senktide robustly induced single LH pulses, while neither the GPR54 antagonist, Kp-234, nor the Dyn agonist and antagonist (U50488 and nor-BNI, respectively) had an effect on basal LH levels. However, Kp-234 potently blocked the senktide-induced LH pulses. Modulation of the Dyn tone by U50488 or nor-BNI did not affect the senktide-induced LH pulses. These data demonstrate that the stimulatory effect of NKB on LH secretion in intact female rats is dependent upon kisspeptin/GPR54 signaling, but not on Dyn signaling.

Tachykinins are important mediators of neuroneuronal and neuromuscular transmission in the gastrointestinal tract, however their contribution to colonic peristalsis in mice remains unclear. Therefore, our aim was to characterise the functional role of tachykinins in mediating peristalsis by evaluating the effect of selective tachykinin NK(1), NK(2) and NK(3) receptor agonists and antagonists on in vitro colonic peristaltic activity in mice. Using a modified Trendelenburg set-up, gradual distension of proximal and distal colonic segments evoked rhythmic, aborally migrating contractions. Peristaltic activity was assessed by quantifying the amplitude and interval of the corresponding pressure waves. Stimulation of NK(1) receptors showed regional differences as both the pressure amplitude and interval were enhanced in the distal colon without affecting peristalsis proximally. Blockade of NK(1) receptors reduced the peristaltic pressure amplitude in the proximal and distal colon while the interval was not significantly altered. NK(2) receptor stimulation resulted in a modest enhancement of the amplitude in proximal and distal segments and a slightly prolonged interval distally. Blockade of NK(2) receptors reduced the peristaltic pressure amplitude and interval in the distal colon. NK(3) receptor stimulation significantly augmented the amplitude in both segments and prolonged the interval distally. However, NK(3) receptor blockade had no effect on peristaltic activity. In conclusion, tachykinins contribute to colonic peristalsis in mice by acting mainly on NK(1) and NK(2) receptors and their effects show a proximal-to-distal gradient. NK(3) receptors might play a role in conditions of excess tachykinin release but appear not to be involved under the conditions of the present study., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We have suggested that in the lamprey, a medullary region called the paratrigeminal respiratory group (pTRG), is essential for respiratory rhythm generation and could correspond to the pre-Bötzinger complex (pre-BötC), the hypothesized kernel of the inspiratory rhythm-generating network in mammals. The present study was performed on in vitro brainstem preparations of adult lampreys to investigate whether some functional characteristics of the respiratory network are retained throughout evolution and to get further insights into the recent debated hypotheses on respiratory rhythmogenesis in mammals, such as for instance the "group-pacemaker" hypothesis. Thus, we tried to ascertain the presence and role of neurokinins (NKs) and burst-generating ion currents, such as the persistent Na(+) current (I(NaP)) and the Ca(2+)-activated non-specific cation current (I(CAN)), described in the pre-Bötzinger complex. Respiratory activity was monitored as vagal motor output. Substance P (SP) as well as NK1, NK2 and NK3 receptor agonists (400-800 nM) applied to the bath induced marked increases in respiratory frequency. Microinjections (0.5-1 nl) of SP as well as the other NK receptor agonists (1 microM) into the pTRG increased the frequency and amplitude of vagal bursts. Riluzole (RIL) and flufenamic acid (FFA) were used to block I(NaP) and I(CAN), respectively. Bath application of either RIL or FFA (20-50 microM) depressed, but did not suppress respiratory activity. Coapplication of RIL and FFA at 50 microM abolished the respiratory rhythm that, however, was restarted by SP microinjected into the pTRG. The results show that NKs may have a modulatory role in the lamprey respiratory network through an action on the pTRG and that I(NaP) and I(CAN) may contribute to vagal burst generation. We suggest that the "group-pacemaker" hypothesis is tenable for the lamprey respiratory rhythm generation since respiratory activity is abolished by blocking both I(NaP) and I(CAN), but is restored by enhancing network excitability., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Cyclosporin A (CsA) is an immunosuppressive drug. In human cancer cells substance P (SP) and neurokinin-1 (NK-1) receptor antagonists, respectively, induce cell proliferation and inhibition. CsA is a tachykinin receptor antagonist that showed selectivity for both NK-1 and NK-2 receptors. CsA exerts antitumor action against gastric (AGS) and colon (HT29) carcinoma cell lines. However, the mechanisms involved in this action remain unknown, and it is unknown whether CsA exerts an antitumor action on other human cancer cell lines or not. To demonstrate that CsA exerts a broad-spectrum antitumor action, we carried out an in vitro study of the growth-inhibitory capacity of CsA against seven human cancer cell lines, namely GAMG glioma, SKN-BE(2) neuroblastoma, WERI-Rb-1 retinoblastoma, HEp-2 larynx carcinoma, CAPAN pancreas carcinoma, 23132/87 gastric carcinoma, and SW-403 colon carcinoma. A Coulter counter was used to determine viable cell numbers followed by application of the MTS colorimetric method. Micromolar concentrations of CsA inhibited the growth of these tumor cells, both with and without previous administration of nanomolar concentrations of SP; the inhibition occurred in a dose-dependent manner. Moreover, CsA blocks SP-induced mitogen stimulation of tumor cells, suggesting that the NK-1 receptor is involved in such action. Following administration of CsA apoptosis was observed in the above seven tumor cell lines. These findings suggest that the antitumor action of CsA is at least due to its NK-1 receptor antagonist pharmacological profile, since the involvement of NK-2 receptors in the mentioned action must not be discarded, and that CsA has a broad-spectrum antitumor action., (Copyright 2010 Elsevier Inc. All rights reserved.)

Described in this unit are methods for obtaining, preparing, and testing smooth muscle preparations bearing tachykinin receptors to study the agonist or antagonist properties of test compounds. Concentration-response curves to agonists are constructed to measure their ability to produce smooth muscle contractions and thus evaluate the potency and efficacy of the agonists. Antagonists are tested for their ability to shift the agonist concentration-response curve and to calculate their potency. Two different protocols are described for each of the three tachykinin receptors (NK(1), NK(2), and NK(3)). The NK(1) receptor assays use guinea pig ileum longitudinal muscle myenteric plexus (GPI) and rat urinary bladder (RUB), the NK(2) receptor assays use isolated endothelium-deprived rabbit pulmonary artery (RPA) and hamster trachea (HT), and the NK(3) receptor assays use GPI and rat portal vein (RPV).

We examined the effects of physalaemin, an agonist of tachykinin receptors, on mechanical responses in the rat esophagus to clarify possible regulatory roles of tachykinins in esophageal motility. Exogenous application of physalaemin caused tonic contractions in rat esophageal segments when tension was recorded in the longitudinal direction but not when tension was recorded in the circular direction. The physalaemin-evoked contractions were blocked by pretreatment with nifedipine, a blocker of L-type calcium channels in both striated and smooth muscle cells. However, tetrodotoxin, a blocker of voltage-dependent sodium channels in striated muscle cells and neurons, did not affect the physalaemin-induced contractions. These results indicate that physalaemin might induce contractile responses in longitudinal smooth muscle of the muscularis mucosa via direct actions on muscle cells but not on neurons. Although pretreatment with a tachykinin NK(1) receptor antagonist, N-acetyl-l-tryptophan 3,5-bis (trifluoromethyl) benzyl ester (L-732,138), did not significantly affect the physalaemin-evoked contractions in rat esophageal segments, a tachykinin NK(2) receptor antagonist, (S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl) butyl] benzamide (SR48968), and a tachykinin NK(3) receptor antagonist, (S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl) piperidin-3-yl)propyl)-4-phenylpiperidin-4-yl)-N-methylacetamide (SR142801), significantly inhibited the physalaemin-evoked contractions. These results suggest that tachykinins can activate longitudinal contraction of smooth muscle in the muscularis mucosa, mediated via tachykinin NK(2) and NK(3) receptors on muscle cells, in the rat esophagus.

Background and Purpose: We investigated the ability of natural and synthetic selective NK receptors agonists and antagonists to modulate cyclooxygenase-2 (COX-2) expression in human polymorphonuclear leucocytes (PMNs)., Experimental Approach: The presence of all three tachykinin in PMNs was assessed by Western blot and PCR techniques. Natural and synthetic ligands selective for the tachykinin receptors were used to modulate COX-2 protein (measured with Western blotting) and activity [as prostaglandin E(2) (PGE(2)) output]. Effects of substance P (SP) on phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-kappaB) activation were studied to analyse the signalling pathway involved in COX-2 up-regulation mediated by SP., Key Results: Stimulation of NK receptors with the natural ligands SP, neurokinin A (NKA) and neurokinin B, in the pmol.L(-1)-micromol.L(-1) concentration range, modulated COX-2 expression and PGE(2) release in a concentration- and time-dependent manner. Experiments with synthetic selective agonists [Sar(9), Met(O(2))(11)]SP, [beta-Ala(8)] NKA(4-10), senktide or selective antagonists L703,606, SR48,968 or SR142801, confirmed that COX-2 up-regulation was mediated by NK receptors. We found that mainly p38, p42 and p46 MAPKs were phosphorylated by SP and SB202190, PD98059 and SP600125, which are selective inhibitors of these kinases, blocked SP-induced COX-2 expression. SP also induced nuclear translocation of NF-kappaB concentration-dependently, with a maximum effect at 1 nmol.L(-1)., Conclusions and Implications: Human PMNs possess functional NK(1), NK(2) and NK(3) receptors, which mediate the induction of COX-2 expression and NF-kappaB activation by SP.

By acting on neurokinin 1 (NK1) receptors, neuropeptides of the tachykinin family can powerfully excite rat hippocampal GABAergic interneurons located in the CA1 region and by this way indirectly inhibit CA1 pyramidal neurons. In addition to contact pyramidal neurons, however, GABAergic hippocampal interneurons can also innervate other interneurons. We thus asked whether activation of tachykinin-sensitive interneurons could indirectly inhibit other interneurons. The study was performed in hippocampal slices of young adult rats. Synaptic events were recorded using the whole-cell patch clamp technique. We found that substance P enhanced GABAergic inhibitory postsynaptic currents in a majority of the interneurons tested. Miniature, action potential-independent inhibitory postsynaptic currents were unaffected by substance P, as were evoked inhibitory synaptic currents. This suggests that the peptide acted at the somatodendritic membrane of interneurons, rather than at their axon terminals. The effect of substance P was mimicked by a selective NK1 receptor agonist, but not by neurokinin 2 (NK2) or neurokinin 3 (NK3) receptor agonists, and was suppressed by a NK1 selective receptor antagonist. In contrast to substance P, oxytocin, another peptide capable of activating hippocampal interneurons, had no effect on the inhibitory synaptic drive onto interneurons. We conclude that tachykinins, by acting on NK1 receptors, can influence the hippocampal activity by indirectly inhibiting both pyramidal neurons and GABAergic interneurons. Depending on the precise balance between these effects, tachykinins may either activate or depress hippocampal network activity.

In the brain, tachykinins acting via the three cloned neurokinin (NK) receptors are implicated in stress-related affective disorders. Hemokinin-1 is a novel tachykinin that reportedly prefers NK1 to NK2 or NK3 receptors. Although NK1 and NK3 receptors are abundantly expressed in the brain, NK2-receptor-mediated electrophysiological effects have rarely been described as NK2 receptors are expressed only in a few brain regions such as the nucleus of the medial septum/diagonal band. Medial septal/diagonal band neurons that control hippocampal mnemonic functions also colocalize NK1 and NK3 receptors. Functionally, intraseptal activation of all three NK receptors increases hippocampal acetylcholine release and NK2 receptors have specifically been implicated in stress-induced hippocampal acetylcholine release. Electrophysiological studies on the effects of NKs on septohippocampal cholinergic neurons are lacking and electrophysiological effects of hemokinin-1 have thus far not been reported in brain neurons. In the present study we examined the electrophysiological and pharmacological effects of multiple NKs on fluorescently tagged septohippocampal cholinergic neurons using whole-cell patch-clamp recordings in a rat brain slice preparation. We demonstrate that a vast majority of septohippocampal cholinergic cells are activated by NK1, NK2 and NK3 receptor agonists as well as by hemokinin-1 via direct post-synaptic mechanisms. Pharmacologically, hemokinin-1 recruits not only NK1 but also NK2 and NK3 receptors to activate septohippocampal cholinergic neurons that are the primary source of acetylcholine for the hippocampus.

Previous research has demonstrated that spinally transected rats can acquire a prolonged flexion response to prevent the delivery of shock. However, rats that receive shock irrespective of leg position cannot learn to maintain the same response. The present experiments examined the role of neurokinin receptors in this learning deficit. Results demonstrated that neurokinin (NK1 and NK2) antagonists blocked the induction of the learning deficit, whereas NK agonists induced a learning deficit. The study found that NK agonist administration did not substitute for uncontrollable shock exposure. Finally, administration of an NK1 agonist prior to uncontrollable shock prevented the induction of the deficit. These results provide additional evidence that engaging nociceptive plasticity undermines the capability of spinal neurons to support adaptive changes., ((PsycINFO Database Record (c) 2007 APA, all rights reserved).)

In most invertebrates, multiple species-specific isoforms of tachykinin-related peptide (TRP) are common. In contrast, only a single conserved TRP isoform, APSGFLGMRamide, has been documented in decapod crustaceans, leading to the hypothesis that it is the sole TRP present in this arthropod order. Previous studies of crustacean TRPs have focused on neuronal tissue, but the recent demonstration of TRPs in midgut epithelial cells in Cancer species led us to question whether other TRPs are present in the gut, as is the case in insects. Using direct tissue matrix assisted laser desorption/ionization Fourier transform mass spectrometry, in combination with sustained off-resonance irradiation collision-induced dissociation, we found that at least one additional TRP is present in Cancer irroratus, Cancer borealis, Cancer magister, and Cancer productus. The novel TRP isoform, TPSGFLGMRamide, was present not only in the midgut, but also in the stomatogastric nervous system (STNS). In addition, we identified an unprocessed TRP precursor APSGFLGMRG, which was detected in midgut tissues only. TRP immunohistochemistry, in combination with preadsorption studies, suggests that APSGFLGMRamide and TPSGFLGMRamide are co-localized in the stomatogastric ganglion (STG), which is contained within the STNS. Exogenous application of TPSGFLGMRamide to the STG elicited a pyloric motor pattern that was identical to that elicited by APSGFLGMRamide, whereas APSGFLGMRG did not alter the pyloric motor pattern.

The endokinins represent several species-divergent and peripherally located mammalian tachykinins (hemokinin-1 in mouse and rat, endokinin-1 in rabbit and endokinins A and B in humans) and also the tachykinin gene-related peptides. These peptides are all encoded on the preprotachykinin 4 (TAC4) gene. Their complementary DNA sequences, gene structures and expression profiles have been determined from a number of different mammalian species. They are all flanked by adjacent upstream and downstream dibasic cleavage sites in their respective precursor proteins, except for human EKA/B that instead possesses a N-terminal monobasic cleavage site. Evidence for differential processing in the periphery at the N-terminal cleavage site of the tachykinins could explain why in humans the evolutionary pressure to maintain the N-terminal dibasic cleavage site of EKA/B has been lost. Furthermore, the TAC4 encoded tachykinins all exhibit a remarkable selectivity and potency for the highly species conserved tachykinin NK(1) receptor, similar to that of substance P. Particular consideration is also given to the potential interactions of the endokinins with the short NK(1) receptor isoform and to speculation of whether there could be an "endokinin-sensitive" NK(1) binding site.

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.

Allodynia or hyperalgesia induced by peripheral nerve injury may be involved in changes in the sensitivity of neurotransmitters at the spinal cord level. In order to clarify the functional role of neurotransmitters in peripheral nerve injury, we used rats with nerve injury induced by chronic constriction of the sciatic nerve (CCI rat model) and estimated the effects of the intrathecal injection of drugs known to affect glutamate and tachykinin receptors. In sham-operated rats, the NMDA receptor agonist NMDA and AMPA-kinate receptor agonist RS-(5)-bromowillardin reduced withdrawal latency. The non-competitive NMDA receptor antagonist MK-801, competitive NMDA receptor antagonist AP-5 and AMPA-kinate receptor antagonist NBQX increased withdrawal latency. Substance P (SP) increased the withdrawal latency but only transitorily. The NK1 receptor antagonist RP67580 increased withdrawal latency, but the NK2 receptor antagonist SR48968 did not show an effect. In CCI rats, RS-(5)-bromowillardin reduced withdrawal latency, but NMDA did not show an effect. NBQX increased withdrawal latency, while MK-801 and AP-5 showed little or no effect. SP reduced withdrawal latency, and both RP67580 and SR48968 increased it. These results indicate that the alteration in sensitivity of ionotropic glutamate receptors and tachykinin receptors in the spinal cord contribute to development and maintenance of nerve injury-evoked neuropathic pain.

Background: Although being widespread in the hippocampus, the role tachykinins play in synaptic transmission is unclear. The effect of substance P on field potentials evoked by stimulation of the Schaffer collateral-commissural fibres and recorded from the CA1 region of the rat hippocampal slice were studied., Results: Perfusion of substance P (8 microM) had no effect on the fEPSP or population spike. Substance P did however cause a selective reduction in the paired pulse depression of population spikes evoked by paired stimulation at interpulse intervals of 20-80 msec. A comparison of the actions of other tachykinin receptor agonists gave an order of potency of substance P > [beta-Ala8]-neurokinin A (4-10) > senktide. The effect of substance P was reduced by the neurokinin-1 receptor antagonist SR140333, but not by the neurokinin-2 or neurokinin-3 receptor antagonists, MDL 29,913 or [Trp7, beta-Ala8]-neurokinin A (4-10)., Conclusion: The order of potency of the agonists, and the effects of the antagonists, both indicate that the effect of substance P on paired pulse depression is mediated by neurokinin-1 receptors.

The effect of loperamide on tachykinin NK(2)- and NK(3)-receptor-mediated 5-HT outflow from guinea pig colonic mucosa was investigated in vitro. The selective tachykinin NK(2)-receptor agonist [beta-Ala(8)]-neurokinin A(4-10) (betaAla-NKA) or the selective NK(3)-receptor agonist senktide elicited an increase in 5-HT outflow from whole colonic strips, but not from mucosa-free muscle layer preparations. The enhancing effect of betaAla-NKA and senktide was prevented by the selective NK(2)-receptor antagonist GR94800 or the selective NK(3)-receptor antagonist SB222200. Loperamide concentration-dependently suppressed the senktide-evoked 5-HT outflow, but failed to affect the betaAla-NKA-evoked 5-HT outflow. The kappa-opioid receptor antagonist nor-binaltorphimine or the delta-opioid receptor antagonist naltrindole displaced the concentration-response curve for the suppressant action of loperamide to the right without significant depression of the maximum. However, the mu-opioid receptor antagonist CTOP did not affect the suppressant effect of loperamide. We concluded that the NK(3) receptor-triggered 5-HT release from colonic mucosa is suppressed by loperamide-sensitive mechanisms, whereas the NK(2)-receptor-triggered 5-HT release is loperamide-insensitive. Our data also suggest that the suppressant effect of loperamide is probably mediated by the activation of kappa- and delta-opioid receptors located on intrinsic neurons.

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.

Present investigations were undertaken to study the influence of peptide NK-1 and NK-2 receptor agonists and antagonists as well as substance P and neurokinin A (the natural ligands for these tachykinin receptors) on oxytocin (OT) release from isolated rat hypothalamo-neurohypophysial (H-N) system as well as to determine whether the tachykinin NK-1 and/or NK-2 receptors contribute to the response of oxytocinergic neurons to melatonin. The results show, for the first time, that highly selective NK-1 receptor agonist, i.e., [Sar(9),Met(O(2))(11)]-Substance P, enhances while the NK-1 receptor antagonist (Tyr(6),D-Phe(7),D-His(9))-Substance P (6-11) - sendide - diminishes significantly OT secretion; the latter peptide was also found to antagonize the substance P-induced hormone release from isolated rat H-N system, when used at the concentration of 10(-7) M/L. Melatonin significantly inhibited basal and substance P-stimulated OT secretion. Neurokinin A and the NK-2 receptor selective agonist (beta-Ala(8))-Neurokinin A (4-10) as well as the NK-2 receptor antagonist (Tyr(5),D-Trp(6,8,9),Lys-NH(2)(10))-Neurokinin A (4-10) were essentially inactive in modifying OT release from the rat H-N system in vitro. The present data indicate a distinct role for tachykinin NK-1 (rather than NK-2) receptor in tachykinin-mediated regulation of OT secretion from the rat H-N system. Under present experimental conditions, however, a role of respective tachykinin receptors in the response of oxytocinergic neurons to melatonin has not been found.

A few naturally occurring insect tachykinin-related peptides, such as stomoxytachykinin (Stc-TK), contain an Ala-residue instead of the highly conserved Gly-residue that is present in most other members of this peptide family. Stc-TK is a potent, partial agonist of the stable fly (Stomoxys calcitrans) tachykinin receptor, STKR. By means of synthetic analogues, the Gly/Ala exchange, representing the addition of a single methyl group in the active core region of these peptides, was shown to be fully responsible for the generation of this partial agonism, which was also accompanied by an increase in agonistic potency. Surprisingly, this Ala-dependent reduction in maximal response levels was only observed for the agonist-induced cellular calcium rise. Stomoxytachykinin, Stc-TK, did not display partial agonism for the STKR-mediated cyclic AMP response. A possible explanation for this differential partial agonism is that the Gly-containing and Ala-replaced peptides recognize and stabilize active receptor conformations that differ in their functional coupling efficacies towards these response pathways. Drosotachykinins, Drm-TK, tachykinin-like peptides encoded in the fruit fly genome, were shown to be STKR-agonists. Interestingly, one of these peptides, which contains an Ala-residue instead of the conserved Gly-residue, also proved to be a potent, partial agonist for STKR.

The ability of calcitonin gene-related peptide (CGRP), to alter the outflow of 5-hydroxytryptamine (5-HT) from the guinea-pig proximal colon, was evaluated using three different isolated preparations: whole colon, mucosa-free muscle layer and submucosa/mucosa preparations. In the presence of the monoamine oxidase A inhibitor, clorgyline, CGRP elicited a concentration-dependent increase in 5-HT outflow from the whole colon, but not from mucosa-free muscle layer preparations. The CGRP-evoked 5-HT outflow was sensitive to tetrodotoxin (TTX) or hexamethonium, but was not detectable in submucosa/mucosa preparations. HCGRP8-37 (3 microM) inhibited the submaximal effect of CGRP on the 5-HT outflow. [Cys(ACM)2,7]hCGRP had a slight stimulant influence on the 5-HT outflow. The selective NK2 and NK3 receptor antagonists, SR48968 or SR142801, respectively, prevented the enhancing effect of CGRP. By contrast, a selective NK1 receptor antagonist L703606, failed to block the effect of CGRP. The enhancing effect of CGRP was mimicked by the NK2 receptor agonist [beta-Ala8]-neurokinin A (NKA)4-10 and the NK3 receptor agonist senktide. The effect of [beta-Ala8]-NKA4-10 on the 5-HT outflow was unaffected by TTX, while the effect of senktide was prevented by TTX, hexamethonium or SR48968. The present data also demonstrated a synergistic action of the NK2 and NK3 receptor agonists on the CGRP-evoked 5-HT outflow. We concluded that CGRP facilitates 5-HT release from the guinea-pig colonic mucosa through an action on myenteric neurons and that this effect is mediated by endogenously released tachykinins, acting via tachykinin NK2/NK3 receptors in cascade. British Journal of Pharmacology (2004) 141, 385-390. doi:10.1038/sj.bjp.0705624

With completion of the sequencing of the human and mouse genomes, the primary sequences of close to 400 non-olfactory G protein-coupled receptors (GPCRs) have been determined. There are intensive efforts within the pharmaceutical industry to discover and develop new therapeutic agents acting via GPCRs. In addition, there is a concerted effort to identify potential new drug targets from the remaining 150+orphan GPCRs through the identification of their ligands. Access to functionally expressed recombinant receptors underpins both of these key drug discovery activities. Typically, GPCR drug discovery screening activities are carried out using mammalian cell lines stably expressing the target of interest. The influx of new receptor sequences originating from genomic sequencing efforts has caused a shift toward wider applications of transient rather than stable expression systems, especially in support of assays for orphan receptor ligand screening. Recombinant baculoviruses in which the polyhedrin promoter has been replaced with a mammalian promoter, termed BacMam viruses, were originally designed as potential new gene therapy delivery vehicles. This same technology offers numerous advantages as a transient expression system in the assay of membrane-expressed drug targets, including GPCRs. Data presented show that BacMam can be used rapidly to generate robust and pharmacologically authentic GPCR assays in several formats, with the potential to transform drug discovery screening processes for this gene family.

Based on the experiments on rats, changes of the level of bile secretion and chemical composition of bile following application of substance P, neurokinin A, and selective agonist of the NK-2 tachykinins receptors neurokinin A Fragment (4-10), are interpreted. These results demonstrate that tachykinins (SP, NKA, and NKA Fragment (4-10)) are true holeretics and affect various enzyme systems in the hepatocyte. The alterations of the coefficients of bile confirm that.

(1) Studies were undertaken to determine the nature of the receptors mediating contractile effects of tachykinins in the uteri of nonpregnant women, and to analyse the expression of preprotachykinins (PPT), tachykinin receptors and the cell-surface peptidase, neprilysin (NEP), in the myometrium from pregnant and nonpregnant women. (2) The neurokinin B (NKB) precursor PPT-B was expressed in higher levels in the myometrium from nonpregnant than from pregnant women. Faint expression of PPT-A mRNA was detectable in the myometrium from nonpregnant but not pregnant women. PPT-C, the gene encoding the novel tachykinin peptide hemokinin-1 (HK-1), was present in trace amounts in the uteri from both pregnant and nonpregnant women. (3) Tachykinin NK(2) receptors were more strongly expressed in tissues from nonpregnant than from pregnant women. NK(1) receptor mRNA was present in low levels in tissues from both pregnant and nonpregnant women. A low abundance transcript corresponding to the NK(3) receptor was present only in tissues from nonpregnant women. (4) The mRNA expression of the tachykinin-degrading enzyme NEP was lower in tissues from nonpregnant than from pregnant women. (5) Substance P (SP), neurokinin A (NKA) and NKB, in the presence of the peptidase inhibitors thiorphan, captopril and bestatin, produced contractions of myometrium from nonpregnant women. The order of potency was NKA>>SP>/=NKB. The potency of NKA was unchanged in the absence of peptidase inhibitors. (6) The tachykinin NK(2) receptor-selective agonist [Lys(5)MeLeu(9)Nle(10)]NKA(4-l0) was approximately equipotent with NKA, but the tachykinin NK(1) and NK(3) receptor-selective agonists [Sar(9)Met(O(2))(11)]SP and [MePhe(7)]NKB were ineffective in the myometrium from nonpregnant women. (7) The uterotonic effects of [Lys(5)MeLeu(9)Nle(10)]NKA(4-10) were antagonized by the tachykinin NK(2) receptor-selective antagonist SR48968. Neither atropine, nor phentolamine nor tetrodotoxin affected responses to [Lys(5)MeLeu(9)Nle(10)]NKA(4-10). (8) These data are consistent with a role of tachykinins in the regulation of human uterine function, and reinforce the importance of NK(2) receptors in the regulation of myometrial contraction.

Peripheral tachykinins (TKs) are believed to play a role in the pathogenesis of inflammatory bowel diseases (IBD). In this study we investigated changes induced by central administration of two natural TK receptor agonists, NK(1) (PG-SPI) and NK(3) (PG-KII), on trinitrobenzene sulphonic acid (TNBS)- and dextran sodium sulphate (DSS)-induced experimental colitis in rats. Colitis was induced by instilling a single intracolonic dose of TNBS 50 mgkg(-1) (0.5 ml in 50% ethanol) or by oral administration of 5% DSS for 7 days. Each group of rats was intracerebroventricularly injected daily with PG-SPI and PG-KII (0.5, 5, and 50 microgkg(-1)). On day 3, TNBS-treated animals were killed and the severity of gut inflammation was evaluated by measuring myeloperoxidase (MPO) activity, interleukin-1beta (IL-1beta) production and by scoring macroscopic and histologic colonic damage. DSS-treated animals were checked daily for the length of survival and for stool consistency and faecal blood. In the TNBS group, PG-SPI and PG-KII increased scores for the severity of colonic damage, stimulated the production of IL-1beta and increased granulocyte infiltration into the colon (MPO activity). In the DSS group, PG-SPI and PG-KII decreased the percentage of surviving animals, and increased the number of rats that developed loose stools and blood in the faeces and the MPO activity. These results indicate that centrally injected NK(1) and NK(3) tachykinin receptor agonists play a proinflammatory role in experimentally-induced colitis in rats.

This study compared the nature and magnitude of the contractile response produced in vitro by selective NK1, NK2 and NK3 tachykinin receptor agonists in circularly and longitudinally oriented strips of myometrium from ovariectomised and ovariectomised oestrogen-treated rats. The nature of the responses produced upon stimulation of the tachykinin receptors varied between the different myometrial preparations and the hormonal environment from which the tissue was taken. Variations included: (i) sustained contraction until washout of agonist; (ii) biphasic contraction until washout of agonist; and (iii) monophasic contraction. The major differences in magnitude of contractions were seen in preparations from oestrogen-treated animals in which responses to stimulation of all tachykinin receptors were reduced in comparison to preparations from non-oestrogen treated animals. Furthermore, the responses in circularly oriented myometrium preparations from oestrogen-treated animals were all markedly reduced compared to responses in longitudinally oriented myometrium preparations. These results suggest that the tachykinin receptors in longitudinally and circularly oriented myometrial layers are differentially regulated, especially in tissue isolated from an oestrogen-dominated environment.

Tachykinins (TKs), which include substance P, neurokinin A and neurokinin B, constitute a group of neuropeptides widely expressed in the CNS where they play several functions connected with neural modulation often in synergy with glutamate excitatory transmission. The aim of this study was to assess whether TKs modulate glutamate response of in vitro cultured cerebellar granule neurons and whether GSA (glutamate-sensitizing activity), a peptide released by these neurons, belongs to the TKs family. Treatment with substance P and other neurokinin 1 receptor (NK1) agonists does not affect the response of cerebellar granule neurons to glutamate toxicity. On the contrary, agonists neurokinin 2 receptor (NK2) and neurokinin 3 receptor (NK3) agonists increase, in a dose and time dependent fashion, the response of the same neurons to glutamate. MEN 10,627, a selective NK2 receptor antagonist, and (Trp(7),betaAla(8)) NKA (4-10), a selective NK3 receptor antagonist inhibit not only the sensitizing action to glutamate of their respective agonists. These antagonists almost equally reduce the glutamate-sensitizing activity of GSA. Such activity is also abolished in the presence of a polyclonal antibody directed against neurokinin B (NKB). These findings indicate that TKs increase glutamate sensitivity in cerebellar granule neurons and that the GSA previously detected in conditioned media of the same cultured neurons belongs to the TK family although its primary structure as compared to known TKs remains to be established.

Mice were acutely intraperitoneally treated with diazepam (DZP), pentylenetetrazol (PTZ) or NaCl 0.9% (control group), and 15 min later, the DZP-treated group received substance P (SP), neurokinin A (NKA; NK1 and NK2 natural preferential agonists), [Trp7 beta-Ala8] NKA(4-10) (Trp-7; NK3 antagonist) or vehicle intracerebroventricularly, whereas the PTZ-treated group was intracerebroventricularly administered with FK 888, SR 48968 (NK1 and NK2 antagonists, respectively) or senktide (SENK--[succinil-Asp6, MePhe8] substance P(6-11); NK3 agonist), or vehicle immediately before they were submitted to the elevated plus-maze (EPM) test. Another group of animals was repeatedly treated with increasing doses of DZP or NaCl 0.9% intraperitoneally for 28 days, and 3 days after the last injection (test day), animals received DZP, FK 888, SR 48968, SENK or vehicle intracerebroventricularly, or DZP (NaCl 0.9%) intraperitoneally before the EPM evaluation. The anxiolytic action of the acute treatment with DZP was inhibited by the central administration of NKA and Trp-7 but not by SP. NK1 and NK2 antagonists, but not NK3 agonist, blocked the anxiogenic action of PTZ, as evaluated in the plus-maze test. Flumazenil (FLM), a benzodiazepine antagonist, was not able to inhibit the anxiolytic profile of action induced by the NK2 antagonist. Central administration of FK 888 and SR 48968 promoted anxiolytic effects in both control and DZP-withdrawn animals, suggesting a clear relationship between the GABAergic and the tachykinergic systems, mostly involving NK1 and NK2 receptors, in the modulation of experimental anxiety in mice.

The tachykinin peptide family certainly represents one of the largest peptide families described in the animal organism. So far, more than 40 tachykinins have been isolated from invertebrate (insects, worms, and molluscs), protochordate, and vertebrate (skin, gastrointestinal tract, peripheral and central nervous system) tissues. Substance P (SP), first identified by bioassay as early as 1931 but sequenced only in 1971, several years after the elucidation of the structure of eledoisin from molluscan tissues and of physalaemin from amphibian skin, may be considered as a prototype of the tachykinins. Hitherto, as many as 19 tachykinins have been isolated from amphibian integument, and eight additional peptides have been isolated from amphibian gut and brain. Counterparts of skin tachykinins in mammalian tissues are SP, neurokinin A, and neurokinin B. Three main receptor subtypes for the tachykinins have been identified (NK1, NK2, and NK3), but their number is probably destined to increase. It is obvious that the peripheral and central effects of the tachykinins may substantially vary depending on the activation of different receptor subtypes. Matters are further complicated by the frequent capacity of the single tachykinins to bind, although with different affinity, to more receptors. It has been recognized that tachykinins have a variety of effects in physiological and pathological conditions, and there is evidence suggesting intrinsic neuroprotective and neurodegenerative properties of these neuropeptides. This review provides an update on the current body of knowledge regarding tachykinin occurrence and distribution in the animal kingdom, from the lowest invertebrates to man, and the physiological and pharmacological actions of tachykinins outlining the pregnant importance of this large peptide family.

This study investigated the cellular basis for the enhanced ganglionic responsiveness to NK1 agonists in the spontaneously hypertensive rat (SHR) in comparison to their normotensive counterpart, the Wistar-Kyoto (WKY) rat. Rats for in vivo studies were anesthetized with pentobarbital and treated with chlorisondamine (10.5 micromol/kg). Extracellular recordings from the external carotid nerve showed a greater responsiveness of decentralized SHR superior cervical ganglia (SCG) to intravenous injection of SP (32 nmol/kg). Blood pressure and heart rate were increased in SHRs, whereas WKY rats responded with a decrease in blood pressure and only slight tachycardia. Membrane properties of SCG neurons, as shown by intracellular microelectrode recordings, were similar between strains. Picospritzer application of the NK1 agonist GR-73632 (100 microM, 1 s) evoked slow depolarization and increased neuron excitability. Spontaneous firing was evoked only in some neurons. Depolarization amplitudes were similar between strains; however, the NK1 agonist depolarized a greater number of neurons in hypertensive rats. In conclusion, SHRs are more responsive to ganglion stimulation by NK1 agonists due to a greater number of responsive cells within the SCG rather than an enhanced responsiveness of individual neurons.

The bioluminescent Ca(2+)-sensitive reporter protein, aequorin, was employed to develop an insect cell-based functional assay system for monitoring receptor-mediated changes of intracellular Ca(2)(+)-concentrations. Drosophila Schneider 2 (S2) cells were genetically engineered to stably express both apoaequorin and the insect tachykinin-related peptide receptor, STKR. Lom-TK III, an STKR agonist, was shown to elicit concentration-dependent bioluminescent responses in these S2-STKR-Aeq cells. The EC(50) value for the calcium effect detected by means of aequorin appeared to be nearly identical to the one that was measured by means of Fura-2, a fluorescent Ca(2)(+)-indicator. In addition, this aequorin-based method was also utilised to study receptor antagonists. Experimental analysis of the effects exerted by spantide I, II and III, three potent substance P antagonists, on Lom-TK III-stimulated S2-STKR-Aeq cells showed that these compounds antagonise STKR-mediated responses in a concentration-dependent manner. The rank order of inhibitory potencies was spantide III > spantide II > spantide I.

1. The effects of the novel mammalian tachykinin, hemokinin 1 (HEK-1), have been investigated by radioligand binding and functional in vitro and in vivo experiments. 2. Similar to SP (K(i)=0.13 nM), HEK-1 inhibited in a concentration-dependent manner and with high affinity [(3)H]-substance P (SP) binding to human NK(1) receptor (K(i)=0.175 nM) while its affinity for [(125)I]-neurokinin A (NKA) binding at human NK(2) receptor was markedly lower (K(i)=560 nM). 3. In isolated bioassays HEK-1 was a full agonist at tachykinin NK(1), NK(2) and NK(3) receptors. In the rat urinary bladder (RUB) HEK-1 was about 3 fold less potent than SP. In the rabbit pulmonary artery (RPA) HEK-1 and in the guinea-pig ileum (GPI), HEK-1 was about 500 fold less potent than NKA and NKB, respectively. 4. The responses to HEK-1 were antagonized by GR 82334 in RUB (pK(B)=5.6+/-0.07), by nepadutant in RPA (pK(B)=8.6+/-0.04) and by SR 142801 in GPI (pK(B)=9.0+/-0.2) with apparent affinities comparable to that measured against tachykinin NK(1), NK(2) and NK(3) receptor-selective agonists, respectively. 5. Intravenous HEK-1 produced dose-related decrease of blood pressure in anaesthetized guinea-pigs (ED(50)=0.1 nmol kg(-1)) and salivary secretion in anaesthetized rats (ED(50)=6 nmol kg(-1)) with potencies similar to that of SP. All these effects were blocked by the selective tachykinin NK(1) receptor antagonist, SR 140333. 6. We conclude that HEK-1 is a full agonist at tachykinin NK(1), NK(2) and NK(3) receptors, possesses a remarkable selectivity for NK(1) as compared to NK(2) or NK(3) receptors and acts in vivo experiments with potency similar to that of SP.

1. The effects of tachykinin NK(1), NK(2) and NK(3) receptor agonists and antagonists were measured on blood pressure (MAP) and heart rate (HR) after bilateral microinjection into the substantia nigra (SN) of awake, unrestrained rats. 2. Increasing doses (25 pmol - 1 nmol) of selective agonists at NK(1) ([Sar(9),Met(O(2))(11)]SP), NK(2) ([beta-Ala(8)]NKA(4 - 10)) and NK(3) (senktide) receptors into the SN produced tachycardia which was selectively and reversibly blocked by the prior injection of tachykinin antagonists at NK(1) (RP67580, 250 pmol), NK(2) (SR48968, 250 pmol) and NK(3) (R-820, 500 pmol) receptor. A rapid fall in MAP followed by a pressor response was seen with 1 nmol of [Sar(9),Met(O(2))(11)]SP. Behavioural activity was elicited by 1 nmol of [Sar(9),Met(O(2)(11)]SP (sniffing > face washing = grooming) and senktide (sniffing > wet dog shake > rearing = locomotion). Tachykinin antagonists had no direct cardiovascular or behavioural effects. 3. The tachycardia produced by 100 pmol of [beta-Ala(8)]NKA(4 - 10) or senktide was abolished by an i.v. treatment with atenolol (beta(1)-adrenoceptor antagonist, 5 mg kg(-1)) while that evoked by [Sar(9),Met(O(2))(11)]SP was reduced. A combination of atenolol (5 mg kg(-1)) and atropine (muscarinic antagonist, 1 mg kg(-1)) blocked the response evoked by [Sar(9),Met(O(2))(11)]SP. 4. These data suggest that the SN is a potential site of modulation of cardiac activity by tachykinins. In addition to the withdrawal of the cardiovagal activity by NK(1) receptor, the three tachykinin receptors appear to increase the sympatho/adrenal drive to the heart. This occurs independently of changes in MAP and behaviour. Hence, this study highlights a new central regulatory mechanism of cardiac autonomic activity.

Substance P, the principal neurokinin peptide in the mammalian brain and the natural ligand for the NK(1) tachykinin receptor, plays an integrative role in the regulation of cardiovascular, neuroendocrine and behavioural responses to stress. In rats, stimulation of periventricular NK(1) receptors in the forebrain induces a distinct pattern of cardiovascular responses which is accompanied by intense grooming behaviour. Ligands for NK(3) receptors induce a different pattern of cardiovascular and behavioural responses which comprises an increased release of vasopressin from the posterior pituitary and wet-dog shakes behaviour. To define the brain areas in the rat which respond to stimulation of forebrain NK(1) and NK(3) receptors and participate in the generation of these responses, the induction of c-Fos immunoreactivity was examined in brains following intracerebroventricular injections of substance P and neurokinin B in conscious rats. Stimulation of central NK(1) receptors by substance P (25, 100 and 500 pmol) injected into the lateral ventricle elicited grooming behaviour (face washing and hind limb grooming) and resulted in a marked c-Fos expression in the paraventricular, dorsomedial and parabrachial nuclei and in the medial thalamus. At 25 pmol, substance P did not significantly increase c-Fos expression, at 100 pmol, maximal c-Fos activation was induced in all four brain regions which responded to the peptide. Intracerebroventricular pretreatment of rats with the selective and high-affinity, non-peptide NK(1) receptor antagonist, RP 67580 (500 pmol), but not with its inactive enantiomer, RP 68651, completely abolished the behavioural response to substance P and reduced the substance P-induced c-Fos expression in all brain areas to nearly control levels. Intracerebroventricular injection of the natural ligand for NK(3) receptors, neurokinin B (500 pmol), elicited wet-dog shakes behaviour and activated c-Fos expression in localized regions of the forebrain including the organum vasculosum laminae terminalis, subfornical organ, median preoptic nucleus, paraventricular, supraoptic and anterior hypothalamic nuclei, medial thalamus and in the ventral tegmental area. These results demonstrate that the neurokinins, substance P and neurokinin B, induce specific and different patterns of c-Fos expression in distinct regions of the rat brain. Brain areas which selectively responded to substance P have been traditionally linked to the central regulation of cardiovascular and neuroendocrine reactions to stress or involved in the processing of nociceptive responses. On the other side, brain areas activated by neurokinin B are known to be involved in the central regulation of blood pressure, water and salt homeostasis or control of behaviour.

Tachykinin receptors in chicken intestine were studied using radioligand binding and functional techniques. Mechanisms of tachykinin-induced contraction were also investigated. Binding of [125I]Bolton-Hunter substance P ([125I]BH-SP) to chicken ileal membranes was rapid, saturable, of high affinity and to a single population of binding sites with Kd 0.72 nM and Bmax 0.48 fmol/ wet weight tissue. The rank order of agonists competing for [125I]BH-SP binding sites was [Sar9]SP > [Arg3]SP (natural tachykinin in chickens) > SP > [Pro9]SP > or = NKA > eledoisin > [Sar9,Met(O2)11]SP >> [Lys5,MeLeu9,Nle10]-NKA(4-10) >> senktide, suggesting similarities to the mammalian NK1 receptor. The NK1 receptor antagonist CP 99994, and NK2 receptor antagonist SR 48968 were weak competitors while spantide, RP 67580, GR 82334, GR 94800 and MEN 11420 were ineffective. The radioligand [125I]NKA showed no specific binding to ileal membranes. The potency order of most tachykinins in contacting isolated ileal longitudinal segments was in good agreement with that obtained from competition binding studies. Contractions to [Arg3]SP, NKA and senktide were greatly reduced by tetrodotoxin, suggesting that neurally-mediated responses were primarily involved. [Arg3]SP and NKA acted mainly by increasing release of acetylcholine, prostaglandins and probably tachykinins. Responses to [Arg3]SP were virtually abolished by nifedipine but were unaffected by NK1 receptor antagonists. Senktide-induced contraction was inhibited by the NK3 receptor antagonist, SR 142801, but was unaffected by atropine or L-NAME. The study provides evidence for a tachykinin receptor with similarities to the NK1 receptor in the chicken small intestine. In addition, senktide may act on a receptor similar to the mammalian NK3 receptor.

Intracellular recording techniques were used to test whether tachykinins could be mediators of slow excitatory postsynaptic potentials (EPSPs) in guinea pig sphincter of Oddi (SO) ganglia. Application of the tachykinin substance P (SP) onto SO neurons caused a prolonged membrane depolarization that was reminiscent of the slow EPSP in these cells. Pressure ejection of the neurokinin 3 (NK3) receptor-specific agonist senktide caused a similar depolarization; however, no responses were detected on application of NK1 or NK2 receptor agonists. The NK3 receptor antagonist SR-142801 (100 nM) significantly inhibited both SP-induced depolarization and the stimulation-evoked slow EPSP, as did NK3 receptor desensitization with senktide. Capsaicin, which causes the release of SP from small-diameter afferent fibers, induced a depolarization that was similar to the evoked slow EPSP in both amplitude and duration. The capsaicin-induced depolarization was significantly attenuated in the presence of SR-142801. These data indicate that tachykinins, released from extrinsic afferent fibers, act via NK3 receptors to provide slow excitatory synaptic input to SO neurons.

In the mammalian female reproductive tract, tachykinin neuropeptides, such as substance P (SP), are localized to a population of sensory fibers and their precise physiological role is still unknown. The aim of the present study was to characterize the population of tachykinin receptors in the pregnant rat uterus and to assess their regulation during the course of pregnancy and after delivery. The expression of the tachykinin NK(1) receptor (NK(1)R), the tachykinin NK(2) receptor (NK(2)R), and the tachykinin NK(3) receptor (NK(3)R) in uteri from rats at different stages of pregnancy and on Day 1 postpartum was investigated by using a semiquantitative reverse transcription-polymerase chain reaction. The contractile effect of tachykinin receptor agonists acting selectively on the NK(1)R, the NK(2)R, or the NK(3)R was investigated by conventional organ bath techniques. Serum levels of estrogen and progesterone were measured by RIA. Our data show that the expression and function of NK(1)R and NK(3)R varied along the course of pregnancy and at postpartum. Uterine NK(2)R mRNA levels remain stable during the course of pregnancy and at Day 1 postpartum; and the contractions elicited by activating selectively the NK(2) receptor in the presence of the neutral endopeptidase inhibitor phosphoramidon (1 microM) were similar in early, mid, or late pregnancy. These results show that the expression and function of tachykinin receptors within the uterus vary with reproductive state and length of gestation, supporting a role for tachykinins in pregnancy and/or parturition in the rat.

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.

1. Vagal nerve stimulation of the guinea-pig isolated oesophagus produced a triphasic tetrodotoxin (TTX)-sensitive contractile response. The third phase, which was resistant to ganglion blocking drugs, was selectively abolished by capsaicin, suggesting the involvement of one or more neuropeptides released from afferent neurons. Receptors on cholinergic neurons were subsequently activated because the response was atropine sensitive. Contractile responses resulting from exogenous substance P were abolished by atropine and TTX and enhanced by physostigmine. These findings suggest that the third phase may be mediated by the action of a substance P-like neuropeptide released from sensory nerve endings that subsequently activated cholinergic neurons. 2. The tachykinin receptors in the body of the guinea-pig oesophagus were characterized by determining the relative agonist potencies of natural tachykinins as well as tachykinin receptor-selective analogues. Antagonist affinities were also determined. The results indicated the presence of both NK2 and NK3 receptors. In addition, the effects of a cocktail of peptidase inhibitors (captopril, thiorphan and amastatin) on responses to various tachykinins and synthetic analogues were determined. The results indicate that one or more peptidases are present in this preparation. 3. Experiments using various tachykinin receptor antagonists were performed to determine whether the activation of tachykinin receptors played a role in the mediation of the third phase of the response to vagal nerve stimulation. While this response was unaffected by NK1 and NK2 receptor-selective antagonists, it was only partially inhibited (23%) by the NK3 receptor antagonist SR 142801. Thus, in the guinea-pig oesophagus, it appears that NK3 receptors play only a minor role in mediating a contractile response when afferent neurons are excited by vagal nerve stimulation.

The tachykinin, neurokinin A (NKA), contracts guinea-pig airways both in vitro and in vivo, preferentially activating smooth muscle NK(2) receptors, although smooth muscle NK(1) receptors may also contribute. In vitro evidence suggests that NKA activates epithelial NK(1) receptors, inducing the release of nitric oxide (NO) and subsequent smooth muscle relaxation. A number of selective NK(1) receptor agonists have been reported to activate both smooth muscle and epithelial NK(1) receptors, however septide appears only to activate smooth muscle NK(1) receptors. The aim of the present study was to investigate whether NKA-induced bronchoconstriction in guinea-pigs in vivo may be limited by NO release via NK(1) receptor activation, and whether selective NK(1) receptor agonists may activate this mechanism differently. Aerosolized NKA caused an increase in total pulmonary resistance (RL) that was markedly reduced by the NK(2) receptor antagonist, SR 48968, and abolished by the combination of SR 48968 and the NK(1) receptor antagonist, CP-99, 994. The increase in RL evoked by NKA was potentiated by pretreatment with the NO synthase (NOs) inhibitor, L-NAME, but not by the inactive enantiomer D-NAME. Potentiation by L-NAME of NKA-induced increase in RL was reversed by L-Arginine, but not by D-Arginine. Pretreatment with L-NAME did not affect the increase in RL induced by the selective NK(2) receptor agonist, [beta-Ala(8)]NKA(4-10), and by the selective NK(1) receptor agonist, septide, whereas it markedly potentiated the increase in RL caused by a different NK(1) selective agonist, [Sar(9),Met(O(2))(11)]SP. Dose-response curves showed that septide was a more potent bronchoconstrictor than [Sar(9),Met(O(2))(11)]SP to cause bronchoconstriction. Pretreatment with the NK(1) receptor antagonist, CP-96,994, abolished the ability of L-NAME to increase bronchoconstriction to aerosolized NKA. Bronchoconstriction to aerosolized NKA was increased by L-NAME, after pretreatment with the NK(3) receptor antagonist, SR 142801. The present study shows that in vivo bronchoconstriction in response to the aerosolized naturally occurring tachykinin, NKA, is limited by its own ability to release relaxant NO via NK(1) receptor activation. This receptor is apparently insensitive to septide, thus justifying, at least in part, the high potency of septide to cause bronchoconstriction in guinea-pigs.

1. The respiratory response to microinjection of tachykinins and analogues into the commissural nucleus of the solitary tract (cNTS) of urethane-anaesthetized rats was investigated in the presence and absence of selective tachykinin NK(1), NK(2) and NK(3) antagonists (RP 67580, SR 48968 and SR 142801, respectively). 2. All tachykinins, except for the selective NK(2) agonist, [Nle(10)]-NKA(4-10), increased tidal volume (VT). The rank potency order of naturally-occurring tachykinins was neurokinin A (NKA)> or =substance P (SP)>>NKB, whereas the rank order for selective analogues was senktide> or = septide>> [Sar(9),Met(O(2))(11)]-SP>>[Nle(10)]-NKA(4-10). Septide (NK(1)-selective) and senktide (NK(3)-selective) were 22 fold more potent (pD(2) approximately 12) at stimulating VT than SP (pD(2) approximately 10.5). 3. Tachykinin agonists produced varying degrees of respiratory slowing, independent of changes in VT. At doses producing maximum stimulation of VT, agonists induced either a mild (<10 breaths min(-1) decrease; SP and septide), moderate (10 - 25 breaths min(-1) decrease; NKA, NKB and [Sar(9),Met(O(2)]-SP) or severe ( approximately 40 breaths min(-1) decrease; senktide) bradypnoea. [Nle(10)]-NKA(4-10) produced a dose-dependent bradypnoea without affecting VT. 4. RP 67580 significantly attenuated the VT response to SP (33 pmol) and NKA (10 pmol) but not NKB (100 pmol). In the presence of RP 67580, the mild bradypnoeic response to NKB was significantly enhanced whereas SP and NKA induced a bradyapnea which was not observed in the absence of RP 67580. SR 48968 had no effect on the VT response to SP or NKB, markedly enhanced the VT response to NKA and completely blocked the bradypnoeic response to [Nle(10)]-NKA(4-10). Only SR142801 attenuated the VT response to NKB. 5. The present data suggest that all three tachykinin receptors (NK(1), NK(2) and NK(3)) are present in the cNTS and are involved in the central control of respiration.

The effect of selective tachykinin receptor agonists and antagonists on human isolated intralobar pulmonary arterial rings was investigated. Neither Substance P (SP) nor neurokinin A (NKA) contracted the arteries. Both of these agonists, however, were potent and efficacious at relaxing the arteries that were precontracted with phenylephrine. The negative log (M) EC(50) values for SP and NKA were 9.0 and 8.3, respectively. The neurokinin (NK)-3 selective agonist, senktide-analog, and the NK-2 receptor selective agonist, [beta-Ala(8)]NKA(4-10), caused neither contractions nor relaxations of the arteries, whereas the NK-1 receptor agonist Ac-[Arg6, Sar9, Met(O2)11]SP(6-11) (ASM-SP) relaxed the tissue with a potency similar to SP. The relaxations to SP, NKA, and ASM-SP were competitively antagonized by the selective NK-1 receptor antagonist CP 99994, with a pK(b) in the nanomolar range. Antagonism of the ASM-SP-induced relaxations was also noted with FK 888, RP 67580, and L 732,138, although these antagonists were much less potent than CP 99994 in this regard. Another NK-1 receptor selective antagonist, SR 140333, caused an insurmountable antagonism of the SP-induced relaxations. The NK-1 receptor-mediated relaxations could be blocked by removing the endothelium, or by a combination of N-nitro-L-arginine and indomethacin. Measurement of prostanoid generation revealed that in endothelial-intact but not endothelial-denuded tissue, ASM-SP caused a selective increase in the production of 6-keto-PGF1alpha, the stable metabolite of prostacyclin. The results indicate that stimulation of NK-1 receptors leads to relaxation of human intralobar pulmonary arteries, which is mediated largely by nitric oxide and prostacyclin released from the endothelium of these vessels.

We have analyzed, by the sucrose gap method, the action of otilonium bromide, a quaternary ammonium derivative in use for the symptomatic therapy of irritable bowel syndrome, on the electrical and mechanical responses initiated by different stimuli in the circular muscle of the guinea-pig proximal colon. Otilonium bromide produced a concentration-dependent inhibition of membrane depolarization (IC50 4.1 microM), action potentials (APs) and contraction (IC50 3.7 microM) produced by the muscarinic receptor agonist, methacholine. It also produced a concentration-dependent inhibition of APs and accompanying contraction (IC50 31 microM) produced by KCl (30 mM), and had a biphasic effect on the cholinergic excitatory junction potential (e.j.p.) produced by single pulse electrical field stimulation: at low concentrations (0.1-0.3 microM) otilonium bromide enhanced the e.j.p. and, at higher concentrations (IC50 22 microM and 16 microM toward depolarization and contraction), produced a concentration-dependent inhibition. Otilonium bromide eliminated the APs superimposed on the depolarization induced by the tachykinin NK1 receptor agonist, [Sar9]substance P-sulphone and suppressed the corresponding contraction (IC50 43 microM) but had little effect on the sustained membrane depolarization induced by this agonist. On the other hand, otilonium bromide produced a similar inhibitory effect on both membrane depolarization and contraction (IC50 38 microM and 45 microM, respectively) induced by the tachykinin NK2 receptor agonist [betaAla8]neurokinin A (4-10). When tested in the presence of nifedipine (1 microM), otilonium bromide had no effect on the membrane depolarization induced by [Sar9]substance P-sulphone but inhibited in a concentration-dependent manner the depolarization induced by [betaAla8]neurokinin A (4-10) (IC50 41 microM). In contrast, the blocker of receptor-operated cation channels, SKF 96365, inhibited with similar potency the depolarization induced by both [Sar9]substance P-sulphone and [betaAla8]neurokinin A (4-10) (IC50 60 microM and 54 microM, respectively). In radioligand binding experiments otilonium bromide produced a concentration-dependent inhibition of the binding of both an agonist ([125I]neurokinin A, Ki 7.2 microM) and an antagonist ([3H]SR 48968, Ki 2.2 microM) to membranes of Chinese hamster ovary cells transfected with the human tachykinin NK2 receptor. In conclusion, the present findings demonstrate that, in the microM range of concentrations, otilonium bromide acts as a muscarinic and tachykinin NK2 receptor antagonist and as a calcium channel blocker. The latter property is likely to account for its ability to suppress contraction initiated by the tachykinin NK1 receptor agonist. Therefore multiple mechanisms of action account for the ability of otilonium bromide to reduce stimulated motility of intestinal smooth muscle.

In the wild-type tachykinin NK3A receptor histidyl residues are present at two positions in TM-V, V:01 and V:05, at which Zn2+ functions as an antagonist in NK1 and kappa-opioid receptors with engineered metal-ion sites. Surprisingly, in the NK3A receptor Zn2+ instead increased the binding of the agonist 125I-[MePhe7]neurokinin B to 150%. [MePhe7]neurokinin B bound to the NK3A receptor in a two-component mode of which Zn2+ eliminated the subnanomolar binding mode but induced a higher binding capacity of the nanomolar binding mode. Signal transduction was not induced by ZnCl2 but 10 microM ZnCl2 enhanced the effect of neurokinin B. Ala-substitution of HisV:01 eliminated the enhancing effect of Zn2+ on peptide binding. It is concluded that physiological concentrations of Zn2+ have a positive modulatory effect on the binding and function of neurokinin B on the NK3A receptor through a bis-His site in TM-V.

In the present study, tachykinin receptors (designated NK 1, NK2 and NK3) involved in regulation of ion transport in porcine jejunum were characterised. Stripped tissue preparations were mounted in Ussing chambers and short-circuited. Substance P produced a concentration dependent increase in short-circuit current, the relationship showing a double sigmoidal form. The non-peptide NK1 receptor antagonist, CP 99,994 ((2S,3S)-3-(2-methoxybenzyl)amino-2-phenylpiperidine), caused a dextral shift of the first sigmoidal response, indicating the involvement of an NK1 receptor. This was further supported by a concentration-dependent response of the NK1 receptor agonist [Sar9Met(O2)11]substance P with an EC50 value of 235.0+/-53.9 nM. Increasing concentrations of CP 99,994 (0.1, 0.3 and 1 microM) produced a parallel dextral shift of the [Sar9Met(O2)11]substance P curve with a slope of the Schild regression significantly different from unity (1.59). The neurokinin A concentration-response curve, with an EC50 value of 68.87+/-16.23 nM, was not significantly changed by the non-peptide NK2 receptor antagonist SR 48,968 ((S)-N-methyl-N-(4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophe nyl)butyl)bezamide). In additional studies, the peptide NK2 receptor antagonists, GR 94,800 (PhCO-Ala-Ala-DTrp-Phe-DPro-Pro-NleNH2) and PD 147,714 ((2,3-diOMeZ)-(S)Trp(S)alphaMePheGlyNH2), did not change the response to neurokinin A. However, CP 99,994 totally inhibited neurokinin A responses at 0.5 microM and above. The NK2 receptor agonist, [beta-Ala8]neurokinin A-(4-10), caused only an increase in short-circuit current in microM concentrations, whereas the NK3 receptor agonist, senktide, did not elicit a response. These results indicate, that substance P and neurokinin A mediate ion transport in porcine jejunum through NK1 receptors. However, tachykinins seem to activate another receptor. Two active conformers of the NK1 receptor might be present.

Three types of tachykinin receptors, namely NK1, NK2 and NK3, are known to preferentially interact with substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), respectively. Experimental evidence indicates that SP and NKA modulate the activity of inflammatory and immune cells, including mononuclear ones. This study evaluated the effects of mammalian tachykinins and selective tachykinin agonists and antagonists on human monocytes isolated from healthy donors: SP, NKA and NKB all evoked a dose-dependent superoxide anion (O2-) production and the NK2 selective agonist [beta-Ala8]-NKA(4-10) induced a full response. The NK3 selective agonist senktide was inactive, while the NK1 selective agonists septide and [Sar9Met(O2)11]SP displayed some effects. These results indicate that NK2 and also some NK1 receptors are present in monocytes isolated from healthy donors. The role of tachykinin receptor activation in rheumatoid arthritis was also investigated, by measuring O2- production and TNF-alpha mRNA expression in monocytes isolated from rheumatoid patients. Tachykinins enhanced the expression of this cytokine in both control and rheumatoid monocytes and NK2 receptor stimulation was shown to trigger an enhanced respiratory burst in monocytes from rheumatoid patients. In conclusion, these results indicate that NK2 and NK1 receptors are present on human monocytes, the former being preferentially involved in rheumatoid arthritis.

The major pulmonary effects of tachykinins are produced by activation of both NK1- and NK2-receptors. A variety of animal models have been used to profile activity of the tachykinins, particularly rodents and guinea pigs, but little information exists regarding methods to evaluate NK1- and NK2-receptor antagonist activity in dogs. This study describes a simple method in dogs to measure NK1- and NK2-receptor agonist and antagonist activity of drugs in the same preparation. We measured pulmonary resistance (RL), dynamic lung compliance (CDyn), minute volume (MV), and mean arterial blood pressure (MAP) before and after challenge with aerosolized NKA (1%) and i.v. SP (100 ng/kg) to quantify responses to the tachykinin challenge. Challenge with NKA produced an increase in RL and a decrease in CDyn, and this bronchospasm was inhibited by the NK2-antagonist SR 48968 (ID50 RL=1.3 mg/kg and ID50 CDyn=1.3 mg/kg, p.o.). The NK1-antagonist, CP 99994 was inactive against NKA-induced bronchospasm at doses up to 10 mg/kg, p.o. When the dogs were challenged with SP, there was a fall in MAP and an increase in MV and both responses were inhibited by CP 99994 (ID50 MV=2.3 mg/kg and ID50 BP=4.5 mg/kg, p.o.), but not by SR 48968 at doses up to 3 mg/kg, p.o. These results identify that NK2-receptors mediate the bronchoconstrictor effect of NKA, and NK1-receptors mediate the hypotension and respiratory stimulation due to SP in dogs. This method offers many advantages for evaluating the effects of tachykinin antagonists including the fact that it is relatively simple to perform and has the capacity to assess both NK1 and NK2 antagonist activity in the same preparation.